Structured Review

Qiagen igg antibodies
Schematic overview of selected results from the employed discovery process. Here, demonstrated for human IgGs against the antigen, Dp8. a Polyclonal phage ELISA signals against different venom fractions and negative control antigens (Cbtx α-cobratoxin, b-Gal β-galactosidase, Strep Streptavidin). b Monoclonal scFv ELISA signals against Dp8. c Summary of <t>DNA</t> sequencing results. Sequences are defined as unique based on V H and V L CDR3 sequences. d Monoclonal <t>IgG</t> ELISA signals for converted clones demonstrating retained binding for the majority of the clones upon conversion from the scFv format
Igg Antibodies, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 11705 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies"

Article Title: In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies

Journal: Nature Communications

doi: 10.1038/s41467-018-06086-4

Schematic overview of selected results from the employed discovery process. Here, demonstrated for human IgGs against the antigen, Dp8. a Polyclonal phage ELISA signals against different venom fractions and negative control antigens (Cbtx α-cobratoxin, b-Gal β-galactosidase, Strep Streptavidin). b Monoclonal scFv ELISA signals against Dp8. c Summary of DNA sequencing results. Sequences are defined as unique based on V H and V L CDR3 sequences. d Monoclonal IgG ELISA signals for converted clones demonstrating retained binding for the majority of the clones upon conversion from the scFv format
Figure Legend Snippet: Schematic overview of selected results from the employed discovery process. Here, demonstrated for human IgGs against the antigen, Dp8. a Polyclonal phage ELISA signals against different venom fractions and negative control antigens (Cbtx α-cobratoxin, b-Gal β-galactosidase, Strep Streptavidin). b Monoclonal scFv ELISA signals against Dp8. c Summary of DNA sequencing results. Sequences are defined as unique based on V H and V L CDR3 sequences. d Monoclonal IgG ELISA signals for converted clones demonstrating retained binding for the majority of the clones upon conversion from the scFv format

Techniques Used: Enzyme-linked Immunosorbent Assay, Negative Control, DNA Sequencing, Clone Assay, Binding Assay

2) Product Images from "Microbial Community Composition in Take-All Suppressive Soils"

Article Title: Microbial Community Composition in Take-All Suppressive Soils

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02198

Correlation between biodiversity index S (species), d (individual), H′ (Shannon), and Simpson (expressed as 1- λ) of endophytic bacteria and rhizosphere and endosphere ascomycetes.
Figure Legend Snippet: Correlation between biodiversity index S (species), d (individual), H′ (Shannon), and Simpson (expressed as 1- λ) of endophytic bacteria and rhizosphere and endosphere ascomycetes.

Techniques Used:

Dendrograms and distance based redundancy analyses (dbRDA) plots of the endosphere of wheat plants grown in conducive and suppressive soils, based on DGGE profiles of total bacteria (A) , actinomycetes (B) , total fungi (C) , and ascomycete (D) and soil chemical parameters (P, K, OM, Al sat, CICE, and Σ basis). Soil parameters are represented with black lines in the dbRDA plots, the length and position represent the correlation ( P
Figure Legend Snippet: Dendrograms and distance based redundancy analyses (dbRDA) plots of the endosphere of wheat plants grown in conducive and suppressive soils, based on DGGE profiles of total bacteria (A) , actinomycetes (B) , total fungi (C) , and ascomycete (D) and soil chemical parameters (P, K, OM, Al sat, CICE, and Σ basis). Soil parameters are represented with black lines in the dbRDA plots, the length and position represent the correlation ( P

Techniques Used: Denaturing Gradient Gel Electrophoresis

3) Product Images from "Naturally Fermented Milk From Northern Senegal: Bacterial Community Composition and Probiotic Enrichment With Lactobacillus rhamnosus"

Article Title: Naturally Fermented Milk From Northern Senegal: Bacterial Community Composition and Probiotic Enrichment With Lactobacillus rhamnosus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02218

Heat map of the average top 50 bacterial genera for each community. The heat map is based on normalized 16S rRNA V4 amplicon sequence data obtained from bacterial DNA isolated from 120 lait caillé samples from six communities in northern Senegal. Samples were collected from plastic buckets in boutiques from Gaé ( n = 26), and from lahals in households from Didjiery ( n = 10), Keur Mbaye Peuhl ( n = 19), Pathé Badio ( n = 19), Guilado ( n = 23), and Medina Cheikh Mountaga ( n = 22). The number of observations for each bacterial genus was plotted in a rainbow scale from black to blue to green to red. Saturation was set at a value of 60 (1% of the maximum).
Figure Legend Snippet: Heat map of the average top 50 bacterial genera for each community. The heat map is based on normalized 16S rRNA V4 amplicon sequence data obtained from bacterial DNA isolated from 120 lait caillé samples from six communities in northern Senegal. Samples were collected from plastic buckets in boutiques from Gaé ( n = 26), and from lahals in households from Didjiery ( n = 10), Keur Mbaye Peuhl ( n = 19), Pathé Badio ( n = 19), Guilado ( n = 23), and Medina Cheikh Mountaga ( n = 22). The number of observations for each bacterial genus was plotted in a rainbow scale from black to blue to green to red. Saturation was set at a value of 60 (1% of the maximum).

Techniques Used: Amplification, Sequencing, Isolation, Northern Blot

4) Product Images from "Polychromophilus spp. (Haemosporida) in Malagasy bats: host specificity and insights on invertebrate vectors"

Article Title: Polychromophilus spp. (Haemosporida) in Malagasy bats: host specificity and insights on invertebrate vectors

Journal: Malaria Journal

doi: 10.1186/s12936-018-2461-8

Bayesian reconstruction showing Polychromophilus spp. infecting Malagasy bats (in blue) and Nycteribiidae (in red) based on cytochrome b . Only values in the major nodes were represented for higher posterior probabilities ( > 0.9). Polychromophilus melanipherus identified in Paratriaenops furculus are followed by an asterisk. Mad: Madagascar, Gui: Guinea, Sw: Switzerland, Gab: Gabon, Ple: Penicillidia leptothrinax , Psp: Penicillidia sp. (cf. fulvida ), Nsty: Nycteribia stylidiopsis
Figure Legend Snippet: Bayesian reconstruction showing Polychromophilus spp. infecting Malagasy bats (in blue) and Nycteribiidae (in red) based on cytochrome b . Only values in the major nodes were represented for higher posterior probabilities ( > 0.9). Polychromophilus melanipherus identified in Paratriaenops furculus are followed by an asterisk. Mad: Madagascar, Gui: Guinea, Sw: Switzerland, Gab: Gabon, Ple: Penicillidia leptothrinax , Psp: Penicillidia sp. (cf. fulvida ), Nsty: Nycteribia stylidiopsis

Techniques Used:

5) Product Images from "Comparison of initial oral microbiomes of young adults with and without cavitated dentin caries lesions using an in situ biofilm model"

Article Title: Comparison of initial oral microbiomes of young adults with and without cavitated dentin caries lesions using an in situ biofilm model

Journal: Scientific Reports

doi: 10.1038/s41598-018-32361-x

Richness ( a ) and alpha diversity ( b ) measures of different time points (2 h-, 4 h-, and 8 h-biofilms) and for saliva and indications (CC: cavitated caries, NC: no cavitated caries). OTUs present in the NTC were removed and richness and Shannon’s alpha diversity measures are shown. The individual points represent individual diversity estimates.
Figure Legend Snippet: Richness ( a ) and alpha diversity ( b ) measures of different time points (2 h-, 4 h-, and 8 h-biofilms) and for saliva and indications (CC: cavitated caries, NC: no cavitated caries). OTUs present in the NTC were removed and richness and Shannon’s alpha diversity measures are shown. The individual points represent individual diversity estimates.

Techniques Used:

Relative abundances of individual taxa per sample at phylum ( a ) and family ( b ) level, and summarized per indication at phylum ( c ) and family ( d ) level. The abundances were computed over all OTUs before filtering out OTUs present in controls. Samples were grouped by indication (CC: cavitated caries, NC: no cavitated caries) and time point (2 h-, 4 h- and 8 h-biofilms, and saliva) in ( a ) and ( b ); in ( c ) and ( d ), the indication is shown per time point. Taxa with a relative abundance below 5% per sample ( a , b ) and indication ( c , d ) were grouped into one category (“rel. abund.
Figure Legend Snippet: Relative abundances of individual taxa per sample at phylum ( a ) and family ( b ) level, and summarized per indication at phylum ( c ) and family ( d ) level. The abundances were computed over all OTUs before filtering out OTUs present in controls. Samples were grouped by indication (CC: cavitated caries, NC: no cavitated caries) and time point (2 h-, 4 h- and 8 h-biofilms, and saliva) in ( a ) and ( b ); in ( c ) and ( d ), the indication is shown per time point. Taxa with a relative abundance below 5% per sample ( a , b ) and indication ( c , d ) were grouped into one category (“rel. abund.

Techniques Used:

6) Product Images from "Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells"

Article Title: Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells

Journal: eLife

doi: 10.7554/eLife.36187

GFP-positive FACS-sorted cells from P7 Tie2-GFP mice represent pure populations of ECs. ( A ) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing performed on GFP-negative FACS-sorted cells, and GFPpos indicates sequencing performed on GFP-positive FACS-sorted cells. R1 and R2 indicate biological replicates. ( B ) Expression levels (TPMs) based on RNA-seq for the indicated genes. The top row of genes are known EC-expressed genes. EC-specific transcripts comprise ~15% of total lung transcripts. The middle row of genes are known immune or mural cell-expressed genes. The bottom row of genes are known abundant parenchymal-expressed genes. In this and subsequent figures, cell or tissue fractions are indicated by the following symbols: GFP-negative, circle; GFP-positive, triangle; Total, square. GFP-positive represents FACS-purified ECs.
Figure Legend Snippet: GFP-positive FACS-sorted cells from P7 Tie2-GFP mice represent pure populations of ECs. ( A ) Heatmap indicating pairwise Pearson correlations for RNA-seq TPMs for protein-coding genes. Total indicates sequencing performed on total dissociated tissue, GFPneg indicates sequencing performed on GFP-negative FACS-sorted cells, and GFPpos indicates sequencing performed on GFP-positive FACS-sorted cells. R1 and R2 indicate biological replicates. ( B ) Expression levels (TPMs) based on RNA-seq for the indicated genes. The top row of genes are known EC-expressed genes. EC-specific transcripts comprise ~15% of total lung transcripts. The middle row of genes are known immune or mural cell-expressed genes. The bottom row of genes are known abundant parenchymal-expressed genes. In this and subsequent figures, cell or tissue fractions are indicated by the following symbols: GFP-negative, circle; GFP-positive, triangle; Total, square. GFP-positive represents FACS-purified ECs.

Techniques Used: FACS, Mouse Assay, RNA Sequencing Assay, Sequencing, Expressing, Purification

7) Product Images from "Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats"

Article Title: Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats

Journal: International Journal of Obesity (2005)

doi: 10.1038/s41366-018-0094-1

Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p
Figure Legend Snippet: Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p

Techniques Used: Expressing, DNA Methylation Assay, Quantitative RT-PCR, Functional Assay, Methylation

8) Product Images from "Identification and comparison of key RNA interference machinery from western corn rootworm, fall armyworm, and southern green stink bug"

Article Title: Identification and comparison of key RNA interference machinery from western corn rootworm, fall armyworm, and southern green stink bug

Journal: PLoS ONE

doi: 10.1371/journal.pone.0203160

Properties of core mi- and si-RNA pathway RNaseIII-domain containing proteins in WCR, FAW, and SGSB. (A) The predicted protein domains encoded by the Drosha, Dicer, Dicer-1, and Dicer-2 transcripts of A . pisum ( Api : XP_003247913.1, XP_001944314.2, XP_016665103.1), B . mori ( Bmo : XM_004928209.1, XM_004922309.1, XP_012551309.1), C . elegans ( Cel : NP_492599 . 1 , NP_498761 . 2 ), D . melanogaster ( Dme : NP_477436 . 1 , NP_524453 . 1 , NP_523778 . 2 ), D . virgifera virgifera (WCR– Dvi : MG225416, MG225417, MG225418), H . sapiens ( Hsa : NP_001093882.1, NP_001258211.1), N . viridula (SGSB– Nvi : MG225445, MG225446, MG225447), S . frugiperda (FAW– Sfr : MG225429, MG225430, MG225431), and T . castaneum ( Tca : XP_967454.2, XP_008199045.1, XP_008201496.1). Predicted domains include two RNaseIIIs (PF00636 and PF14622), a DCR dimer motif (PF03368), a DSRM (PF00035), a helicase C (PF00271), a PAZ (PF02170), and either a ResIII (PF04851) or DEAD-box helicase (PF00270) domain. E-values for domains predicted in the WCR, FAW, and SGSB proteins range from 4.0×10 −10 to 1.1×10 −36 , with the exception of the DCR-1 and DCR-2 C-terminal DSRMs. (B) Maximum likelihood phylogenetic tree topology of translated RNaseIII protein-coding sequences (1000 bootstrap replications). Black symbols above each entry indicate phylogenetic order as follows: circles (●) for Diptera, squares (■) for Lepidoptera, diamonds (◆) for Coleoptera, teardrops (💧) for Hemiptera, xrhombus (❖) for Primate, and sunburst (✸) for Rhabditida.
Figure Legend Snippet: Properties of core mi- and si-RNA pathway RNaseIII-domain containing proteins in WCR, FAW, and SGSB. (A) The predicted protein domains encoded by the Drosha, Dicer, Dicer-1, and Dicer-2 transcripts of A . pisum ( Api : XP_003247913.1, XP_001944314.2, XP_016665103.1), B . mori ( Bmo : XM_004928209.1, XM_004922309.1, XP_012551309.1), C . elegans ( Cel : NP_492599 . 1 , NP_498761 . 2 ), D . melanogaster ( Dme : NP_477436 . 1 , NP_524453 . 1 , NP_523778 . 2 ), D . virgifera virgifera (WCR– Dvi : MG225416, MG225417, MG225418), H . sapiens ( Hsa : NP_001093882.1, NP_001258211.1), N . viridula (SGSB– Nvi : MG225445, MG225446, MG225447), S . frugiperda (FAW– Sfr : MG225429, MG225430, MG225431), and T . castaneum ( Tca : XP_967454.2, XP_008199045.1, XP_008201496.1). Predicted domains include two RNaseIIIs (PF00636 and PF14622), a DCR dimer motif (PF03368), a DSRM (PF00035), a helicase C (PF00271), a PAZ (PF02170), and either a ResIII (PF04851) or DEAD-box helicase (PF00270) domain. E-values for domains predicted in the WCR, FAW, and SGSB proteins range from 4.0×10 −10 to 1.1×10 −36 , with the exception of the DCR-1 and DCR-2 C-terminal DSRMs. (B) Maximum likelihood phylogenetic tree topology of translated RNaseIII protein-coding sequences (1000 bootstrap replications). Black symbols above each entry indicate phylogenetic order as follows: circles (●) for Diptera, squares (■) for Lepidoptera, diamonds (◆) for Coleoptera, teardrops (💧) for Hemiptera, xrhombus (❖) for Primate, and sunburst (✸) for Rhabditida.

Techniques Used:

Properties of core mi- and siRNA pathway double-stranded RNA binding proteins in WCR, FAW, and SGSB. (A) The predicted protein domains encoded by the Pasha-PA, LOQS-PB, and R2D2 transcripts of A . pisum (Api: XP_001947403.1, XP_016657757.1), B . mori ( Bmo : XP_012552270.1, XP_012550849.1, NP_001182007.1), C . elegans ( Cel : NP_001293461.1, NP_499265.1), D . melanogaster ( Dme : NP_651879.1, NP_609646.1, NP_609152.1), D . virgifera virgifera (WCR– Dvi : MG225419, MG225420, MG225423), H . sapiens ( Has : NP_073557.3, NP_599150.1), N . viridula (SGSB– Nvi : MG225448, MG225451, MG225453), S . frugiperda (FAW– Sfr : MG225432, MG225435, MG225438), and T . castaneum ( Tca : XP_971282.1, XP_966668.1, NP_001128425.1). Predicted domains include two to three DSRMs (PF00035) and a WW (PF00397). E-values for domains predicted in WCR, FAW, and SGSB range from 1.5×10 −3 to 9.2×10 −15 . (B) Maximum likelihood phylogenetic tree topology of translated dsRBP protein-coding sequences (1000 bootstrap replications). Black symbols above each entry indicate phylogenetic order as follows: circles (●) for Diptera, squares (■) for Lepidoptera, diamonds (◆) for Coleoptera, teardrops (💧) for Hemiptera, xrhombus (❖) for Primate, and sunburst (✸) for Rhabditida.
Figure Legend Snippet: Properties of core mi- and siRNA pathway double-stranded RNA binding proteins in WCR, FAW, and SGSB. (A) The predicted protein domains encoded by the Pasha-PA, LOQS-PB, and R2D2 transcripts of A . pisum (Api: XP_001947403.1, XP_016657757.1), B . mori ( Bmo : XP_012552270.1, XP_012550849.1, NP_001182007.1), C . elegans ( Cel : NP_001293461.1, NP_499265.1), D . melanogaster ( Dme : NP_651879.1, NP_609646.1, NP_609152.1), D . virgifera virgifera (WCR– Dvi : MG225419, MG225420, MG225423), H . sapiens ( Has : NP_073557.3, NP_599150.1), N . viridula (SGSB– Nvi : MG225448, MG225451, MG225453), S . frugiperda (FAW– Sfr : MG225432, MG225435, MG225438), and T . castaneum ( Tca : XP_971282.1, XP_966668.1, NP_001128425.1). Predicted domains include two to three DSRMs (PF00035) and a WW (PF00397). E-values for domains predicted in WCR, FAW, and SGSB range from 1.5×10 −3 to 9.2×10 −15 . (B) Maximum likelihood phylogenetic tree topology of translated dsRBP protein-coding sequences (1000 bootstrap replications). Black symbols above each entry indicate phylogenetic order as follows: circles (●) for Diptera, squares (■) for Lepidoptera, diamonds (◆) for Coleoptera, teardrops (💧) for Hemiptera, xrhombus (❖) for Primate, and sunburst (✸) for Rhabditida.

Techniques Used: RNA Binding Assay

9) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky667

TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

10) Product Images from "Analysis of Syndecan-2 Methylation in Bowel Lavage Fluid for the Detection of Colorectal Neoplasm"

Article Title: Analysis of Syndecan-2 Methylation in Bowel Lavage Fluid for the Detection of Colorectal Neoplasm

Journal: Gut and Liver

doi: 10.5009/gnl17357

Methylation assessment of SDC2 gene in colorectal tissues by bisulfite pyrosequencing. The methylation level of the SDC2 gene was evaluated in normal mucosa (N), hyperplastic polyp (HP), tubular adenoma (TA) and villous adenoma and high-grade dysplasia tissues (VA HGD). The methylation indexes (MtIs) of each sample are represented with box-and-whisker plots. The difference in the MtI of SDC2 was statistically significant at p
Figure Legend Snippet: Methylation assessment of SDC2 gene in colorectal tissues by bisulfite pyrosequencing. The methylation level of the SDC2 gene was evaluated in normal mucosa (N), hyperplastic polyp (HP), tubular adenoma (TA) and villous adenoma and high-grade dysplasia tissues (VA HGD). The methylation indexes (MtIs) of each sample are represented with box-and-whisker plots. The difference in the MtI of SDC2 was statistically significant at p

Techniques Used: Methylation, Whisker Assay

11) Product Images from "Detection of the MYD88 p.L265P Mutation in the CSF of a Patient With Secondary Central Nervous System Lymphoma"

Article Title: Detection of the MYD88 p.L265P Mutation in the CSF of a Patient With Secondary Central Nervous System Lymphoma

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2018.00382

Droplet digital PCR results from examination of the CNS tissue and the CSF-ctDNA for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.
Figure Legend Snippet: Droplet digital PCR results from examination of the CNS tissue and the CSF-ctDNA for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.

Techniques Used: Digital PCR, Mutagenesis

12) Product Images from "Inhibitory effects of fluoroquinolone antibiotics on Babesia divergens and Babesia microti, blood parasites of veterinary and zoonotic importance"

Article Title: Inhibitory effects of fluoroquinolone antibiotics on Babesia divergens and Babesia microti, blood parasites of veterinary and zoonotic importance

Journal: Infection and Drug Resistance

doi: 10.2147/IDR.S159519

PCR of the ss-rRNA gene in different organs of Babesia microti- infected mice treated with DDW (positive control), 25 mg⋅kg –1 diminazene aceturate (DA), and DA (10 mg⋅kg –1 ) combined with enoxacin (50 mg⋅kg –1 ). Note: M indicates a 100 bp DNA ladder. Abbreviations: NC, negative control; PC, positive control; DDW, double-distilled water; S, spleen; L, lung; H, heart; K, kidney; DA, diminazene aceturate; PCR, polymerase chain reaction.
Figure Legend Snippet: PCR of the ss-rRNA gene in different organs of Babesia microti- infected mice treated with DDW (positive control), 25 mg⋅kg –1 diminazene aceturate (DA), and DA (10 mg⋅kg –1 ) combined with enoxacin (50 mg⋅kg –1 ). Note: M indicates a 100 bp DNA ladder. Abbreviations: NC, negative control; PC, positive control; DDW, double-distilled water; S, spleen; L, lung; H, heart; K, kidney; DA, diminazene aceturate; PCR, polymerase chain reaction.

Techniques Used: Polymerase Chain Reaction, Infection, Mouse Assay, Positive Control, Negative Control

13) Product Images from "Ultra-long-acting removable drug delivery system for HIV treatment and prevention"

Article Title: Ultra-long-acting removable drug delivery system for HIV treatment and prevention

Journal: Nature Communications

doi: 10.1038/s41467-018-06490-w

In vitro inhibition of HIV-1 infection with serum from ultra-LA dolutegravir-treated mice. Serum from female NSG mice administered with ultra-LA dolutegravir (250 mg/kg) collected at days 7 ( n = 3), 28 ( n = 4), and 84 ( n = 2) was used for a TZM-bl cell-based assay (measured in duplicates). a Inhibition of HIV-1 infection (%) with various dilution of serum. Solid lines indicate nonlinear curve fit for the data. b Comparison of in vitro inhibitory activity of 1% serum collected from ultra-LA dolutegravir-treated NSG mice at the indicated time points (means ± s.e.m). c Comparison of in vitro inhibitory activity (%) of serum from ultra-LA dolutegravir-treated mice and log 10 concentration of dolutegravir. A non-parametric rank-based correlation analysis accounting for clustered observations (Kendall’s tau) was used
Figure Legend Snippet: In vitro inhibition of HIV-1 infection with serum from ultra-LA dolutegravir-treated mice. Serum from female NSG mice administered with ultra-LA dolutegravir (250 mg/kg) collected at days 7 ( n = 3), 28 ( n = 4), and 84 ( n = 2) was used for a TZM-bl cell-based assay (measured in duplicates). a Inhibition of HIV-1 infection (%) with various dilution of serum. Solid lines indicate nonlinear curve fit for the data. b Comparison of in vitro inhibitory activity of 1% serum collected from ultra-LA dolutegravir-treated NSG mice at the indicated time points (means ± s.e.m). c Comparison of in vitro inhibitory activity (%) of serum from ultra-LA dolutegravir-treated mice and log 10 concentration of dolutegravir. A non-parametric rank-based correlation analysis accounting for clustered observations (Kendall’s tau) was used

Techniques Used: In Vitro, Inhibition, Infection, Mouse Assay, Cell Based Assay, Activity Assay, Concentration Assay

A single dose of ultra-LA dolutegravir protects against multiple high dose HIV-1 challenges. a Experimental design. BLT mice were treated with ultra-LA dolutegravir ( n = 5) or placebo ( n = 5) and vaginally exposed to HIV CH040 or HIV THRO 1 week, and 7 weeks later. The implant was removed from two mice 8 weeks after second HIV challenge. Cell-associated HIV-DNA in multiple tissues was analyzed at the end of the experiment. Plasma HIV-RNA concentration in control ( b ) and treated mice ( c ). d Time-to-events plot illustrating the estimated probability of protection ( p = 0.02). e Plasma concentration of dolutegravir. f Plasma concentration of dolutegravir ( n = 2) after the implant removal relative to dolutegravir concentration immediately before the implant removal (dotted line). Individual measurements and median ± range is shown. Experiment was conducted once
Figure Legend Snippet: A single dose of ultra-LA dolutegravir protects against multiple high dose HIV-1 challenges. a Experimental design. BLT mice were treated with ultra-LA dolutegravir ( n = 5) or placebo ( n = 5) and vaginally exposed to HIV CH040 or HIV THRO 1 week, and 7 weeks later. The implant was removed from two mice 8 weeks after second HIV challenge. Cell-associated HIV-DNA in multiple tissues was analyzed at the end of the experiment. Plasma HIV-RNA concentration in control ( b ) and treated mice ( c ). d Time-to-events plot illustrating the estimated probability of protection ( p = 0.02). e Plasma concentration of dolutegravir. f Plasma concentration of dolutegravir ( n = 2) after the implant removal relative to dolutegravir concentration immediately before the implant removal (dotted line). Individual measurements and median ± range is shown. Experiment was conducted once

Techniques Used: Mouse Assay, Concentration Assay

Suppression of systemic HIV infection by ultra-LA dolutegravir. a Experimental design. BLT mice infected with HIV-1 JRC-SF were subcutaneously administered ultra-LA dolutegravir ( n = 4) or placebo ( n = 4) and monitored for plasma dolutegravir and HIV-RNA concentrations. b Concentration of dolutegravir in plasma from ultra-LA dolutegravir-treated mice. Plasma HIV-RNA concentration in individual mice c or means ± s.e.m. d HIV-RNA in cervico-vaginal secretions (CVS) of individual mice ( e ) or means ± s.e.m. f CD4 + T cells (relative amount of CD3 + cells) in CVS of individual mice ( g ) or means ± s.e.m. h Yellow lines in c , e , and g ultra-long-acting dolutegravir-treated mice, blue lines placebo-treated mice. Experiment was conducted once
Figure Legend Snippet: Suppression of systemic HIV infection by ultra-LA dolutegravir. a Experimental design. BLT mice infected with HIV-1 JRC-SF were subcutaneously administered ultra-LA dolutegravir ( n = 4) or placebo ( n = 4) and monitored for plasma dolutegravir and HIV-RNA concentrations. b Concentration of dolutegravir in plasma from ultra-LA dolutegravir-treated mice. Plasma HIV-RNA concentration in individual mice c or means ± s.e.m. d HIV-RNA in cervico-vaginal secretions (CVS) of individual mice ( e ) or means ± s.e.m. f CD4 + T cells (relative amount of CD3 + cells) in CVS of individual mice ( g ) or means ± s.e.m. h Yellow lines in c , e , and g ultra-long-acting dolutegravir-treated mice, blue lines placebo-treated mice. Experiment was conducted once

Techniques Used: Infection, Mouse Assay, Concentration Assay

Implant formation and dolutegravir concentration in plasma and female reproductive tract. a Solidified ultra-LA dolutegravir surgically removed 7 days after administration. Scale bar represents 1 cm. b Pharmacological profile of NSG and BLT mice administered ultra-LA dolutegravir (250 mg/kg), five independent experiments, n = 21. c Dolutegravir concentrations in plasma and parts of female reproductive tract 7, 28, and 84 days after subcutaneous administration of ultra-LA dolutegravir (250 mg/kg). The data from two independent experiments are shown, n = 6 for each time point. Shown are individual animals and the means ± s.e.m. (standard error of the mean). Tissue density of 1 g/ml was used to compare dolutegravir concentration between tissues and plasma. d Plasma concentration of dolutegravir in 2 rhesus macaques administered 1 ml of formulation used in ( b ) and c (100 mg of dolutegravir)
Figure Legend Snippet: Implant formation and dolutegravir concentration in plasma and female reproductive tract. a Solidified ultra-LA dolutegravir surgically removed 7 days after administration. Scale bar represents 1 cm. b Pharmacological profile of NSG and BLT mice administered ultra-LA dolutegravir (250 mg/kg), five independent experiments, n = 21. c Dolutegravir concentrations in plasma and parts of female reproductive tract 7, 28, and 84 days after subcutaneous administration of ultra-LA dolutegravir (250 mg/kg). The data from two independent experiments are shown, n = 6 for each time point. Shown are individual animals and the means ± s.e.m. (standard error of the mean). Tissue density of 1 g/ml was used to compare dolutegravir concentration between tissues and plasma. d Plasma concentration of dolutegravir in 2 rhesus macaques administered 1 ml of formulation used in ( b ) and c (100 mg of dolutegravir)

Techniques Used: Concentration Assay, Mouse Assay

14) Product Images from "Metal Transformation by a Novel Pelosinus Isolate From a Subsurface Environment"

Article Title: Metal Transformation by a Novel Pelosinus Isolate From a Subsurface Environment

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01689

Maximum Likelihood tree showing phylogenetic relationships of the 16S rRNA genes in the Class Negativicutes; the types species for each genus in the four families within this class is included. Branch supports determined by Bayesian estimation ≥80 are shown at branch points. Scale bar indicates 0.1 changes per nucleotide. Tree was rooted with 4 species (not shown): Bacillus subtilis (AB042061), Clostridium acetobutylicum (AE001437), Desulfotomaculum acetoxidans (Y11566), and Heliobacillus mobilis (AB100835).
Figure Legend Snippet: Maximum Likelihood tree showing phylogenetic relationships of the 16S rRNA genes in the Class Negativicutes; the types species for each genus in the four families within this class is included. Branch supports determined by Bayesian estimation ≥80 are shown at branch points. Scale bar indicates 0.1 changes per nucleotide. Tree was rooted with 4 species (not shown): Bacillus subtilis (AB042061), Clostridium acetobutylicum (AE001437), Desulfotomaculum acetoxidans (Y11566), and Heliobacillus mobilis (AB100835).

Techniques Used:

15) Product Images from "NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease"

Article Title: NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease

Journal: Nature Communications

doi: 10.1038/s41467-018-06125-0

Vancomycin treatment or supplementation with butyrate ablates the Nlrp1 phenotype. a Stool from WT and Nlrp1 −/− mice was harvested before and after vancomycin (50 mg/L) treatment for 4 weeks. Bacterial DNA from stool was isolated from untreated (UT) and vancomycin-treated mice, and depletion of the Coccoides group (belonging to the Clostridiales phylum) was confirmed using specific 16S primers by quantitative PCR. Results were normalized to the total bacteria present in the stool. b Vancomycin-treated mice were subjected to 3% (w/v) DSS for 6 days and disease severity was measured according to percentage weight loss and c colon length. Short chain fatty acid analysis was performed on fecal samples collected from WT and Nlrp1 −/− mice before (UT) and after vancomycin treatment. The concentration of d butyrate and e propionate was measured by gas chromatography mass spectrometry. f WT and Nlrp1 −/− mice were supplemented with 2% butyrate in drinking water ad libitum for 28 days, and then subjected to 2.5% (w/v) DSS for 6 days and disease severity measured according to percentage weight loss or g colon length. Data are representative of 3 independent experiments with 3–6 mice per group. Means ± SEM; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. A two-tailed unpaired t- test was used to determine statistical significance between two groups; and a two-way ANOVA with Tukey’s post-hoc comparisons was performed on data that involved more than two comparisons
Figure Legend Snippet: Vancomycin treatment or supplementation with butyrate ablates the Nlrp1 phenotype. a Stool from WT and Nlrp1 −/− mice was harvested before and after vancomycin (50 mg/L) treatment for 4 weeks. Bacterial DNA from stool was isolated from untreated (UT) and vancomycin-treated mice, and depletion of the Coccoides group (belonging to the Clostridiales phylum) was confirmed using specific 16S primers by quantitative PCR. Results were normalized to the total bacteria present in the stool. b Vancomycin-treated mice were subjected to 3% (w/v) DSS for 6 days and disease severity was measured according to percentage weight loss and c colon length. Short chain fatty acid analysis was performed on fecal samples collected from WT and Nlrp1 −/− mice before (UT) and after vancomycin treatment. The concentration of d butyrate and e propionate was measured by gas chromatography mass spectrometry. f WT and Nlrp1 −/− mice were supplemented with 2% butyrate in drinking water ad libitum for 28 days, and then subjected to 2.5% (w/v) DSS for 6 days and disease severity measured according to percentage weight loss or g colon length. Data are representative of 3 independent experiments with 3–6 mice per group. Means ± SEM; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. A two-tailed unpaired t- test was used to determine statistical significance between two groups; and a two-way ANOVA with Tukey’s post-hoc comparisons was performed on data that involved more than two comparisons

Techniques Used: Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Concentration Assay, Gas Chromatography, Mass Spectrometry, Two Tailed Test

16) Product Images from "Germline-activating mutations in PIK3CD compromise B cell development and function"

Article Title: Germline-activating mutations in PIK3CD compromise B cell development and function

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20180010

Pik3cd GOF B cells show defective switching but normal expansion and affinity maturation in vivo. WT or Pik3cd GOF SWHEL cells were transferred to WT congenic hosts, which were then immunized with HEL-2x-SRBC. (A) The expansion of SW HEL cells was tracked over time (mean ± SEM, n = 3–4 mice per group, representative experiment shown). (B) Percentage of cells with a plasmablast or GC phenotype was determined. (C) Percentage of cells that switched to IgG1 or were unswitched (IgM + ) was determined in the plasmablast and GC populations. (D) Levels of HEL-specific serum Ig of various classes at day 5.5 were determined by ELISA. (B–D, each linked point shows mean ± SEM [ n = 3–5] of single experiment). (E) Donor GC IgG1 + cells were sorted on day 10 and sequenced to identify mutations. Significant differences were determined by paired t tests. **, P
Figure Legend Snippet: Pik3cd GOF B cells show defective switching but normal expansion and affinity maturation in vivo. WT or Pik3cd GOF SWHEL cells were transferred to WT congenic hosts, which were then immunized with HEL-2x-SRBC. (A) The expansion of SW HEL cells was tracked over time (mean ± SEM, n = 3–4 mice per group, representative experiment shown). (B) Percentage of cells with a plasmablast or GC phenotype was determined. (C) Percentage of cells that switched to IgG1 or were unswitched (IgM + ) was determined in the plasmablast and GC populations. (D) Levels of HEL-specific serum Ig of various classes at day 5.5 were determined by ELISA. (B–D, each linked point shows mean ± SEM [ n = 3–5] of single experiment). (E) Donor GC IgG1 + cells were sorted on day 10 and sequenced to identify mutations. Significant differences were determined by paired t tests. **, P

Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay

A p110δ inhibitor partially alleviates the defects in B cell differentiation due to hyperactive PI3K signaling. (A–C) Sort-purified transitional B cells from healthy donors or PIK3CD GOF patients ( n = 5) were cultured for 5 d with CD40L plus IL-21 in the absence or presence of the PI3K p110δ inhibitor leniolisib. Secretion of (A) IgM, (B) IgG, and (C) IgA was then determined. Data are expressed as mean percentage of Ig secreted (± SEM) by B cells stimulated with CD40L/IL-21. (D–G) Follicular B cells (B220 + CD93 − CD23 + CD21 lo ) were sorted from the spleens of WT or Pik3cd GOF mice. Cells were labeled with CFSE and stimulated with anti-CD40 + IL-4 in the absence or presence of leniolisib for 4 d. (D and E) Cells were stained for expression of IgG1 and the total percentage of IgG1+ B cells detected (D) and the % of IgG1+ B cells in each division as determined by CFSE dilution (E) were determined. (F) Expression of Aicda in WT or Pik3cd GOF B cells cultured with anti-CD40 + IL-4 was determined by qPCR. Plots show mean ± SEM, n = 3. (G) Plot of Aicda expression versus percentage IgG1+ cells generated following culture of WT or Pik3cd GOF B cells with anti-CD40 + IL-4 in the absence or presence of leniolisib. Significant differences were determined by two-way ANOVA. **, P
Figure Legend Snippet: A p110δ inhibitor partially alleviates the defects in B cell differentiation due to hyperactive PI3K signaling. (A–C) Sort-purified transitional B cells from healthy donors or PIK3CD GOF patients ( n = 5) were cultured for 5 d with CD40L plus IL-21 in the absence or presence of the PI3K p110δ inhibitor leniolisib. Secretion of (A) IgM, (B) IgG, and (C) IgA was then determined. Data are expressed as mean percentage of Ig secreted (± SEM) by B cells stimulated with CD40L/IL-21. (D–G) Follicular B cells (B220 + CD93 − CD23 + CD21 lo ) were sorted from the spleens of WT or Pik3cd GOF mice. Cells were labeled with CFSE and stimulated with anti-CD40 + IL-4 in the absence or presence of leniolisib for 4 d. (D and E) Cells were stained for expression of IgG1 and the total percentage of IgG1+ B cells detected (D) and the % of IgG1+ B cells in each division as determined by CFSE dilution (E) were determined. (F) Expression of Aicda in WT or Pik3cd GOF B cells cultured with anti-CD40 + IL-4 was determined by qPCR. Plots show mean ± SEM, n = 3. (G) Plot of Aicda expression versus percentage IgG1+ cells generated following culture of WT or Pik3cd GOF B cells with anti-CD40 + IL-4 in the absence or presence of leniolisib. Significant differences were determined by two-way ANOVA. **, P

Techniques Used: Cell Differentiation, Purification, Cell Culture, Mouse Assay, Labeling, Staining, Expressing, Real-time Polymerase Chain Reaction, Generated

Peripheral B cells in PIK3CD GOF patients have an immature phenotype. Expression of (A) CD5, (B) CD38, (C) CD21, (D) CD23, (E) CD44, (F) CD19, (G) CCR7, (H) CXCR4, (I) CXCR5, or (J) BCL2 was determined on transitional, naive, and memory B cells in peripheral blood of healthy donors or patients with PIK3CD GOF mutations ( n = 6–30). Histogram plots are representative of healthy donors (upper panels) or PIK3CD GOF patients (lower panels). The bar graphs depict the geometric mean fluorescence intensity (± SEM) of each indicated molecule. Significant differences were determined by unpaired Student's t tests. *, P
Figure Legend Snippet: Peripheral B cells in PIK3CD GOF patients have an immature phenotype. Expression of (A) CD5, (B) CD38, (C) CD21, (D) CD23, (E) CD44, (F) CD19, (G) CCR7, (H) CXCR4, (I) CXCR5, or (J) BCL2 was determined on transitional, naive, and memory B cells in peripheral blood of healthy donors or patients with PIK3CD GOF mutations ( n = 6–30). Histogram plots are representative of healthy donors (upper panels) or PIK3CD GOF patients (lower panels). The bar graphs depict the geometric mean fluorescence intensity (± SEM) of each indicated molecule. Significant differences were determined by unpaired Student's t tests. *, P

Techniques Used: Expressing, Fluorescence

Pik3cd GOF B cells show a division-related decrease in switching to multiple IgG isotypes. (A–C) Follicular B cells (B220 + CD93 − CD23 + CD21 lo ) were sorted from the spleens of WT or Pik3cd GOF mice. Cells were labeled with CFSE and stimulated with anti-CD40 + IL-4 (A), LPS + TGF-β (B) or LPS alone (C) for 4 d after which they were stained for IgG1, IgG2b and IgG3 as shown. Left panels show total percentage of cells of each isotype. Right panels show the percentage of switched cells in each division as determined by CFSE. Plots show mean ± SEM, n = 4–6. Significant differences were determined by paired t tests. **, P
Figure Legend Snippet: Pik3cd GOF B cells show a division-related decrease in switching to multiple IgG isotypes. (A–C) Follicular B cells (B220 + CD93 − CD23 + CD21 lo ) were sorted from the spleens of WT or Pik3cd GOF mice. Cells were labeled with CFSE and stimulated with anti-CD40 + IL-4 (A), LPS + TGF-β (B) or LPS alone (C) for 4 d after which they were stained for IgG1, IgG2b and IgG3 as shown. Left panels show total percentage of cells of each isotype. Right panels show the percentage of switched cells in each division as determined by CFSE. Plots show mean ± SEM, n = 4–6. Significant differences were determined by paired t tests. **, P

Techniques Used: Mouse Assay, Labeling, Staining

PIK3CD GOF transitional B cells have impaired molecular responses to IL-21. (A and B) Transitional B cells from healthy donors and PIK3CD GOF patients were cultured with CD40L alone or together with IL-21 and gene expression profiles for each were obtained by microarray. (A) Heat map of log 2 -transformed expression values for genes induced by IL-21 and involved in plasmablast generation. (B) Heat map of log 2 -transformed expression values for key driver genes that distinguish CD40L/IL-21–treated transitional B cells from healthy donors and PIK3CD GOF patients identified by NMF. (C and D) qPCR analysis of human tonsil B cell subsets (N, naive; M, memory; GC, germinal center; PC, plasma cells) showing differential expression of genes identified in B that are down-regulated (C) or up-regulated (D) in human PCs relative to other B cell populations. Values represent the mean ± SEM of experiments using tonsils from 4 different donors. (E and F) Transitional B cells from healthy donors or PIK3CD GOF patients were cultured with CD40L alone or together with IL-21 for 5 d. (E) Proportions of CD27 hi CD38 hi plasmablasts generated in these cultures were determined by flow cytometry. Contour plots are representative of data obtained from two to three independent experiments using transitional B cells isolated from different healthy donors or PIK3CD GOF patients. (F) Relative expression levels of the indicated genes were determined in transitional B cells by qPCR. Data are expressed as mean fold change (±SEM; n = 3) of gene expression in transitional B cells stimulated with CD40L/IL-21 relative to transitional B cells stimulated with CD40L alone. The dashed line represents a fold-change of 1.0, indicating no change in the presence versus the absence of IL-21 by CD40L-stimulated transitional B cells.
Figure Legend Snippet: PIK3CD GOF transitional B cells have impaired molecular responses to IL-21. (A and B) Transitional B cells from healthy donors and PIK3CD GOF patients were cultured with CD40L alone or together with IL-21 and gene expression profiles for each were obtained by microarray. (A) Heat map of log 2 -transformed expression values for genes induced by IL-21 and involved in plasmablast generation. (B) Heat map of log 2 -transformed expression values for key driver genes that distinguish CD40L/IL-21–treated transitional B cells from healthy donors and PIK3CD GOF patients identified by NMF. (C and D) qPCR analysis of human tonsil B cell subsets (N, naive; M, memory; GC, germinal center; PC, plasma cells) showing differential expression of genes identified in B that are down-regulated (C) or up-regulated (D) in human PCs relative to other B cell populations. Values represent the mean ± SEM of experiments using tonsils from 4 different donors. (E and F) Transitional B cells from healthy donors or PIK3CD GOF patients were cultured with CD40L alone or together with IL-21 for 5 d. (E) Proportions of CD27 hi CD38 hi plasmablasts generated in these cultures were determined by flow cytometry. Contour plots are representative of data obtained from two to three independent experiments using transitional B cells isolated from different healthy donors or PIK3CD GOF patients. (F) Relative expression levels of the indicated genes were determined in transitional B cells by qPCR. Data are expressed as mean fold change (±SEM; n = 3) of gene expression in transitional B cells stimulated with CD40L/IL-21 relative to transitional B cells stimulated with CD40L alone. The dashed line represents a fold-change of 1.0, indicating no change in the presence versus the absence of IL-21 by CD40L-stimulated transitional B cells.

Techniques Used: Cell Culture, Expressing, Microarray, Transformation Assay, Real-time Polymerase Chain Reaction, Generated, Flow Cytometry, Cytometry, Isolation

PIK3CD GOF mutations block B cell development in the bone marrow at the pre-BII stage. BM aspirates from healthy donors ( n = 6), patients with PIK3CD GOF mutations ( n = 3), or one patient following hematopoietic stem cell transplant were labeled with mAbs against CD34, CD19, CD20, CD10, IgM, and CD27. Proportions of pro-B (CD19 + CD34 + CD10 + CD20 − IgM − ), pre-BI (CD19 + CD34 − CD10 + CD20 − IgM − ), pre-BII (CD19 + CD34 − CD10 + CD20 dim IgM − ), immature (CD19 + CD34CD10 + CD20 + IgM + ), and recirculating mature (CD19 + CD34 − CD10 − CD20 + ) B cells were determined. (A–C) Contour plots in show CD34 versus CD10 staining to identify pro-B cells, which were then further analyzed for pre-BI, pre-BII, immature, and recirculating mature according to differential expression of CD20 and CD10. (D) Mean ± SEM of the different subsets of B cells in the BM. (E) Representative contour plots showing CD10 versus CD27 staining on CD20 + B cells, and (F) mean ± SEM of CD10 + CD27 − , naive (CD20 + CD10 − CD27 − ) and memory (CD20 + CD27 + ) B cells in the BM. Significant differences were determined by Student's t test. **, P
Figure Legend Snippet: PIK3CD GOF mutations block B cell development in the bone marrow at the pre-BII stage. BM aspirates from healthy donors ( n = 6), patients with PIK3CD GOF mutations ( n = 3), or one patient following hematopoietic stem cell transplant were labeled with mAbs against CD34, CD19, CD20, CD10, IgM, and CD27. Proportions of pro-B (CD19 + CD34 + CD10 + CD20 − IgM − ), pre-BI (CD19 + CD34 − CD10 + CD20 − IgM − ), pre-BII (CD19 + CD34 − CD10 + CD20 dim IgM − ), immature (CD19 + CD34CD10 + CD20 + IgM + ), and recirculating mature (CD19 + CD34 − CD10 − CD20 + ) B cells were determined. (A–C) Contour plots in show CD34 versus CD10 staining to identify pro-B cells, which were then further analyzed for pre-BI, pre-BII, immature, and recirculating mature according to differential expression of CD20 and CD10. (D) Mean ± SEM of the different subsets of B cells in the BM. (E) Representative contour plots showing CD10 versus CD27 staining on CD20 + B cells, and (F) mean ± SEM of CD10 + CD27 − , naive (CD20 + CD10 − CD27 − ) and memory (CD20 + CD27 + ) B cells in the BM. Significant differences were determined by Student's t test. **, P

Techniques Used: Blocking Assay, Labeling, Staining, Expressing

Intact differentiation to plasmablasts but impaired class switching in PIK3CD GOF naive and transitional B cells. (A–G) Naive and/or transitional B cells from healthy donors or PIK3CD GOF patients were cultured for 5 d with CD40L alone or together with IL-21. (A and B) Proportions of CD38 hi CD27 hi plasmablasts generated from naive B cells were determined by flow cytometry ( n = 7–8). (C and D) Expression of Blimp-1 was determined by (C) qPCR in naive and transitional B cells cultured with CD40L or CD40L/IL-21 ( n = 5) and (D) flow cytometry of activated B cell blasts (CD27 − CD38 -/lo ) and plasmablasts generated from naive B cells cultured with CD40L or CD40L/IL-21 ( n = 4–5). (E) Expression of AICDA was determined by qPCR in naive and transitional B cells cultured with CD40L or CD40L/IL-21 ( n = 5). (F and G) Expression of intracellular IgM, IgG, and IgA by plasmablasts was determined by flow cytometry ( n = 5–6). The FACS plots in A, D, and F are representative of cultured naive B cells from healthy donors or PIK3CD GOF patients; the graphs in B, C, E, and G depict the mean ± SEM of independent experiments using naive B cells from different donors and patients. (H and I) Sort-purified naive (H; n = 9–12) and transitional (I; n = 11–14) B cells from healthy donors or PIK3CD GOF patients were cultured for 5 or 7 d with CD40L and IL-21. Ig secretion was then determined. Values represent the mean ± SEM. Significant differences were determined by Student's t tests. *, P
Figure Legend Snippet: Intact differentiation to plasmablasts but impaired class switching in PIK3CD GOF naive and transitional B cells. (A–G) Naive and/or transitional B cells from healthy donors or PIK3CD GOF patients were cultured for 5 d with CD40L alone or together with IL-21. (A and B) Proportions of CD38 hi CD27 hi plasmablasts generated from naive B cells were determined by flow cytometry ( n = 7–8). (C and D) Expression of Blimp-1 was determined by (C) qPCR in naive and transitional B cells cultured with CD40L or CD40L/IL-21 ( n = 5) and (D) flow cytometry of activated B cell blasts (CD27 − CD38 -/lo ) and plasmablasts generated from naive B cells cultured with CD40L or CD40L/IL-21 ( n = 4–5). (E) Expression of AICDA was determined by qPCR in naive and transitional B cells cultured with CD40L or CD40L/IL-21 ( n = 5). (F and G) Expression of intracellular IgM, IgG, and IgA by plasmablasts was determined by flow cytometry ( n = 5–6). The FACS plots in A, D, and F are representative of cultured naive B cells from healthy donors or PIK3CD GOF patients; the graphs in B, C, E, and G depict the mean ± SEM of independent experiments using naive B cells from different donors and patients. (H and I) Sort-purified naive (H; n = 9–12) and transitional (I; n = 11–14) B cells from healthy donors or PIK3CD GOF patients were cultured for 5 or 7 d with CD40L and IL-21. Ig secretion was then determined. Values represent the mean ± SEM. Significant differences were determined by Student's t tests. *, P

Techniques Used: Cell Culture, Generated, Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction, FACS, Purification

GOF mutations in PIK3CD arrest peripheral B cell development and differentiation. PBMCs from healthy donors ( n = 45–60) and patients with PIK3CD GOF mutations ( n = 21–39) were labeled with mAbs against CD20, CD10, CD27, IgG, or IgA. The proportions of (A) B (CD20 + ) cells within the lymphocyte gate, (B) transitional, naive, and memory cells within the B cell population, and (C) IgG + and IgA + cells within the memory population were determined by flow cytometry. Histogram and contour plots are representative of healthy donors or PIK3CD GOF patients. Each symbol in the summary graphs corresponds to an individual donor or patient; horizontal bars represent the mean. Significant differences were determined by unpaired Student's t tests. ****, P
Figure Legend Snippet: GOF mutations in PIK3CD arrest peripheral B cell development and differentiation. PBMCs from healthy donors ( n = 45–60) and patients with PIK3CD GOF mutations ( n = 21–39) were labeled with mAbs against CD20, CD10, CD27, IgG, or IgA. The proportions of (A) B (CD20 + ) cells within the lymphocyte gate, (B) transitional, naive, and memory cells within the B cell population, and (C) IgG + and IgA + cells within the memory population were determined by flow cytometry. Histogram and contour plots are representative of healthy donors or PIK3CD GOF patients. Each symbol in the summary graphs corresponds to an individual donor or patient; horizontal bars represent the mean. Significant differences were determined by unpaired Student's t tests. ****, P

Techniques Used: Labeling, Flow Cytometry, Cytometry

Mice with overactive PI3K show aberrant B cell development. (A) B cells were stained intracellularly for phosphorylated Akt (T308 and S473) and S6 (S235/236). Histograms show representative staining from WT (black), Pik3cd E1020K heterozygous ( Pik3cd GOF , red), or Pik3cd E1020K homozygous ( Pik3cd homGOF blue) mice and graphs give MFI relative to WT controls (mean ± SEM, n = 5). (B–D) BM and spleens from WT and Pik3cd E1020K heterozygous GOF mice aged 8–12 wk were stained to identify different B cell populations. (B) B cell development in the BM. Flow cytometry plots showing representative staining of IgD versus IgM on B220 + cells. Numbers are percent IgM − IgD − , IgM hi , and IgD hi cells. IgM − IgD − cells were further gated on CD24 and CD43 to identify pre-pro–, pro- and pre-B cells. Graphs give mean ± SEM ( n = 9–12). (C) Transitional cells in the spleen. Flow plots show IgM versus CD23 on B220 + CD93 + cells. Graphs show absolute numbers of T1 and T2/T3 cells as well as the percentage of each population that is IgM hi (mean ± SEM, n = 9–12). (D) Percentages of follicular (CD23 + CD21 lo ) and MZ (CD21 hi CD23 lo ) B cells were determined in the mature B cell population (CD93 − ) of the spleen. Flow plots show representative staining of CD21 versus CD23. Graphs show absolute numbers of follicular and MZ cells (mean ± SEM, n = 10–13). (E) Percentages of B1a (CD19 + B220 lo CD5 + ) or B1b (CD19 + B220 lo CD5 − ) cells were determined. Flow plots show representative staining of B220 versus CD19 gated on CD19 + cells (upper panel) and CD19 versus CD5 gated on CD19 + B220 lo cells (lower panel). Graphs show absolute numbers of B1a and B1b cells in the spleen (mean ± SEM, n = 6–8). Significant differences were determined by unpaired Student's t tests. *, P
Figure Legend Snippet: Mice with overactive PI3K show aberrant B cell development. (A) B cells were stained intracellularly for phosphorylated Akt (T308 and S473) and S6 (S235/236). Histograms show representative staining from WT (black), Pik3cd E1020K heterozygous ( Pik3cd GOF , red), or Pik3cd E1020K homozygous ( Pik3cd homGOF blue) mice and graphs give MFI relative to WT controls (mean ± SEM, n = 5). (B–D) BM and spleens from WT and Pik3cd E1020K heterozygous GOF mice aged 8–12 wk were stained to identify different B cell populations. (B) B cell development in the BM. Flow cytometry plots showing representative staining of IgD versus IgM on B220 + cells. Numbers are percent IgM − IgD − , IgM hi , and IgD hi cells. IgM − IgD − cells were further gated on CD24 and CD43 to identify pre-pro–, pro- and pre-B cells. Graphs give mean ± SEM ( n = 9–12). (C) Transitional cells in the spleen. Flow plots show IgM versus CD23 on B220 + CD93 + cells. Graphs show absolute numbers of T1 and T2/T3 cells as well as the percentage of each population that is IgM hi (mean ± SEM, n = 9–12). (D) Percentages of follicular (CD23 + CD21 lo ) and MZ (CD21 hi CD23 lo ) B cells were determined in the mature B cell population (CD93 − ) of the spleen. Flow plots show representative staining of CD21 versus CD23. Graphs show absolute numbers of follicular and MZ cells (mean ± SEM, n = 10–13). (E) Percentages of B1a (CD19 + B220 lo CD5 + ) or B1b (CD19 + B220 lo CD5 − ) cells were determined. Flow plots show representative staining of B220 versus CD19 gated on CD19 + cells (upper panel) and CD19 versus CD5 gated on CD19 + B220 lo cells (lower panel). Graphs show absolute numbers of B1a and B1b cells in the spleen (mean ± SEM, n = 6–8). Significant differences were determined by unpaired Student's t tests. *, P

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry

17) Product Images from "Susceptibility-related differences in the quantity of developmental stages of Myxobolus spp. (Myxozoa) in fish blood"

Article Title: Susceptibility-related differences in the quantity of developmental stages of Myxobolus spp. (Myxozoa) in fish blood

Journal: PLoS ONE

doi: 10.1371/journal.pone.0204437

Prevalence and intensity of Myxobolus pseudodispar in host blood in exposure trial #1. (A) Prevalence of the parasite in host species in the relation of sampling time. (B) Boxplot of infection intensity in examined fish species. Significant differences: a: p = 0.043, b: p = 0.041. (C) Boxplot of infection intensity by fish species and at different sampling time. Significant or borderline differences: a: p = 0.087, b: p = 0.016. Rr: common roach Rutilus rutilus ; Cg: gibel carp Carassius gibelio ; Se: rudd Scardinius erythrophthalmus ; Log 10 CNRQ: log10 transformed, calibrated normalized relative quantities of parasite DNA based on qPCR measurements.
Figure Legend Snippet: Prevalence and intensity of Myxobolus pseudodispar in host blood in exposure trial #1. (A) Prevalence of the parasite in host species in the relation of sampling time. (B) Boxplot of infection intensity in examined fish species. Significant differences: a: p = 0.043, b: p = 0.041. (C) Boxplot of infection intensity by fish species and at different sampling time. Significant or borderline differences: a: p = 0.087, b: p = 0.016. Rr: common roach Rutilus rutilus ; Cg: gibel carp Carassius gibelio ; Se: rudd Scardinius erythrophthalmus ; Log 10 CNRQ: log10 transformed, calibrated normalized relative quantities of parasite DNA based on qPCR measurements.

Techniques Used: Sampling, Infection, Fluorescence In Situ Hybridization, Transformation Assay, Real-time Polymerase Chain Reaction

18) Product Images from "Knockout of Mpv17-Like Protein (M-LPH) Gene in Human Hepatoma Cells Results in Impairment of mtDNA Integrity through Reduction of TFAM, OGG1, and LIG3 at the Protein Levels"

Article Title: Knockout of Mpv17-Like Protein (M-LPH) Gene in Human Hepatoma Cells Results in Impairment of mtDNA Integrity through Reduction of TFAM, OGG1, and LIG3 at the Protein Levels

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2018/6956414

Measurement of the intracellular 8-OHdG level for M-LPH-WT and -KO HepG2 cells. The bars represent the mean ± SD of the results from four independent experiments. Differences at p
Figure Legend Snippet: Measurement of the intracellular 8-OHdG level for M-LPH-WT and -KO HepG2 cells. The bars represent the mean ± SD of the results from four independent experiments. Differences at p

Techniques Used:

19) Product Images from "Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice"

Article Title: Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice

Journal: Scientific Reports

doi: 10.1038/s41598-018-33930-w

Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P
Figure Legend Snippet: Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P

Techniques Used: Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Mouse Assay

20) Product Images from "Characterisation of the novel deleterious RAD51C p.Arg312Trp variant and prioritisation criteria for functional analysis of RAD51C missense changes"

Article Title: Characterisation of the novel deleterious RAD51C p.Arg312Trp variant and prioritisation criteria for functional analysis of RAD51C missense changes

Journal: British Journal of Cancer

doi: 10.1038/bjc.2017.286

Spanish non-BRCA1/2 high-risk breast and ovarian cancer family selected for Whole Exome Sequencing (WES) study. ( A ) Proband is highlighted by a black arrow. Individuals with tumours and ages of diagnosis are shown. ( B ) Electropherograms of RAD51C sequence spanning the c.934C > T variant show the presence of the c.934C > T variant in the proband’s blood sample. Sequence traces confirms loss of heterozygosity (LOH) in the proband’s ( C ) and her sister’s ( D ) ovarian tumours and RAD51C wild-type sequence in normal tissue from the proband’s father ( E ). A full colour version of this figure is available at the British Journal of Cancer journal online.
Figure Legend Snippet: Spanish non-BRCA1/2 high-risk breast and ovarian cancer family selected for Whole Exome Sequencing (WES) study. ( A ) Proband is highlighted by a black arrow. Individuals with tumours and ages of diagnosis are shown. ( B ) Electropherograms of RAD51C sequence spanning the c.934C > T variant show the presence of the c.934C > T variant in the proband’s blood sample. Sequence traces confirms loss of heterozygosity (LOH) in the proband’s ( C ) and her sister’s ( D ) ovarian tumours and RAD51C wild-type sequence in normal tissue from the proband’s father ( E ). A full colour version of this figure is available at the British Journal of Cancer journal online.

Techniques Used: Sequencing, Variant Assay

21) Product Images from "Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells"

Article Title: Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky548

A dual gRNA approach for CRISPR/Cas9-mediated correction of DM1-iPSC Myo and evidence for trinucleotide CTG repeat excision. ( A ) Diagrammatic representation for targeting of the 3 ‘UTR region of the DMPK gene using a dual gRNA approach for CRISPR/Cas9-mediated gene correction. The dual gRNAs ( 5′ 3′-CTG repeat -gRNA ) target Cas9 on either side of the CTG repeat region for excision of the expanded trinucleotide repeat. ( B ) Cas9 immunofluorescence staining of CRISPR/Cas9 treated DM1-iPSC-Myo cells, at 1-week post transduction. The upper panel shows representative images of DM1-iPSC-Myo cells stained for Cas9 (in red) and co-stained with DAPI for nuclei (in blue) (scale bar = 50 μm). The lower panel shows the graph for the quantitation of microscopy data for Cas9 positive cells. ( C ) Representative electropherograms of Triplet Repeat Primed PCR (TP) products from DM1-iPSC-Myo after CRISPR/Cas9-mediated gene editing from three independent experiments for each of the three treatments (Cas9 and 5′ 3′-CTG repeat -gRNA; Cas9 and scrambled gRNA; 5′-CTG repeat -gRNA, 3′-CTG repeat -gRNA and no Cas9) and untreated control conditions (WT-iPSC-Myo and DM1-iPSC-Myo). ( D ) Sanger sequencing results of on-target activity. The DMPK target locus was amplified by primers flanking the 2 SNPs [ C > T ; G > A : mutant > wild-type allele] and the CTG repeat region [ (CTG) ∼1371 /(CTG) 5 ]. The SNPs allowed discrimination of mutant ( C G ) and wild-type alleles (T A). Analysis of CRISPR/Cas9 activity on the targeted mutant allele showed a large deletion [(–) ∼4188 bp] between the 5′-CTG repeat -gRNA and 3′-CTG repeat -gRNA target sites. CRISPR/Cas9 activity on wild type allele was also detected by deletions between the corresponding gRNA target sites. Representative sequences of the wild-type allele with commonly found deletions and insertions are depicted in the figure. SNPs marked in red are seen in the mutant allele and those in blue are present in the wild type allele. Insertions are indicated by (+) and deletions are indicated by (–). Small letters represent the inserted nucleotides.
Figure Legend Snippet: A dual gRNA approach for CRISPR/Cas9-mediated correction of DM1-iPSC Myo and evidence for trinucleotide CTG repeat excision. ( A ) Diagrammatic representation for targeting of the 3 ‘UTR region of the DMPK gene using a dual gRNA approach for CRISPR/Cas9-mediated gene correction. The dual gRNAs ( 5′ 3′-CTG repeat -gRNA ) target Cas9 on either side of the CTG repeat region for excision of the expanded trinucleotide repeat. ( B ) Cas9 immunofluorescence staining of CRISPR/Cas9 treated DM1-iPSC-Myo cells, at 1-week post transduction. The upper panel shows representative images of DM1-iPSC-Myo cells stained for Cas9 (in red) and co-stained with DAPI for nuclei (in blue) (scale bar = 50 μm). The lower panel shows the graph for the quantitation of microscopy data for Cas9 positive cells. ( C ) Representative electropherograms of Triplet Repeat Primed PCR (TP) products from DM1-iPSC-Myo after CRISPR/Cas9-mediated gene editing from three independent experiments for each of the three treatments (Cas9 and 5′ 3′-CTG repeat -gRNA; Cas9 and scrambled gRNA; 5′-CTG repeat -gRNA, 3′-CTG repeat -gRNA and no Cas9) and untreated control conditions (WT-iPSC-Myo and DM1-iPSC-Myo). ( D ) Sanger sequencing results of on-target activity. The DMPK target locus was amplified by primers flanking the 2 SNPs [ C > T ; G > A : mutant > wild-type allele] and the CTG repeat region [ (CTG) ∼1371 /(CTG) 5 ]. The SNPs allowed discrimination of mutant ( C G ) and wild-type alleles (T A). Analysis of CRISPR/Cas9 activity on the targeted mutant allele showed a large deletion [(–) ∼4188 bp] between the 5′-CTG repeat -gRNA and 3′-CTG repeat -gRNA target sites. CRISPR/Cas9 activity on wild type allele was also detected by deletions between the corresponding gRNA target sites. Representative sequences of the wild-type allele with commonly found deletions and insertions are depicted in the figure. SNPs marked in red are seen in the mutant allele and those in blue are present in the wild type allele. Insertions are indicated by (+) and deletions are indicated by (–). Small letters represent the inserted nucleotides.

Techniques Used: CRISPR, CTG Assay, Immunofluorescence, Staining, Transduction, Quantitation Assay, Microscopy, Polymerase Chain Reaction, Sequencing, Activity Assay, Amplification, Mutagenesis

Generation of DM1-iPS cells (DM1-iPSCs) and DM1-iPSC derived inducible myogenic cells (DM1-iPSC-Myo). ( A ) Schematic overview showing CRISPR/Cas9 based correction of DM1 patient iPSCs derived myogenic cells (DM1-iPSC-Myo). ( B ) Representative image of DM1-iPSC clones and healthy control iPSCs stained for RNA foci by fluorescent in situ hybridization (FISH). An antisense Cy3-labeled probe was used against trinucleotide CUG expanded repeat. Arrowheads indicated ribonuclear foci. Upper panel represents stained nuclei at lower magnification (scale bar = 20μm) and lower panel represents higher magnification of selected region (scale bar = 2μm). Nuclei were counter-stained with DAPI. ( C ) Southern blot analysis to detect the length of trinucleotide CTG repeats in five DM1-iPSC clones from two DM1 patients (L22, L81 and L23; FL8 and FL5) and healthy control iPSCs. EcoR I digested genomic DNA was subjected to agarose gel electrophoresis and probed to detect the DMPK locus. (mut = mutant; wt = wild type) . ( D ) Representative image of FISH staining on DM1-iPSC-Myo for detection of ribonuclear foci. Arrowheads indicate multiple RNA foci in nuclei of DM1-iPSC-Myo. Healthy iPSC-Myo were used as a negative control. Upper panel represents stained nuclei at lower magnification (scale bar = 20 μm) and lower panel represents higher magnification of selected region (scale bar = 2 μm). Nuclei were counter-stained with DAPI. ( E ) Myogenic conversion of DM1-iPSC-Myo (L81 and L23) and healthy iPSC-Myo post MyoD induction were stained for a mature muscle marker, myosin heavy chain (MyHC) (scale bar = 100 μm). Nuclei were counter-stained with DAPI. ( F ) Southern blot analysis of trinucleotide CTG repeats length in DM1-iPSC-Myo (L81 and L23; FL8 and FL5) and healthy-iPSC-Myo to check the length of triplet repeats post-differentiation (mut = mutant; wt = wild type) .
Figure Legend Snippet: Generation of DM1-iPS cells (DM1-iPSCs) and DM1-iPSC derived inducible myogenic cells (DM1-iPSC-Myo). ( A ) Schematic overview showing CRISPR/Cas9 based correction of DM1 patient iPSCs derived myogenic cells (DM1-iPSC-Myo). ( B ) Representative image of DM1-iPSC clones and healthy control iPSCs stained for RNA foci by fluorescent in situ hybridization (FISH). An antisense Cy3-labeled probe was used against trinucleotide CUG expanded repeat. Arrowheads indicated ribonuclear foci. Upper panel represents stained nuclei at lower magnification (scale bar = 20μm) and lower panel represents higher magnification of selected region (scale bar = 2μm). Nuclei were counter-stained with DAPI. ( C ) Southern blot analysis to detect the length of trinucleotide CTG repeats in five DM1-iPSC clones from two DM1 patients (L22, L81 and L23; FL8 and FL5) and healthy control iPSCs. EcoR I digested genomic DNA was subjected to agarose gel electrophoresis and probed to detect the DMPK locus. (mut = mutant; wt = wild type) . ( D ) Representative image of FISH staining on DM1-iPSC-Myo for detection of ribonuclear foci. Arrowheads indicate multiple RNA foci in nuclei of DM1-iPSC-Myo. Healthy iPSC-Myo were used as a negative control. Upper panel represents stained nuclei at lower magnification (scale bar = 20 μm) and lower panel represents higher magnification of selected region (scale bar = 2 μm). Nuclei were counter-stained with DAPI. ( E ) Myogenic conversion of DM1-iPSC-Myo (L81 and L23) and healthy iPSC-Myo post MyoD induction were stained for a mature muscle marker, myosin heavy chain (MyHC) (scale bar = 100 μm). Nuclei were counter-stained with DAPI. ( F ) Southern blot analysis of trinucleotide CTG repeats length in DM1-iPSC-Myo (L81 and L23; FL8 and FL5) and healthy-iPSC-Myo to check the length of triplet repeats post-differentiation (mut = mutant; wt = wild type) .

Techniques Used: Derivative Assay, CRISPR, Clone Assay, Staining, In Situ Hybridization, Fluorescence In Situ Hybridization, Labeling, Southern Blot, CTG Assay, Agarose Gel Electrophoresis, Mutagenesis, Negative Control, Marker

Analysis of target region in the CRISPR/Cas9-corrected DM1-iPSC-Myo and ribonuclear foci staining of corrected DM1-iPSC-Myo and DM1 primary myoblasts. ( A ) Graph representing distribution of SMRT sequencing reads based on the various amplicon sizes ∼633 bp (excised fragments) and ∼723bp (WT fragments). The sequences ranging between ∼723 bp and ∼4000 bp were fragments with indels and partially deleted repeat regions. Each bar represents distribution of reads from each of the three conditions (Cas9 + 5′ 3′-CTG repeat -gRNA , Cas9 control and gRNA control) and untreated DM1-iPSC-Myo control. ( B ) Representative image of CRISPR/Cas9-corrected DM1-iPSC-Myo (L81) stained for ribonuclear foci. Cas9 and scrambled gRNA; 5′-CTG repeat -gRNA, 3′-CTG repeat -gRNA and no Cas9 were used as negative controls. An antisense Cy3-labeled probe was used to detect the presence of ribonuclear foci (NF). The ribonuclear foci negative and positive nuclei were denoted as NF − (white) and NF + (red), respectively. Each representative image is a maximum intensity z projection of the z slice images. For all the conditions (Cas9 + 3′ 5′-CTG repeat -gRNA , scrambled gRNA and no Cas9) enlarged z slices of selected ribonuclear foci negative (NF-) and positive (NF+) nucleus are represented. Nuclei were counter-stained with DAPI (scale bar = 20 μm). ( C ) Quantification of ribonuclear foci (NF) in CRISPR/Cas9-corrected DM1-iPSC Myo. The total number of ribonuclear foci per total number of nuclei was calculated. Total of nuclei counted is 6500. The data is represented as mean ± SEM. The statistics were performed using two-tailed unpaired Student's t -test (*** P
Figure Legend Snippet: Analysis of target region in the CRISPR/Cas9-corrected DM1-iPSC-Myo and ribonuclear foci staining of corrected DM1-iPSC-Myo and DM1 primary myoblasts. ( A ) Graph representing distribution of SMRT sequencing reads based on the various amplicon sizes ∼633 bp (excised fragments) and ∼723bp (WT fragments). The sequences ranging between ∼723 bp and ∼4000 bp were fragments with indels and partially deleted repeat regions. Each bar represents distribution of reads from each of the three conditions (Cas9 + 5′ 3′-CTG repeat -gRNA , Cas9 control and gRNA control) and untreated DM1-iPSC-Myo control. ( B ) Representative image of CRISPR/Cas9-corrected DM1-iPSC-Myo (L81) stained for ribonuclear foci. Cas9 and scrambled gRNA; 5′-CTG repeat -gRNA, 3′-CTG repeat -gRNA and no Cas9 were used as negative controls. An antisense Cy3-labeled probe was used to detect the presence of ribonuclear foci (NF). The ribonuclear foci negative and positive nuclei were denoted as NF − (white) and NF + (red), respectively. Each representative image is a maximum intensity z projection of the z slice images. For all the conditions (Cas9 + 3′ 5′-CTG repeat -gRNA , scrambled gRNA and no Cas9) enlarged z slices of selected ribonuclear foci negative (NF-) and positive (NF+) nucleus are represented. Nuclei were counter-stained with DAPI (scale bar = 20 μm). ( C ) Quantification of ribonuclear foci (NF) in CRISPR/Cas9-corrected DM1-iPSC Myo. The total number of ribonuclear foci per total number of nuclei was calculated. Total of nuclei counted is 6500. The data is represented as mean ± SEM. The statistics were performed using two-tailed unpaired Student's t -test (*** P

Techniques Used: CRISPR, Staining, Sequencing, Amplification, CTG Assay, Labeling, Two Tailed Test

Biological effects of CRSIPR/Cas9 mediated correction of DM1-iPSC-Myo. ( A ) Dual staining for MBNL1 and ribonuclear foci co-localization in the CRSIPR/Cas9-corrected versus control conditions (Cas9 and scrambled gRNA; 5′-CTG repeat -gRNA, 3′-CTG repeat -gRNA and no Cas9). Representative image of DM1-iPSC-Myo stained for MBNL1 and Ribonuclear foci by combined immunostaining-FISH staining. Each representative image is a maximum intensity z projection of the z slices. For control conditions, enlarged image of selected nuclei are represented under different filters. For the condition (Cas9 + 3′ 5′-CTG repeat -gRNA ) enlarged z slices of selected ribonuclear foci negative (NF-) and positive (NF+) nucleus are represented under different filters. Nuclei were counterstained with DAPI. ( B ) Quantification of the microscopy data is represented in term of ratio between the total dual positive (MBNL1 + RNA + foci)/total number of nuclei observed in each condition for the L23, L81, FL8 and FL5 DM1-iPSC-Myo cells. The data is represented as mean ± SEM. The statistics were performed using two-tailed unpaired Student's t -test (*** P
Figure Legend Snippet: Biological effects of CRSIPR/Cas9 mediated correction of DM1-iPSC-Myo. ( A ) Dual staining for MBNL1 and ribonuclear foci co-localization in the CRSIPR/Cas9-corrected versus control conditions (Cas9 and scrambled gRNA; 5′-CTG repeat -gRNA, 3′-CTG repeat -gRNA and no Cas9). Representative image of DM1-iPSC-Myo stained for MBNL1 and Ribonuclear foci by combined immunostaining-FISH staining. Each representative image is a maximum intensity z projection of the z slices. For control conditions, enlarged image of selected nuclei are represented under different filters. For the condition (Cas9 + 3′ 5′-CTG repeat -gRNA ) enlarged z slices of selected ribonuclear foci negative (NF-) and positive (NF+) nucleus are represented under different filters. Nuclei were counterstained with DAPI. ( B ) Quantification of the microscopy data is represented in term of ratio between the total dual positive (MBNL1 + RNA + foci)/total number of nuclei observed in each condition for the L23, L81, FL8 and FL5 DM1-iPSC-Myo cells. The data is represented as mean ± SEM. The statistics were performed using two-tailed unpaired Student's t -test (*** P

Techniques Used: Staining, CTG Assay, Immunostaining, Fluorescence In Situ Hybridization, Microscopy, Two Tailed Test

22) Product Images from "De novo genome assembly of Oryza granulata reveals rapid genome expansion and adaptive evolution"

Article Title: De novo genome assembly of Oryza granulata reveals rapid genome expansion and adaptive evolution

Journal: Communications Biology

doi: 10.1038/s42003-018-0089-4

Timing of the bursts of the three retrotransposons, RIRE2 , ATLANTYS , and COPIA , in O. granulata . a Timing of the burst of RIRE2 . b Timing of the burst of ATLANTYS and COPIA . For each LTR retrotransposons in O. granulata genome, the curves represent the date of divergence in MYA translated from the observed divergence, using the molecular clock (1.3×10 − 8 substitutions per site per year from Ma and Bennetzen 47 ). The groups of paralogs used to compute the pairwise distances are defined with EMBOSS. The y -axis represents the total number of copy equivalents
Figure Legend Snippet: Timing of the bursts of the three retrotransposons, RIRE2 , ATLANTYS , and COPIA , in O. granulata . a Timing of the burst of RIRE2 . b Timing of the burst of ATLANTYS and COPIA . For each LTR retrotransposons in O. granulata genome, the curves represent the date of divergence in MYA translated from the observed divergence, using the molecular clock (1.3×10 − 8 substitutions per site per year from Ma and Bennetzen 47 ). The groups of paralogs used to compute the pairwise distances are defined with EMBOSS. The y -axis represents the total number of copy equivalents

Techniques Used:

Venn diagram showing the distribution of gene families between O. granulata , O. brachyantha , O. sativa , and B. distachyon . The intersections between species indicate the numbers of shared gene families, while unique family numbers are shown in species-specific areas. The center represents the number of families shared by all species simultaneously. Only families with four or more protein members were considered. Analysis of families showed 12,585 families shared by all species simultaneously
Figure Legend Snippet: Venn diagram showing the distribution of gene families between O. granulata , O. brachyantha , O. sativa , and B. distachyon . The intersections between species indicate the numbers of shared gene families, while unique family numbers are shown in species-specific areas. The center represents the number of families shared by all species simultaneously. Only families with four or more protein members were considered. Analysis of families showed 12,585 families shared by all species simultaneously

Techniques Used:

FISH mapping of centromeric and pericentromeric probes on pachytene chromosomes of O. granulata and phenotypic characterization of its pachytene chromosomes. a Immunodetection of rabbit antibody against rice CENH3 on pachytene chromosomes of O. granulata . b FISH identification of precipitated DNA isolated by ChIP using rice anti-CENH3 antibodies. c Dual color FISH of GCS1 cloned from ChIP DNAs and RIRE2 . GCS1 was labeled with digoxigenin-dUTP (magenta), while RIRE2 was labeled with biotin-dUTP (cyan). d Immuno-FISH detection of RIRE2 . Rice anti-CENH3 antibodies were used to label centromeres. e Pachytene chromosome morphology of O. granulata . Arrows indicate the 12 centromere constrictions on each pachytene chromosome of O. granulata . f Pachytene chromosome morphology of O. sativa . Chromosomes were counterstained with DAPI. Scale bars = 5 μm
Figure Legend Snippet: FISH mapping of centromeric and pericentromeric probes on pachytene chromosomes of O. granulata and phenotypic characterization of its pachytene chromosomes. a Immunodetection of rabbit antibody against rice CENH3 on pachytene chromosomes of O. granulata . b FISH identification of precipitated DNA isolated by ChIP using rice anti-CENH3 antibodies. c Dual color FISH of GCS1 cloned from ChIP DNAs and RIRE2 . GCS1 was labeled with digoxigenin-dUTP (magenta), while RIRE2 was labeled with biotin-dUTP (cyan). d Immuno-FISH detection of RIRE2 . Rice anti-CENH3 antibodies were used to label centromeres. e Pachytene chromosome morphology of O. granulata . Arrows indicate the 12 centromere constrictions on each pachytene chromosome of O. granulata . f Pachytene chromosome morphology of O. sativa . Chromosomes were counterstained with DAPI. Scale bars = 5 μm

Techniques Used: Fluorescence In Situ Hybridization, Immunodetection, Isolation, Chromatin Immunoprecipitation, Clone Assay, Labeling

Characterization of 1597 collinear gene blocks between O. granulata and O. sativa genome. Chromosome plots of the O. sativa genome (left), comprising 1019 contigs with collinear gene blocks of O. granulata (right) that cover ~45.86 % of the assembly size. Chromosomes and contigs filled with density of repeat elements for every 100 kb window. Syntenic regions are shaded in gray boxes between them. Black circles show the percentage masked by RIRE2 in a window, and only where the proportion is more than 0.2 here
Figure Legend Snippet: Characterization of 1597 collinear gene blocks between O. granulata and O. sativa genome. Chromosome plots of the O. sativa genome (left), comprising 1019 contigs with collinear gene blocks of O. granulata (right) that cover ~45.86 % of the assembly size. Chromosomes and contigs filled with density of repeat elements for every 100 kb window. Syntenic regions are shaded in gray boxes between them. Black circles show the percentage masked by RIRE2 in a window, and only where the proportion is more than 0.2 here

Techniques Used:

23) Product Images from "Occurrence of cagA+vacA s1a m1 i1 Helicobacter pylori in farm animals in Egypt and ability to survive in experimentally contaminated UHT milk"

Article Title: Occurrence of cagA+vacA s1a m1 i1 Helicobacter pylori in farm animals in Egypt and ability to survive in experimentally contaminated UHT milk

Journal: Scientific Reports

doi: 10.1038/s41598-018-32671-0

Number of PCR positive samples that contained the spiral viable culturable form of Helicobacter pylori as determined by bacteriological culture and identification. Milk and fecal samples from the three species of animals that were positive using PCR were subjected to bacteriological culture and biochemical identification. The results are shown as total numbers of milk samples (Milk) and fecal samples (Feces) that were tested positive in PCR (PCR) and numbers of PCR positive samples that showed positive colonies of H . pylori (Bacteriological Culture).
Figure Legend Snippet: Number of PCR positive samples that contained the spiral viable culturable form of Helicobacter pylori as determined by bacteriological culture and identification. Milk and fecal samples from the three species of animals that were positive using PCR were subjected to bacteriological culture and biochemical identification. The results are shown as total numbers of milk samples (Milk) and fecal samples (Feces) that were tested positive in PCR (PCR) and numbers of PCR positive samples that showed positive colonies of H . pylori (Bacteriological Culture).

Techniques Used: Polymerase Chain Reaction

Evolutionary relationship between the strains isolated in the present study and those retrieved from the gene bank based on Helicobacter genus-specific 16s. The phylogenetic tree was constructed by using the neighbor-joining method and the evolutionary analysis was conducted in MEGA7. The analysis involved nine nucleotide sequences, two represents the strains of the present study and eight that had 90–100% identity with our sequences. The evolutionary distances were calculated and expressed in the units of the number of base differences per sequence. The numbers on the nodes (shown on the left next to the branches) indicate the number of times (percentage) the species grouped together.
Figure Legend Snippet: Evolutionary relationship between the strains isolated in the present study and those retrieved from the gene bank based on Helicobacter genus-specific 16s. The phylogenetic tree was constructed by using the neighbor-joining method and the evolutionary analysis was conducted in MEGA7. The analysis involved nine nucleotide sequences, two represents the strains of the present study and eight that had 90–100% identity with our sequences. The evolutionary distances were calculated and expressed in the units of the number of base differences per sequence. The numbers on the nodes (shown on the left next to the branches) indicate the number of times (percentage) the species grouped together.

Techniques Used: Isolation, Construct, Sequencing

Survival of Helicobacter pylori in experimentally contaminated UHT milk. UHT milk was artificially inoculated with cytotoxic spiral viable culturable form (SVCF) of H . pylori strains (1.5 × 10 7 cells/ml, 0 hour) of the genotype cagA + vacA + s1a m1 i1 isolated from cow fecal samples. The milk was incubated at 4 °C, 37 °C and 40 °C for a time period from 1 to 30 days. After each time period, the numbers of the spiral viable culturable form (SVCF) and coccoid viable non-culturable form (CVNCF) present in milk were counted using a haemocytometer. *a indicates a significant difference in the bacterial count between 0 hour and all other time points, * with horizontal lines indicates significant differences between day 1 as well as day 2 and the subsequent days (day 3 to 30 days) using Repeated Measures ANOVA. The milk was further subjected to PCR, bacterial culture and reverse transcription PCR (RT-PCR); P indicates positive, n means negative.
Figure Legend Snippet: Survival of Helicobacter pylori in experimentally contaminated UHT milk. UHT milk was artificially inoculated with cytotoxic spiral viable culturable form (SVCF) of H . pylori strains (1.5 × 10 7 cells/ml, 0 hour) of the genotype cagA + vacA + s1a m1 i1 isolated from cow fecal samples. The milk was incubated at 4 °C, 37 °C and 40 °C for a time period from 1 to 30 days. After each time period, the numbers of the spiral viable culturable form (SVCF) and coccoid viable non-culturable form (CVNCF) present in milk were counted using a haemocytometer. *a indicates a significant difference in the bacterial count between 0 hour and all other time points, * with horizontal lines indicates significant differences between day 1 as well as day 2 and the subsequent days (day 3 to 30 days) using Repeated Measures ANOVA. The milk was further subjected to PCR, bacterial culture and reverse transcription PCR (RT-PCR); P indicates positive, n means negative.

Techniques Used: Isolation, Incubation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Load of Helicobacter pylori in gastric mucosa of the experimentally infected groups of balb c/mice as measured by quantitative real-time polymerase chain reaction (qPCR). Four groups of mice were fed orally uninfected UHT milk ( Negative control Group ) or UHT milk containing SS1 reference strain ( Positive control Group ), spiral viable culturable form ( SVCF Group ) or coccoid viable non-culturable form of H . pylori ( CVNCF Group ). Each dot represents an individual mouse, the middle horizontal lines indicate mean values. Bacterial load was measured in the gastric mucosa using qPCR and the results are shown as number of bacterial cells per 200 ng of mouse genomic DNA. Homogenates of the gastric mucosa were also examined for mRNA expression of H . pylori virulence genes using RT-PCR and were further subjected to bacterial culture.
Figure Legend Snippet: Load of Helicobacter pylori in gastric mucosa of the experimentally infected groups of balb c/mice as measured by quantitative real-time polymerase chain reaction (qPCR). Four groups of mice were fed orally uninfected UHT milk ( Negative control Group ) or UHT milk containing SS1 reference strain ( Positive control Group ), spiral viable culturable form ( SVCF Group ) or coccoid viable non-culturable form of H . pylori ( CVNCF Group ). Each dot represents an individual mouse, the middle horizontal lines indicate mean values. Bacterial load was measured in the gastric mucosa using qPCR and the results are shown as number of bacterial cells per 200 ng of mouse genomic DNA. Homogenates of the gastric mucosa were also examined for mRNA expression of H . pylori virulence genes using RT-PCR and were further subjected to bacterial culture.

Techniques Used: Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Expressing, Reverse Transcription Polymerase Chain Reaction

24) Product Images from "Src-mediated phosphorylation converts FHL1 from tumor suppressor to tumor promoter"

Article Title: Src-mediated phosphorylation converts FHL1 from tumor suppressor to tumor promoter

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201708064

Kindlin-2 and c-Src regulate the subcellular localization of FHL1. (A) HeLa cells transfected with the control siRNA or kindlin-2 siRNA were replated on FN-coated coverslips for 4 h and stained with anti–kindlin-2 and anti-FHL1 antibodies. The nuclei were stained with DAPI. (B) Kindlin-2 depletion of kindlin-2 +/− MEF cells was confirmed by Western blotting. (C) WT and kindlin-2 +/− MEF cells were replated on FN-coated coverslips for 4 h and immunoreacted with anti–kindlin-2, anti-FHL1, and antipaxillin. Localization of kindlin-2, FHL1, and paxillin were observed by confocal microscopy under a 63× objective. KO, knockout. (D) Indicated plasmids were transfected into HeLa cells for 48 h, and then cells were immunoreacted with anti-HA and anti-Flag antibodies. The nuclei were stained with DAPI. Expression and localization of HA-Src and Flag-FHL1 were observed under a confocal microscope with a 63× objective. (E and F) HeLa cells were transfected with indicated plasmids for 48 h. Then, cells were fractionated and blotted with the indicated antibodies. Tubulin and YY1 were examined to indicate the cytoplasmic and nuclear extracts, respectively. (G) Indicated plasmids were transfected into HeLa cells for 48 h, and then cells were immunoreacted with anti-HA or anti–p-Tyr149–FHL1 or anti–p-Tyr272–FHL1 antibodies. The nuclei were stained with DAPI. (H) H1299 cells were pretreated with DMSO or PP2 for 12 h followed by staining with anti-FHL1 and anti–kindlin-2 antibodies. Then, the expression and localization of FHL1 and kindlin-2 were determined by confocal microscopy under a 63× objective. Bars, 10 µm. (I) H1299 cells were pretreated with DMSO or PP2 for 12 h followed by immunoblotting with indicated antibodies.
Figure Legend Snippet: Kindlin-2 and c-Src regulate the subcellular localization of FHL1. (A) HeLa cells transfected with the control siRNA or kindlin-2 siRNA were replated on FN-coated coverslips for 4 h and stained with anti–kindlin-2 and anti-FHL1 antibodies. The nuclei were stained with DAPI. (B) Kindlin-2 depletion of kindlin-2 +/− MEF cells was confirmed by Western blotting. (C) WT and kindlin-2 +/− MEF cells were replated on FN-coated coverslips for 4 h and immunoreacted with anti–kindlin-2, anti-FHL1, and antipaxillin. Localization of kindlin-2, FHL1, and paxillin were observed by confocal microscopy under a 63× objective. KO, knockout. (D) Indicated plasmids were transfected into HeLa cells for 48 h, and then cells were immunoreacted with anti-HA and anti-Flag antibodies. The nuclei were stained with DAPI. Expression and localization of HA-Src and Flag-FHL1 were observed under a confocal microscope with a 63× objective. (E and F) HeLa cells were transfected with indicated plasmids for 48 h. Then, cells were fractionated and blotted with the indicated antibodies. Tubulin and YY1 were examined to indicate the cytoplasmic and nuclear extracts, respectively. (G) Indicated plasmids were transfected into HeLa cells for 48 h, and then cells were immunoreacted with anti-HA or anti–p-Tyr149–FHL1 or anti–p-Tyr272–FHL1 antibodies. The nuclei were stained with DAPI. (H) H1299 cells were pretreated with DMSO or PP2 for 12 h followed by staining with anti-FHL1 and anti–kindlin-2 antibodies. Then, the expression and localization of FHL1 and kindlin-2 were determined by confocal microscopy under a 63× objective. Bars, 10 µm. (I) H1299 cells were pretreated with DMSO or PP2 for 12 h followed by immunoblotting with indicated antibodies.

Techniques Used: Transfection, Staining, Western Blot, Confocal Microscopy, Knock-Out, Expressing, Microscopy

Kindlin-2 competes with Src to interact with FHL1. (A) HeLa cells were transfected with indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag M2 beads followed by immunoblotting (IB) with indicated antibodies. (B) H1299 cells were pretreated with DMSO or PP2 for 12 h, and the interaction between kindlin-2 and FHL1 was analyzed. (C) HeLa cells were transfected with indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag M2 beads followed by immunoblotting with indicated antibodies. (D) H1299 cells were transfected with control siRNA or kindlin-2 siRNA for 48 h, the interaction between Src and FHL1 was analyzed, and p-Tyr of FHL1 was determined. WCL, whole-cell lysate. (E) HeLa cells were transfected with indicated plasmids and siRNAs. Cell lysates were immunoprecipitated with anti-Flag M2 beads followed by immunoblotting with indicated antibodies. (F) HeLa cells were transfected with FHL1-WT, FHL1-Y149-272F (YF; phosphorylation-deficient mutant), or FHL1-Y149-272D (YD; phosphomimetic mutant) expression vectors together with GFP–kindlin-2. Cell lysates were immunoprecipitated with anti-Flag M2 beads that were immunoblotted with kindlin-2 antibody. (G–I) H1299 cells were starved and kept in suspension for 30 min or replated on FN-coated dishes for 1 h. Then, cells were lysed and immunoprecipitated with kindlin-2, Src, or FHL1 antibodies followed by immunoblotting with indicated antibodies. (J) Visualization of endogenous FHL1, kindlin-2, and endogenous Src in H1299 cells. FHL1 (green) was mainly colocalized with kindlin-2 (red) and Src (orange) in focal adhesion sites. Bars, 10 µm.
Figure Legend Snippet: Kindlin-2 competes with Src to interact with FHL1. (A) HeLa cells were transfected with indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag M2 beads followed by immunoblotting (IB) with indicated antibodies. (B) H1299 cells were pretreated with DMSO or PP2 for 12 h, and the interaction between kindlin-2 and FHL1 was analyzed. (C) HeLa cells were transfected with indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag M2 beads followed by immunoblotting with indicated antibodies. (D) H1299 cells were transfected with control siRNA or kindlin-2 siRNA for 48 h, the interaction between Src and FHL1 was analyzed, and p-Tyr of FHL1 was determined. WCL, whole-cell lysate. (E) HeLa cells were transfected with indicated plasmids and siRNAs. Cell lysates were immunoprecipitated with anti-Flag M2 beads followed by immunoblotting with indicated antibodies. (F) HeLa cells were transfected with FHL1-WT, FHL1-Y149-272F (YF; phosphorylation-deficient mutant), or FHL1-Y149-272D (YD; phosphomimetic mutant) expression vectors together with GFP–kindlin-2. Cell lysates were immunoprecipitated with anti-Flag M2 beads that were immunoblotted with kindlin-2 antibody. (G–I) H1299 cells were starved and kept in suspension for 30 min or replated on FN-coated dishes for 1 h. Then, cells were lysed and immunoprecipitated with kindlin-2, Src, or FHL1 antibodies followed by immunoblotting with indicated antibodies. (J) Visualization of endogenous FHL1, kindlin-2, and endogenous Src in H1299 cells. FHL1 (green) was mainly colocalized with kindlin-2 (red) and Src (orange) in focal adhesion sites. Bars, 10 µm.

Techniques Used: Transfection, Immunoprecipitation, Mutagenesis, Expressing

25) Product Images from "DNA-methylation-mediated silencing of miR-486-5p promotes colorectal cancer proliferation and migration through activation of PLAGL2/IGF2/β-catenin signal pathways"

Article Title: DNA-methylation-mediated silencing of miR-486-5p promotes colorectal cancer proliferation and migration through activation of PLAGL2/IGF2/β-catenin signal pathways

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1105-9

MiR-486-5p expression is regulated by DNA methylation of its promoter. a MethPrimer website predicted that a CpG island is located at the promoter region of miR-486-5p. b , c BSP analysis demonstrated that methylated CG sites in CRC cell lines were more than that in FHC. d MSP analysis uncovered hypermethylation of the miR-486-5p promoter in CRC tissues when compared to ANTs. e The expression of miR-486-5p restored in CRC cell lines using 5-aza-2′-deoxycytidine. Data are represented as means ± S.D. from at least three independent experiments. * p
Figure Legend Snippet: MiR-486-5p expression is regulated by DNA methylation of its promoter. a MethPrimer website predicted that a CpG island is located at the promoter region of miR-486-5p. b , c BSP analysis demonstrated that methylated CG sites in CRC cell lines were more than that in FHC. d MSP analysis uncovered hypermethylation of the miR-486-5p promoter in CRC tissues when compared to ANTs. e The expression of miR-486-5p restored in CRC cell lines using 5-aza-2′-deoxycytidine. Data are represented as means ± S.D. from at least three independent experiments. * p

Techniques Used: Expressing, DNA Methylation Assay, Methylation

26) Product Images from "The Xenopus animal cap transcriptome: building a mucociliary epithelium"

Article Title: The Xenopus animal cap transcriptome: building a mucociliary epithelium

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky771

Region and stage-specific expression of repetitive DNA elements via RNA in situ hybridisation analysis of ERV1_4-LTR, LTRX1_LTR_Xt and Rem2b. Pictures display results from sense and antisense probe hybridisations performed and stained in parallel. Pictures of NF10.5 embryos represent sagittal sections; inserts show whole mount view of the animal hemisphere.
Figure Legend Snippet: Region and stage-specific expression of repetitive DNA elements via RNA in situ hybridisation analysis of ERV1_4-LTR, LTRX1_LTR_Xt and Rem2b. Pictures display results from sense and antisense probe hybridisations performed and stained in parallel. Pictures of NF10.5 embryos represent sagittal sections; inserts show whole mount view of the animal hemisphere.

Techniques Used: Expressing, In Situ, Hybridization, Staining

27) Product Images from "Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies"

Article Title: Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky251

NaME-PrO application on BAT25 microsatellite using circulating DNA from colon cancer samples. ( A ) Comparison between NaME-PrO-treated and untreated screening for BAT25 microsatellites on plasma circulating DNA following HRM analysis: examples from three positive samples (#230, 236, 266) plus cfDNA from a healthy control patient (#38) are shown. ( B ) Capillary electrophoresis analysis for the same samples (red = variant, blue = WT).
Figure Legend Snippet: NaME-PrO application on BAT25 microsatellite using circulating DNA from colon cancer samples. ( A ) Comparison between NaME-PrO-treated and untreated screening for BAT25 microsatellites on plasma circulating DNA following HRM analysis: examples from three positive samples (#230, 236, 266) plus cfDNA from a healthy control patient (#38) are shown. ( B ) Capillary electrophoresis analysis for the same samples (red = variant, blue = WT).

Techniques Used: Electrophoresis, Variant Assay

28) Product Images from "A hPSC-based platform to discover gene-environment interactions that impact human β-cell and dopamine neuron survival"

Article Title: A hPSC-based platform to discover gene-environment interactions that impact human β-cell and dopamine neuron survival

Journal: Nature Communications

doi: 10.1038/s41467-018-07201-1

Midbrain dopamine neurons are hypersensitive to propargite-induced cell toxicity. a Characterization of cortical neuron and mDA neuron derived from H9 hESCs. Upper panel represents bright field images of cortical- and mDA-neurons. Lower panel shows cortical neurons stained for MAP2 (red) and CTIP2 (green) while mDA neurons were stained for TH (red) and FOXA2 (blue). Scale bars, 50 μm. b Relative cell survival rate of cortical- and mDA-neurons treated with DMSO or different doses of propargite. Relative cell survival was quantified by dividing propargite-treated cells to the DMSO control ( n = 3). c , d Representative image ( c ) and relative cell survival rate ( d ) of mDA neurons treated with DMSO or propargite (1 μM) in the presence or absence of GSH (2 mM). mDA cells were stained for TH (red) and FOXA2 (gray), and all cells were counterstained with DAPI (blue). Scale bars, 50 μm. Relative cell survival rate was analyzed by quantification of FOXA2 + (gray) cells ( n = 3). e Representative image of mDA cells treated with DMSO, DMSO+2 mM GSH, 1 μM propargite, or 1 μM propargite+2 mM GSH. White arrows indicate propargite-treated mDA cells (TH; red) co-stained with the DNA damage marker (rH2AX; green), and all cells were counterstained with DAPI (blue). Scale bars, 50 μm. f Western blotting analysis of necrosis marker (extracellular HMGB1) in DMSO or propargite (1 μM) treated mDA cells with/without GSH (2 mM). Only propargite-treated mDA cell had high extracellular HMGB1 level ( n = 3). g Relative cell survival rate, quantified by the expression of FOXA2+ cells ( n = 3), of mDA cells derived from isogenic wild type, GSTT1 −/− , and GSTM1 −/− H1 hESCs treated with DMSO and propargite (3 μM). h GSTT1 , but not GSTM1 expression, in substantia nigra region of postmortem brains is significantly downregulated in Parkinson’s disease patients compared to age-matched controls. Values for RNA expression used here are from a published gene expression data and selected values except absent its detection 38 . Values presented as mean ± S.D. p -value was calculated by unpaired two-tailed Student’s t -test were * p
Figure Legend Snippet: Midbrain dopamine neurons are hypersensitive to propargite-induced cell toxicity. a Characterization of cortical neuron and mDA neuron derived from H9 hESCs. Upper panel represents bright field images of cortical- and mDA-neurons. Lower panel shows cortical neurons stained for MAP2 (red) and CTIP2 (green) while mDA neurons were stained for TH (red) and FOXA2 (blue). Scale bars, 50 μm. b Relative cell survival rate of cortical- and mDA-neurons treated with DMSO or different doses of propargite. Relative cell survival was quantified by dividing propargite-treated cells to the DMSO control ( n = 3). c , d Representative image ( c ) and relative cell survival rate ( d ) of mDA neurons treated with DMSO or propargite (1 μM) in the presence or absence of GSH (2 mM). mDA cells were stained for TH (red) and FOXA2 (gray), and all cells were counterstained with DAPI (blue). Scale bars, 50 μm. Relative cell survival rate was analyzed by quantification of FOXA2 + (gray) cells ( n = 3). e Representative image of mDA cells treated with DMSO, DMSO+2 mM GSH, 1 μM propargite, or 1 μM propargite+2 mM GSH. White arrows indicate propargite-treated mDA cells (TH; red) co-stained with the DNA damage marker (rH2AX; green), and all cells were counterstained with DAPI (blue). Scale bars, 50 μm. f Western blotting analysis of necrosis marker (extracellular HMGB1) in DMSO or propargite (1 μM) treated mDA cells with/without GSH (2 mM). Only propargite-treated mDA cell had high extracellular HMGB1 level ( n = 3). g Relative cell survival rate, quantified by the expression of FOXA2+ cells ( n = 3), of mDA cells derived from isogenic wild type, GSTT1 −/− , and GSTM1 −/− H1 hESCs treated with DMSO and propargite (3 μM). h GSTT1 , but not GSTM1 expression, in substantia nigra region of postmortem brains is significantly downregulated in Parkinson’s disease patients compared to age-matched controls. Values for RNA expression used here are from a published gene expression data and selected values except absent its detection 38 . Values presented as mean ± S.D. p -value was calculated by unpaired two-tailed Student’s t -test were * p

Techniques Used: Multiple Displacement Amplification, Derivative Assay, Staining, Marker, Western Blot, Expressing, RNA Expression, Two Tailed Test

A hPSC-based population study discovers that GSTT1 -null pancreatic β-like cells are hypersensitive to propargite-induced cell death. a Survival rate of INS + cells derived from 10 different hESC or iPSC lines cultured in the presence of 1.6 μM propargite ( n = 3), and genotype analysis of GSTM1 and GSTT1 in those hESCs and iPSCs. b , c Correlation of INS + cell survival rate in the presence of 1.6 μM propargite in cells lacking both GSTM1 and GSTT1 ( b ), or lacking only GSTM1 ( c ). n.s. indicates a non-significant difference. d Western blotting analysis of GSTT1 or GSTM1 protein expression in INS + cells derived from isogenic wild type, GSTT1 −/− or GSTM1 −/− H1 hESCs. The −/− null clones were CRSIPR-induced biallelic frameshift mutants. The two GSTT1 knockout clones were both homozygous null mutants, and the two GSTM1 knockout clones were both compound-null mutants. e Flow cytometry analysis of C-peptide + cells in isogenic GSTT1 −/− or GSTM1 −/− hESC-derived D18 cells. f Inhibition curve of propargite on INS + cells derived from GSTT1 +/+ or GSTT1 −/− H1 hESCs ( n = 3). g , h Representative images ( g ) and DNA damage rate ( h ) of GSTT1 +/+ and GSTT1 −/− β-like cells ( n = 3). Scale bars, 800 μm. γ-H2A.X + /INS + cells are highlighted with yellow arrows. i Western blot analysis of GSTT1 protein in EndoC-βH1 cells carrying sgGSTT1 . Two CRISPR gRNAs ( sgGSTT1 -1 and sgGSTT1 -2) were used for generating GSTT1 −/− EndoC-βH1 cells. j , k Representative images ( j ) and cell death rate ( k ) of GSTT1 −/− EndoC-βH1 cells treated with 1.6 μM propargite ( n = 3). Scale bars, 200 μm. Values presented as mean ± S.D. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t -test were * p
Figure Legend Snippet: A hPSC-based population study discovers that GSTT1 -null pancreatic β-like cells are hypersensitive to propargite-induced cell death. a Survival rate of INS + cells derived from 10 different hESC or iPSC lines cultured in the presence of 1.6 μM propargite ( n = 3), and genotype analysis of GSTM1 and GSTT1 in those hESCs and iPSCs. b , c Correlation of INS + cell survival rate in the presence of 1.6 μM propargite in cells lacking both GSTM1 and GSTT1 ( b ), or lacking only GSTM1 ( c ). n.s. indicates a non-significant difference. d Western blotting analysis of GSTT1 or GSTM1 protein expression in INS + cells derived from isogenic wild type, GSTT1 −/− or GSTM1 −/− H1 hESCs. The −/− null clones were CRSIPR-induced biallelic frameshift mutants. The two GSTT1 knockout clones were both homozygous null mutants, and the two GSTM1 knockout clones were both compound-null mutants. e Flow cytometry analysis of C-peptide + cells in isogenic GSTT1 −/− or GSTM1 −/− hESC-derived D18 cells. f Inhibition curve of propargite on INS + cells derived from GSTT1 +/+ or GSTT1 −/− H1 hESCs ( n = 3). g , h Representative images ( g ) and DNA damage rate ( h ) of GSTT1 +/+ and GSTT1 −/− β-like cells ( n = 3). Scale bars, 800 μm. γ-H2A.X + /INS + cells are highlighted with yellow arrows. i Western blot analysis of GSTT1 protein in EndoC-βH1 cells carrying sgGSTT1 . Two CRISPR gRNAs ( sgGSTT1 -1 and sgGSTT1 -2) were used for generating GSTT1 −/− EndoC-βH1 cells. j , k Representative images ( j ) and cell death rate ( k ) of GSTT1 −/− EndoC-βH1 cells treated with 1.6 μM propargite ( n = 3). Scale bars, 200 μm. Values presented as mean ± S.D. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t -test were * p

Techniques Used: Derivative Assay, Cell Culture, Western Blot, Expressing, Clone Assay, Knock-Out, Flow Cytometry, Cytometry, Inhibition, CRISPR, Two Tailed Test

29) Product Images from "Orthogonal Cas9–Cas9 chimeras provide a versatile platform for genome editing"

Article Title: Orthogonal Cas9–Cas9 chimeras provide a versatile platform for genome editing

Journal: Nature Communications

doi: 10.1038/s41467-018-07310-x

Cas9–Cas9 fusions expand the targeting range of SpCas9 allowing deletion of the GATA1-binding element in the BCL11A enhancer+58 kb. a Lesion rates and types at tandem target sites, with suboptimal SpCas9 but canonical SaCas9 PAMs (Supplementary Fig. 8) are determined by deep sequencing with bulk analysis. SaCas9 generates robust editing whereas SpCas9 displays low or no activity. In Cas9–Cas9 fusion format, SpCas9 cuts effectively at these protospacers as observed from the SpCas9–dSaCas9 fusions or the fused wild-type nucleases. b Sequence information of the four target sites chosen for more detailed assessment of the application of Cas9–Cas9 fusions for the deletion of the GATA1-binding element in the functional core of the BCL11A enhancer+58 kb (highlighted in gray 21 ). c Lesion rates and types at four target sites spanning the GATA1 element are determined by deep sequencing after UMI-correction. Deep sequencing data are from three independent biological replicates performed on different days in HEK293T cells (Supplementary Data 1 ). Error bars indicate ± s.e.m
Figure Legend Snippet: Cas9–Cas9 fusions expand the targeting range of SpCas9 allowing deletion of the GATA1-binding element in the BCL11A enhancer+58 kb. a Lesion rates and types at tandem target sites, with suboptimal SpCas9 but canonical SaCas9 PAMs (Supplementary Fig. 8) are determined by deep sequencing with bulk analysis. SaCas9 generates robust editing whereas SpCas9 displays low or no activity. In Cas9–Cas9 fusion format, SpCas9 cuts effectively at these protospacers as observed from the SpCas9–dSaCas9 fusions or the fused wild-type nucleases. b Sequence information of the four target sites chosen for more detailed assessment of the application of Cas9–Cas9 fusions for the deletion of the GATA1-binding element in the functional core of the BCL11A enhancer+58 kb (highlighted in gray 21 ). c Lesion rates and types at four target sites spanning the GATA1 element are determined by deep sequencing after UMI-correction. Deep sequencing data are from three independent biological replicates performed on different days in HEK293T cells (Supplementary Data 1 ). Error bars indicate ± s.e.m

Techniques Used: Binding Assay, Sequencing, Activity Assay, Functional Assay

Cas9–Cas9 fusions achieve robust and specific genome editing. a Summary of the GUIDE-seq genome-wide off-target analysis of SpCas9 WT , Sa/NmCas9 WT , and SpCas9 WT -Sa/NmCas9 WT at four GATA1 target sites (Supplementary Data 2 ). b Deep sequencing determined lesion rates for these nucleases at a subset of off-target sites discovered by the GUIDE-seq data or predicted by CasOFFinder (Supplementary Data 3 ). The names of SpCas9, NmCas9, and SaCas9 off-target sites are colored in dark red, blue, and green. The GUIDE-seq result is from single experiment, and amplicon deep sequencing data are from three independent biological replicates performed on different days in HEK293T cells (Supplementary Data 1 ). Error bars indicate ± s.e.m. Statistical significance is determined by one-way analysis of variance (ANOVA), *, **, ***, and NS denote BH-adjusted P -values of
Figure Legend Snippet: Cas9–Cas9 fusions achieve robust and specific genome editing. a Summary of the GUIDE-seq genome-wide off-target analysis of SpCas9 WT , Sa/NmCas9 WT , and SpCas9 WT -Sa/NmCas9 WT at four GATA1 target sites (Supplementary Data 2 ). b Deep sequencing determined lesion rates for these nucleases at a subset of off-target sites discovered by the GUIDE-seq data or predicted by CasOFFinder (Supplementary Data 3 ). The names of SpCas9, NmCas9, and SaCas9 off-target sites are colored in dark red, blue, and green. The GUIDE-seq result is from single experiment, and amplicon deep sequencing data are from three independent biological replicates performed on different days in HEK293T cells (Supplementary Data 1 ). Error bars indicate ± s.e.m. Statistical significance is determined by one-way analysis of variance (ANOVA), *, **, ***, and NS denote BH-adjusted P -values of

Techniques Used: Genome Wide, Sequencing, Amplification

Development of a functional Cas9–Cas9 nuclease framework. a Schematic of SpCas9 MT –dNm/SaCas9 fusions. PAM-interaction attenuated SpCas9 15 (yellow star) is C-terminally fused to a nuclease-dead Cas9 from N. meningiditis or S. aureus . Each Cas9 is loaded with its cognate sgRNA. b Top, schematic of parameters tested for target site organization. Four composite target site configurations are tested (D1:D4). The red arrow and rectangle represent the SpCas9 protospacer (in 5′–3′ orientation) and PAM, respectively, whereas the blue arrow and rectangle represent the Nm/SaCas9 protospacer (in 5′–3′ orientation) and PAM, respectively. Two dashed lines indicate the edges of each orthogonal Cas9-binding site, and x represents the number of intervening nucleotides. Bottom, activity profiles of SpCas9 (blue), SpCas9 MT3 (R1335K; gray), and C-terminal fusions for SpCas9 MT3 –dNmCas9 (pink), and SpCas9 MT3 -dSaCas9 (purple) in the GFP reporter assay. GFP reporter assay data are from three independent biological replicates performed on different days in HEK293T cells. c Changes in the activity profile of SpCas9 MT3 -dSaCas9 nuclease as a function of the distance between sgRNA-binding sites at the AAVS1 locus. Top, schematic of the orientation of the target sites, where the SpCas9 site is fixed and the SaCas9 site is shifted away various distances to examine its distance-dependent activity (Supplementary Fig. 2 ) Bottom, deep sequencing data are from three independent biological replicates performed on different days in HEK293T cells, where the orientation and spacing between the orthogonal Cas9 sites is indicated below the x -axis (Supplementary Data 1 ). Error bars indicate ± s.e.m
Figure Legend Snippet: Development of a functional Cas9–Cas9 nuclease framework. a Schematic of SpCas9 MT –dNm/SaCas9 fusions. PAM-interaction attenuated SpCas9 15 (yellow star) is C-terminally fused to a nuclease-dead Cas9 from N. meningiditis or S. aureus . Each Cas9 is loaded with its cognate sgRNA. b Top, schematic of parameters tested for target site organization. Four composite target site configurations are tested (D1:D4). The red arrow and rectangle represent the SpCas9 protospacer (in 5′–3′ orientation) and PAM, respectively, whereas the blue arrow and rectangle represent the Nm/SaCas9 protospacer (in 5′–3′ orientation) and PAM, respectively. Two dashed lines indicate the edges of each orthogonal Cas9-binding site, and x represents the number of intervening nucleotides. Bottom, activity profiles of SpCas9 (blue), SpCas9 MT3 (R1335K; gray), and C-terminal fusions for SpCas9 MT3 –dNmCas9 (pink), and SpCas9 MT3 -dSaCas9 (purple) in the GFP reporter assay. GFP reporter assay data are from three independent biological replicates performed on different days in HEK293T cells. c Changes in the activity profile of SpCas9 MT3 -dSaCas9 nuclease as a function of the distance between sgRNA-binding sites at the AAVS1 locus. Top, schematic of the orientation of the target sites, where the SpCas9 site is fixed and the SaCas9 site is shifted away various distances to examine its distance-dependent activity (Supplementary Fig. 2 ) Bottom, deep sequencing data are from three independent biological replicates performed on different days in HEK293T cells, where the orientation and spacing between the orthogonal Cas9 sites is indicated below the x -axis (Supplementary Data 1 ). Error bars indicate ± s.e.m

Techniques Used: Functional Assay, Binding Assay, Activity Assay, Reporter Assay, Sequencing

SpCas9 MT –dSa/NmCas9 fusions improve the specificity of editing. a Sequences of dual Cas9 genomic target sites at the VEGFA locus. The SpCas9 protospacer is bold underlined with its PAM is in red, the SaCas9 protospacer is double underlined with its PAM is green, and the NmCas9 protospacer is wavy underlined with its PAM in blue. b Lesion rates of the nuclease platforms are determined by deep sequencing. c , d Genome-wide off-target analysis of the nuclease platforms determined via GUIDE-seq 12 (Supplementary Data 2 ). c The number of off-target peaks detected for the given nuclease. d Fold improvement of the specificity ratio of the Cas9 MT –dCas9 framework relative to SpCas9 WT . e Deep sequencing determined lesion rates of the nucleases at a small set of off-target sites discovered within the GUIDE-seq data. GUIDE-seq result is from a single experiment, whereas amplicon deep sequencing data are from three independent biological replicates performed on different days in HEK293T cells (Supplementary Data 1 ). Error bars indicate ± s.e.m
Figure Legend Snippet: SpCas9 MT –dSa/NmCas9 fusions improve the specificity of editing. a Sequences of dual Cas9 genomic target sites at the VEGFA locus. The SpCas9 protospacer is bold underlined with its PAM is in red, the SaCas9 protospacer is double underlined with its PAM is green, and the NmCas9 protospacer is wavy underlined with its PAM in blue. b Lesion rates of the nuclease platforms are determined by deep sequencing. c , d Genome-wide off-target analysis of the nuclease platforms determined via GUIDE-seq 12 (Supplementary Data 2 ). c The number of off-target peaks detected for the given nuclease. d Fold improvement of the specificity ratio of the Cas9 MT –dCas9 framework relative to SpCas9 WT . e Deep sequencing determined lesion rates of the nucleases at a small set of off-target sites discovered within the GUIDE-seq data. GUIDE-seq result is from a single experiment, whereas amplicon deep sequencing data are from three independent biological replicates performed on different days in HEK293T cells (Supplementary Data 1 ). Error bars indicate ± s.e.m

Techniques Used: Sequencing, Genome Wide, Amplification

Cas9–Cas9 dual nucleases display enhanced precise deletions out to ~200 bp site separation. Changes in the activity profile of SpCas9 WT –SaCas9 WT nuclease as a function of the distance between sgRNA-binding sites at the AAVS1 locus. Boxed inset: schematic of the orientation of the target sites, where the SpCas9 site is fixed and the SaCas9 site is shifted to examine the distance-dependent activity of the Cas9–Cas9 fusions (Supplementary Fig. 2 ). Bar graph: deep sequencing data, where the various lesion types and rates at AAVS1 sites are determined by UMI-corrected deep sequencing. Data are from three independent biological replicates performed on different days in HEK293T cells, where the orientation and spacing between the orthogonal Cas9 sites is indicated below the x -axis (Supplementary Data 1 ). Error bars indicate ± s.e.m
Figure Legend Snippet: Cas9–Cas9 dual nucleases display enhanced precise deletions out to ~200 bp site separation. Changes in the activity profile of SpCas9 WT –SaCas9 WT nuclease as a function of the distance between sgRNA-binding sites at the AAVS1 locus. Boxed inset: schematic of the orientation of the target sites, where the SpCas9 site is fixed and the SaCas9 site is shifted to examine the distance-dependent activity of the Cas9–Cas9 fusions (Supplementary Fig. 2 ). Bar graph: deep sequencing data, where the various lesion types and rates at AAVS1 sites are determined by UMI-corrected deep sequencing. Data are from three independent biological replicates performed on different days in HEK293T cells, where the orientation and spacing between the orthogonal Cas9 sites is indicated below the x -axis (Supplementary Data 1 ). Error bars indicate ± s.e.m

Techniques Used: Activity Assay, Binding Assay, Sequencing

30) Product Images from "The cellular phenotype of cytoplasmic incompatibility in Culex pipiens in the light of cidB diversity"

Article Title: The cellular phenotype of cytoplasmic incompatibility in Culex pipiens in the light of cidB diversity

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007364

Wolbachia density in testes. Wolbachia densities in mosquito testes were measured by quantitative PCR as the ratio between the number of copies of the Wolbachia wsp gene and the C . pipiens ace-2 nuclear gene. The colored dots represent the average density of Wolbachia in a pool of three pairs of testes and the red strips represent the average Wolbachia density for each line/ w Pip strain. Letters represent the different statistical groups ( i . e . means with the same letter are not significantly different), showing that Lavar males harbored significantly less Wolbachia in their testes than the other males.
Figure Legend Snippet: Wolbachia density in testes. Wolbachia densities in mosquito testes were measured by quantitative PCR as the ratio between the number of copies of the Wolbachia wsp gene and the C . pipiens ace-2 nuclear gene. The colored dots represent the average density of Wolbachia in a pool of three pairs of testes and the red strips represent the average Wolbachia density for each line/ w Pip strain. Letters represent the different statistical groups ( i . e . means with the same letter are not significantly different), showing that Lavar males harbored significantly less Wolbachia in their testes than the other males.

Techniques Used: Real-time Polymerase Chain Reaction

31) Product Images from "Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA"

Article Title: Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA

Journal: BMC Plant Biology

doi: 10.1186/s12870-018-1506-z

PCR analysis with primers specific for the omega-D4 gene from Chinese Spring. Genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), Chinese Spring (4) and nullisomic tetrasomic lines of Chinese Spring N1AT1B (5), N1AT1D (6), N1BT1A (7), N1BT1D (8), N1DT1A (9) and N1DT1B (10) was amplified. The sizes of molecular weight markers in kb are shown in lane M
Figure Legend Snippet: PCR analysis with primers specific for the omega-D4 gene from Chinese Spring. Genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), Chinese Spring (4) and nullisomic tetrasomic lines of Chinese Spring N1AT1B (5), N1AT1D (6), N1BT1A (7), N1BT1D (8), N1DT1A (9) and N1DT1B (10) was amplified. The sizes of molecular weight markers in kb are shown in lane M

Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight

PCR analysis with primers specific for LMW-GS encoded at the Glu-B3 locus ( a ), omega-5 gliadins ( b ), and repeat junction primers 19S-1.3-2 ( c ) and 143E-1-600 ( d ) located at the ends of a 5.8 Mb region of chromosome 1B in Chinese Spring. In each panel, genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), N1BT1A (4) and N1BT1D (5) nullisomic tetrasomic lines of Chinese Spring or Chinese Spring (6) was amplified. The sizes of molecular weight markers in kb are shown in lane M in each panel. Primer sequences are shown in Table 2
Figure Legend Snippet: PCR analysis with primers specific for LMW-GS encoded at the Glu-B3 locus ( a ), omega-5 gliadins ( b ), and repeat junction primers 19S-1.3-2 ( c ) and 143E-1-600 ( d ) located at the ends of a 5.8 Mb region of chromosome 1B in Chinese Spring. In each panel, genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), N1BT1A (4) and N1BT1D (5) nullisomic tetrasomic lines of Chinese Spring or Chinese Spring (6) was amplified. The sizes of molecular weight markers in kb are shown in lane M in each panel. Primer sequences are shown in Table 2

Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight

32) Product Images from "Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms?"

Article Title: Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms?

Journal: Pediatric research

doi: 10.1038/s41390-018-0060-6

Main genera detected by 16S rRNA sequencing. DNA was purified using a column based assay and the 16S rRNA sequencing method was used to detect the genera present in MEEs. Genera with a percentage of reads higher than 5% are shown in this graph. For each sample, the total reads is added on the top of the bars. Genus NC refers to a genus that couldn’t be characterized.
Figure Legend Snippet: Main genera detected by 16S rRNA sequencing. DNA was purified using a column based assay and the 16S rRNA sequencing method was used to detect the genera present in MEEs. Genera with a percentage of reads higher than 5% are shown in this graph. For each sample, the total reads is added on the top of the bars. Genus NC refers to a genus that couldn’t be characterized.

Techniques Used: Sequencing, Purification

33) Product Images from "Breed Differences in PCV2 Uptake and Disintegration in Porcine Monocytes"

Article Title: Breed Differences in PCV2 Uptake and Disintegration in Porcine Monocytes

Journal: Viruses

doi: 10.3390/v10100562

Fate of the PCV2 genome in blood monocytes. PCV2 was added to monocytes and samples were collected at different time points after virus addition. Afterwards, cells were subjected to DNA extraction. ( A ) The number of PCV2 genome copies at each time point was quantified by qPCR and expressed as a relative percentage to that at 1 h; ( B ) The agarose gel electrophoresis image of PCR amplification results of the full-length PCV2 genome at each time point. The PCR was performed with the primer set wgPCV2-fw/rev which amplifies the full length of PCV2 genome. Data were obtained from three individual pigs (hybrid Piétrain × Hypor Libra).
Figure Legend Snippet: Fate of the PCV2 genome in blood monocytes. PCV2 was added to monocytes and samples were collected at different time points after virus addition. Afterwards, cells were subjected to DNA extraction. ( A ) The number of PCV2 genome copies at each time point was quantified by qPCR and expressed as a relative percentage to that at 1 h; ( B ) The agarose gel electrophoresis image of PCR amplification results of the full-length PCV2 genome at each time point. The PCR was performed with the primer set wgPCV2-fw/rev which amplifies the full length of PCV2 genome. Data were obtained from three individual pigs (hybrid Piétrain × Hypor Libra).

Techniques Used: DNA Extraction, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

34) Product Images from "T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1"

Article Title: T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1

Journal: eLife

doi: 10.7554/eLife.38314

Spi1 expression is controlled by DNA methylation ( A ) Schematic illustrating the procedures involved in cell isolation and RRBS analysis; ( B ) DNA methylation status of genes specifically expressed in the leukemic blast (left) and LSC-enriched (right) subpopulations; ( C ) Spi1 promoter methylation status in normal T cells, LSC-enriched cells and blast-enriched cells.
Figure Legend Snippet: Spi1 expression is controlled by DNA methylation ( A ) Schematic illustrating the procedures involved in cell isolation and RRBS analysis; ( B ) DNA methylation status of genes specifically expressed in the leukemic blast (left) and LSC-enriched (right) subpopulations; ( C ) Spi1 promoter methylation status in normal T cells, LSC-enriched cells and blast-enriched cells.

Techniques Used: Expressing, DNA Methylation Assay, Cell Isolation, Methylation

35) Product Images from "Young parents produce offspring with short telomeres: A study in a long-lived bird, the Black-browed Albatross (Thalassarche melanophrys)"

Article Title: Young parents produce offspring with short telomeres: A study in a long-lived bird, the Black-browed Albatross (Thalassarche melanophrys)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193526

Relationship between offspring telomere length and mean parental age in Black-browed albatrosses. Filled and open circles respectively represent females and males. The solid line represents the relationship between offspring telomere length and parental age. The dotted lines represent the 95% confidence intervals for the relationship between offspring telomere length and mean parental age.
Figure Legend Snippet: Relationship between offspring telomere length and mean parental age in Black-browed albatrosses. Filled and open circles respectively represent females and males. The solid line represents the relationship between offspring telomere length and parental age. The dotted lines represent the 95% confidence intervals for the relationship between offspring telomere length and mean parental age.

Techniques Used:

36) Product Images from "BAK/BAX-Mediated Apoptosis Is a Myc-Induced Roadblock to Reprogramming"

Article Title: BAK/BAX-Mediated Apoptosis Is a Myc-Induced Roadblock to Reprogramming

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2017.12.019

Enhanced Production of iPSCs in the Absence of Apoptosis Yields Cells with DNA and Genome Stability Comparable with that of WT iPSCs Manually picked iPSC clones were cultured prior to assessment of genomic stability. (A) Representative confocal images of iPSC colonies (passage 5) stained for γH2AX, which marks DNA double-strand breaks. Scale bars, 20 μm. (B) Quantification of γH2AX foci was automated using γ-irradiated iPSC colonies as a positive control to set the threshold for fluorescence intensity. The average number of γH2AX foci in the nuclei of WT and Bak −/− ; Bax −/− iPSCs is not significantly different. For this experiment, independent MEF lines derived from different embryos (n = 4 for WT; n = 3 for Bak −/− ; Bax −/− ) were reprogrammed and corresponding iPSC lines established. Each point represents analysis of 1 colony (15–30 nuclei), with 9–12 colonies analyzed per independently derived iPSC line. Bars represent the mean values for each genotype. n.s., not significant. (C) Quantification of CNAs in iPSC clones (passage 2) derived from Bak −/− ; Bax −/− MEFs compared with those derived from WT and p53 −/− MEFs. There is no significant difference in the number of CNAs in WT and Bak;Bax DKO parental MEFs and their clonal iPSC derivatives. In contrast, iPSC clones derived from p53 −/− MEFs exhibit a significantly higher number of CNAs (n = 6 iPSC clones derived from a single MEF line/genotype). Bars represent the mean values for each genotype. ∗∗ p
Figure Legend Snippet: Enhanced Production of iPSCs in the Absence of Apoptosis Yields Cells with DNA and Genome Stability Comparable with that of WT iPSCs Manually picked iPSC clones were cultured prior to assessment of genomic stability. (A) Representative confocal images of iPSC colonies (passage 5) stained for γH2AX, which marks DNA double-strand breaks. Scale bars, 20 μm. (B) Quantification of γH2AX foci was automated using γ-irradiated iPSC colonies as a positive control to set the threshold for fluorescence intensity. The average number of γH2AX foci in the nuclei of WT and Bak −/− ; Bax −/− iPSCs is not significantly different. For this experiment, independent MEF lines derived from different embryos (n = 4 for WT; n = 3 for Bak −/− ; Bax −/− ) were reprogrammed and corresponding iPSC lines established. Each point represents analysis of 1 colony (15–30 nuclei), with 9–12 colonies analyzed per independently derived iPSC line. Bars represent the mean values for each genotype. n.s., not significant. (C) Quantification of CNAs in iPSC clones (passage 2) derived from Bak −/− ; Bax −/− MEFs compared with those derived from WT and p53 −/− MEFs. There is no significant difference in the number of CNAs in WT and Bak;Bax DKO parental MEFs and their clonal iPSC derivatives. In contrast, iPSC clones derived from p53 −/− MEFs exhibit a significantly higher number of CNAs (n = 6 iPSC clones derived from a single MEF line/genotype). Bars represent the mean values for each genotype. ∗∗ p

Techniques Used: Clone Assay, Cell Culture, Staining, Irradiation, Positive Control, Fluorescence, Derivative Assay

37) Product Images from "The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer"

Article Title: The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer

Journal: Cell Death & Disease

doi: 10.1038/s41419-017-0137-x

UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in ( e and g ) (* P
Figure Legend Snippet: UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in ( e and g ) (* P

Techniques Used: Over Expression, Western Blot, Transfection, shRNA

38) Product Images from "CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels"

Article Title: CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels

Journal: Nature microbiology

doi: 10.1038/s41564-017-0019-0

CD81 regulates X4-tropic HIV-1 RT. a) Primary T lymphoblasts pre-treated with 2μM of scramble or CD81pept for 5 days, were infected with HIV-1 NL4-3 strain or HIV-VSV-G. Early or late RT products were measured as in Figure 2a . Data are mean fold change ± SEM of 2 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Primary T lymphoblasts transfected with control or CD81 siRNA were infected with NL4-3 strain, and RT was measured as in Figure 2a . Data are from a representative experiment out of two. In-box shows immunoblots of whole cell lysates from the represented experiment probed for CD81, and ERM as loading control. The CD81/ERM signal ratio is indicated.
Figure Legend Snippet: CD81 regulates X4-tropic HIV-1 RT. a) Primary T lymphoblasts pre-treated with 2μM of scramble or CD81pept for 5 days, were infected with HIV-1 NL4-3 strain or HIV-VSV-G. Early or late RT products were measured as in Figure 2a . Data are mean fold change ± SEM of 2 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Primary T lymphoblasts transfected with control or CD81 siRNA were infected with NL4-3 strain, and RT was measured as in Figure 2a . Data are from a representative experiment out of two. In-box shows immunoblots of whole cell lysates from the represented experiment probed for CD81, and ERM as loading control. The CD81/ERM signal ratio is indicated.

Techniques Used: Infection, Transfection, Western Blot

CD81 negatively regulates cellular dNTP content through SAMHD1. a) Jurkat T cells transfected with control or CD81 siRNA were infected with HIV-1 NL4-3 strain, and early RT products were measured by qPCR at the indicated times. Data are mean fold change ± SEM of 2 independent experiments performed in triplicate. In-box shows representative immunoblots of CD81 and tubulin as loading control, and the CD81/Tubulin signal ratio is indicated. b) Jurkat T cells pre-treated with 2μM of scramble or CD81pept for 5 days were infected with NL4-3 strain, and RT was analysed as in a . c) Mean fold change ± SEM of the dNTP content of Hela/R5 or Hela/R5 CRISPR/Cas9-CD81 cells (left graph, 4 independent experiments), and primary T lymphoblasts transfected with control or CD81 siRNA (right graph, 2 independent experiments) measured by a HIV RT-based dNTP assay. Analysis was performed by paired Student t -test, *** p
Figure Legend Snippet: CD81 negatively regulates cellular dNTP content through SAMHD1. a) Jurkat T cells transfected with control or CD81 siRNA were infected with HIV-1 NL4-3 strain, and early RT products were measured by qPCR at the indicated times. Data are mean fold change ± SEM of 2 independent experiments performed in triplicate. In-box shows representative immunoblots of CD81 and tubulin as loading control, and the CD81/Tubulin signal ratio is indicated. b) Jurkat T cells pre-treated with 2μM of scramble or CD81pept for 5 days were infected with NL4-3 strain, and RT was analysed as in a . c) Mean fold change ± SEM of the dNTP content of Hela/R5 or Hela/R5 CRISPR/Cas9-CD81 cells (left graph, 4 independent experiments), and primary T lymphoblasts transfected with control or CD81 siRNA (right graph, 2 independent experiments) measured by a HIV RT-based dNTP assay. Analysis was performed by paired Student t -test, *** p

Techniques Used: Transfection, Infection, Real-time Polymerase Chain Reaction, Western Blot, CRISPR

39) Product Images from "Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples"

Article Title: Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples

Journal: BMC Research Notes

doi: 10.1186/s13104-018-3300-2

Ct values dot plots of PCR-positive samples for the seven eukaryotic enteric pathogens: Blastocystis spp. (n = 9), Cryptosporidium parvum / hominis (n = 8), Cyclospora cayetanensis (n = 10), Dientamoeba fragilis (n = 9), Giardia intestinalis (n = 8), Cystoisospora belli (n = 10) and Enterocytozoon bieneusi (n = 7) obtained with the two extraction procedures evaluated in the present study: EZ1 ® (EZ1) and QIAamp ® DNA Stool Mini Kit ® (QA). Mean values and standard deviation ranges for each pathogen are represented by large and short horizontal bars, respectively. Statistical significance is represented as **(p
Figure Legend Snippet: Ct values dot plots of PCR-positive samples for the seven eukaryotic enteric pathogens: Blastocystis spp. (n = 9), Cryptosporidium parvum / hominis (n = 8), Cyclospora cayetanensis (n = 10), Dientamoeba fragilis (n = 9), Giardia intestinalis (n = 8), Cystoisospora belli (n = 10) and Enterocytozoon bieneusi (n = 7) obtained with the two extraction procedures evaluated in the present study: EZ1 ® (EZ1) and QIAamp ® DNA Stool Mini Kit ® (QA). Mean values and standard deviation ranges for each pathogen are represented by large and short horizontal bars, respectively. Statistical significance is represented as **(p

Techniques Used: Polymerase Chain Reaction, Standard Deviation

40) Product Images from "Genomic analysis of DNA repair genes and androgen signaling in prostate cancer"

Article Title: Genomic analysis of DNA repair genes and androgen signaling in prostate cancer

Journal: BMC Cancer

doi: 10.1186/s12885-018-4848-x

Comparison of DNA damage response gene expression in prostate cancer cell lines. a Venn diagram of differentially expressed DNA repair genes in LNCaP, VCaP, and PC3-AR cell line models. A total of 450 expert-curated DNA damage repair genes were assessed for androgen-mediated expression changes (2 nM R1881) using the following cut-offs: > 2-fold or
Figure Legend Snippet: Comparison of DNA damage response gene expression in prostate cancer cell lines. a Venn diagram of differentially expressed DNA repair genes in LNCaP, VCaP, and PC3-AR cell line models. A total of 450 expert-curated DNA damage repair genes were assessed for androgen-mediated expression changes (2 nM R1881) using the following cut-offs: > 2-fold or

Techniques Used: Expressing

Mirin inhibition of MRE11 reduces androgen-stimulated gene expression. a Mirin dose response of androgen-mediated transcription (2 nM R1881, 12 h). Relative expression (RT-qPCR) of FKBP5 mRNA in LNCaP, VCaP, and PC3-AR cells. b Relative expression (RT-qPCR) of SOCS2 , HOMER2 , and ABCC4 in PC3-AR cells. c Relative expression (RT-qPCR) of three androgen-mediated genes PSA , FKBP5 , and TMPRSS2 after inhibiting MRE11 with mirin or ATM with KU55933 in LNCaP cells. d Alamar blue cell viability assay measuring mirin effect on cell growth/survival in RWPE-1, LNCaP, VCaP, and PC3-AR after 72 h. e Mirin IC 50 values derived from Alamar blue cell viability assays from three experimental replicates
Figure Legend Snippet: Mirin inhibition of MRE11 reduces androgen-stimulated gene expression. a Mirin dose response of androgen-mediated transcription (2 nM R1881, 12 h). Relative expression (RT-qPCR) of FKBP5 mRNA in LNCaP, VCaP, and PC3-AR cells. b Relative expression (RT-qPCR) of SOCS2 , HOMER2 , and ABCC4 in PC3-AR cells. c Relative expression (RT-qPCR) of three androgen-mediated genes PSA , FKBP5 , and TMPRSS2 after inhibiting MRE11 with mirin or ATM with KU55933 in LNCaP cells. d Alamar blue cell viability assay measuring mirin effect on cell growth/survival in RWPE-1, LNCaP, VCaP, and PC3-AR after 72 h. e Mirin IC 50 values derived from Alamar blue cell viability assays from three experimental replicates

Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Viability Assay, Derivative Assay

Characterization of PC3 cells stably transduced with WT AR. a AR protein expression level in PC3-AR cells compared to LNCaP and VCaP cells. b Immunofluorescence localization of AR in PC3-AR cells before and after treatment with synthetic androgen (2 nM R1881, 15 min), which induces nuclear import of AR. c AR complexes isolated by immunoprecipitation and examined for Hsp90β and Hsp70 content, which is released by androgen. d Immunoblot showing androgen-induced expression of FKBP51 in LNCaP and PC3-AR cells after an overnight treatment of 2 nM R1881. e Androgen-induced G1 cell cycle arrest after a 12 h R1881 treatment
Figure Legend Snippet: Characterization of PC3 cells stably transduced with WT AR. a AR protein expression level in PC3-AR cells compared to LNCaP and VCaP cells. b Immunofluorescence localization of AR in PC3-AR cells before and after treatment with synthetic androgen (2 nM R1881, 15 min), which induces nuclear import of AR. c AR complexes isolated by immunoprecipitation and examined for Hsp90β and Hsp70 content, which is released by androgen. d Immunoblot showing androgen-induced expression of FKBP51 in LNCaP and PC3-AR cells after an overnight treatment of 2 nM R1881. e Androgen-induced G1 cell cycle arrest after a 12 h R1881 treatment

Techniques Used: Stable Transfection, Transduction, Expressing, Immunofluorescence, Isolation, Immunoprecipitation

Commonly used prostate cell lines contain potentially deleterious mutations. Diagrams showing the positions (lollipops) of deleterious mutations in DNA repair proteins. Shown are the deleterious mutations found in RWPE-1, LNCaP, and PC3-AR cells. Red dots correspond to missense mutations and blue dots correspond to missense mutations projected to result in protein truncation
Figure Legend Snippet: Commonly used prostate cell lines contain potentially deleterious mutations. Diagrams showing the positions (lollipops) of deleterious mutations in DNA repair proteins. Shown are the deleterious mutations found in RWPE-1, LNCaP, and PC3-AR cells. Red dots correspond to missense mutations and blue dots correspond to missense mutations projected to result in protein truncation

Techniques Used:

Androgen-dependent transcriptional responses of prostate cancer cell lines. a RT-qPCR shows androgen treatment (2 nM R1881, 12 h) induces FKBP5 , ABCC4 , EAF2 , and PIAS1 expression in PC3-AR cells. b Eigengene expression plots from WGCNA of PC3-AR RNA-sequencing. c Venn diagram of all differentially expressed transcripts in PC3-AR, LNCaP, and VCaP cell lines after 2 nM R1881 androgen treatment. Differential fold change was set to the following parameters: > 1.5-fold or
Figure Legend Snippet: Androgen-dependent transcriptional responses of prostate cancer cell lines. a RT-qPCR shows androgen treatment (2 nM R1881, 12 h) induces FKBP5 , ABCC4 , EAF2 , and PIAS1 expression in PC3-AR cells. b Eigengene expression plots from WGCNA of PC3-AR RNA-sequencing. c Venn diagram of all differentially expressed transcripts in PC3-AR, LNCaP, and VCaP cell lines after 2 nM R1881 androgen treatment. Differential fold change was set to the following parameters: > 1.5-fold or

Techniques Used: Quantitative RT-PCR, Expressing, RNA Sequencing Assay

Related Articles

DNA Extraction:

Article Title: Microbial Community Composition in Take-All Suppressive Soils
Article Snippet: .. DNA from endosphere and rhizosphere soils was extracted in three replicates using Power Soil DNA Isolation Kit (QIAGEN, United States), according to the manufacturer’s instructions. .. DNA was quantified and its purity evaluated using the A260/A280 and A260/A230 ratios provided by MultiskanTM GO software.

Transfection:

Article Title: In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies
Article Snippet: .. In order to express the IgG antibodies in mammalian cells, transfection quality DNA was prepared using Plasmid Plus Kit (Qiagen, 12945). .. A total of 180 μg of the DNA was transfected into 180 mL of Expi293TM cells (Thermo Fisher) using ExpiFectamineTM 293 Transfection Kit (Thermo Fisher, A14525) according to manufacturer’s instructions.

Amplification:

Article Title: Naturally Fermented Milk From Northern Senegal: Bacterial Community Composition and Probiotic Enrichment With Lactobacillus rhamnosus
Article Snippet: .. Amplicon libraries were pooled in equimolar amounts and purified using the Qiaquick Gel extraction kit (Qiagen). .. Paired-end sequencing of amplicons was conducted on the Illumina MiSeq platform (Illumina, Eindhoven, Netherlands).

Sample Prep:

Article Title: Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells
Article Snippet: .. RNA and DNA sample preparation RNA was extracted from an aliquot of dissociated tissue prior to filtering and antibody staining and from GFP-positive and GFP-negative FACS sorted cells using the RNeasy Micro Plus kit (74034, QIAGEN, Venlo, Netherlands). .. DNA for MethylC-seq was prepared from GFP-positive FACS-sorted cells using the DNeasy Blood and Tissue kit (69504, QIAGEN).

Isolation:

Article Title: Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats
Article Snippet: .. DNA methylation analysis Genomic DNA from ARC punches were isolated using Qiagen AllPrep DNA/RNA mini kit (80204) according to the manufacturer’s instructions. ..

Article Title: Identification and comparison of key RNA interference machinery from western corn rootworm, fall armyworm, and southern green stink bug
Article Snippet: .. Total RNA was isolated from whole flash-frozen WCR of each of 14 life stages by first homogenizing in Buffer RLT with 0.01% PEG using the RNeasy Mini Kit (Qiagen N.V., Hilden, Germany) following manufacturer’s instructions. .. Directly following column elution, isolated RNAs were DNase-treated using the Ambion TURBO DNA-free Kit and associated protocol (Thermo Fisher Scientific, Inc. Waltham, MA).

Next-Generation Sequencing:

Article Title: Comparison of initial oral microbiomes of young adults with and without cavitated dentin caries lesions using an in situ biofilm model
Article Snippet: .. DNA preparation and next generation sequencing DNA was extracted from biofilm samples and saliva samples using a commonly used DNA preparation kit (Qiagen DNA Blood and tissue kit, Qiagen, Hilden, G). .. Briefly, enamel slabs were placed in a 2 ml microcentrifuge tube and vortexed for 30 s at 2,500 rpm in 180 µl lysozyme-Triton TE-solution (20 mM Tris-Cl, pH 8.0, 2 mM sodium EDTA, 1.2% Triton® X-100, lysozyme 20 mg/ml) and stored at 37 °C for 30 min. Then, 20 µl of Proteinase K were added and the samples were thoroughly vortexed again for 30 s. After adding 200 µl Buffer AL and incubation at 56 °C for 60 min samples were placed on ice for 2 min.

DNA Methylation Assay:

Article Title: Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats
Article Snippet: .. DNA methylation analysis Genomic DNA from ARC punches were isolated using Qiagen AllPrep DNA/RNA mini kit (80204) according to the manufacturer’s instructions. ..

FACS:

Article Title: Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells
Article Snippet: .. RNA and DNA sample preparation RNA was extracted from an aliquot of dissociated tissue prior to filtering and antibody staining and from GFP-positive and GFP-negative FACS sorted cells using the RNeasy Micro Plus kit (74034, QIAGEN, Venlo, Netherlands). .. DNA for MethylC-seq was prepared from GFP-positive FACS-sorted cells using the DNeasy Blood and Tissue kit (69504, QIAGEN).

Gel Extraction:

Article Title: Naturally Fermented Milk From Northern Senegal: Bacterial Community Composition and Probiotic Enrichment With Lactobacillus rhamnosus
Article Snippet: .. Amplicon libraries were pooled in equimolar amounts and purified using the Qiaquick Gel extraction kit (Qiagen). .. Paired-end sequencing of amplicons was conducted on the Illumina MiSeq platform (Illumina, Eindhoven, Netherlands).

Purification:

Article Title: Naturally Fermented Milk From Northern Senegal: Bacterial Community Composition and Probiotic Enrichment With Lactobacillus rhamnosus
Article Snippet: .. Amplicon libraries were pooled in equimolar amounts and purified using the Qiaquick Gel extraction kit (Qiagen). .. Paired-end sequencing of amplicons was conducted on the Illumina MiSeq platform (Illumina, Eindhoven, Netherlands).

Plasmid Preparation:

Article Title: In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies
Article Snippet: .. In order to express the IgG antibodies in mammalian cells, transfection quality DNA was prepared using Plasmid Plus Kit (Qiagen, 12945). .. A total of 180 μg of the DNA was transfected into 180 mL of Expi293TM cells (Thermo Fisher) using ExpiFectamineTM 293 Transfection Kit (Thermo Fisher, A14525) according to manufacturer’s instructions.

Staining:

Article Title: Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells
Article Snippet: .. RNA and DNA sample preparation RNA was extracted from an aliquot of dissociated tissue prior to filtering and antibody staining and from GFP-positive and GFP-negative FACS sorted cells using the RNeasy Micro Plus kit (74034, QIAGEN, Venlo, Netherlands). .. DNA for MethylC-seq was prepared from GFP-positive FACS-sorted cells using the DNeasy Blood and Tissue kit (69504, QIAGEN).

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  • 91
    Qiagen dna methylation analysis genomic dna
    Gene expression and hypothalamic Pomc <t>DNA</t> methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the <t>ARC.</t> c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p
    Dna Methylation Analysis Genomic Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 91/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Qiagen stranded dna
    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated <t>DNA</t> template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and <t>PCR</t> amplified for the next round of selection.
    Stranded Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Qiagen csf derived circulating tumor dna ctdna analysis formalin fixed paraffin embedded
    Droplet digital PCR results from examination of the CNS tissue and the <t>CSF-ctDNA</t> for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.
    Csf Derived Circulating Tumor Dna Ctdna Analysis Formalin Fixed Paraffin Embedded, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Qiagen human intestinal biopsies bacterial dna
    Vancomycin treatment or supplementation with butyrate ablates the Nlrp1 phenotype. a Stool from WT and Nlrp1 −/− mice was harvested before and after vancomycin (50 mg/L) treatment for 4 weeks. Bacterial <t>DNA</t> from stool was isolated from untreated (UT) and vancomycin-treated mice, and depletion of the Coccoides group (belonging to the Clostridiales phylum) was confirmed using specific <t>16S</t> primers by quantitative PCR. Results were normalized to the total bacteria present in the stool. b Vancomycin-treated mice were subjected to 3% (w/v) DSS for 6 days and disease severity was measured according to percentage weight loss and c colon length. Short chain fatty acid analysis was performed on fecal samples collected from WT and Nlrp1 −/− mice before (UT) and after vancomycin treatment. The concentration of d butyrate and e propionate was measured by gas chromatography mass spectrometry. f WT and Nlrp1 −/− mice were supplemented with 2% butyrate in drinking water ad libitum for 28 days, and then subjected to 2.5% (w/v) DSS for 6 days and disease severity measured according to percentage weight loss or g colon length. Data are representative of 3 independent experiments with 3–6 mice per group. Means ± SEM; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. A two-tailed unpaired t- test was used to determine statistical significance between two groups; and a two-way ANOVA with Tukey’s post-hoc comparisons was performed on data that involved more than two comparisons
    Human Intestinal Biopsies Bacterial Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p

    Journal: International Journal of Obesity (2005)

    Article Title: Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats

    doi: 10.1038/s41366-018-0094-1

    Figure Lengend Snippet: Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p

    Article Snippet: DNA methylation analysis Genomic DNA from ARC punches were isolated using Qiagen AllPrep DNA/RNA mini kit (80204) according to the manufacturer’s instructions.

    Techniques: Expressing, DNA Methylation Assay, Quantitative RT-PCR, Functional Assay, Methylation

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Journal: Nucleic Acids Research

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    doi: 10.1093/nar/gky667

    Figure Lengend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Article Snippet: The amplified double stranded DNA was purified using a PCR cleanup column (Qiagen) and the PEG-functionalized strand was separated from the FAM labeled strand on a denaturing 10% polyacrylamide gel.

    Techniques: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    Droplet digital PCR results from examination of the CNS tissue and the CSF-ctDNA for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.

    Journal: Frontiers in Oncology

    Article Title: Detection of the MYD88 p.L265P Mutation in the CSF of a Patient With Secondary Central Nervous System Lymphoma

    doi: 10.3389/fonc.2018.00382

    Figure Lengend Snippet: Droplet digital PCR results from examination of the CNS tissue and the CSF-ctDNA for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.

    Article Snippet: CSF-derived circulating tumor DNA (CtDNA) analysis Formalin-fixed paraffin-embedded (FFPE) tissue was used to extract DNA using QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA, USA).

    Techniques: Digital PCR, Mutagenesis

    Vancomycin treatment or supplementation with butyrate ablates the Nlrp1 phenotype. a Stool from WT and Nlrp1 −/− mice was harvested before and after vancomycin (50 mg/L) treatment for 4 weeks. Bacterial DNA from stool was isolated from untreated (UT) and vancomycin-treated mice, and depletion of the Coccoides group (belonging to the Clostridiales phylum) was confirmed using specific 16S primers by quantitative PCR. Results were normalized to the total bacteria present in the stool. b Vancomycin-treated mice were subjected to 3% (w/v) DSS for 6 days and disease severity was measured according to percentage weight loss and c colon length. Short chain fatty acid analysis was performed on fecal samples collected from WT and Nlrp1 −/− mice before (UT) and after vancomycin treatment. The concentration of d butyrate and e propionate was measured by gas chromatography mass spectrometry. f WT and Nlrp1 −/− mice were supplemented with 2% butyrate in drinking water ad libitum for 28 days, and then subjected to 2.5% (w/v) DSS for 6 days and disease severity measured according to percentage weight loss or g colon length. Data are representative of 3 independent experiments with 3–6 mice per group. Means ± SEM; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. A two-tailed unpaired t- test was used to determine statistical significance between two groups; and a two-way ANOVA with Tukey’s post-hoc comparisons was performed on data that involved more than two comparisons

    Journal: Nature Communications

    Article Title: NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease

    doi: 10.1038/s41467-018-06125-0

    Figure Lengend Snippet: Vancomycin treatment or supplementation with butyrate ablates the Nlrp1 phenotype. a Stool from WT and Nlrp1 −/− mice was harvested before and after vancomycin (50 mg/L) treatment for 4 weeks. Bacterial DNA from stool was isolated from untreated (UT) and vancomycin-treated mice, and depletion of the Coccoides group (belonging to the Clostridiales phylum) was confirmed using specific 16S primers by quantitative PCR. Results were normalized to the total bacteria present in the stool. b Vancomycin-treated mice were subjected to 3% (w/v) DSS for 6 days and disease severity was measured according to percentage weight loss and c colon length. Short chain fatty acid analysis was performed on fecal samples collected from WT and Nlrp1 −/− mice before (UT) and after vancomycin treatment. The concentration of d butyrate and e propionate was measured by gas chromatography mass spectrometry. f WT and Nlrp1 −/− mice were supplemented with 2% butyrate in drinking water ad libitum for 28 days, and then subjected to 2.5% (w/v) DSS for 6 days and disease severity measured according to percentage weight loss or g colon length. Data are representative of 3 independent experiments with 3–6 mice per group. Means ± SEM; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. A two-tailed unpaired t- test was used to determine statistical significance between two groups; and a two-way ANOVA with Tukey’s post-hoc comparisons was performed on data that involved more than two comparisons

    Article Snippet: 16S metagenomics analysis of human intestinal biopsies Bacterial DNA was isolated from rectal mucosal biopsies using QIAGEN DNeasy Blood & Tissue kit (Hilden, Germany).

    Techniques: Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Concentration Assay, Gas Chromatography, Mass Spectrometry, Two Tailed Test