Journal: The Journal of Infectious Diseases

Article Title: Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood

doi: 10.1093/infdis/jiw330

Figure Lengend Snippet: List of Loop-Mediated Isothermal Amplification Primers Used in the Present Study

Article Snippet: As a positive control for the sample-preparation step, MS2 phage (Zeptomatrix, New York) was included in the lysis buffer at a final concentration of 10 7 PFU/mL and detected with MS2-specific LAMP primers (Table ).

Techniques: Amplification, Sequencing, Virus

Journal: The Journal of Infectious Diseases

Article Title: Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood

doi: 10.1093/infdis/jiw330

Figure Lengend Snippet: Sensitivity of the Ebola virus (EBOV) reverse transcription–loop-mediated isothermal amplification method. Sensitivity was determined against 2 strains of EBOV, EBOV-Kikwit (stock concentration, 1.12 × 107 plaque-forming units [PFU]/mL) and EBOV-Makona (4 × 107 PFU/mL). Ten-fold serial dilutions of each virus were made in human whole blood. Each dilution was added to the lysis buffer at 5%, and 50 µL of this suspension was used to reconstitute lyophilized reagents. Abbreviations: NEG, negative control; POS, positive control (MS2 phage).

Article Snippet: As a positive control for the sample-preparation step, MS2 phage (Zeptomatrix, New York) was included in the lysis buffer at a final concentration of 10 7 PFU/mL and detected with MS2-specific LAMP primers (Table ).

Techniques: Virus, Reverse Transcription, Amplification, Concentration Assay, Lysis, Suspension, Negative Control, Positive Control

Journal: The Journal of Infectious Diseases

Article Title: Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood

doi: 10.1093/infdis/jiw330

Figure Lengend Snippet: Specificity of the Ebola virus (EBOV) reverse transcription–loop-mediated isothermal amplification (RT-LAMP) method. After completion of the reaction (40 minutes) LAMP reaction products were separated on 2% agarose gel. A, Specificity of EBOV RT-LAMP, using RNA extracts. A total of 5 µL of RNA was used as template in a 25-µL reaction mix. The reaction was performed at 72°C for 40 minutes. CCHFV, Crimean-Congo hemorrhagic fever virus; CHIKV: Chikungunya virus; DENV-1, dengue virus serotype 1; DENV-2, dengue virus serotype 2; DENV-3, dengue virus serotype 3; DENV-4, dengue virus serotype 4; EBOV, Ebola virus-Makona; LASV-J, Lassa virus–Josiah; LASV-S, Lassa virus–Sauer; MARV, Marburg virus–Musoke; WNV: West Nile virus. B, Specificity of EBOV RT-LAMP, using different pathogens. Each pathogen was diluted 1:10 in human whole blood. Each dilution was added to the lysis buffer at 5%, and 50 µL of this suspension was used to reconstitute lyophilized reagents. The reaction was performed at 72°C for 40 minutes. Abbreviations: MS2, MS2 phage (positive control); Neg, negative control (no target); rEBOV, recombinant EBOV-Makona complementary DNA plasmid. A list of other pathogens is provided in Table ​Table22.

Article Snippet: As a positive control for the sample-preparation step, MS2 phage (Zeptomatrix, New York) was included in the lysis buffer at a final concentration of 10 7 PFU/mL and detected with MS2-specific LAMP primers (Table ).

Techniques: Virus, Reverse Transcription, Amplification, Agarose Gel Electrophoresis, Lysis, Suspension, Positive Control, Negative Control, Recombinant, Plasmid Preparation

Journal: medRxiv

Article Title: SARS-CoV-2 virus dynamics in recently infected people – data from a household transmission study

doi: 10.1101/2022.03.17.22272516

Figure Lengend Snippet: Ct value curves over time since each participant first tested positive against each target, within age groups. Dots represent mean observed values within age groups, and vertical bars show bootstrapped 95% confidence intervals. Smooth lines represent predicted values from the Generalized Additive Model of Ct values over time, accounting for age and repeated measurements. Panel A shows results from participants age 0-11 (triangle) compared to the reference group, age 18-49 (circle); Panels B and C repeat this comparison with age 12-17 or 50+. Each plot from left to right represents a SARS-CoV-2 target from one of the two included testing platforms (CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel or the ThermoFisher TaqPathTM COVID-19 ComboKit).

Article Snippet: All specimens were tested by RT-PCR using either the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel (EUA CDC-006-00019; with N1 and N2 gene targets and RNase P control; CDC assay) or the ThermoFisher TaqPath™ COVID-19 ComboKit (with S and N gene and ORF1ab targets and MS2 spike control; ThermoFisher assay).

Techniques: Quantitative RT-PCR, Diagnostic Assay

Journal: Nature Communications

Article Title: Epigenetic regulation of the circadian gene Per1 contributes to age-related changes in hippocampal memory

doi: 10.1038/s41467-018-05868-0

Figure Lengend Snippet: Overexpression of Per1 in the dorsal hippocampus ameliorates age-related impairments in object location memory. a Schematic of lentivirus construct used to overexpress v5-tagged Per1 (pLVX-v5Per1) compared to the empty vector control (pLVX-EV). b Per1 mRNA was significantly increased 24 and 48 h after transfection of pLVX-v5Per1 in HT22 cells compared to cells transfected with EV (Two-way ANOVA: Group x Timepoint interaction ( F (1,8) = 52.8, p < 0.001), Sidak’s post hoc tests, *** p < 0.001, n = 3, 3, 3, 3). c 18-m.o. mice given hippocampal infusions of pLVX-v5Per1 showed significantly better memory for OLM than mice given pLVX-EV control virus (Two-way ANOVA: virus x session interaction, ( F (1,29) = 7.15, p < 0.05), Sidak’s post hoc tests, ** p < 0.01, *** p < 0.001, n = 16, 15, all males). d Total exploration was similar for both groups at test ( t (29) = 0.57, p = 0.57). e Schematic of CRISPR/dCas9 Synergistic Activation Mediator (SAM) system used to drive Per1 transcription. Top: Individual components of SAM. Bottom: Components assembled at the Per1 promoter, driving Per1 transcription. f Per1 mRNA was significantly increased 48 h after transfection of CRISPR-SAM components in HT22 cells compared to cells transfected with the control sgRNA (Two-way ANOVA: Group x Timepoint interaction ( F (1,8) = 14.77, p < 0.01), Sidak’s post hoc tests, ** p < 0.001, n = 3,3,3,3). g 18-m.o. mice given hippocampal infusions of the CRISPR-SAM system with sgRNA targeting Per1 showed significantly better memory for OLM compared to EV control mice with control sgRNA (Two-way ANOVA: Virus x Session interaction ( F (1,30) = 5.83, p < 0.05), Sidak’s post hoc tests, *** p < 0.001, n = 17, 15, all males). h Total exploration was similar for both groups at test ( t (30) = 0.41, p = 0.68). Data are presented as mean ± SEM

Article Snippet: Similarly, to verify that the CRISPR-SAM system can effectively drive Per1 transcription, HT22 cells were transfected with dCas9-VP64 (lenti MS2-P65_HSF1_Blast was a gift from Feng Zhang Addgene plasmid #61425), MS2-P65-HSF1 (lenti dCAS-VP64_Blast was a gift from Feng Zhang, Addgene plasmid #61426), and either Per1 sgRNA (Per1 sgRNA) or the non-targeting control sgRNA (ctrl sgRNA) (lenti sgRNA (MS2)_zeo backbone was a gift from Feng Zhang, Addgene plasmid #61427) using Lipofectamine LTX (Invitrogen).

Techniques: Over Expression, Construct, Plasmid Preparation, Control, Transfection, Virus, CRISPR, Activation Assay

Journal: Cancer Research Communications

Article Title: Netrin-1 and UNC5B Cooperate with Integrins to Mediate YAP-Driven Cytostasis

doi: 10.1158/2767-9764.CRC-24-0101

Figure Lengend Snippet: A CRISPR screen identifies UNC5B as a YAP effector. A, Z -scores comparing YAP-expressing (day 25) to Empty (day 25) cells for each sgRNA in the CRISPR screen. Select genes are indicated. n = 4. B, TEAD1 knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing TEAD1 knockout efficiency and expression of ectopic YAP (right). *, P < 0.05 compared with sgControl cells, n = 3. C, UNC5B knockout rescues YAP-induced cytostasis in Y79 cells (left). Western blot showing UNC5B knockout efficiency and expression of ectopic YAP (right). ***, P < 0.001 compared with sgControl cells, n = 4. D, RT-qPCR for YAP target genes ( AJUBA, AMOTL2, CYR61 and AHNAK ) or a control gene ( RER1 ) in control or UNC5B knockout Y79 cells ± ectopic YAP expression. n = 3.

Article Snippet: Briefly, a pooled CRISPR sgRNA library was constructed by cloning sgRNA sequences (4/gene plus 50 nontargeting controls) into the LentiCRISPR v2 lentiviral backbone (Addgene plasmid #52961; RRID:Addgene_52961).

Techniques: CRISPR, Expressing, Knock-Out, Western Blot, Quantitative RT-PCR, Control

Journal: Lab on a chip

Article Title: LabDisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza A H3N2 virus.

doi: 10.1039/c5lc00871a

Figure Lengend Snippet: Fig. 3 Fluorescence signal for parallel RT-PCR reactions acquired after the annealing step in each reaction cavity. Except reaction cavity 4 (parainfluenza panel) and reaction cavity 8 (atypical bacteria panel) each reaction cavity contained primers and fluorescence probe also detecting RNA bacteriophage MS2 as internal control. Missing primer and fluorescence probes for internal control led to the negative signal in these two reaction cavities.

Article Snippet: As initial sample material we used the RNA bacteriophage MS2 (DSM-13767, type: Levivirus, species: Enterobacteria phage MS2, DSMZ GmbH, Germany) acting as internal control of the used respiratory panels and was diluted with SM buffer, pH 7.5 (Alfa Aesar GmbH & Co KG, Germany) and applied with the concentration of 75 pfu ml−1.

Techniques: Fluorescence, Reverse Transcription Polymerase Chain Reaction, Bacteria, Control