Journal: NPJ Parkinson's Disease

Article Title: Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity

doi: 10.1038/s41531-023-00444-w

Figure Lengend Snippet: a Spontaneous excitatory (sEPSC) and inhibitory postsynaptic currents (sIPSC) measured in DIV14-18 hippocampal neurons from SNCA −/− rats transduced with αS WT or αS S129A (schematics created with BioRender.com). b Representative sEPSC traces. c Representative sIPSC traces. Scale bars, X -axis = 1 s and Y -axis = 100 pA. d sEPSC frequency in SNCA −/− neurons expressing WT αS or αS S129A. e Cumulative frequency distribution of data shown in d , expressed as percentage. f sEPSC amplitude in SNCA −/− neurons expressing αS WT or αS S129A. g Cumulative frequency distribution plot of data shown in f , expressed as percentage. Total number of individual events analyzed in panels e and g : αS WT = 1228; αS S129A = 1701. Total number of individual cells across two independent neuronal cultures ( N = 2 ) recorded in sEPSC experiments: WT αS = 21 and αS S129A n = 12. h , i sIPSC frequency between conditions as bar charts and cumulative distribution, respectively. j sIPSC amplitude in SNCA −/− neurons expressing WT or S129A αS. k Cumulative frequency distribution of data shown in j . Total number of individual events analyzed in panels i and k : αS WT = 302; αS S129A = 585. Total number of individual cells across two independent neuronal cultures recorded in sIPSC experiments: αS WT = 19 and αS S129A = 15. l , m The excitatory/inhibitory ratio of the amplitude as a bar chart and cumulative frequency distribution, respectively. E/I amplitude ratios were derived from f and j . Respective averages in f (αS WT or αS S129A) were divided by respective individual values (αS WT or αS S129A) in j to obtain E/I amplitude ratios. Each circle represents an individual cell. Recordings performed on at least three different days. Unpaired t -tests with Welch’s correction; two-tailed; mean ± SD; ns not significant; *** p < 0.001; **** p < 0.0001.

Article Snippet: Briefly, 293-T cells were transfected with αS WT or S129A plasmids along with pMD2.G and psPAX2 (packaging plasmids: Addgene #12259 and #12260, respectively).

Techniques: Transduction, Expressing, Derivative Assay, Two Tailed Test

Journal: NPJ Parkinson's Disease

Article Title: Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity

doi: 10.1038/s41531-023-00444-w

Figure Lengend Snippet: a – f Generation of the S129A knock-in mutation in mice. a Genomic structure of the mouse SNCA gene. Exons are depicted after transcript variant SNCA-201 (ENSMUST00000114268.5), with coding and non-coding regions shown as filled or open boxes, respectively. Exon 5 is boxed with red dashed line. b A knock-in strategy using CRISPR-Cas9 and ssODN was employed to generate the S129A mutation in Exon 5 of the endogenous SNCA gene. Relative positions of sgRNA (orange horizontal line), ssODN (blue horizontal line), and the S129A mutation (red vertical line) are indicated. c – f ES cell clone screening and mouse genotyping. A 3-primer PCR strategy (primers indicated with black and red arrows) was designed to distinguish WT and mutant alleles ( c , d ). For genotyping, a common pair of primers (indicated with green arrows) was used for PCR followed by sequencing to distinguish different genotypes ( c , e , f ). g Total mouse brain homogenates from indicated genotypes. WB for total αS, pS129 (D1R1R) and GAPDH. h Input and out current curve from hippocampal slices (CA1 region) of indicated mouse genotypes. N = 3 animals each, n = 16 (WT) or 17 (S129A KI ) individual slices. i Paired-pulse facilitation of WT and S129A KI hippocampal slices. Inter-stimulation intervals as indicated. N = 3 animals each, n = 16 (WT) or 17 (S129A KI ) individual slices. Unpaired t -tests for 20, 40, 60, 100, 200, and 500 ms with Welch’s correction; two-tailed; mean ± SD; ns not significant; * p < 0.05; ** p < 0.01. j Short-term plasticity assessed by multi-pulse events. Values normalized to first excitatory post synaptic current (EPSP). N = 3 animals each, n = 16 (WT) or 17 (S129A KI ) individual slices. Unpaired t -tests for pulses 2, 3, 4, 5, 6, 7, and 8 with Welch’s correction; two-tailed; mean ± SD; ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. k , l Long Term Potentiation (LTP) of WT and S129A KI . LTP induced by standard 100 Hz stimulation. N = 3 animals each, n = 8 (WT) or 10 (S129A KI ) individual slices. Unpaired t -tests for panel l (values at 60 min from K) with Welch’s correction; two-tailed; mean ± SD; ** p < 0.01.

Article Snippet: Briefly, 293-T cells were transfected with αS WT or S129A plasmids along with pMD2.G and psPAX2 (packaging plasmids: Addgene #12259 and #12260, respectively).

Techniques: Knock-In, Mutagenesis, Variant Assay, CRISPR, Sequencing, Two Tailed Test

Journal: Disease Markers

Article Title: Molecular Markers for Detecting Schistosoma Species by Loop-Mediated Isothermal Amplification

doi: 10.1155/2020/8042705

Figure Lengend Snippet: TD-PCR F3-B3 for amplifying S. bovis NADH-1. A 58-53°C temperature range and 53°C × 15 cycles were used. Sm: S. mansoni DNA; Sh: S. haematobium DNA; Sj: S. japonicum DNA; Si: S. intercalatum DNA; Sb: S. bovis DNA; N: negative control (ultrapure water, no DNA). M: molecular weight marker (100 bp PLUS BLUE DNA ladder).

Article Snippet: Genomic DNA from adult male and female S. haematobium (Egyptian Strain; NR-31682) and genomic DNA from adult male and female S. japonicum (Chinese Strain; NR-36066) were obtained from the Schistosomiasis Resource Centers for distribution by BEI Resources, NIAID, NIH ( https://www.beiresources.org/Collection/51/Schistosome-Resource-Centers.aspx ).

Techniques: Negative Control, Molecular Weight, Marker

Journal: Disease Markers

Article Title: Molecular Markers for Detecting Schistosoma Species by Loop-Mediated Isothermal Amplification

doi: 10.1155/2020/8042705

Figure Lengend Snippet: TD-PCR F3-B3 for amplifying genus Schistosoma ITS-1. A 61-57°C temperature range, and 57°C × 30 cycles were used. Sm: S. mansoni DNA; Sh: S. haematobium DNA; Sj: S. japonicum DNA; Si: S. intercalatum DNA; Sb: S. bovis DNA; N: negative control (ultrapure water, no DNA). M: molecular weight marker (100 bp PLUS BLUE DNA ladder).

Article Snippet: Genomic DNA from adult male and female S. haematobium (Egyptian Strain; NR-31682) and genomic DNA from adult male and female S. japonicum (Chinese Strain; NR-36066) were obtained from the Schistosomiasis Resource Centers for distribution by BEI Resources, NIAID, NIH ( https://www.beiresources.org/Collection/51/Schistosome-Resource-Centers.aspx ).

Techniques: Negative Control, Molecular Weight, Marker

Journal: Disease Markers

Article Title: Molecular Markers for Detecting Schistosoma Species by Loop-Mediated Isothermal Amplification

doi: 10.1155/2020/8042705

Figure Lengend Snippet: LAMP assay for amplifying S. bovis NADH-1. (a). Specificity assessment. Only NADH-1 was amplified using S. bovis DNA. Sm: S. mansoni DNA; Sh: S. haematobium DNA; Sj: S. japonicum DNA; Si: S. intercalatum DNA; Sb: S. bovis DNA; N: negative controls (ultrapure water, no DNA). (b). Sensitivity assessment. Sb: S. bovis genomic DNA (10 ng/ μ L); lanes 10 −1 -10 −9 , 10-fold serially dilutions.

Article Snippet: Genomic DNA from adult male and female S. haematobium (Egyptian Strain; NR-31682) and genomic DNA from adult male and female S. japonicum (Chinese Strain; NR-36066) were obtained from the Schistosomiasis Resource Centers for distribution by BEI Resources, NIAID, NIH ( https://www.beiresources.org/Collection/51/Schistosome-Resource-Centers.aspx ).

Techniques: Lamp Assay, Amplification

Journal: Disease Markers

Article Title: Molecular Markers for Detecting Schistosoma Species by Loop-Mediated Isothermal Amplification

doi: 10.1155/2020/8042705

Figure Lengend Snippet: LAMP for amplifying the genus Schistosoma ITS-1 sequence. Lanes Sm, Sh, Sj, Si, and Sb mean S. mansoni , S. haematobium , S. japonicum , S. intercalatum , and S. bovis DNAs, respectively; Lanes N: negative controls (ultrapure water, no DNA).

Article Snippet: Genomic DNA from adult male and female S. haematobium (Egyptian Strain; NR-31682) and genomic DNA from adult male and female S. japonicum (Chinese Strain; NR-36066) were obtained from the Schistosomiasis Resource Centers for distribution by BEI Resources, NIAID, NIH ( https://www.beiresources.org/Collection/51/Schistosome-Resource-Centers.aspx ).

Techniques: Sequencing

Journal: Disease Markers

Article Title: Molecular Markers for Detecting Schistosoma Species by Loop-Mediated Isothermal Amplification

doi: 10.1155/2020/8042705

Figure Lengend Snippet: Sensitivity assessment of LAMP for amplifying Schistosoma ITS-1 sequence using DNA from different schistosome species. (a) Detection limit for S. mansoni (1 pg). (b) Detection limit for S. haematobium (0.1 pg). (c) Detection limit for S. intercalatum (1 pg). (d) Detection limit for S. bovis (10 pg). Lanes Sm, Sh, Si, and Sb mean S. mansoni , S. haematobium , S. intercalatum , and S. bovis DNAs, respectively. Lanes 10 −1 -10 −9 : 10-fold serial dilutions of DNA. Lanes N: negative controls (no DNA template).

Article Snippet: Genomic DNA from adult male and female S. haematobium (Egyptian Strain; NR-31682) and genomic DNA from adult male and female S. japonicum (Chinese Strain; NR-36066) were obtained from the Schistosomiasis Resource Centers for distribution by BEI Resources, NIAID, NIH ( https://www.beiresources.org/Collection/51/Schistosome-Resource-Centers.aspx ).

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Non-canonical NFκB activation promotes chemokine expression in podocytes

doi: 10.1038/srep28857

Figure Lengend Snippet: ( A ) NIK siRNA downregulates NIK protein and mRNA expression. *p < 0.005 vs Scramble siRNA. ( B ) NIK siRNA prevents the upregulation of CCL21 mRNA in response to TWEAK. ( C ) NIK siRNA prevents the upregulation of CCL21 protein in response to TWEAK at 24 h. ( D ) NIK siRNA does not prevent the upregulation of CCL19 mRNA in response to TWEAK. ( E ) NIK siRNA does not prevent the upregulation of RANTES mRNA in response to TWEAK. Expression of mRNA was assessed by real time RT-PCR and protein by Western blot. Mean ± SEM of three independent experiments, *p < 0.005 vs Scramble siRNA, **p < 0.005 vs Scramble siRNA+TWEAK.

Article Snippet: Cells were seeded at a 3 × 10 5 in 6-wells plates and transfected with 25 nM NIK siRNA (Santa Cruz), Opti-MEM I Reduced Serum Medium and Lipofectamine 2000 (Invitrogen) .

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Non-canonical NFκB activation promotes chemokine expression in podocytes

doi: 10.1038/srep28857

Figure Lengend Snippet: In splenocytes, both CCL19 and CCL21 are transcriptional targets of the non-canonical pathway for NFκB activation involving NFκB2/RelB heterodimers . ( A ) Murine renal tubular cells . TWEAK-induced RANTES expression is inhibited by parthenolide, suggesting canonical NFκB activation. By contrast, CCL21 expression is prevented by NIK siRNA, but not by parthenolide. While not specifically studied by functional inhibitors, the time-course of CCL19 expression and the fact that CCL19 was upregulated by TWEAK but not by TNF suggests that CCL19 is a transcriptional target for non-canonical NFκB activation in tubular cells. ( B ) Podocytes. Note the different time-course of TWEAK-induced RANTES and CCL19 mRNA expression between tubular cells and podocytes. The increased CCL19 mRNA and the early increase in RANTES mRNA is inhibited by parthenolide, suggesting dependence on canonical NFκB activation. CCL21 is the only chemokine that depends on non-canonical NFκB activation in splenocytes, tubular cells and podocytes. The late increase of RANTES mRNA expression in podocytes could not be inhibited by targeting the canonical or non-canonical pathways suggesting NFκB-independence or activation of alternative NFκB pathways .

Article Snippet: Cells were seeded at a 3 × 10 5 in 6-wells plates and transfected with 25 nM NIK siRNA (Santa Cruz), Opti-MEM I Reduced Serum Medium and Lipofectamine 2000 (Invitrogen) .

Techniques: Activation Assay, Expressing, Functional Assay