Journal: medRxiv

Article Title: Validation of an unbiased metagenomic detection assay for RNA viruses in viral transport media and plasma

doi: 10.1101/2024.03.26.24304688

Figure Lengend Snippet: Number of MS2 Phage Reads in Different Sample Matrices.

Article Snippet: RNA from contrived and remnant clinical samples was extracted using the Qiagen RNeasy Plus Micro Kit (Qiagen #74034) with minor changes to the manufacturer’s standard protocol adapting to the specific sample matrix and input volume of 140 µL sample material augmented with 5 µL of MS2 Phage internal control (ZeptoMetrix, 0810274; 145 µL total input).

Techniques:

Journal: Nature Communications

Article Title: Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway

doi: 10.1038/s41467-023-39337-0

Figure Lengend Snippet: a Population gene expression levels of MAP1B transcripts in the dorsal lateral prefrontal cortical region (BA9) among several psychiatric disorders and healthy controls collected by PsychENCODE Consortium. Y -axis: Gene expression value of MAP1B mRNA (RNA-seq TPM). ASD autism spectrum disorder ( n = 31). BPD bipolar disorder ( N = 172). SCZ schizophrenia ( N = 497). CTR, health ( N = 1166). The gene expression levels of MAP1B in ASD are significantly higher than others by one-side t -test. p < 8.66e-7 for CTR, p < 1.42e-3 for SCZ, and p < 4.83e-8 for BPD. b Experimental strategy for assessing cognitive functions of targeted Map1b gene activation in CAMK2A-expressing excitatory neurons in the PFC of mice. Activation of endogenous Map1b gene in the PFC was achieved through stereotaxic injection of AAV expressing Map1b -targeting guide RNA (AAV8- sgMap1b -hSyn1-flex-mCherry) and AAV expressing Cre driven by Camk2a promoter (AAV9- pCamK2a -GFP-Cre) into the medial PFC of dCas9Activator mice. c Representative confocal images of neurons in the prefrontal cortex (PFC) expressing GFP-Cre (green), sgRNA-mCherry (red), MAP1B (white) in AAV-injected dCas9Activator mice, assessed after behavioral tests. Scale bars: 10 μm. d Quantification of MAP1B intensity in GFP+/mCherry+ neurons in mPFC. Two-tailed, unpaired Student’s t -test, p = 0.0045. N = 4 individual mice. e Experimental scheme of testing social interest (SI) and social novelty (SN). f , g MAP1B-EE mice exhibited reduced social interest (SI, f ), p = 0.0195 and social recognition (SN, g ), p = 0.0090. Two-tailed, unpaired Student’s t -test. sgCtrl : N = 8 mice, sgMap1b : N = 7 mice. Data are presented as box plot in ( a ) (center line, median; box limits, upper and lower quartiles; whiskers, min to max). Data are presented as mean ± s.e.m in other panels. Source data are provided as a Source Data file.

Article Snippet: Five sgRNAs targeting the proximal promoter of mouse Map1b gene ( sgMap1b #1–5), between −305 bp and −24 bp relative to the TSS, were designed in Benchling based on published scoring methods (sequences are included in Supplementary Data ) and cloned to the same lentiviral sgRNA vector (Addgene #61427).

Techniques: Gene Expression, RNA Sequencing, Activation Assay, Expressing, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway

doi: 10.1038/s41467-023-39337-0

Figure Lengend Snippet: a Experimental scheme for assessing the autophagy in MAP1B-EE neurons. Hippocampal neurons were isolated from dCas9Activator mice and infected with LV-Cre-GFP and LV-sgRNA-mCherry. b Representative confocal images of neurons infected with either sgMap1b (MAP1B-EE) or sgCtrl (Control) expressing Cre-GFP (green), sgRNA-mCherry (red), LC3 (white). Scale bars, 10 μm. c LC3 intensity. Two-tailed, unpaired Student’s t -test with unequal variances, p = 0.0476. Ctrl: N = 3 independent biological replicates. d Sample Western blot analysis of mouse neurons with MAP1B-EE (LV- sgMap1b infected ) and controls ( sgCtrl ). e Quantitative analysis of LC3-I: p = 0.1597; LC3-II: p = 0.0297; LC3-II/I (LC3-II/LC3-I): p = 0.0434; two-tailed, unpaired Student’s t -test with unequal variances. f Sample Western blot analysis of hippocampal neurons with MAP1B-EE and controls treated with BafA1 or vehicle. g Quantitative analysis of proteins treated with BafA1 before harvest. LC3I: p = 0.1359; LC3-II: p = 0.0049; LC3-II/I: p = 0.0107; two-tailed, unpaired Student’s t -test with unequal variances. For ( e ) and ( g ), protein amounts were normalized to GAPDH and subsequently normalized to control cells. N = 3 biological replicates. h , i Experimental scheme for assessing autophagy flux. Hippocampal neurons from dCas9Activator mice were transfected with an autophagy reporter (CAG-mCherry/GFP/LC3-IRES-Cre) together with either sgCtrl or sgMap1b . j Representative confocal images of autophagy reporter-transfected cells showing autophagosomes (yellow puncta) and autophagolysosomes (red puncta) in neurons. Scale bars, 10 μm. k – n Number of total puncta, p = 0.0038 ( k ), autophagosomes, p = 0.0188 ( l ), and autophagolysosomes, p = 0.0035 ( m ), and calculated ratio of autophagolysosomes over total puncta, p = 0.3070 ( n ). Quantification of puncta was done after 3D reconstruction, two-tailed, unpaired Student’s t -test was used. N = 3 biological replicates. o Representative confocal images of p62 (white) puncta localized in hippocampal neurons. Scale bars, 10 μm. p Quantification of p62 puncta was done after 3D reconstruction. Two-tailed, unpaired Student’s t -test, p = 0.0026. N = 3 biological replicates. All data are presented as mean ± s.e.m. Source data are provided as a Source Data file.

Article Snippet: Five sgRNAs targeting the proximal promoter of mouse Map1b gene ( sgMap1b #1–5), between −305 bp and −24 bp relative to the TSS, were designed in Benchling based on published scoring methods (sequences are included in Supplementary Data ) and cloned to the same lentiviral sgRNA vector (Addgene #61427).

Techniques: Isolation, Infection, Control, Expressing, Two Tailed Test, Western Blot, Transfection

Journal: Nature Communications

Article Title: Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway

doi: 10.1038/s41467-023-39337-0

Figure Lengend Snippet: a , b Analysis of LC3 intensity of human neurons with MAP1B-EE. Representative confocal images of human neurons ( a ) stained with dCas9-GFP (green), sgRNA-mCherry (red), and LC3 (white). Scale bars, 10 μm. b Two-tailed, unpaired Student’s t -test, p = 0.0155. n = 3 independent neuronal differentiations, N = 1. c Representative confocal images of neurons in ex vivo human cortical slices infected with LV-shRNA-mCherry (red) and stained with DAPI (blue) and LC3 (white). Scale bars: 20 μm. d Quantification of LC3 intensity in mCherry+ neurons in human cortical slices. Two-tailed, unpaired Student’s t -test, p < 0.0001. N = 3 individual cortices. e Representative confocal images of neurons in the rhesus macaque cortex expressing shRNA-mCherry (red), LC3 (white) in lentivirus-infected ex vivo macaque cortical slices. Scale bars: 20 μm. f Quantification of LC3 intensity in mCherry+ neurons in macaque cortical slices. Two-tailed, unpaired Student’s t -test, p = 0.0238. N = 3 individual cortices. g , h Analysis of LC3 intensity of 5q13.2trip ASD neurons with or without MAP1B knockdown. Representative confocal images ( g ) of neurons stained with DAPI (blue), shRNA-GFP (green) and LC3 (red). Scale bars, 10 μm. h Two-tailed, unpaired Student’s t -test, p = 0.0434. n = 3 independent neuronal differentiation, N = 1. Data are presented as mean ± s.e.m. Source data are provided as a Source Data file.

Article Snippet: Five sgRNAs targeting the proximal promoter of mouse Map1b gene ( sgMap1b #1–5), between −305 bp and −24 bp relative to the TSS, were designed in Benchling based on published scoring methods (sequences are included in Supplementary Data ) and cloned to the same lentiviral sgRNA vector (Addgene #61427).

Techniques: Staining, Two Tailed Test, Ex Vivo, Infection, shRNA, Expressing, Knockdown

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: The journal of physiological sciences : JPS

Article Title: ADAM8 promotes alcoholic liver fibrosis through the MAPK signaling pathway.

doi: 10.1186/s12576-024-00943-2

Figure Lengend Snippet: Fig. 1 Successful construction of ADAM8-sgRNA3 plasmid for inhibiting murine ADAM8 gene expression. A Agarose gel electrophoresis. WT: wild-type C2C12 cells, ADAM8-sgRNA: C2C12 cells transfected with the ADAM8-sgRNA, and M: molecular weight marker. B Genotype comparison results between wild-type and mutant cells. C Immunoblotting and quantitative analysis of ADAM8 protein expression in both wild-type and ADAM8-sgRNA cells (compared with WT, **P < 0.01)

Article Snippet: Primary antibodies against PDGF-B, ADAM8, p-ERK1/2, PCNA, TNF-α, p-c-Jun, p–p38 MAPK, and HSP27 were purchased from Santa Cruz Biotechnology, Inc. (USA). α-SMA antibody was from Wailei Biotechnology Co., Ltd (Chenyang, China), Collagen-I antibody was from Proteintech Group, Inc (Wuhan, China), TGF-β1 antibody was from Zhengneng Biotechnology Co., Ltd (Chengdu, China), GAPDH and Bcl-2 antibodies were from Hua’an Biotechnology Co., Ltd, and HRP-conjugated goat anti-mouse or rabbit antibodies were purchased from Beijing Abway Antibody Technology Co., Ltd (Beijing, China).

Techniques: Plasmid Preparation, Gene Expression, Agarose Gel Electrophoresis, Transfection, Molecular Weight, Marker, Comparison, Mutagenesis, Western Blot, Expressing

Journal: The journal of physiological sciences : JPS

Article Title: ADAM8 promotes alcoholic liver fibrosis through the MAPK signaling pathway.

doi: 10.1186/s12576-024-00943-2

Figure Lengend Snippet: Fig. 4 Effect of inhibiting ADAM8 expression in alcoholic liver fibrosis mice. Detection of liver tissue damage (A), lipid droplet content (B), glycogen content (C), and TNF-α and IL-1 (D) in each group of mice. Staining charts for the control group (a), alcohol group (b), and ADAM8-sgRNA3 plasmid group (plasmid group) (c); Quantitative statistical charts for the liver tissue of mice in each group (d) (compared with the control group, *P < 0.05 or **P < 0.01; compared with the alcohol group, #P < 0.05 or ##P < 0.01)

Article Snippet: Primary antibodies against PDGF-B, ADAM8, p-ERK1/2, PCNA, TNF-α, p-c-Jun, p–p38 MAPK, and HSP27 were purchased from Santa Cruz Biotechnology, Inc. (USA). α-SMA antibody was from Wailei Biotechnology Co., Ltd (Chenyang, China), Collagen-I antibody was from Proteintech Group, Inc (Wuhan, China), TGF-β1 antibody was from Zhengneng Biotechnology Co., Ltd (Chengdu, China), GAPDH and Bcl-2 antibodies were from Hua’an Biotechnology Co., Ltd, and HRP-conjugated goat anti-mouse or rabbit antibodies were purchased from Beijing Abway Antibody Technology Co., Ltd (Beijing, China).

Techniques: Expressing, Staining, Control, Plasmid Preparation

Journal: The journal of physiological sciences : JPS

Article Title: ADAM8 promotes alcoholic liver fibrosis through the MAPK signaling pathway.

doi: 10.1186/s12576-024-00943-2

Figure Lengend Snippet: Fig. 7 Expression of ADAM8 protein and proliferation of LX-2 cells in each group. A Immunoblot bands of ADAM8 protein in LX-2 cells for each group. B Quantitative statistical chart of ADAM8 protein expression changes in LX-2 cells for each group. C Proliferation of LX-2 cells in each group (compared with the NC group, **P < 0.01; compared with the alcohol group, ##P < 0.01 or #P < 0.05)

Article Snippet: Primary antibodies against PDGF-B, ADAM8, p-ERK1/2, PCNA, TNF-α, p-c-Jun, p–p38 MAPK, and HSP27 were purchased from Santa Cruz Biotechnology, Inc. (USA). α-SMA antibody was from Wailei Biotechnology Co., Ltd (Chenyang, China), Collagen-I antibody was from Proteintech Group, Inc (Wuhan, China), TGF-β1 antibody was from Zhengneng Biotechnology Co., Ltd (Chengdu, China), GAPDH and Bcl-2 antibodies were from Hua’an Biotechnology Co., Ltd, and HRP-conjugated goat anti-mouse or rabbit antibodies were purchased from Beijing Abway Antibody Technology Co., Ltd (Beijing, China).

Techniques: Expressing, Western Blot

Journal: The journal of physiological sciences : JPS

Article Title: ADAM8 promotes alcoholic liver fibrosis through the MAPK signaling pathway.

doi: 10.1186/s12576-024-00943-2

Figure Lengend Snippet: Fig. 9 Mechanism diagram about how ADAM8 contribute to alcoholic liver fibrosis (ALF). Chronic alcohol consumption leads to the activation of hepatic stellate cells (HSCs), transitioning them from a quiescent state to an active state. In their active state, these HSCs begin producing pro-inflammatory and pro-fibrogenic cytokines, including IL-1, TGFβ1, PDGF, and TNF-α. ADAM8 expression is upregulated in response to these inflammatory signals, which subsequently activates the MAPK signaling pathway, including the phosphorylation of ERK, JNK, and p38 MAPK. This signaling cascade promotes further activation and proliferation of HSCs, which accelerates the fibrogenic process and contributes to the development of alcoholic liver fibrosis

Article Snippet: Primary antibodies against PDGF-B, ADAM8, p-ERK1/2, PCNA, TNF-α, p-c-Jun, p–p38 MAPK, and HSP27 were purchased from Santa Cruz Biotechnology, Inc. (USA). α-SMA antibody was from Wailei Biotechnology Co., Ltd (Chenyang, China), Collagen-I antibody was from Proteintech Group, Inc (Wuhan, China), TGF-β1 antibody was from Zhengneng Biotechnology Co., Ltd (Chengdu, China), GAPDH and Bcl-2 antibodies were from Hua’an Biotechnology Co., Ltd, and HRP-conjugated goat anti-mouse or rabbit antibodies were purchased from Beijing Abway Antibody Technology Co., Ltd (Beijing, China).

Techniques: Activation Assay, Expressing, Phospho-proteomics

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: Nucleic Acids Research

Article Title: Protein-responsive ribozyme switches in eukaryotic cells

doi: 10.1093/nar/gku875

Figure Lengend Snippet: Effect of ligand localization on protein-responsive ribozyme switch activity. ( A ) Gene-regulatory activity of MS2-responsive ribozyme switches in human cells for unlocalized (2MS2mut-DsRed), nuclear localized (NLS-2MS2mut-DsRed) and cytoplasmic localized (2MS2mut-DsRed-NES) ligand. Reported BFP values are the geometric mean ± SD from biological duplicates and normalized to the non-cleaving control (sTRSVctrl). ( B and C ) Gene-regulatory activity of (B) MS2-A11 and (C) MS2-B1 for unlocalized, nuclear localized and cytoplasmic localized ligand as a function of doxycycline concentration (0–50 μg/l). Reported BFP values are the geometric mean ± SD from biological duplicates, except MS2-B1 at 0.8 μg/l, unlocalized (singlet). ( D and E ) Gene-regulatory activity of (D) MS2-A11 and (E) MS2-B1 for unlocalized, nuclear localized and cytoplasmic localized ligand as a function of ligand concentration. Ligand levels are reported as the DsRed geometric mean ± SD from biological duplicates, except MS2-B1 at 0.8 μg/l, unlocalized (singlet). BFP and DsRed levels are reported for stably integrated constructs encoding the indicated ribozyme switch and ligand expression cassettes at the indicated doxycycline or ligand levels.

Article Snippet: A DNA fragment encoding 2MS2mut (MS2 V75E/A81G head-to-tail fused dimer) was synthesized by GeneArt (Life Technologies) and inserted into pCS2595 between NotI/ApaI to form pCS2686.

Techniques: Activity Assay, Control, Concentration Assay, Stable Transfection, Construct, Expressing

Journal: Wellcome Open Research

Article Title: A blueprint for the implementation of a validated approach for the detection of SARS-Cov2 in clinical samples in academic facilities

doi: 10.12688/wellcomeopenres.15937.2

Figure Lengend Snippet: ( A ) Amplification plot of cloned SARS-Cov2 template plasmid in 5 10-fold dilutions with FAM reporter. The x-axis displays the number of PCR cycles and the y-axis show the magnitude of normalized fluorescence signal generated by the reporter at each cycle during the PCR amplification in the form of ∆Rn. ( B ) Amplification plot of cloned MS2 control from spiked test samples with ROX reporter. The x-axis displays the number of PCR cycles and the y-axis show the magnitude of normalized fluorescence signal generated by the reporter at each cycle during the PCR amplification in the form of ∆Rn. Data analysed using QuantStudio 6 and 7 Flex Realtime PCR System Software colours correspond to plate location.

Article Snippet: Per reaction, the master mix is made up of: 12.5 µl 2X Luna Universal Probe One-Step reaction mix, 0.5 µl of 20 pmoles/µl Wu forward primer (ATGGGTTGGGATTATCC T AAATGTGA), 0.5 µl of 20 pmoles/μl Wu reverse primer (GCAGTTGT G GCATC T CC T GATGA G ), 0.3 µl of 10pmoles/µl MGB Probe 3 FAM (ATGCTTAG A AT T ATGGCCTC A C), 0.5 µl of 10 pmoles/μl of internal control forward primer (MS2) (supplied by Eurogentec), 0.5 µl of 10 pmoles/µl internal control reverse primer (MS2), 0.3 µl of 10 pmoles/µl internal probe (MS2 ROX), 1 µl of Luna WarmStart RT Enzyme Mix (New England Biolabs) and 3.9 µl water.

Techniques: Amplification, Clone Assay, Plasmid Preparation, Fluorescence, Generated, Software