Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: Loss of NDRG1 enhances HCV infection. (A) Knockdown of NDRG1. Huh7.5.1 cells were transfected with a pool of NDRG1-specific, no-target control siRNAs (siNT), and/or the NDRG1 plasmid (pNDRG1) for 72 h. Cells were infected with HCV for 48 h, and total intracellular and extracellular RNA was isolated and quantified by RT-qPCR using gene-specific primers. NDRG1 knockdown and overexpression were also detected by Western blotting using the Wes system. (B) HCV assembly is increased in NDRG1-depleted cells. Huh7.5.1 cells were treated as described above for panel A and infected with HCV. At 48 h postinfection, extracellular and intracellular virus was isolated and used to infect naive Huh7.5.1 cells. Infectivity (50% tissue culture infective dose [TCID50] per milliliter) was calculated by limiting dilution and detection of the HCV core protein by immunofluorescence. (C and D) NDRG1 depletion does not affect HCV entry. Knockdown in Huh7.5.1 cells was carried out as described above, and two different viral luciferase reporter constructs were utilized to examine entry (HCVpp and HCVsc). Cells were lysed, and luciferase activity was measured 48 h later by using a luminometer. CD81 knockdown was used as a positive control for entry. (E and F) NDRG1 knockdown does not alter HCV RNA replication. Huh7.5.1 cells were treated with siRNA for 72 h and transfected with the HCV SGR (E), while Huh7-SGR cells (F) were treated with siRNAs only. Knockdown of PI4KCA was used as a positive control in each system. Luciferase activity was measured 72 h after treatment. All error bars denote standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. **, P ≤ 0.01; *, P ≤ 0.05 (comparison to the negative controls).

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Infection, Knockdown, Transfection, Control, Plasmid Preparation, Isolation, Quantitative RT-PCR, Over Expression, Western Blot, Virus, Immunofluorescence, Luciferase, Construct, Activity Assay, Positive Control, Comparison

Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: HCV infection reduces NDRG1 mRNA and protein levels. (A) HCV infection reduces NDRG1 protein levels. Huh7.5.1 cells were infected in 12-well plates with HCV, and total protein was isolated at the indicated time points. The indicated proteins were detected by Western blotting (film) using protein-specific antibodies and HRP-conjugated species-specific secondary antibodies. β-Tubulin was used as a loading control. (B) NDRG1 staining is reduced in HCV-positive cells. Knockdown or control uninfected cells were infected with HCV for 48 h and then fixed and stained by using NDRG1-specific antibodies (NDRG1 [top three panels] and phosphospecific NDRG1 Ser330 [bottom panel]) and Alexa Fluor 568 secondary antibody (red). HCV-infected cells were detected with anticore antibody and Alexa Fluor 488 secondary antibody (green). White boxes denote areas magnified three times from the original images. The fluorescent signal in both uninfected and HCV-positive cells (n = 15) was quantified by using ImageJ. (C) NDRG1 mRNA expression is diminished by HCV. Huh7.5.1 cells were infected with HCV, and total RNA was isolated at 48 and 72 h postinfection. NDRG1 mRNA was detected by using RT-qPCR, and the values were normalized to the GAPDH mRNA level. (D) HCV infection reduces NDRG1 promoter activity. Huh7.5.1 cells were transfected with the indicated promoter luciferase constructs for 24 h and infected with HCV for 48 h. Cells were lysed and measured for luciferase activity on a luminometer. Uninfected cells were treated and measured in parallel. The GAPDH promoter was used as a normalization control. In panel A, the image is of one of three independent blots. For panel B, n = 15, and for panels A to D, error bars denote standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. **, P ≤ 0.01; *, P ≤ 0.05 (comparison to the negative controls). Bars, 10 μm.

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Infection, Isolation, Western Blot, Control, Staining, Knockdown, Expressing, Quantitative RT-PCR, Activity Assay, Transfection, Luciferase, Construct, Comparison

Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: NDRG1 knockdown alters HCV infection in primary human hepatocytes (PHHs). (A) Efficient knockdown of NDRG1 in PHHs. Knockdown was performed by transfecting PHHs with a pool of 4 NDRG1-specific siRNAs. Control cells were transfected with scrambled nontargeting siRNAs (siNT). At 72 h posttransfection, total RNA was harvested, and the NDRG1 mRNA level was measured by RT-qPCR. Expression was normalized to GAPDH mRNA detection. (B) HCV infection is enhanced in NDRG1-depleted PHHs. Knockdown was performed as described above for panel A, and both control and knockdown cells were infected with HCV. Total intracellular and extracellular RNAs were harvested 48 h later, and HCV RNA was quantified by RT-qPCR. (C) NDRG1 levels are reduced in HCV-infected PHHs. HCV-dependent fluorescence relocalization (HDFR) was used to detect HCV infection of PHHs. PHHs were transduced and incubated with MAVS-RFP reporter lentivirus for 24 h and then infected with HCV for 48 h. NDRG1 staining was detected by using an anti-NDRG1 antibody and anti-mouse Alexa Fluor 488. Cells with red nuclei indicate HCV-infected cells. Sec. Ab ctrl (secondary antibody control) indicates the absence of primary antibody incubation. Error bars denote standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. **, P ≤ 0.01; *, P ≤ 0.05 (compared to the negative controls). Bars, 10 μm.

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Knockdown, Infection, Control, Transfection, Quantitative RT-PCR, Expressing, Fluorescence, Incubation, Staining

Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: The effects of NDRG1 knockdown on lipid droplet (LD) formation. (A) NDRG1 depletion increases lipid droplet formation. Huh7.5.1 cells were treated with a pool of 4 NDRG1-specific siRNAs for 72 h and prepared for staining as described in the legend of Fig. 2B. Cells were fixed and incubated with NDRG1 mouse antibody and HCS LipidTOX followed by Alexa Fluor 488 anti-mouse secondary antibody. Nuclei were stained with Hoechst dye. Panels on the far right are magnified three times to show greater detail. Lipid droplets were quantified for 849 cells in the siNT control-treated sample and 1,514 cells in the siNDRG1-treated sample. The LD area, number of LDs per cell, and LD area per cell are shown on the right. (B) The lipid droplet size/intensity negatively correlates with NDRG1 levels. Cells were fixed, incubated with NDRG1 antibodies followed by Alexa Fluor 488 secondary antibodies (green), and stained for lipid droplets with HCS LipidTOX stain. In siNDRG1-treated samples, clusters of cells with or without NDRG1 expression are demarcated (in white). Cells with undetectable NDRG1 showed much higher levels of lipid droplet formation, and quantifications of the average LD size, average number of LDs per cell, and LD area per cell are shown on the right. (C) NDRG1 knockdown increases the lipid droplet size and intensity. Knockdown was performed by using a pool of 4 NDRG1-specific siRNAs as described above for panel A. Cells were fixed and incubated with an NDRG1 primary mouse antibody followed by Alexa Fluor 488 anti-mouse antibody (green). Lipid droplets were stained by using an adipophilin primary rabbit antibody and Alexa Fluor 568 anti-rabbit second antibody (red). Nuclei were stained with Hoechst dye (blue). The far-right panels are zoomed three times to show more details. The fluorescence intensity of lipid droplets was determined by using ImageJ software. All error bars denote standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. **, P ≤ 0.01; *, P ≤ 0.05 (compared to the negative controls). Bars, 10 mm.

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Knockdown, Staining, Incubation, Control, Expressing, Fluorescence, Software

Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: Evaluation of the effect of NDRG1 depletion on lipid metabolism. (A) Effect of NDRG1 knockdown on the intracellular lipid composition. Huh7.5.1 cells were transfected with siNT or siNDRG1 pools as described in Materials & Methods. At 72 h posttransfection, the contents of triglyceride, total cholesterol, and cholesteryl ester were determined. NS, not significant. (B) Density gradient analysis of secreted HCV particles. The viral supernatants from NDRG1 knockdown and control infected Huh7.5.1 cells producing HCV were subjected to isopycnic centrifugation through iodixanol gradients, as described in Materials and Methods. Representative profiles are shown. Fractions were harvested from the gradients, and the relative HCV RNA level was calculated by RT-qPCR. RNA levels are expressed as HCV RNA international units (IU) per microliter. The inset graph shows an enlarged view of fractions 1 to 10, where the low-density or highly lipidated viral particles are located. (C) Heat map displaying the normalized gene expression levels of the top genes identified in the analysis. Differentially expressed genes were filtered by a P value of less than 0.0001 and a log 2-fold change of more than 2 or less than −2. The x axis dendrogram shows hierarchical clustering between samples, and the y axis dendrogram shows hierarchical clustering between genes. The range of relative expression levels is represented by green for low expression, through black, to red for high expression levels. (D) Bar chart representing the top canonical pathways identified by IPA. The top x axis shows the significance [−log 10 (P value)] of the pathways as determined by the right-tailed Fisher exact test. The length of each bar denotes the significance of the pathway. The bottom x axis refers to the orange squares connected by a thin line and represents the ratio (the number of genes in a given pathway from the data set, divided by the total number of genes that make up that pathway). The gray dotted line shows the significance cutoff for pathways (P value of less than 0.05). Data were analyzed by using Qiagen IPA. fMLP, formyl Met-Leu-Phe receptor. (E) NDRG1 depletion does not affect SREBP1 or SREBP2 expression. Huh7.5.1 cells were transfected with siNDRG1 or siNT pools for 72 h. Total intracellular RNA was isolated, and SREBP1 and SREBP2 expression was detected by using RT-qPCR. Error bars denote standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. *, P ≤ 0.05 compared to the negative controls.

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Knockdown, Transfection, Control, Infection, Centrifugation, Quantitative RT-PCR, Gene Expression, Expressing, Isolation

Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: HCV infection upregulates MYC and reduces NDRG1 phosphorylation. (A) HCV replication upregulates MYC. Huh7.5.1 cells were infected with HCV, and total RNA was harvested at the indicated intervals. MYC expression was measured by RT-qPCR. Uninfected cells were used as a control. (B) HCV replication does not upregulate NMYC. Huh7.5.1 cells were infected with HCV and harvested as described above for panel A. NMYC expression was detected by RT-qPCR. Uninfected cells were used as a control. (C) Quantitative Western blot analysis of HCV-infected cells. Huh7.5.1 cells were infected with HCV, and total protein was harvested at the indicated time points. Protein levels were quantified by using the Wes system, except for NS3, which was quantified by a traditional Western method. Uninfected cells were harvested in parallel as a control. Error bars denote standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. **, P ≤ 0.01; *, P ≤ 0.05 (compared to the negative controls). In panel C, graphs represent total peak intensity as measured by the instrument.

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Infection, Phospho-proteomics, Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: Roles of MYC and CTNNB1 in regulating NDRG1 levels. (A) Knockdown of MYC. Huh7.5.1 cells were transfected with siRNAs targeting MYC or the control siNT, total protein was isolated 72 h later, and the MYC expression level was measured by Western blotting. (B) HCV-mediated reduction of NDRG1 levels is dependent on MYC. Huh7.5.1 cells were transfected with MYC-specific siRNAs and infected with HCV 48 h later. Total protein was isolated at 72 h postinfection. Proteins were detected by using the indicated antibodies. (C) Quantitation of Western blots. Data from panel B were quantified and normalized to the value for the β-tubulin control. (D and E) Knockdown of STAT3. Huh7.5.1 cells were transfected with control and STAT3-specific siRNAs and infected with HCV. STAT3 and MYC mRNA levels were quantified by RT-qPCR. (F and G) CTNNB1 knockdown. Huh7.5.1 cells were transfected with control and CTNNB1-specific siRNAs and infected with HCV. CTNNB1 and MYC mRNA levels were quantified by RT-qPCR. Error bars denote standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. **, P ≤ 0.01; *, P ≤ 0.05 (compared to the negative controls).

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Knockdown, Transfection, Control, Isolation, Expressing, Western Blot, Infection, Quantitation Assay, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: MYC reduces NDRG1 phosphorylation by downregulating SGK1 expression. (A) MYC overexpression. Huh7.5.1 cells were transfected with either control or MYC overexpression vectors, and protein was isolated at 48 h posttransfection. Protein was detected by using quantitative Western blotting (Wes) with the indicated antibodies. (B) MYC overexpression further reduces NDRG1 levels in HCV-infected cells. Huh7.5.1 cells were transfected with control or MYC expression vectors as described above for panel A. At 24 h posttransfection, the cells were HCV infected for an additional 72 h. The cells and proteins were then prepared as described above for panel A. Uninfected control cells were harvested in parallel. (C) HCV infection decreases SGK1 mRNA expression. A HCV time course was performed as described in the legend of Fig. 2, and SGK1 mRNA levels were monitored by RT-qPCR. An uninfected control was performed in parallel. (D) HCV infection reduces SGK1 protein levels. A HCV time course was performed as described in the legend of Fig. 2. SGK1 and GSK3β levels were monitored with the indicated primary antibodies and secondary HRP conjugates. Uninfected controls were run in parallel. (E) HCV alters the promoter activity of NDRG1, MYC, and SGK1. Promoter constructs containing a luciferase reporter under the control of NDRG1, MYC, and SGK1 promoter regions were transfected into Huh7.5.1 cells. At 6 h posttransfection, the cells were infected with HCV. Luciferase activity was measured at 48 h postinfection. The GAPDH promoter construct was used as a control. (F) Knockdown of SGK1 increases HCV RNA levels. Knockdown was performed as described in the text, using a pool of siRNAs. Seventy-two hours later, cells were infected with HCV, total intracellular and extracellular RNAs were harvested at 48 h postinfection, and HCV RNA was detected by RT-qPCR. (G) Overexpression of MYC increases HCV RNA levels. Cells were transfected with control or MYC expression plasmids for 24 h and infected with HCV. Total intracellular and extracellular RNAs were harvested at 48 h postinfection, and HCV RNA was detected by RT-qPCR. Error bars indicate standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. **, P ≤ 0.01; *, P ≤ 0.05 (compared to the negative controls).

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Phospho-proteomics, Expressing, Over Expression, Transfection, Control, Isolation, Western Blot, Infection, Quantitative RT-PCR, Activity Assay, Construct, Luciferase, Knockdown

Journal: Journal of Virology

Article Title: N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

doi: 10.1128/JVI.01166-17

Figure Lengend Snippet: Model for HCV, MYC, and NDRG1 interactions. HCV infection induces MYC expression probably via the activation of β-catenin and concomitantly decreases SGK1 and NDRG1 expression levels. The loss of SGK1 further diminishes the phosphorylation of NDRG1, and this results in increased lipid droplet formation. See Discussion for more details.

Article Snippet: The NDRG1 (entire open reading frame [ORF]) Flag-Myc-tagged vector, the MYC vector (untagged), and the empty parental vector were obtained commercially (Origene).

Techniques: Infection, Expressing, Activation Assay, Phospho-proteomics

Journal: Metabolites

Article Title: Evolution of Inflammatory and Oxidative Stress Markers in Romanian Obese Male Patients with Type 2 Diabetes Mellitus after Laparoscopic Sleeve Gastrectomy: One Year Follow-Up

doi: 10.3390/metabo10080308

Figure Lengend Snippet: Comparison of the hormonal levels determined at V1, V2 and V3 for the conventional treatment (CTG) versus LSG group; 2 way-ANOVA with Bonferroni post hoc (data shown as median with IQR, p -value shows interaction effect, significant results are marked with *, ** or *** and show the significant differences observed at V2 and V3 compared to V1, if not marked, results are not statistically significant).

Article Snippet: Concentrations of hormones involved in the regulation of food intake, including glucagon-like peptide 1 (GLP-1) with anorexic role and ghrelin with orexigenic role, have also been determined by using commercially available ELISA kits (MBS760336 MyBioSource, Inc., BioZyme for GLP-1 kits and EIA-4710 kit for the active (acylated) form, human ghrelin (active) (DRG Instruments, Germany).

Techniques:

Journal: Experimental neurology

Article Title: Transcriptomic Analyses of Genes and Tissues in Inherited Sensory Neuropathies

doi: 10.1016/j.expneurol.2016.06.023

Figure Lengend Snippet: Transcriptomics data were filtered for genes >3 fold differentially expressed in sciatic relative to DRG, and ordered by RPKM value. Genes >10 fold differentially expressed are marked with an asterisk. Given that Schwann cells and nerve tracks are a subset of DRG, very few genes are more than 3 fold differentially expressed in sciatic, and generally have a high SEM (error bars). DRG n =8, sciatic n = 4.

Article Snippet: Human L3 DRGs (N=4) were purchased from Anabios (San Diego, CA).

Techniques:

Journal: Experimental neurology

Article Title: Transcriptomic Analyses of Genes and Tissues in Inherited Sensory Neuropathies

doi: 10.1016/j.expneurol.2016.06.023

Figure Lengend Snippet: Genes strongly differential between DRG and sciatic were additionally probed for enrichment relative to 32 tissues present in the Human Protein Atlas. These genes were normalized to a % of maximum expression in any tissue and colored according to numeric value. Genes that are strongly enriched in DRG or sciatic relative to a broad complement of tissues are likely to be molecular signatures of this tissue. Genes are sorted by maximum expression (RPKM/FPKM) in any of the 34 tissues. Several CMT genes (NEFL, KIF5A in DRG and MPZ, PMP22 and PRX in sciatic) are among the most highly differential genes in DRG or sciatic. Both DRG and sciatic show enhanced similarity to the cerebral cortex, the only brain tissue presented in the Atlas data

Article Snippet: Human L3 DRGs (N=4) were purchased from Anabios (San Diego, CA).

Techniques: Expressing

Journal: Experimental neurology

Article Title: Transcriptomic Analyses of Genes and Tissues in Inherited Sensory Neuropathies

doi: 10.1016/j.expneurol.2016.06.023

Figure Lengend Snippet: DRG tissue was stained for either neurofilamen light chain (left) or myelin protein zero (right), which are markers for neurons and Schwann cells respectively. In the case of neurofilament, these proteins are made in the soma, but functional assembly is also required for axonal structure. Because of this, this protein stains both neuronal cell bodies and axonal bundles. These genes are among the most highly expressed, highly differential genes in each tissue. The presence of myelin protein tracks in the DRG is indicative of Schwann cell coated myelinated axonal sheaths extending out from the DRG into the dorsal roots, and to the periphery and the spinal cord. These cells represent a major non-neuronal component of DRGs. Scale bar represents 200μm.

Article Snippet: Human L3 DRGs (N=4) were purchased from Anabios (San Diego, CA).

Techniques: Staining, Functional Assay

Journal: Experimental neurology

Article Title: Transcriptomic Analyses of Genes and Tissues in Inherited Sensory Neuropathies

doi: 10.1016/j.expneurol.2016.06.023

Figure Lengend Snippet: Genes from transcriptomic data were ranked by RPKM values and binned according to level of expression. In both tissue types, number of detectable annotated genes decreases as RPKM value increases, with a small number of genes expressed at extremely high levels. Compared with the DRG, which is a more diverse tissue containing several types of neurons, satellite cells, fibroblasts, and Schwann cells, the sciatic nerve is more homogeneous containing mostly Schwann cells and some adipose. The extremely high level of expression of myelin-related proteins in Schwann exceeds the level of any transcript from DRG.

Article Snippet: Human L3 DRGs (N=4) were purchased from Anabios (San Diego, CA).

Techniques: Expressing

Journal: Experimental neurology

Article Title: Transcriptomic Analyses of Genes and Tissues in Inherited Sensory Neuropathies

doi: 10.1016/j.expneurol.2016.06.023

Figure Lengend Snippet: Significance of differential expression was calculated from raw reads using DESEQ (R, Bioconductor) normalization and testing. Separately, RPKM values were calculated as an abundance estimate. A. All genes were plotted according to average RPKM between the two tissues examined and fold difference (DRG/sciatic). Significantly differentially expressed genes (p < .01) are colored by enrichment in the DRG (blue) or sciatic (red), with genes below 1 RPKM shown in a lighter shade. In general large fold change genes and highly expressed genes were significant with 4615 genes enriched in the DRG and 4277 genes enriched in the sciatic nerve. B Non-significant genes using the same color scheme to denote genes below 1 RPKM, which are in general more variable than more highly expressed genes. C, D. Control plots of average expression vs fold change expression were generated by dividing each group of samples into two halves, and plotting them against each other, and are plotted on the same Y-axis scale as in the cross-tissue comparions. Panels C and D span 2 orders of magnitude differential expression, whereas panel A spans >7.

Article Snippet: Human L3 DRGs (N=4) were purchased from Anabios (San Diego, CA).

Techniques: Quantitative Proteomics, Control, Expressing, Generated

Journal: Experimental neurology

Article Title: Transcriptomic Analyses of Genes and Tissues in Inherited Sensory Neuropathies

doi: 10.1016/j.expneurol.2016.06.023

Figure Lengend Snippet: Transcriptomics data were filtered for genes >3 fold differentially expressed in DRG relative to sciatic, and ordered by RPKM value. Genes >10 fold differentially expressed are marked with an asterisk. Error bars show SEM. DRG n =8, sciatic nerve n = 4.

Article Snippet: Human L3 DRGs (N=4) were purchased from Anabios (San Diego, CA).

Techniques:

Journal: Experimental neurology

Article Title: Transcriptomic Analyses of Genes and Tissues in Inherited Sensory Neuropathies

doi: 10.1016/j.expneurol.2016.06.023

Figure Lengend Snippet: (A) DRG and sciatic nerve tissue were collected, and RNA was extracted, sequenced and mapped. Genes are plotted by fold difference between tissues vs. average RPKM, and colored according to significance values. Grey line at 1 indicates no change. Nefl, MT-Atp6, Mpz and Pmp22 are plotted on a separate axis because of their high level of expression, and are among the highest expressed genes in DRG and/or sciatic nerve. Five genes (Hsbp3, Slc5a7, Hodx10, Trpv4 and Ngf) are expressed below 1 RPKM in the adult rat. In general, some peripheral neuropathy genes are either highly expressed or highly differential between sciatic nerve and DRG, although the majority of the genes are common to both tissues. (B) All pain channelopathy genes are highly enriched in the DRG. DRG n = 8, sciatic nerve n =4.

Article Snippet: Human L3 DRGs (N=4) were purchased from Anabios (San Diego, CA).

Techniques: Expressing

Journal: Experimental neurology

Article Title: Transcriptomic Analyses of Genes and Tissues in Inherited Sensory Neuropathies

doi: 10.1016/j.expneurol.2016.06.023

Figure Lengend Snippet: This figure was adapted from Rossor, et al (2013) with the addition of quantitative data (RPKM values) from transcriptomics experiments. This provides a quantitative measure of the level of expression of these genes between the two major tissues involved in CMT and nociception. In general these genes can be divided into the sciatic nerve/Schwann cell enriched population and the DRG population.

Article Snippet: Human L3 DRGs (N=4) were purchased from Anabios (San Diego, CA).

Techniques: Expressing

Journal: F1000Research

Article Title: The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

doi: 10.12688/f1000research.19804.3

Figure Lengend Snippet: a ) Cortisol (C) enzyme-linked immunosorbent assay (ELISA) kit: serum of post-transportation / sample one (white circle) and serum of pre-transportation / sample two (black triangle down) were diluted 1:2 to 1:16 in assay buffer and measured with serial C standards (black circle) ranging from 10 to 400 ng/ml. The curve of sample one, sample two, and C standards produced almost identical R 2 values: 0.987, 0.945, and 0.992, respectively. b ) Testosterone (T) ELISA kit: serum of bull 1 / sample one (white circle) and serum of bull 2 / sample two (black triangle down) were diluted 1:2 to 1:16 in assay buffer and measured with serial T standards (black circle) ranging from 0.5 to 16 ng/ml. The curve of sample one, sample two, and T standards produced almost identical R 2 values: 0.964, 0.993, and 0.945, respectively.

Article Snippet: The validation of commercial ELISA kits for measuring hormones in animals has been successfully conducted in several species, such as progesterone in serum of cattle using human progesterone ELISA kit (Clinpro International Co. LLC, Union City, CA 94587, USA) , estradiol-17-beta and inhibin A in plasma of buffalo using human ELISA kit (For estradiol: Cat No. EIA-2693, DRG Instruments GmbH, Marburg, Germany; For Inhibin A: Inhibin A DSL-10-28100, Diagnostic System Laboratories Inc, Webster, Texas, USA) , C in plasma of horses using Immunotech kit , and T in serum of Kacang goats using T ELISA kit (Cat No. EIA-1559, DRG Instruments GmbH, Marburg, Germany) developed for human .

Techniques: Enzyme-linked Immunosorbent Assay, Produced

Journal: F1000Research

Article Title: The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

doi: 10.12688/f1000research.19804.3

Figure Lengend Snippet: Results of the analytical validation of the commercial cortisol (DRG, Cat. No. EIA-1887) and testosterone (DRG, Cat. No. EIA-1559) enzyme-linked immunosorbent assay (ELISA) kits.

Article Snippet: The validation of commercial ELISA kits for measuring hormones in animals has been successfully conducted in several species, such as progesterone in serum of cattle using human progesterone ELISA kit (Clinpro International Co. LLC, Union City, CA 94587, USA) , estradiol-17-beta and inhibin A in plasma of buffalo using human ELISA kit (For estradiol: Cat No. EIA-2693, DRG Instruments GmbH, Marburg, Germany; For Inhibin A: Inhibin A DSL-10-28100, Diagnostic System Laboratories Inc, Webster, Texas, USA) , C in plasma of horses using Immunotech kit , and T in serum of Kacang goats using T ELISA kit (Cat No. EIA-1559, DRG Instruments GmbH, Marburg, Germany) developed for human .

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Intra Assay, Control, Inter Assay

Journal: F1000Research

Article Title: The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

doi: 10.12688/f1000research.19804.3

Figure Lengend Snippet: a ) Concentrations of cortisol (C) before (pre-trans) and after (post-trans) transportation measured using a commercial C ELISA kit (DRG, Cat. No. EIA-1887). b ) Concentrations of testosterone (T) in bulls and cows measured using a commercial T ELISA kit (DRG, Cat. No. EIA-1559). Different superscripts above line symbols and histogram indicate a significant difference between groups (p < 0.05).

Article Snippet: The validation of commercial ELISA kits for measuring hormones in animals has been successfully conducted in several species, such as progesterone in serum of cattle using human progesterone ELISA kit (Clinpro International Co. LLC, Union City, CA 94587, USA) , estradiol-17-beta and inhibin A in plasma of buffalo using human ELISA kit (For estradiol: Cat No. EIA-2693, DRG Instruments GmbH, Marburg, Germany; For Inhibin A: Inhibin A DSL-10-28100, Diagnostic System Laboratories Inc, Webster, Texas, USA) , C in plasma of horses using Immunotech kit , and T in serum of Kacang goats using T ELISA kit (Cat No. EIA-1559, DRG Instruments GmbH, Marburg, Germany) developed for human .

Techniques: Enzyme-linked Immunosorbent Assay