Construction and analysis of circular RNA molecular regulatory networks in liver cancer

<p>Liver cancer is the sixth most prevalent cancer, and the third most frequent cause of cancer-related deaths. Circular RNAs (circRNAs), a kind of special endogenous ncRNAs, have been coming back to the forefront of cancer genomics research. In this study, we used a <i>systems biology approach</i> to construct and analyze the circRNA molecular regulatory networks in the context of liver cancer. We detected a total of 127 differentially expressed circRNAs and 3,235 differentially expressed mRNAs. We selected the top-5 upregulated circRNAs to construct a circRNA-miRNA-mRNA network. We enriched the pathways and gene ontology items and determined their participation in cancer-related pathways such as p53 signaling pathway and pathways involved in angiogenesis and cell cycle. Quantitative real-time PCR was performed to verify the top-five circRNAs. ROC analysis showed circZFR, circFUT8, circIPO11 could significantly distinguish the cancer samples, with an AUC of 0.7069, 0.7575, and 0.7103, respectively. Our results suggest the circRNA-miRNA-mRNA network may help us further understand the molecular mechanisms of tumor progression in liver cancer, and reveal novel biomarkers and therapeutic targets.</p

data_sheet_1_Brain Structural and Perfusion Signature of Amyotrophic Lateral Sclerosis With Varying Levels of Cognitive Deficit.DOCX

Objective<p>To characterize the patterns of brain atrophy and perfusion as measured by arterial spin labeling (ASL)-MRI, in amyotrophic lateral sclerosis (ALS) patients with varying levels of cognitive deficit, including ALS with frontotemporal dementia (FTD).</p>Methods<p>A total of 55 ALS patients and 20 healthy controls (HCs) were included, and all participants underwent neuropsychological assessments and MRI scans. According to their cognitive performance, ALS patients were further subclassified into ALS with normal cognition (ALS-Cn, n = 27), ALS with cognitive impairment (ALS-Ci, n = 17), and ALS-FTD (n = 11). Voxel-based comparisons of gray matter (GM) changes and cerebral blood flow (CBF) were conducted among the subgroups.</p>Results<p>The whole-brain comparisons of GM changes and CBF among ALS-Ci, ALS-Cn, and HCs were not significantly different. However, the ALS-FTD patients demonstrated a similar pattern of GM loss and hypoperfusion with more significant alterations in the left frontal and temporal lobe compared with the HCs, ALS-Cn, and ALS-Ci patients. Decreased CBF was found in many of the same brain areas wherein structural alterations occurred, although isolated GM loss and hypoperfusion were also observed. In addition, for both GM and CBF abnormalities, a similar pattern of changes was found in the comparisons of ALS-FTD vs. ALS-Ci, ALS-FTD vs. ALS-Cn, and ALS-FTD vs. HCs, with the differences being most significant between ALS-FTD and HCs.</p>Conclusion<p>The cognitive status of ALS patients is associated with different patterns of GM changes and cerebral perfusion. ASL-MRI might be a useful tool with which to investigate the pathological burden of ALS and to disclose the early signature of possible cognitive impairment.</p

data_sheet_2_Brain Structural and Perfusion Signature of Amyotrophic Lateral Sclerosis With Varying Levels of Cognitive Deficit.DOCX

Objective<p>To characterize the patterns of brain atrophy and perfusion as measured by arterial spin labeling (ASL)-MRI, in amyotrophic lateral sclerosis (ALS) patients with varying levels of cognitive deficit, including ALS with frontotemporal dementia (FTD).</p>Methods<p>A total of 55 ALS patients and 20 healthy controls (HCs) were included, and all participants underwent neuropsychological assessments and MRI scans. According to their cognitive performance, ALS patients were further subclassified into ALS with normal cognition (ALS-Cn, n = 27), ALS with cognitive impairment (ALS-Ci, n = 17), and ALS-FTD (n = 11). Voxel-based comparisons of gray matter (GM) changes and cerebral blood flow (CBF) were conducted among the subgroups.</p>Results<p>The whole-brain comparisons of GM changes and CBF among ALS-Ci, ALS-Cn, and HCs were not significantly different. However, the ALS-FTD patients demonstrated a similar pattern of GM loss and hypoperfusion with more significant alterations in the left frontal and temporal lobe compared with the HCs, ALS-Cn, and ALS-Ci patients. Decreased CBF was found in many of the same brain areas wherein structural alterations occurred, although isolated GM loss and hypoperfusion were also observed. In addition, for both GM and CBF abnormalities, a similar pattern of changes was found in the comparisons of ALS-FTD vs. ALS-Ci, ALS-FTD vs. ALS-Cn, and ALS-FTD vs. HCs, with the differences being most significant between ALS-FTD and HCs.</p>Conclusion<p>The cognitive status of ALS patients is associated with different patterns of GM changes and cerebral perfusion. ASL-MRI might be a useful tool with which to investigate the pathological burden of ALS and to disclose the early signature of possible cognitive impairment.</p

DataSheet_3_Reliable Estimation of CD8 T Cell Inhibition of In Vitro HIV-1 Replication.xlsx

The HIV-1 viral inhibition assay (VIA) measures CD8 T cell-mediated inhibition of HIV replication in CD4 T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. The VIA has multiple sources of variability arising from in vitro HIV infection and co-culture of two T cell populations. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence interval. An Excel template is provided to facilitate calculations. Virus inhibition was higher in people living with HIV suppressed on antiretroviral therapy (n=14, mean: 40.0%, median: 43.8%, range: 8.2 to 73.3%; p < 0.0001, two-tailed, exact Mann-Whitney test) compared to HIV-seronegative donors (n = 21, mean: -13.7%, median: -14.4%, range: -49.9 to 20.9%) and was stable over time (n = 6, mean %COV 9.4%, range 0.9 to 17.3%). Cross-sectional data were used to define 8% inhibition as the threshold to confidently detect specific CD8 T cell activity and determine the minimum number of culture replicates and p24+ cells needed to have 90% statistical power to detect this threshold. Last, we note that, in HIV seronegative donors, the addition of CD8 T cells to HIV infected CD4 T cells consistently increased HIV replication, though the level of increase varied markedly between donors. This co-culture effect may contribute to the weak correlations observed between CD8 T cell VIA and other measures of HIV-specific CD8 T cell function.</p

DataSheet_2_Reliable Estimation of CD8 T Cell Inhibition of In Vitro HIV-1 Replication.docx

The HIV-1 viral inhibition assay (VIA) measures CD8 T cell-mediated inhibition of HIV replication in CD4 T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. The VIA has multiple sources of variability arising from in vitro HIV infection and co-culture of two T cell populations. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence interval. An Excel template is provided to facilitate calculations. Virus inhibition was higher in people living with HIV suppressed on antiretroviral therapy (n=14, mean: 40.0%, median: 43.8%, range: 8.2 to 73.3%; p < 0.0001, two-tailed, exact Mann-Whitney test) compared to HIV-seronegative donors (n = 21, mean: -13.7%, median: -14.4%, range: -49.9 to 20.9%) and was stable over time (n = 6, mean %COV 9.4%, range 0.9 to 17.3%). Cross-sectional data were used to define 8% inhibition as the threshold to confidently detect specific CD8 T cell activity and determine the minimum number of culture replicates and p24+ cells needed to have 90% statistical power to detect this threshold. Last, we note that, in HIV seronegative donors, the addition of CD8 T cells to HIV infected CD4 T cells consistently increased HIV replication, though the level of increase varied markedly between donors. This co-culture effect may contribute to the weak correlations observed between CD8 T cell VIA and other measures of HIV-specific CD8 T cell function.</p

DataSheet_1_Reliable Estimation of CD8 T Cell Inhibition of In Vitro HIV-1 Replication.docx

The HIV-1 viral inhibition assay (VIA) measures CD8 T cell-mediated inhibition of HIV replication in CD4 T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. The VIA has multiple sources of variability arising from in vitro HIV infection and co-culture of two T cell populations. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence interval. An Excel template is provided to facilitate calculations. Virus inhibition was higher in people living with HIV suppressed on antiretroviral therapy (n=14, mean: 40.0%, median: 43.8%, range: 8.2 to 73.3%; p < 0.0001, two-tailed, exact Mann-Whitney test) compared to HIV-seronegative donors (n = 21, mean: -13.7%, median: -14.4%, range: -49.9 to 20.9%) and was stable over time (n = 6, mean %COV 9.4%, range 0.9 to 17.3%). Cross-sectional data were used to define 8% inhibition as the threshold to confidently detect specific CD8 T cell activity and determine the minimum number of culture replicates and p24+ cells needed to have 90% statistical power to detect this threshold. Last, we note that, in HIV seronegative donors, the addition of CD8 T cells to HIV infected CD4 T cells consistently increased HIV replication, though the level of increase varied markedly between donors. This co-culture effect may contribute to the weak correlations observed between CD8 T cell VIA and other measures of HIV-specific CD8 T cell function.</p

DataSheet_4_Reliable Estimation of CD8 T Cell Inhibition of In Vitro HIV-1 Replication.xlsx

The HIV-1 viral inhibition assay (VIA) measures CD8 T cell-mediated inhibition of HIV replication in CD4 T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. The VIA has multiple sources of variability arising from in vitro HIV infection and co-culture of two T cell populations. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence interval. An Excel template is provided to facilitate calculations. Virus inhibition was higher in people living with HIV suppressed on antiretroviral therapy (n=14, mean: 40.0%, median: 43.8%, range: 8.2 to 73.3%; p < 0.0001, two-tailed, exact Mann-Whitney test) compared to HIV-seronegative donors (n = 21, mean: -13.7%, median: -14.4%, range: -49.9 to 20.9%) and was stable over time (n = 6, mean %COV 9.4%, range 0.9 to 17.3%). Cross-sectional data were used to define 8% inhibition as the threshold to confidently detect specific CD8 T cell activity and determine the minimum number of culture replicates and p24+ cells needed to have 90% statistical power to detect this threshold. Last, we note that, in HIV seronegative donors, the addition of CD8 T cells to HIV infected CD4 T cells consistently increased HIV replication, though the level of increase varied markedly between donors. This co-culture effect may contribute to the weak correlations observed between CD8 T cell VIA and other measures of HIV-specific CD8 T cell function.</p

Image_1_Reliable Estimation of CD8 T Cell Inhibition of In Vitro HIV-1 Replication.tif

The HIV-1 viral inhibition assay (VIA) measures CD8 T cell-mediated inhibition of HIV replication in CD4 T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. The VIA has multiple sources of variability arising from in vitro HIV infection and co-culture of two T cell populations. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence interval. An Excel template is provided to facilitate calculations. Virus inhibition was higher in people living with HIV suppressed on antiretroviral therapy (n=14, mean: 40.0%, median: 43.8%, range: 8.2 to 73.3%; p < 0.0001, two-tailed, exact Mann-Whitney test) compared to HIV-seronegative donors (n = 21, mean: -13.7%, median: -14.4%, range: -49.9 to 20.9%) and was stable over time (n = 6, mean %COV 9.4%, range 0.9 to 17.3%). Cross-sectional data were used to define 8% inhibition as the threshold to confidently detect specific CD8 T cell activity and determine the minimum number of culture replicates and p24+ cells needed to have 90% statistical power to detect this threshold. Last, we note that, in HIV seronegative donors, the addition of CD8 T cells to HIV infected CD4 T cells consistently increased HIV replication, though the level of increase varied markedly between donors. This co-culture effect may contribute to the weak correlations observed between CD8 T cell VIA and other measures of HIV-specific CD8 T cell function.</p

Hypermethylation status and clinical outcome in patients with juvenile myelomonocytic leukemia (JMML).

<p>(A) Kaplan–Meier curves represent the probability of transplantation-free survival (TFS) in the 92 patients with JMML. TFS was defined as the probability of being alive and transplantation free. Both death and transplantation were considered events. The probability of 5-year TFS in the aberrant methylation score (AMS) 0 cohort (solid line) was significantly higher than that in the AMS 1–2 (long dashed line) and AMS 3–4 cohorts (dashed line), p < 0.001. (B) Kaplan–Meier curves represent the probability of overall survival (OS) in the 92 patients with JMML. Death was considered an event. The probability of OS in both the AMS 0 (solid line) and 1–2 cohorts (long dashed line) was significantly higher than that in the AMS 3–4 cohort (dashed line), p < 0.001.</p

Profile of genetic mutations and aberrant methylation.

<p>(A) Mutation status of RAS pathway genes and secondary genes (<i>SETBP1</i> and <i>JAK3</i>) identified as gene targets. Aberrant methylation scores (AMS) in a cohort of 92 patients with juvenile myelomonocytic leukemia are summarized. A rhombus denotes a patient with Noonan syndrome-associated myeloproliferative disorder. (B) Mutations in <i>SETBP1</i> and <i>JAK3</i> were associated with a higher AMS. The mean AMS of patients with <i>SETBP1</i> and/or <i>JAK3</i> mutations was higher than that of patients without secondary mutations (p = 0.03).</p