Sirtuin 3 Attenuates Amyloid-Beta Induced Neuronal Hypometabolism

Alzheimer\u27s disease (AD) is manifested by regional cerebral hypometabolism. Sirtuin 3 (Sirt3) is localized in mitochondria and regulates cellular metabolism, but the role of Sirt3 in AD-related hypometabolism remains elusive. We used expression profiling and weighted gene co-expression network analysis (WGCNA) to analyze cortical neurons from a transgenic mouse model of AD (APPSwInd). Based on WGCNA results, we measured NAD+ level, NAD+/ NADH ratio, Sirt3 protein level and its deacetylation activity, and ATP production across both in vivo and in vitro models. To investigate the effect of Sirt3 on amyloid-β (Aβ)-induced mitochondria damage, we knocked down and over-expressed Sirt3 in hippocampal cells. WGCNA revealed Sirt3 as a key player in Aβ-related hypometabolism. In APP mice, the NAD+ level, NAD+/ NADH ratio, Sirt3 protein level and activity, and ATP production were all reduced compared to the control. As a result, learning and memory performance were impaired in 9-month-old APP mice compared to wild type controls. Using hippocampal HT22 cells model, Sirt3 overexpression increased Sirt3 deacetylation activity, rescued mitochondria function, and salvaged ATP production, which were damaged by Aβ. Sirt3 plays an important role in regulating Aβ-induced cerebral hypometabolism. This study suggests a potential direction for AD therapy

Cerebral small vessel disease burden is associated with poststroke depressive symptoms: A 15-month prospective study

Objective: All types of cerebral small vessel disease (SVD) markers including lacune, white matter hyperintensities (WMH), cerebral microbleeds, and perivascular spaces were found to be associated with poststroke depressive symptoms (PDS). This study explored whether the combination of the four markers constituting an overall SVD burden was associated with PDS. Methods: A cohort of 563 patients with acute ischemic stroke were followed over a 15-month period after the index stroke. A score of _7 on the 15-item Geriatric Depression Scale was defined as clinically significant PDS. Scores of the four SVD markers ascertained on magnetic resonance imaging were summed up to represent total SVD burden. The association between SVD burden and PDS was assessed with generalized estimating equation models. Results: The study sample had a mean age of 67.0 _ 10.2 years and mild-moderate stroke [National Institutes of Health Stroke Scale score: 3, interquartile, 1–5]. PDS were found in 18.3%, 11.6%, and 12.3% of the sample at 3, 9, and 15 months after stroke, respectively. After adjusting for demographic characteristics, vascular risk factors, social support, stroke severity, physical and cognitive functions, and size and locations of stroke, the SVD burden was associated with an increased risk of PDS [odds ratio = 1.30; 95% confidence interval = 1.07–1.58; p = 0.010]. Other significant predictors of PDS were time of assessment, female sex, smoking, number of acute infarcts, functional independence, and social support. Conclusion: SVD burden was associated with PDS examined over a 15-month follow-up in patients with mild to moderate acute ischemic stroke

Evaluation of pre-analytical factors affecting plasma DNA analysis.

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies

Therapeutic targets for HIV-1 infection in the host proteome

BACKGROUND: Despite the success of HAART, patients often stop treatment due to the inception of side effects. Furthermore, viral resistance often develops, making one or more of the drugs ineffective. Identification of novel targets for therapy that may not develop resistance is sorely needed. Therefore, to identify cellular proteins that may be up-regulated in HIV infection and play a role in infection, we analyzed the effects of Tat on cellular gene expression during various phases of the cell cycle. RESULTS: SOM and k-means clustering analyses revealed a dramatic alteration in transcriptional activity at the G1/S checkpoint. Tat regulates the expression of a variety of gene ontologies, including DNA-binding proteins, receptors, and membrane proteins. Using siRNA to knock down expression of several gene targets, we show that an Oct1/2 binding protein, an HIV Rev binding protein, cyclin A, and PPGB, a cathepsin that binds NA, are important for viral replication following induction from latency and de novo infection of PBMCs. CONCLUSION: Based on exhaustive and stringent data analysis, we have compiled a list of gene products that may serve as potential therapeutic targets for the inhibition of HIV-1 replication. Several genes have been established as important for HIV-1 infection and replication, including Pou2AF1 (OBF-1), complement factor H related 3, CD4 receptor, ICAM-1, NA, and cyclin A1. There were also several genes whose role in relation to HIV-1 infection have not been established and may also be novel and efficacious therapeutic targets and thus necessitate further study. Importantly, targeting certain cellular protein kinases, receptors, membrane proteins, and/or cytokines/chemokines may result in adverse effects. If there is the presence of two or more proteins with similar functions, where only one protein is critical for HIV-1 transcription, and thus, targeted, we may decrease the chance of developing treatments with negative side effects

Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas.

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated data from protein, genomic and MR imaging from 20 treatment-naïve glioma cases and 16 recurrent GBM cases. Multiplexed immunofluorescence (MxIF) was used to generate single cell data for 43 protein markers representing all cancer hallmarks, Genomic sequencing (exome and RNA (normal and tumor) and magnetic resonance imaging (MRI) quantitative features (protocols were T1-post, FLAIR and ADC) from whole tumor, peritumoral edema and enhancing core vs equivalent normal region were also collected from patients. Based on MxIF analysis, 85,767 cells (glioma cases) and 56,304 cells (GBM cases) were used to generate cell-level data for 24 biomarkers. K-means clustering was used to generate 7 distinct groups of cells with divergent biomarker profiles and deconvolution was used to assign RNA data into three classes. Spatial and molecular heterogeneity metrics were generated for the cell data. All features were compared between IDH mt and IDHwt patients and were finally combined to provide a holistic/integrated comparison. Protein expression by hallmark was generally lower in the IDHmt vs wt patients. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion also differed between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. A coherent picture of enhanced angiogenesis in IDHwt tumors was derived from multiple platforms (genomic, proteomic and imaging) and scales from individual proteins to cell clusters and heterogeneity, as well as bulk tumor RNA and imaging features. Longer overall survival for IDH1mt glioma patients may reflect mutation-driven alterations in cellular, molecular, and spatial heterogeneity which manifest in discernable radiological manifestations

Upfront Biology-Guided Therapy in Diffuse Intrinsic Pontine Glioma: Therapeutic, Molecular, and Biomarker Outcomes from PNOC003

PURPOSE PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG). PATIENTS AND METHODS Patients (3-25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN). RESULTS Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2-18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7-14; TP53wt 13.3 mo; 95% CI, 11.8-NA; P = 3.4e-2), genome instability (P = 3.1e-3), and RT resistance (P = 6.4e-4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome. CONCLUSIONS Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs

Genome-wide characterization of pancreatic adenocarcinoma patients using next generation sequencing

Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P<0.05), along with tumor-stroma interactions and tumor suppressive pathways. While sequencing of more patients is needed, the high resolution genomic and transcriptomic information we have acquired here provides valuable information on the molecular composition of PAC and helps to establish a foundation for improved therapeutic selection

Look, the World is Watching How We Treat Migrants! The Making of the Anti-Trafficking Legislation during the Ma Administration

Employing the spiral model, this research analyses how anti-human trafficking legislation was promulgated during the Ma Ying-jeou (Ma Yingjiu) presidency. This research found that the gov- ernment of Taiwan was just as accountable for the violation of mi- grants’ human rights as the exploitive placement agencies and abusive employers. This research argues that, given its reliance on the United States for political and security support, Taiwan has made great ef- forts to improve its human rights records and meet US standards for protecting human rights. The reform was a result of multilevel inputs, including US pressure and collaboration between transnational and domestic advocacy groups. A major contribution of this research is to challenge the belief that human rights protection is intrinsic to dem- ocracy. In the same light, this research also cautions against Taiwan’s subscription to US norms since the reform was achieved at the cost of stereotyping trafficking victimhood, legitimising state surveillance, and further marginalising sex workers

Leveraging Spatial Variation in Tumor Purity for Improved Somatic Variant Calling of Archival Tumor Only Samples

Archival tumor samples represent a rich resource of annotated specimens for translational genomics research. However, standard variant calling approaches require a matched normal sample from the same individual, which is often not available in the retrospective setting, making it difficult to distinguish between true somatic variants and individual-specific germline variants. Archival sections often contain adjacent normal tissue, but this tissue can include infiltrating tumor cells. As existing comparative somatic variant callers are designed to exclude variants present in the normal sample, a novel approach is required to leverage adjacent normal tissue with infiltrating tumor cells for somatic variant calling. Here we present lumosVar 2.0, a software package designed to jointly analyze multiple samples from the same patient, built upon our previous single sample tumor only variant caller lumosVar 1.0. The approach assumes that the allelic fraction of somatic variants and germline variants follow different patterns as tumor content and copy number state change. lumosVar 2.0 estimates allele specific copy number and tumor sample fractions from the data, and uses a to model to determine expected allelic fractions for somatic and germline variants and to classify variants accordingly. To evaluate the utility of lumosVar 2.0 to jointly call somatic variants with tumor and adjacent normal samples, we used a glioblastoma dataset with matched high and low tumor content and germline whole exome sequencing data (for true somatic variants) available for each patient. Both sensitivity and positive predictive value were improved when analyzing the high tumor and low tumor samples jointly compared to analyzing the samples individually or in-silico pooling of the two samples. Finally, we applied this approach to a set of breast and prostate archival tumor samples for which tumor blocks containing adjacent normal tissue were available for sequencing. Joint analysis using lumosVar 2.0 detected several variants, including known cancer hotspot mutations that were not detected by standard somatic variant calling tools using the adjacent tissue as presumed normal reference. Together, these results demonstrate the utility of leveraging paired tissue samples to improve somatic variant calling when a constitutional sample is not available

Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory Neuroblastoma

Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects.This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations