Robust Enrichment Research Articles

On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons

SummaryRibosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.

Optimization of Salmonella detection in garlic, onion, cinnamon, red chili pepper powders and green tea

Salmonella is the causative agent of many outbreaks related to spice consumption. However, because of the antimicrobial properties of various spices which hinders recovery and detection, Salmonella detection in spices remains a challenge. The objective of this study was to optimize an enrichment broth for Salmonella growth in different spices and tea, in order to maintain an adequate pH and decrease the antimicrobial effects of spices during Salmonella enrichment and subsequent detection. Salmonella contaminated spice and tea dried samples were prepared and the detection of Salmonella was assessed using the developed broth and automated DNA extraction and RT-PCR. Double strength Buffered Peptone Water (BPW) was used to maintain pH, and L-cysteine and DL-serine were added to the broth to reduce the effects of antimicrobial compounds in spices. The modified enrichment broth allowed the growth of Salmonella from each spice sample. Sample to broth ratios varied from 1:9 (garlic powder, chili peppers and tea), to 1:20 (cinnamon). The pH value of each enrichment varied but remained above 4.8. The addition of L-cysteine (30 mmol/L) allowed Salmonella recovery and growth in garlic and onion samples and the addition of DL-serine (11.23 mmol/L) allowed the recovery and growth in cinnamon. The results indicated that Salmonella detection was achieved in <24 h in the modified (BPW + L-cysteine and DL-serine) enrichment broth followed by detection by RT-PCR. This protocol could allow for a more rapid, robust, and sensitive enrichment method for Salmonella in spices.

Continuous flow, large‐scale, microbial fuel cell system for the sustained treatment of swine waste

Microbial fuel cells (MFCs) have long held the promise of being a cost-effective technology for the energy-neutral treatment of wastewater. However, successful pilot-scale demonstrations for this technology are still limited to very few. Here, we present a large-scale MFC system, composed of 12 MFCs with a total volume of 110L, successfully treating swine wastewater at a small educational farm. The system was operated for over 200days in continuous mode with hydraulic residence time of 4hr. Very stable electrochemical and waste treatment performance was observed with up to 65% of chemical oxygen demand (COD) removed and a maximum treatment rate of 5.0kgCOD/m3 .day. Robust microbial enrichment was performed and adapted to metabolize and transform a diversity of compounds present. The Net Energy Recovery (NER=0.11kWhr/kg COD) is not only competitive with conventional cogeneration processes, but is in fact sufficient to sustain the operational energy requirements of the system. PRACTITIONER POINTS: This study demonstrates the design and operation of a large-scale microbial fuel cells (MFC) system for continuous treatment of swine wastewater. The system achieved a high chemical oxygen demand removal rate within a short hydraulic residence time. This study moves one-step closer to applying MFC technology for real wastewater treatment.

Loss of the tumor suppressor, Tp53, enhances the androgen receptor-mediated oncogenic transformation and tumor development in the mouse prostate.

Recent genome analysis of human prostate cancers demonstrated that both AR gene amplification and TP53 mutation are among the most frequently observed alterations in advanced prostate cancer. However, the biological role of these dual genetic alterations in prostate tumorigenesis is largely unknown. In addition, there are no biologically relevant models that can be used to assess the molecular mechanisms for these genetic abnormalities. Here, we report a novel mouse model, in which elevated transgenic AR expression and Trp53 deletion occur simultaneously in mouse prostatic epithelium to mimic human prostate cancer cells. These compound mice developed an earlier onset of high-grade prostatic intraepithelial neoplasia and accelerated prostate tumors in comparison to mice harboring only the AR transgene. Histological analysis showed prostatic sarcomatoid and basaloid carcinomas with massive squamous differentiation in the above compound mice. RNA-sequencing analyses identified a robust enrichment of the signature genes for human prostatic basal cell carcinomas in the above prostate tumors. Master Regulator Analysis revealed SOX2 as a transcriptional regulator in prostatic basal cell tumors. Elevated expression of SOX2 and its downstream target genes were detected in prostatic tumors of the compound mice. Chromatin immunoprecipitation analyses implicate a co-regulatory role of AR and SOX2 in the expression of prostatic basal cell signature genes. Our data demonstrate a critical role of SOX2 in prostate tumorigenesis and provide mechanistic insight into prostate tumor aggressiveness and progression mediated by aberrant AR and p53 signaling pathways.

Abstract 373: Dysregulated RPB1 is a key driver of overgrowth and drug resistance in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a group of highly aggressive malignancies with a 5-year overall survival (OS) of &amp;lt;40%. Cell overgrowth with apoptosis resistance is a hallmark of AML, but little is known about how it occurs. In this study, we show that dysregulated the largest subunit of RNA polymerase II (RPB1) encoded by POLR2A gene is critically involved in this hallmark in AML. qRT-PCR and RNA-seq results showed that POLR2A mRNA were significantly overexpressed in 76.87% (113/147) compared with normal hematopoietic cells(1.5526 versus 0.8447, P&amp;lt;0.001). Western blotting analysis revealed that RPB1 protein is also aberrantly activated in 68.14%(67/113) of AML patients but rare or even absent in normal hematopoietic cells, (0.7889 versus 0.0509, P&amp;lt;0.01). TCGA data analysis indicated that aberrant activation of POLR2A mRNA occurred predominantly in AML, CHOL and THYM, but not in most other types of cancers. Importantly, we observed that RPB1 expression levels were closely associated with tumor burden and poor prognosis. Notably, we found that POLR2A inhibition induced potent apoptosis and growth inhibition of refractory AML cells. Global transcriptomic analysis reveals that POLR2A knockout selectively depleted an array of AML-related oncogenic and anti-apoptosis genes such as c-MYC, RUNX2, MEIS1, FLT3, CDC25A and BCL-2. Conversely, enforced overexpression of RPB1 exhibits robust enrichment of oncogenes and anti-apoptosis genes, leading to cell overgrowth and drug resistance. Of particular interesting, targeting RPB1 by both genetic and pharmacological molecules could lead complete regression of refractory human AML in mouse models. In conclusion, our findings for the first time reveal that dysregulated RPB1 is a central oncogenic hub that drives overgrowth and drug resistance by coordinately hijacking an array of oncogenic genes and anti-apoptosis factors. Importantly, RPB1 is not absolutely required for the survival of normal hematopoietic cells, which is contrary to the commonly accepted belief that RPB1, the heart of the cellular transcription machinery, is required for all protein-coding genes. These data suggest that targeting RPB1 might be a potential therapeutics for treating refractory/relapse AML. Note: This abstract was not presented at the meeting. Citation Format: Qingfeng Yu, Shu Zheng, Ying Xu, Rongzhen Xu. Dysregulated RPB1 is a key driver of overgrowth and drug resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 373.

Organoid-based ex vivo reconstitution of Kras-driven pancreatic ductal carcinogenesis.

The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model.

An unbiased in vitro screen for activating epidermal growth factor receptor mutations

Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites, yet only a few of these mutations have been functionally tested. Here, we describe an unbiased platform for the functional characterization of thousands of variants of a single receptor tyrosine kinase (RTK) gene in a single assay. Our in vitroscreen for activating mutations (iSCREAM) platform enabled rapid analysis of mutations conferring gain-of-function RTK activity promoting clonal growth. The screening strategy included a somatic model of cancer evolution and utilized a library of 7,216 randomly mutated epidermal growth factor receptor (EGFR) single-nucleotide variants that were tested in murine lymphoid Ba/F3 cells. These cells depend on exogenous interleukin-3 (IL-3) for growth, but this dependence can be compensated by ectopic EGFR overexpression, enabling selection for gain-of-function EGFR mutants. Analysis of the enriched mutants revealed EGFR A702V, a novel activating variant that structurally stabilized the EGFR kinase dimer interface and conferred sensitivity to kinase inhibition by afatinib. As proof of concept for our approach, we recapitulated clinical observations and identified the EGFR L858R as the major enriched EGFR variant. Altogether, iSCREAM enabled robust enrichment of 21 variants from a total of 7,216 EGFR mutations. These findings indicate the power of this screening platform for unbiased identification of activating RTK variants that are enriched under selection pressure in a model of cancer heterogeneity and evolution.

One-Step SH2 Superbinder-Based Approach for Sensitive Analysis of Tyrosine Phosphoproteome.

Tyrosine phosphorylation plays a major role in regulating cell signaling pathways governing diverse biological functions such as proliferation and differentiation. Systemically mapping phosphotyrosine (pTyr) sites is the key to understanding molecular mechanisms underlining pTyr-dependent signaling. Although mass spectrometry-based technologies have been widely used for pTyr site profiling and quantification, their applications are often hindered by the poor efficiency in current multistep enrichment procedures for inherently low abundance pTyr peptides, especially under physiological conditions. Taking advantage of the sequence-independent high affinity of SH2 superbinder toward pTyr residues, we have developed a simplified one-step pTyr peptide enrichment method that uses immobilized SH2 superbinder for unbiased and robust enrichment of endogenous pTyr peptides from biological samples. By eliminating the prerequisite global phosphopeptide enrichment step in our previously developed two-step method, we minimized sample loss and improved peptide capture efficiency. Applying this method to Jurkat cells at resting state, where the tyrosine phosphorylation level is low, both the number of identified pTyr peptides and sites are increased by three folds compared to the two-step method. Specifically, we were able to identify 511 nonredundant pTyr peptides, corresponding to 403 high confidence pTyr sites, from Jurkat cells with high level technical reproducibility (Pearson's correlation coefficient as high as 0.94). Further applying this method to two human breast cancer cell lines, BT474 and HCC1954, before and after EGF stimulation, we demonstrated that this approach could be a powerful tool for illustrating pTyr-dependent signaling network controlling cellular behaviors such as drug resistance.

Cocaine'omics: Genome-wide and transcriptome-wide analyses provide biological insight into cocaine use and dependence.

We investigated the genetic and molecular architecture of cocaine dependence (CD) and cocaine use by integrating genome-/transcriptome-wide analyses. To prioritize candidates for follow-up investigation, we also sought to translate gene expression findings across species. Using data from the largest genome-wide association study (GWAS) of CD to date (n=3176, 74% with CD), we assessed genomic heritability, gene-based associations, and tissue enrichment. We detected a significant single-nucleotide polymorphism heritability of 28% for CD and identified three genes (two loci) underlying this predisposition: the C1qL2 (complement component C1 q like 2), KCTD20 (potassium channel tetramerization domain containing 20), and STK38 (serine/threonine kinase 38) genes. Tissue enrichment analyses indicated robust enrichment in numerous brain regions, including the hippocampus. We used postmortem human hippocampal RNA-sequencing data from previous study (n=15, seven chronic cocaine users) to follow up genome-wide results and to identify differentially expressed genes/transcripts and gene networks underlying cocaine use. Cross-species analyses utilized hippocampal gene expression from a mouse model of cocaine use. Differentially expressed genes/transcripts in humans were enriched for the genes nominally associated with CD via GWAS (P<0.05) and for differentially expressed genes in the hippocampus of cocaine-exposed mice. We identified KCTD20 as a central component of a hippocampal gene network strongly associated with human cocaine use, and this gene network was conserved in the mouse hippocampus. We outline a framework to map and translate genome-wide findings onto tissue-specific gene expression, which provided biological insight into cocaine use/dependence.

Combinatorial Peptide Ligand Library-Based Photoaffinity Probe for the Identification of Phosphotyrosine-Binding Domain Proteins.

Phosphotyrosine (pY) serves as a docking site for the recognition proteins containing pY-binding (pYB) modules, such as the SH2 domain, to mediate cell signal transduction. Thus, it is vital to profile these binding proteins for understanding of signal regulation. However, identification of pYB proteins remains a significant challenge due to their low abundance and typically weak and transient interactions with pY sites. Herein, we designed and prepared a pY-peptide photoaffinity probe for the robust and specific enrichment and identification of its binding proteins. Using SHC1-pY317 as a paradigm, we showed that the developed probe enables to capture target protein with high selectivity and remarkable specificity even in a complex context. Notably, we expanded the strategy to a combinatorial pY-peptide-based photoaffinity probe by using combinatorial peptide ligand library (CPLL) technique and identified 24 SH2 domain proteins, which presents a deeper profiling of pYB proteins than previous reports using affinity probes. Moreover, the method can be used to mine putative pYB proteins and confirmed PKN2 as a selective binder to pY, expanding the repertoire of known domain proteins. Our approach provides a general strategy for rapid and robust interrogating pYB proteins and will promote the understanding of the signal transduction mechanism.

Ti (IV) attached-phosphonic acid functionalized capillary monolith as a stationary phase for in-syringe-type fast and robust enrichment of phosphopeptides.

In this study, poly(vinylphosphonic acid-co-ethylene dimethacrylate), poly(VPA-co-EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 μm. The phosphopeptides from different sources were enriched on Ti (IV)-attached poly(VPA-co-EDMA) monolith using a syringe-pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of β-casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA-co-EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of β-casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal-to-noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA-co-EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.

Activation of hepatocyte growth factor/MET signaling initiates oncogenic transformation and enhances tumor aggressiveness in the murine prostate

Emerging evidence has shown that the hepatocyte growth factor (HGF) and its receptor, MET proto-oncogene, receptor tyrosine kinase (MET), promote cell proliferation, motility, morphogenesis, and angiogenesis. Whereas up-regulation of MET expression has been observed in aggressive and metastatic prostate cancer, a clear understanding of MET function in prostate tumorigenesis remains elusive. Here, we developed a conditional Met transgenic mouse strain, H11Met/+:PB-Cre4, to mimic human prostate cancer cells with increased MET expression in the prostatic luminal epithelium. We found that these mice develop prostatic intraepithelial neoplasia after HGF administration. To further assess the biological role of MET in prostate cancer progression, we bred H11Met/+/PtenLoxP/LoxP:PBCre4 compound mice, in which transgenic Met expression and deletion of the tumor suppressor gene Pten occurred simultaneously only in prostatic epithelial cells. These compound mice exhibited accelerated prostate tumor formation and invasion as well as increased metastasis compared with PtenLoxP/LoxP:PB-Cre4 mice. Moreover, prostatic sarcomatoid carcinomas and lesions resembling the epithelial-to-mesenchymal transition developed in tumor lesions of the compound mice. RNA-Seq and qRT-PCR analyses revealed a robust enrichment of known tumor progression and metastasis-promoting genes in samples isolated from H11Met/+/PtenLoxP/LoxP:PB-Cre4 compound mice compared with those from PtenLoxP/LoxP:PB-Cre4 littermate controls. HGF-induced cell proliferation and migration also increased in mouse embryonic fibroblasts (MEFs) from animals with both Met transgene expression and Pten deletion compared with Pten-null MEFs. The results from these newly developed mouse models indicate a role for MET in hastening tumorigenesis and metastasis when combined with the loss of tumor suppressors.

Robust Cell Selections and Depletions across Various Hematopoietic Cell Fractions on the Clinimacs Plus Instrument

Introduction: Cell enrichment and/or depletion by selection is critical for graft engineering in cellular and gene therapies. The semi-automated CliniMACS Plus Instrument (Miltenyi Biotec) is used in clinical cell processing to enrich or deplete a variety of hematopoietic cells including T, B, monocytes, NK cells, and/or CD34+ hematopoietic stem/progenitor cells (HSPCs). Antibody-conjugated paramagnetic beads attach to the desired cells which are separated from the rest of the apheresis or bone marrow product in a closed-system magnetic separation column. Either the selected cells retained in the separation column (positive selection) or those in the “flow-through” fraction (negative selection) may be used for downstream processing. There is sparse data comparing cell selection efficiency and purity across the various selection methods on the CliniMACS Plus Instrument. Further, the effect of donor demographics and other upstream processing events on final viable cell recovery (VCR)/ cell purity (CP) are unknown. This study systematically compared these parameters across various selection methods.Methods: From 2008 to 2018, data were obtained from 45 clinical processing protocols involving 991 consecutive adult and pediatric donor/patient apheresis products or bone marrow harvests, which underwent cell selections on the Miltenyi CliniMACS Plus device. Cell selections included 1.CD34+ (n=669), 2.CD4+ (n=179), 3.CD3-/56+ (n=79), 4.CD3+/19- (n=6), 5.CD4+/8+ (n=21), and 6.CD25- (n=37) cells. Based on protocol design or logistical needs, apheresis or bone marrow collections were processed/selected on the same day or held overnight for next day processing. Products were also either washed to remove platelets (PW) or loaded onto the selection device directly. Data on donor age, BMI, donor type (autologous vs. allogeneic), sex, race, pre-apheresis peripheral blood cell counts and concentrations, as well as apheresis bag blood counts were evaluated. Outcome variables included post-selection VCR and CP. Correlations and multivariate regression analyses were performed for the largest selection type, i.e CD34+ cell selections.Results: Summary data (means ± 1SD) on method-specific enrichments and depletions are shown in the Table. Median post-selection VCR and CP varied by selection method. VCR was the highest for the CD34+ cell selections (Fig. a). CP was the highest and most consistent for CD4+ selections (Fig. b). Platelet and red cell depletions also varied by selection method and averaged 3.9 ± 1.63 and 2.06 ± 0.74 logs, respectively. Selection procedures used for cell depletion resulted in near complete removal of the undesired cell fractions (Table).In multivariate regression analyses, products processed on the same day (compared to those held overnight) (Fig. c), those that underwent a PW (Fig. d), and had higher pre-apheresis peripheral blood CD34+ absolute cell counts and concentrations (Fig. e) were associated significantly with higher CD34+ CP (adjusted r2=0.2; p<0.0001). Higher pre-apheresis CD34+ concentrations (Fig. f) were associated with a lower CD34+ VCR (15%, CI: 5.6-25.3). Conversely, PW (Fig. g) correlated with higher CD34+ VCR (3.5%; CI: 0.8-6.28). Trends across the study period demonstrated a significant improvement in CD34+ VCR from 2008 to 2017 (Fig. h).Conclusions: This is the largest study, to our knowledge, to systematically summarize and analyze Miltenyi CliniMACS Plus selections for clinical graft engineering. Cell selections on the Miltenyi CliniMACS Plus resulted in robust enrichment with near complete depletion of the undesired cell fractions. CD34+ CP was higher in patients with higher CD34+ cells counts following mobilization. Same day processing, as well as PW further improved CD34+ CP. PW also improved CD34+ VCR. This was likely due to a reduction in non-specific platelet binding to the paramagnetic beads. Higher pre-selection CD34+ cell concentrations in the product marginally worsened VCR. This was possibly due to bead saturation in each selection method. Improved CD34+ VCR over time was likely due to increased platelet washes in the latter years. Further studies are ongoing to assess impact of cell selection variations on downstream manufacturing steps (cell transduction, expansion) and clinical (cell engraftment, GVHD incidence) outcomes. [Display omitted] DisclosuresLarochelle:Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Differentiation Kit.

A stable generalized/eXtended FEM with discontinuous interpolants for fracture mechanics

This paper presents numerical studies with three classes of quadratic Generalized FEM (GFEM) approximations and shows that all of them lead to errors that are orders of magnitude smaller than the FEM with quarter-point elements, provided that appropriate enrichments are selected. However, all of them lead to severely ill-conditioned systems of equations, with a condition number up to O(h−10) in the case of the GFEM based on a quadratic partition of unity. Enrichment modifications able to address the ill-conditioning of quadratic GFEM approximations while preserving their optimal convergence are proposed. A robust enrichment modification strategy based on a discontinuous FE interpolant is proposed to control the conditioning of branch function enrichment. The discontinuous FE interpolant is a generalization of the continuous one used with the Stable GFEM (SGFEM). We show that SGFEM spaces based on p-hierarchical FEM enrichments are the same as their GFEM counterparts. This guarantees that both GFEM and SGFEM spaces will lead to the same solution. This is not the case for the other classes of second-order spaces. The robustness of the proposed approximation spaces with respect to the crack location in the mesh is also demonstrated.

Nanoparticle Delivery of RIG-I Agonist Enables Effective and Safe Adjuvant Therapy in Pancreatic Cancer.

Local immunomodulation can be a promising strategy to augment the efficacy and decrease off-target toxicities associated with cancer treatment. Pancreatic cancer is resistant to immunotherapies due to the immunosuppressive tumor microenvironment. Herein, we investigated a therapeutic approach involving delivery of a short interfering double-stranded RNA (dsRNA), specific to Bcl2, with 5' triphosphate ends, by lipid calcium phosphate nanoparticles, in an orthotopic allograft KPC model of pancreatic cancer. Retinoic acid-inducible gene I (RIG-I)-like receptors can bind to 5' triphosphate dsRNA (ppp dsRNA), a pathogen-associated molecular pattern, producing type I interferon, while Bcl2 silencing can drive apoptosis of cancer cells. Our approach demonstrated a robust enrichment of tumor tissue with therapeutic nanoparticles and enabled a significant tumor growth inhibition, prolonging median overall survival. Nanoparticles encapsulating dual-therapeutic ppp dsRNA allowed strong induction in levels of pro-inflammatory Th1 cytokines, further increasing proportions of CD8+ Tcells over regulatory Tcells, M1 over M2 macrophages, and decreased levels of immunosuppressive B regulatory and plasma cells in the tumor microenvironment. Thus, these results provide a new immunotherapy approach for pancreatic cancer.

Dissecting the genetic relationship between cardiovascular risk factors and Alzheimer's disease.

Cardiovascular (CV)- and lifestyle-associated risk factors (RFs) are increasingly recognized as important for Alzheimer's disease (AD) pathogenesis. Beyond the ε4 allele of apolipoprotein E (APOE), comparatively little is known about whether CV-associated genes also increase risk for AD. Using large genome-wide association studies and validatedtools to quantify genetic overlap, we systematically identified single nucleotide polymorphisms (SNPs) jointly associated with AD and one or more CV-associated RFs, namely body mass index (BMI), type 2 diabetes (T2D), coronary artery disease (CAD), waist hip ratio (WHR), total cholesterol (TC), triglycerides (TG), low-density (LDL) and high-density lipoprotein (HDL). In fold enrichment plots, we observed robust genetic enrichment in AD as a function of plasma lipids (TG, TC, LDL, and HDL); we found minimal AD genetic enrichment conditional on BMI, T2D, CAD, and WHR. Beyond APOE, at conjunction FDR < 0.05 we identified 90 SNPs on 19 different chromosomes that were jointly associated with AD and CV-associated outcomes. In meta-analyses across three independent cohorts, we found four novel loci within MBLAC1 (chromosome 7, meta-p = 1.44 × 10-9), MINK1 (chromosome 17, meta-p = 1.98 × 10-7) and two chromosome 11 SNPs within the MTCH2/SPI1 region (closest gene = DDB2, meta-p = 7.01 × 10-7 and closest gene = MYBPC3, meta-p = 5.62 × 10-8). In a large 'AD-by-proxy' cohort from the UK Biobank, we replicated three of the four novel AD/CV pleiotropic SNPs, namely variants within MINK1, MBLAC1, and DDB2. Expression of MBLAC1, SPI1, MINK1 and DDB2 was differentially altered within postmortem AD brains. Beyond APOE, we show that the polygenic component of AD is enriched for lipid-associated RFs. We pinpoint a subset of cardiovascular-associated genes that strongly increase the risk for AD. Our collective findings support a disease model in which cardiovascular biology is integral to the development of clinical AD in a subset of individuals.

A metagenomic study of the rumen virome in domestic caprids.

This project sought to investigate the domestic caprid rumen virome by developing a robust viral DNA isolation and enrichment protocol (utilizing membrane filtration, ultra-centrifugation, overnight PEG treatment and nuclease treatment) and using RSD-PCR and high throughput sequencing (HTS) techniques. 3.53% of the reads obtained were analogous to those of viruses denoting Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, Microviridae, Poxviridae, Tectiviridae and Marseillevirus. Most of the sequenced reads from the rumen were similar to those of phages, which are critical in maintaining the rumen microbial populations under its carrying capacity. Though identified in the rumen, most of these viruses have been reported in other environments as well. Improvements in the viral DNA enrichment and isolation protocol are required to obtain data that are more representative of the rumen virome. The 102,130 unknown reads (92.31%) for the goat and 36,241 unknown reads (93.86%) for the sheep obtained may represent novel genomes that need further study.

Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. Here, we performed droplet-based single-cell RNA sequencing (scRNA-seq) of the human iPSCs after iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of NO. Nonendothelial cell populations resulting from the differentiation protocol were identified, which included immature cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with 4 major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes, respectively. Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of nonendothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.

Abstract 472: Large-Scale Single-Cell RNA-Seq Identifies Heterogeneous Populations of Human Primary and Induced Pluripotent Stem Cell-Derived Endothelial Cells

Rationale: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. Objective: Here we performed droplet-based single-cell RNA-sequencing (scRNA-seq) of the human iPSCs following iPSC-EC differentiation and of primary endothelial cells isolated from human coronary arteries. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Methods and Results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs sorted by CD144 antibody-conjugated magnetic sorting exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of nitric oxide. Non-endothelial cell populations resulting from the differentiation protocol were identified, which included immature and atrial-like cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with four major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes respectively. These findings were then cross-analyzed with the primary EC data. Conclusions: Massively parallel scRNA-seq allowed analysis of thousands of human iPSCs subjected to EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of non-endothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity. Cross-analysis with scRNA-seq data of human coronary artery ECs reveals transcriptional similarities and dissimilarities of iPSC-ECs to the primary ECs that will be addressed in subsequent studies.

DMCM: a Data-adaptive Mutation Clustering Method to identify cancer-related mutation clusters.

Functional somatic mutations within coding amino acid sequences confer growth advantage in pathogenic process. Most existing methods for identifying cancer-related mutations focus on the single amino acid or the entire gene level. However, gain-of-function mutations often cluster in specific protein regions instead of existing independently in the amino acid sequences. Some approaches for identifying mutation clusters with mutation density on amino acid chain have been proposed recently. But their performance in identification of mutation clusters remains to be improved. Here we present a Data-adaptive Mutation Clustering Method (DMCM), in which kernel density estimate (KDE) with a data-adaptive bandwidth is applied to estimate the mutation density, to find variable clusters with different lengths on amino acid sequences. We apply this approach in the mutation data of 571 genes in over twenty cancer types from The Cancer Genome Atlas (TCGA). We compare the DMCM with M2C, OncodriveCLUST and Pfam Domain and find that DMCM tends to identify more significant clusters. The cross-validation analysis shows DMCM is robust and cluster cancer type enrichment analysis shows that specific cancer types are enriched for specific mutation clusters. DMCM is written in Python and analysis methods of DMCM are written in R. They are all released online, available through https://github.com/XinguoLu/DMCM. Supplementary data are available at Bioinformatics online.