Yao, Janet

Abstract Extracellular vesicles (EV) are phospholipid‐encapsulated nanoparticles secreted by cells into their surrounding environment. EVs can transfer a variety of biomolecules that mediate intercellular communication and play a key role in physiological and pathological processes. Therefore, EVs are emerging as new biomarkers for diseases, therapeutic targets, and drug delivery vehicles. The isolation and detection of EVs requires time‐consuming and labor‐intensive processes, first to extract EVs from biological and physiological fluids and then to detect EV‐associated molecules with high sensitivity. The methodologies and instruments commonly used for EV analysis are not widely accessible outside of dedicated research laboratories, creating practical barriers for the study of EV‐associated molecules in biology research and clinical applications. To bridge this gap, we developed a proof‐of‐concept digital microfluidic device that can automatedly extract EVs from 20 µL of culture and human plasma samples within 25 min and detect EV‐bound proteins (e.g PD‐L1) on‐chip using an electrochemical sensor. This work serves as a framework for the development of streamlined EV analysis in both research and diagnostics.

Background: Primary sclerosing cholangitis (PSC) is a major risk factor for cholangiocarcinoma (CCA). We investigated biliary and fecal microbiota to determine whether specific microbes in the bile or stool are associated with PSC or CCA. Methods: Bile was obtained from 32 patients with PSC, 23 with CCA with PSC, 26 with CCA without PSC, and 17 controls. Over 90% of bile samples were from patients with perihilar CCA. Stool was obtained from 31 patients with PSC (11 were matched to bile), 16 with CCA with PSC (10 matched to bile), and 11 with CCA without PSC (6 matched to bile). Microbiota composition was assessed using 16SrRNA-marker-based sequencing and was compared between groups. Results: Bile has a unique microbiota distinguished from negative DNA controls and stool. Increased species richness and abundance of Fusobacteria correlated with duration of PSC and characterized the biliary microbiota in CCA. Stool microbiota composition showed no significant differences between groups. Conclusions: We identified a unique microbial signature in the bile of patients with increased duration of PSC or with CCA, suggesting a role for microbiota-driven inflammation in the pathogenesis and or progression to perihilar CCA. Further studies are needed to test this hypothesis.

Mechanisms that mediate migration, homing and integration of blood outgrowth endothelial cells (BOEC) to sites of vascular injury are incompletely understood, although matrix metalloproteinases (MMPs) have been implicated in this process. Farnesoid X receptor (FXR) signaling pathway has recently been recognized to regulate vascular wall signaling. The aim of this study was to characterize the expression of FXR in human BOEC and examine signaling pathways relevant to BOEC migration. FXR mRNA and protein levels were detected by reverse‐transcriptase PCR and Western blot. Microarray analysis using a focused human endothelial cell biology array (Superarray) suggested that natural (Chenodeoxycholic acid (CDCA), 0.1–100μM) and synthetic (6‐ethylchenodeoxycholic acid (6‐ECDCA) 0.01–10μM) FXR ligands significantly increased MMP9 transcription which was confirmed by real time PCR and gelatin zymography. SiRNA based silencing of FXR prevented CDCA induced increases in MMP9 mRNA, while retroviral over‐expression of FXR in BOEC enhanced MMP9 mRNA levels. CDCA induced expression of Small Heterodimeric Partner (SHP), a downstream target of FXR. Retroviral over‐expression of SHP increased, whereas siRNA silencing of SHP inhibited CDCA induced MMP9 mRNA levels. Migration analysis using Boyden assay demonstrated that CDCA stimulated migration of BOEC which was blocked by transfection with FXR or MMP9 siRNA (p<0.05; n=5). These studies identify a novel signaling pathway by which a key protein important in cell homing and migration, MMP9, is regulated through a previously unrecognized FXR‐SHP pathway in BOEC. Funded by NIH Grant # DK59615

Metagenomic shotgun sequencing for the identification of pathogens is being increasingly utilized as a diagnostic method. Interpretation of large and complicated data sets is a significant challenge, for which multiple commercial tools have been developed. Three commercial metagenomic shotgun sequencing tools, CosmosID, One Codex, and IDbyDNA, were compared to determine whether they result in similar interpretations of the same sequencing data.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory, cholestatic liver disease of unknown etiopathogenesis which is recognized as a major risk factor for cholangiocarcinoma (CCA). We aimed to investigate the roles of the biliary and intestinal microflora in the inflammatory process of PSC and to determine if the microflora is associated with progression to CCA. METHODS: We studied the bile and stool microbiomes in samples obtained from patients with PSC (31 stool and 35 bile samples), sporadic CCA (11 stool and 26 bile samples), and PSC coexisting with CCA (16 stool and bile 17 samples). Control bile was obtained from patients with cholelithiasis or choledocholithiasis (n = 17). DNA extraction was followed by amplification and high-throughput next generation sequencing (MiSeq) of the 16S rDNA (V3-V5) region to identify the microbiota present. We performed: 1. analysis of the stool microbiome between PSC and CCA patients; 2. analysis of the biliary microbiome in PSC and CCA patients; 3. comparison of the biliary microbiome collected before and after biliary stent placement. RESULTS: Although bile samples from study patients had significantly lower sequencing depths than stool, their sequencing depths were much higher than bile from negative controls. The microbiomes of stool and bile samples from the same patients were significantly correlated ( P = 0.022). CCA bile samples had significant difference of alpha diversity from control bile samples with less Streptococcaceae and Desulfovibrionaceae (FDR < 10%) than PSC whereas no significant change of stool microbiome was identified between CCA and PSC. Fusobacterium and Gemella were correlated to PSC duration. An increased abundance of Fusobacteria after stent placement was revealed. Fusobacteria, which is inflammation-promoting, was also increased with the number of stents in the bile duct. CONCLUSION: Our study suggests that the bile microbiome is potentially a more distinctive biomarker than the stool microbiome for PSC and CCA development. There is increased inhabitance of inflammation-promoting bacteria in the bile of CCA patients and after biliary stent placement. Increased PSC duration also increases the microbial burden in the bile, which may also promote CCA development. We further detected specific bile microbes statistically associated with CCA or stent presence in the bile ducts, which raises the possibility of their role in the CCA progression or stent obstruction. Additional analyses with more samples are needed.

Single cell sequencing is a technology capable of analyzing the genome of a single cell within a population. This technology is mostly integrated with microfluidics for precise cell manipulation and fluid handling. So far, most of the microfluidic-based single cell genomic studies have been focused on lab-cultured species or cell lines that are relatively easy to handle following standard microfluidic-based protocols without additional adjustments. The major challenges for performing single cell sequencing on clinical samples is the complex nature of the samples which requires additional sample processing steps to obtain intact single cells of interest without using amplification-inhibitive agents. Fluorescent-activated cell sorting is a common option to obtain single cells from clinical samples for single cell applications but requires &gt;100 000 viable cells in suspension and the need for specialized laboratory and personnel. In this work, we present a protocol that can be used to obtain intact epithelial cells from snap-frozen postsurgical human endometrial tissues for single cell whole genome amplification. Our protocol includes sample thawing, cell dissociation, and labeling for genome amplification of targeted cells. Between 80% and 100% of single cell replicates lead to &gt;25 ng of DNA after amplification with no measurable contamination, sufficient for downstream sequencing.

Metagenomic shotgun sequencing has the potential to transform how serious infections are diagnosed by offering universal, culture-free pathogen detection. This may be especially advantageous for microbial diagnosis of prosthetic joint infection (PJI) by synovial fluid analysis since synovial fluid cultures are not universally positive and since synovial fluid is easily obtained preoperatively.

In carbon tetrachloride-induced liver cirrhosis, diminution of hepatic endothelial nitric oxide synthase (eNOS) activity may contribute to impaired hepatic vasodilation and portal hypertension. The mechanisms responsible for these events remain unknown; however, a role for the NOS-associated proteins caveolin and calmodulin has been postulated. The purpose of this study is to characterize the expression and cellular localization of the NOS inhibitory protein caveolin-1 in normal rat liver and to then examine the role of caveolin in conjunction with calmodulin in regulation of NOS activity in cholestatic portal hypertension. In normal liver, caveolin protein is expressed preferentially in nonparenchymal cells compared with hepatocytes as assessed by Western blot analysis of isolated cell preparations. Additionally, within the nonparenchymal cell populations, caveolin expression is detected within both liver endothelial cells and hepatic stellate cells. Next, studies were performed 4 wk after bile duct ligation (BDL), a model of portal hypertension characterized by prominent cholestasis, as evidenced by a significant increase in serum cholesterol in BDL animals. After BDL, caveolin protein levels from detergent-soluble liver lysates are significantly increased as assessed by Western blot analysis. Immunoperoxidase staining demonstrates that this increase is most prominent within sinusoids and venules. Additionally, caveolin-1 upregulation is associated with a significant reduction in NOS catalytic activity in BDL liver lysates, an event that is corrected with provision of excess calmodulin, a protein that competitively binds eNOS from caveolin. We conclude that, in cholestatic portal hypertension, caveolin may negatively regulate NOS activity in a manner that is reversible by excess calmodulin.

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) contributes to hepatic vascular homeostasis. The aim of this study was to examine whether delivery of an adenoviral vector encoding eNOS gene to liver affects vasomotor function in vivo and the mechanism of NO production in vitro. Rats were administered adenoviruses encoding β-galactosidase (AdCMVLacZ) or eNOS (AdCMVeNOS) via tail vein injection and studied 1 wk later. In animals transduced with AdCMVLacZ, β-galactosidase activity was increased in the liver, most prominently in hepatocytes. In AdCMVeNOS-transduced animals, eNOS protein levels and catalytic activity were significantly increased. Overexpression of eNOS diminished baseline perfusion pressure and constriction in response to the α1-agonist methoxamine in the perfused liver. Transduction of cultured hepatocytes with AdCMVeNOS resulted in the targeting of recombinant eNOS to a perinuclear distribution and binding with the NOS-activating protein heat shock protein 90. These events were associated with increased ionomycin-stimulated NO release. In summary, this is the first study to demonstrate successful delivery of the recombinant eNOS gene to liver in vivo and in vitro with ensuing NO production.