Publications by authors named "Meng Pu"

24 Publications

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Role of gut microbiota in regulating gastrointestinal dysfunction and motor symptoms in a mouse model of Parkinson's disease.

Gut Microbes 2021 Jan;13(1):1866974

Division of Gastroenterology and Hepatology, Mayo Clinic , Rochester, MN, USA.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized primarily by motor and non-motor gastrointestinal (GI) deficits. GI symptoms' including compromised intestinal barrier function often accompanies altered gut microbiota composition and motor deficits in PD. Therefore, in this study, we set to investigate the role of gut microbiota and epithelial barrier dysfunction on motor symptom generation using a rotenone-induced mouse model of PD. We found that while six weeks of 10 mg/kg of chronic rotenone administration by oral gavage resulted in loss of tyrosine hydroxylase (TH) neurons in both germ-free (GF) and conventionally raised (CR) mice, the decrease in motor strength and coordination was observed only in CR mice. Chronic rotenone treatment did not disrupt intestinal permeability in GF mice but resulted in a significant change in gut microbiota composition and an increase in intestinal permeability in CR mice. These results highlight the potential role of gut microbiota in regulating barrier dysfunction and motor deficits in PD.
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http://dx.doi.org/10.1080/19490976.2020.1866974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833732PMC
January 2021

Bacterially Derived Tryptamine Increases Mucus Release by Activating a Host Receptor in a Mouse Model of Inflammatory Bowel Disease.

iScience 2020 Dec 13;23(12):101798. Epub 2020 Nov 13.

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905, USA.

Recent studies emphasize the role of microbial metabolites in regulating gastrointestinal (GI) physiology through activation of host receptors, highlighting the potential for inter-kingdom signaling in treating GI disorders. In this study, we show that tryptamine, a tryptophan-derived bacterial metabolite, stimulates mucus release from goblet cells via activation of G-protein-coupled receptor (GPCR) 5-HT4R. Germ-free mice colonized with engineered optimized to produce tryptamine (Trp D+) exhibit decreased weight loss and increased mucus release following dextran sodium sulfate treatment when compared with mice colonized with control (Trp D-). Additional beneficial effects in preventing barrier disruption and lower disease activity index were seen only in female mice, highlighting sex-specific effects of the bacterial metabolite. This study demonstrates potential for the precise modulation of mucus release by microbially produced 5-HT4 GPCR agonist as a therapeutic strategy to treat inflammatory conditions of the GI tract.
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http://dx.doi.org/10.1016/j.isci.2020.101798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702010PMC
December 2020

Plasmid Acquisition Alters Vancomycin Susceptibility in Clostridioides difficile.

Gastroenterology 2021 02 14;160(3):941-945.e8. Epub 2020 Nov 14.

Division of Gastroenterology and Hepatology, Department of Medicine, Rochester, Minnesota; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota. Electronic address:

The increasing incidence of primary and recurring Clostridioides difficile infections (CDI), which evade current treatment strategies, reflects the changing biology of C difficile. Here, we describe a putative plasmid-mediated mechanism potentially driving decreased sensitivity of C difficile to vancomycin treatment. We identified a broad host range transferable plasmid in a C difficile strain associated with lack of adequate response to vancomycin treatment. The transfer of this plasmid to a vancomycin-susceptible C difficile isolate decreased its susceptibility to vancomycin in vitro and resulted in more severe disease in a humanized mouse model. Our findings suggest plasmid acquisition in the gastrointestinal tract to be a possible mechanism underlying vancomycin treatment failure in patients with CDI, but further work is needed to characterize the mechanism by which plasmid genes determine vancomycin susceptibility in C difficile.
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http://dx.doi.org/10.1053/j.gastro.2020.10.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878333PMC
February 2021

The mRNA of TCTP functions as a sponge to maintain homeostasis of TCTP protein levels in hepatocellular carcinoma.

Cell Death Dis 2020 11 12;11(11):974. Epub 2020 Nov 12.

Department of Hepatobiliary Surgery, Xijing Hospital, Air Force Medical University, Xi'an, Shaanxi Province, 710032, China.

Translationally controlled tumor protein (TCTP) is a highly conserved protein that accumulated in the tumorigenesis of various malignancies. Despite the important role of TCTP protein in tumor progression, the precise function and underlying mechanistic regulation of TCTP mRNA in hepatocellular carcinoma (HCC) remain unclear. In this study, we found that TCTP protein was overexpressed in HCC patients but TCTP mRNA expression levels were reversed. TCTP knockout HCC cells exhibited attenuated abilities of proliferation, migration, and invasion. The knockdown of TCTP by siRNA effectively reduced TCTP mRNA levels but not protein levels in HCC cells. Moreover, although the constitutive knockdown of TCTP inhibited almost 80% of TCTP protein expression levels in tumors of wildtype transgenic mice (TCTP KD/WT), partial restoration of TCTP protein expression was observed in the tumors of heterozygous TCTP mice (TCTP KD/TCTP±). The blockage of mRNA synthesis with ActD stimulated TCTP protein expression in HCC cells. In contrast, combined treatment with ActD and CHX or MG132 treatment alone did not lead to the TCTP protein accumulation in cells. Furthermore, following the introduction of exogenous TCTP in cells and orthotopic HCC tumor models, the endogenous TCTP protein did not change with the recombinational TCTP expression and kept a rather stable level. Dual-luciferase assays revealed that the coding sequence of TCTP mRNA functions as a sponge to regulate the TCTP protein expression. Collectively, our results indicated that the TCTP mRNA and protein formed a closed regulatory circuit and works as a buffering system to keep the homeostasis of TCTP protein levels in HCC.
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http://dx.doi.org/10.1038/s41419-020-03149-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665032PMC
November 2020

Vibrio cholerae Type VI Activity Alters Motility Behavior in Mucin.

J Bacteriol 2020 11 19;202(24). Epub 2020 Nov 19.

Department of Biology, Indiana University Bloomington, Bloomington, Indiana, USA

Motility is required for many bacterial pathogens to reach and colonize target sites. traverses a thick mucus barrier coating the small intestine to reach the underlying epithelium. We screened a transposon library in motility medium containing mucin to identify factors that influence mucus transit. Lesions in structural genes of the type VI secretion system (T6SS) were among those recovered. Two-dimensional (2D) and 3D single-cell tracking was used to compare the motility behaviors of wild-type cells and a mutant that collectively lacked three essential T6SS structural genes (T6SS). In the absence of mucin, wild-type and T6SS cells exhibited similar speeds and run-reverse-flick (RRF) swimming patterns, in which forward-moving cells briefly backtrack before stochastically reorienting (flicking) in a new direction upon resuming forward movement. We show that mucin induced T6SS expression and activity in wild-type bacteria but significantly decreased their swimming speed and flicking, yielding curvilinear or near-surface circular traces for many cells. Conversely, mucin slowed T6SS cells to a lesser extent, and many continued to flick and produce RRF-like traces. Δ cells, which exclusively swim in the forward direction and thus cannot flick, also produced curvilinear traces with or without mucin present and, on occasion, near-surface circular traces in the presence of mucin. The dependence of flicking on swimming speed suggested that mucin-induced T6SS activity further decreased motility and thereby reduced flicking probability during reverse-to-forward transitions. We propose that this encourages cells to continue on their current trajectory rather than reorienting, which may benefit those tracking toward the epithelial surface. deploys an arsenal of virulence factors as it attempts to traverse a protective mucus layer and reach the epithelial surface of the distal small intestine. The T6SS used to cull bacterial competition during infection is induced by mucus. We show that this activity may serve an additional purpose by further decreasing motility in the presence of mucin, thereby reducing the probability of speed-dependent, near-perpendicular directional changes. We posit that this encourages cells to maintain course rather than change direction, which may aid those attempting to reach and colonize the epithelial surface.
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http://dx.doi.org/10.1128/JB.00261-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685549PMC
November 2020

Clostridioides difficile Whole-genome Sequencing Differentiates Relapse With the Same Strain From Reinfection With a New Strain.

Clin Infect Dis 2021 03;72(5):806-813

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA.

Background: Current approaches in tracking Clostridioides difficile infection (CDI) and individualizing patient management are incompletely defined.

Methods: We recruited 468 subjects with CDI at Mayo Clinic Rochester between May and December 2016 and performed whole-genome sequencing (WGS) on C. difficile isolates from 397. WGS was also performed on isolates from a subset of the subjects at the time of a recurrence of infection. The sequence data were analyzed by determining core genome multilocus sequence type (cgMLST), with isolates grouped by allelic differences and the predicted ribotype.

Results: There were no correlations between C. difficile isolates based either on cgMLST or ribotype groupings and CDI outcome. An epidemiologic assessment of hospitalized subjects harboring C. difficile isolates with ≤2 allelic differences, based on standard infection prevention and control assessment, revealed no evidence of person-to-person transmission. Interestingly, community-acquired CDI subjects in 40% of groups with ≤2 allelic differences resided within the same zip code. Among 18 subjects clinically classified as having recurrent CDI, WGS revealed 14 with initial and subsequent isolates differing by ≤2 allelic differences, suggesting a relapse of infection with the same initial strain, and 4 with isolates differing by >50 allelic differences, suggesting reinfection. Among the 5 subjects classified as having a reinfection based on the timing of recurrence, 3 had isolates with ≤2 allelic differences between them, suggesting a relapse, and 2 had isolates differing by >50 allelic differences, suggesting reinfection.

Conclusions: Our findings point to potential transmission of C. difficile in the community. WGS better differentiates relapse from reinfection than do definitions based on the timing of recurrence.
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http://dx.doi.org/10.1093/cid/ciaa159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935391PMC
March 2021

Cathelicidin Peptides Restrict Bacterial Growth via Membrane Perturbation and Induction of Reactive Oxygen Species.

mBio 2019 09 10;10(5). Epub 2019 Sep 10.

Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.

All metazoans produce antimicrobial peptides (AMPs) that have both broad antimicrobial and immunomodulatory activity. Cathelicidins are AMPs that preferentially kill Gram-negative bacteria , purportedly by assembling into higher-order structures that perforate the membrane. We utilized high-resolution, single-cell fluorescence microscopy to examine their mechanism of action in real time. Engineered cathelicidins rapidly bound to Gram-negative and Gram-positive cells and penetrated the cytoplasmic membrane. Rapid failure of the peptidoglycan superstructure in regions of active turnover caused leakage of cytoplasmic contents and the formation of membrane-bound blebs. A mutation anticipated to destabilize interactions between cathelicidin subunits had no effect on bactericidal activity, suggesting that cathelicidins have activities beyond perforating the membrane. Nanomolar concentrations of cathelicidins, although not bactericidal, reduced the growth rate of Gram-negative and Gram-positive bacteria. The cells exhibited expression changes in multiple essential processes, including protein synthesis, peptidoglycan biosynthesis, respiration, and the detoxification of reactive oxygen species (ROS). Time-lapse imaging revealed that ROS accumulation preceded bleb formation, and treatments that reduced cellular ROS levels overcame these bactericidal effects. We propose that that the primary effect of cathelicidins is to induce the production of ROS that damage bacterial molecules, leading to slowed growth or cell death. Given their low circulating levels , AMPs may serve to slow bacterial population expansion so that cellular immunity systems can respond to and battle the infection. Antimicrobial peptides (AMPs) are an important part of the mammalian innate immune system in the battle against microbial infection. How AMPs function to control bacteria is not clear, as nearly all activity studies use nonphysiological levels of AMPs. We monitored peptide action in live bacterial cells over short time frames with single-cell resolution and found that the primary effect of cathelicidin peptides is to increase the production of oxidative molecules that cause cellular damage in Gram-positive and Gram-negative bacteria.
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http://dx.doi.org/10.1128/mBio.02021-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737244PMC
September 2019

Calcium-dependent site-switching regulates expression of the atypical iam pilus locus in Vibrio vulnificus.

Environ Microbiol 2020 10 16;22(10):4167-4182. Epub 2019 Aug 16.

Department of Biology, Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA.

The opportunistic human pathogen Vibrio vulnificus inhabits warm coastal waters and asymptomatically colonizes seafood, most commonly oysters. We previously characterized an isolate that exhibited greater biofilm formation, aggregation and oyster colonization than its parent. This was due, in part, to the production of a Type IV Tad pilus (Iam). However, the locus lacked key processing and regulatory genes required for pilus production. Here, we identify a pilin peptidase iamP, and LysR-type regulator (LRTR) iamR, that fulfil these roles and show that environmental calcium, which oysters enrich for shell repair and growth, regulates iam expression. The architecture of the iam locus differs from the classical LRTR paradigm and requires an additional promoter to be integrated into the regulatory network. IamR specifically recognized the iamR promoter (P ) and the intergenic iamP-iamA region (P ). P exhibited classical negative auto-regulation but, strikingly, IamR inversely regulated the divergent iamP and iamA promoters in a calcium-dependent manner. Moreover, expression of the c-di-GMP and calcium-regulated, biofilm-promoting brp exopolysaccharide was IamA-dependent. These results support a scenario in which the calcium-enriched oyster environment triggers IamP-mediated processing of prepilin amassed in the periplasm for rapid pilin elaboration and subsequent BRP production to promote colonization.
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http://dx.doi.org/10.1111/1462-2920.14763DOI Listing
October 2020

Microneedle Patch-Mediated Treatment of Bacterial Biofilms.

ACS Appl Mater Interfaces 2019 Apr 12;11(16):14640-14646. Epub 2019 Apr 12.

Current treatments of bacterial biofilms are limited by the poor penetration of antibiotics through their physical barrier as well as significant off-target toxicity of antibiotics and the induction of antibiotic resistance. Here we report a microneedle patch-mediated treatment for the effective elimination of biofilms by penetrating the biofilm and specifically delivering antibiotics to regions of active growth. We fabricated patches with self-dissolvable microneedles and needle tips loaded with chloramphenicol (CAM)-bearing and gelatinase-sensitive gelatin nanoparticles ([email protected]). During the microneedle patch-mediated treatment, arrays of 225 microneedles simultaneously penetrate the biofilm matrix. Once inside, the microneedles dissolve and uniformly release [email protected] into the surrounding area. In response to the gelatinase produced by the active bacterial community, the [email protected] disassemble and release CAM into these active regions of the biofilm. Moreover, [email protected] exhibited minimal off-target toxicity compared to direct CAM administration, which in turn favors wound healing. Importantly, we found that our microneedle-mediated treatment is more effective in treating Vibrio vulnificus biofilms than drug in free solution. We believe this new treatment strategy can be used to improve the delivery of a wide range of antimicrobial agents to biofilm-contaminated sites.
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http://dx.doi.org/10.1021/acsami.9b02578DOI Listing
April 2019

ERRATUM.

Oncol Res 2019 Feb;27(2):281-282

Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.

MicroRNA-200a (miR-200a) is frequently downregulated in most cancer types and plays an important role in carcinogenesis and cancer progression. In this study, we determined that miR-200a was downregulated in hepatocellular carcinoma (HCC) tissues and cell lines, consistent with the results of our previous study. Because a previous study suggested that downregulation of miR-200a is correlated with HCC metastasis, we aimed to elucidate the mechanism underlying the role of miR-200a in metastasis in HCC. Here we observed that overexpression of miR-200a resulted in suppression of HCC metastatic ability, including HCC cell migration, invasion, and metastasis, in vitro and in vivo. Furthermore, bioinformatics and luciferase reporter assays indicated that GAB1 is a direct target of miR-200a. Inhibition of GAB1 resulted in substantially decreased cell invasion and migration similar to that observed with overexpression of miR-200a in HCC cell lines, whereas restoration of GAB1 partially rescued the inhibitory effects of miR-200a. Taken together, these data provide novel information for comprehending the tumor-suppressive role of miR-200a in HCC pathogenesis through inhibition of GAB1 translation.
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http://dx.doi.org/10.3727/096504019X15476499940873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848442PMC
February 2019

Environmental Calcium Initiates a Feed-Forward Signaling Circuit That Regulates Biofilm Formation and Rugosity in Vibrio vulnificus.

mBio 2018 08 28;9(4). Epub 2018 Aug 28.

Department of Biology, Indiana University-Bloomington, Bloomington, Indiana, USA

Poor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic human pathogen , as well as the bivalves (oysters) it naturally colonizes, is indigenous to estuaries and human-inhabited coastal regions and must endure constantly changing environmental conditions as freshwater and seawater enter, mix, and exit the water column. Elevated cellular c-di-GMP levels trigger biofilm formation, but relatively little is known regarding the environmental signals that initiate this response. Here, we show that calcium is a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers expression of the extracellular polysaccharide that enhances biofilm formation. A transposon screen for the loss of calcium-induced expression revealed CysD, an enzyme in the sulfate assimilation pathway. Targeted disruption of the pathway indicated that the production of a specific metabolic intermediate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), was required for calcium-induced expression and that PAPS was separately required for development of the physiologically distinct rugose phenotype. Thus, PAPS behaves as a second messenger in Moreover, c-di-GMP and BrpT (the activator of expression) acted in concert to bias expression of the sulfate assimilation pathway toward PAPS and c-di-GMP accumulation, establishing a feed-forward regulatory loop to boost expression. Thus, this signaling network links extracellular calcium and sulfur availability to the intracellular second messengers PAPS and c-di-GMP in the regulation of biofilm formation and rugosity, survival phenotypes underpinning its evolution as a resilient environmental organism. The second messenger c-di-GMP is a key regulator of bacterial physiology. The genome encodes nearly 100 proteins predicted to make, break, and bind c-di-GMP. However, relatively little is known regarding the environmental signals that regulate c-di-GMP levels and biofilm formation in Here, we identify calcium as a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers -mediated biofilm formation. We show that PAPS, a metabolic intermediate of the sulfate assimilation pathway, acts as a second messenger linking environmental calcium and sulfur source availability to the production of another intracellular second messenger (c-di-GMP) to regulate biofilm and rugose colony formation, developmental pathways that are associated with environmental persistence and efficient bivalve colonization by this potent human pathogen.
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http://dx.doi.org/10.1128/mBio.01377-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113621PMC
August 2018

Silencing of CDCA5 inhibits cancer progression and serves as a prognostic biomarker for hepatocellular carcinoma.

Oncol Rep 2018 Oct 17;40(4):1875-1884. Epub 2018 Jul 17.

Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.

Cell division cycle associated 5 (CDCA5) has been associated with the progression of several types of cancers. However, its possible role and mechanism in hepatocellular carcinoma (HCC) remain unknown. In the present study, immunohistochemical staining and real‑time PCR were used to assess CDCA5 protein and mRNA levels in clinical samples. Statistical analysis was performed to explore the clinical correlation between CDCA5 protein expression and clinicopathological features and overall survival in HCC patients. Cell counting and colony formation assays were employed to analyse the effect of CDCA5 on cell proliferation, and flow cytometry was used to study the role of CDCA5 in cell cycle progression and apoptosis. Moreover, subcutaneous xenograft tumour models were implemented to predict the efficacy of targeting CDCA5 in HCC in vivo. We found that CDCA5 expression was significantly higher in HCC tumour tissues, was associated with clinicopathological characteristics, and predicted poor overall survival in HCC patients. Silencing of CDCA5 with small interfering RNA (siRNA) inhibited cell proliferation and induced G2/M cell cycle arrest in vitro. The xenograft growth assay revealed that CDCA5 downregulation impeded HCC growth in vivo. Further study indicated that CDCA5 depletion decreased the levels of ERK1/2 and AKT phosphorylation in vitro and in vivo. Taken together, these results indicate that CDCA5 may act as a novel prognostic biomarker and therapeutic target for HCC.
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http://dx.doi.org/10.3892/or.2018.6579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6111608PMC
October 2018

A Tad pilus promotes the establishment and resistance of biofilms to mechanical clearance.

NPJ Biofilms Microbiomes 2018 23;4:10. Epub 2018 Apr 23.

1Department of Molecular and Cellular Biochemistry, Indiana University of Bloomington, Bloomington, IN USA.

is autochthonous to estuaries and warm coastal waters. Infection occurs via open wounds or ingestion, where its asymptomatic colonization of seafood, most infamously oysters, provides a gateway into the human food chain. Colonization begins with initial surface contact, which is often mediated by bacterial surface appendages called pili. Type IV Tad pili are widely distributed in the Vibrionaceae, but evidence for a physiological role for these structures is scant. The genome codes for three distinct loci. Recently, a positive correlation was demonstrated between the expression of and the phenotypes of a descendent (NT) that exhibited increased biofilm formation, auto-aggregation, and oyster colonization relative to its parent. However, the mechanism by which pilus expression promoted these phenotypes was not determined. Here, we show that deletion of the pilin gene () altered the near-surface motility profile of NT cells from high curvature, orbital retracing patterns characteristic of cells actively probing the surface to low curvature traces indicative of wandering and diminished bacteria-surface interactions. The NT pilin mutant also exhibited decreased initial surface attachment, attenuated auto-aggregation and formed fragile biofilms that disintegrated under hydrodynamic flow. Thus, the locus, designated , promoted nitial surface attachment, uto-aggregation and resistance to echanical clearance of biofilms. The prevalence of loci in the Vibrionaceae suggests that they may play equally important roles in other family members.
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http://dx.doi.org/10.1038/s41522-018-0052-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913241PMC
April 2018

A conserved tad pilus promotes Vibrio vulnificus oyster colonization.

Environ Microbiol 2018 02 29;20(2):828-841. Epub 2017 Dec 29.

Molecular and Cellular Biochemistry, Indiana University Bloomington, IN, USA.

Vibrio vulnificus has the highest death rate (>35%) and per-case economic burden ($3.3 million) of any foodborne pathogen in the United States. Infections occur via open wounds or following ingestion of contaminated seafood, most infamously oysters. We isolated a 1000th generation descendant, designated NT that exhibited increased biofilm and aggregate formation relative to its parent. We identified two significant causal changes underlying these phenotypes. First, the entire 24-kb capsular polysaccharide biosynthesis locus, which is essential for virulence but inhibits biofilm formation, had been purged from the genome. However, NT formed more extensive biofilms and aggregates than a defined cps mutant, suggesting that additional factor(s) contributed to its phenotypes. Second, the expression of a tight adherence (tad) pilus locus was elevated in NT. Deletion of the associated pilin (flp) decreased NT biofilm and aggregate formation. Furthermore, NTΔflp strains were deficient relative to NT in an oyster colonization model, demonstrating a positive correlation between the biofilm and aggregation phenotypes associated with Tad pilus production and efficient bacterial retention by feeding oysters. Despite being widely distributed in the Vibrionaceae, this is the first demonstration of a bona fide physiological role for a Tad pilus in this bacterial family.
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http://dx.doi.org/10.1111/1462-2920.14025DOI Listing
February 2018

High MRPS23 expression contributes to hepatocellular carcinoma proliferation and indicates poor survival outcomes.

Tumour Biol 2017 Jul;39(7):1010428317709127

1 Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an, China.

Hepatocellular carcinoma is one of the most prevalent neoplasms and the leading cause of cancer-related mortality worldwide. Mitochondrial ribosomal protein S23 is encoded by a nuclear gene and participates in mitochondrial protein translation. Mitochondrial ribosomal protein S23 overexpression has been found in many types of cancer. In this study, we explored mitochondrial ribosomal protein S23 expression in primary hepatocellular carcinoma tissues compared with matched adjacent non-tumoral liver tissues using mitochondrial ribosomal protein S23 messenger RNA and protein levels collected from public databases and clinical samples. Immunohistochemistry was performed to analyze the relationship between mitochondrial ribosomal protein S23 and various clinicopathological features. The results indicated that mitochondrial ribosomal protein S23 was significantly overexpressed in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 expression was correlated with the tumor size and tumor-metastasis-node stage. Moreover, patients with high mitochondrial ribosomal protein S23 expression levels presented poorer survival rates. Mitochondrial ribosomal protein S23 was an independent prognostic factor for survival, especially at the early stage of hepatocellular carcinoma. In addition, the downregulation of mitochondrial ribosomal protein S23 decreased the proliferation of hepatocellular carcinoma in vitro and in vivo. In conclusion, we verified for the first time that mitochondrial ribosomal protein S23 expression was upregulated in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 levels can predict poor clinical outcomes in hepatocellular carcinoma, and this protein plays a key role in tumor proliferation. Therefore, mitochondrial ribosomal protein S23 may be a potential therapeutic target for hepatocellular carcinoma.
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http://dx.doi.org/10.1177/1010428317709127DOI Listing
July 2017

The Proline Variant of the W[F/L/M][T/S]R Cyclic Di-GMP Binding Motif Suppresses Dependence on Signal Association for Regulator Function.

J Bacteriol 2017 Oct 5;199(19). Epub 2017 Sep 5.

Department of Biology, Indiana University, Bloomington, Indiana, USA

is an estuarine bacterium and potent opportunistic human pathogen. It enters the food chain by asymptomatically colonizing a variety of marine organisms, most notably oysters. Expression of the -encoded extracellular polysaccharide, which enhances cell-surface adherence, is regulated by cyclic di-GMP (c-di-GMP) and the activator BrpT. The and homologs VpsT and CpsQ, directly bind c-di-GMP via a novel W[F/L/M][T/S]R motif, and c-di-GMP binding is absolutely required for activity. Notably, BrpT belongs to a distinct subclass of VpsT-like regulators that harbor a proline in the third position of the c-di-GMP binding motif (WLPR), and the impact of this change on activity is unknown. We show that the locus is organized as two linked operons with BrpT specifically binding to promoters upstream of and Expression data and structural modeling suggested that BrpT might be less dependent on c-di-GMP binding for activity than VpsT or CpsQ. We show that the affinity of BrpT for c-di-GMP is low and that signal binding is not a requisite for BrpT function. Furthermore, a BrpT mutant engineered to carry a canonical WLTR motif (BrpT) bound c-di-GMP with high affinity and its activity was now c-di-GMP dependent. Conversely, introduction of the WLPR motif into VpsT suppressed its dependence on c-di-GMP for activity. This is the first demonstration of reduced dependence on signal association for regulator function within this motif family. Thus, BrpT defines a new class of VpsT-like transcriptional regulators, and the WLPR motif variant may similarly liberate the activity of other subclass members. A genome may encode nearly 100 proteins that make, break, and bind c-di-GMP, underscoring its central role in the physiology of these bacteria. The activity of the biofilm regulators VpsT of and CpsQ of is regulated by the direct binding of c-di-GMP via a novel W[F/L/M][T/S]R motif. The homolog, BrpT, bears an unusual WLPR variant and remains active at low intracellular c-di-GMP levels. This suggests that the WLPR motif may also liberate the activity of other members of this subclass. A single point mutation at the 3rd position of the motif was sufficient to moderate dependence on c-di-GMP binding for activator function, highlighting the simplicity with which complex bacterial signaling networks can be rewired.
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http://dx.doi.org/10.1128/JB.00344-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585709PMC
October 2017

Tg737 regulates epithelial-mesenchymal transition and cancer stem cell properties via a negative feedback circuit between Snail and HNF4α during liver stem cell malignant transformation.

Cancer Lett 2017 08 20;402:52-60. Epub 2017 May 20.

Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China. Electronic address:

Determining the origin of liver cancer stem cells is important for treating hepatocellular carcinoma. Tg737 deficiency plays an important role in the malignant transformation of liver stem cells, but the underlying mechanism remains unclear. Here we established a chemical-induced mouse hepatoma model and found that Tg737 and hepatocyte nuclear factor 4-alpha (HNF4α) expression decreased and epithelial-mesenchymal transition (EMT)-related marker expression increased during liver cancer development. To investigate the underlying mechanism, we knocked down Tg737 in WB-F344 (WB) rat hepatic oval cells. Loss of Tg737 resulted in nuclear β-catenin accumulation and activation of the Wnt/β-catenin pathway, which further promoted EMT and the malignant phenotype. XAV939, a β-catenin inhibitor, attenuated WB cell malignant transformation due to Tg737 knockdown. To clarify the relationships of Tg737, the β-catenin pathway, and HNF4α, we inhibited Snail and overexpressed HNF4α after Tg737 knockdown in WB cells and found that Snail and HNF4α comprise a negative feedback circuit. Taken together, the results showed that Tg737 regulates a Wnt/β-catenin/Snail-HNF4α negative feedback circuit, thereby blocking EMT and the malignant transformation of liver stem cells to liver cancer stem cells.
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http://dx.doi.org/10.1016/j.canlet.2017.05.005DOI Listing
August 2017

Loss of exosomal miR-320a from cancer-associated fibroblasts contributes to HCC proliferation and metastasis.

Cancer Lett 2017 07 10;397:33-42. Epub 2017 Mar 10.

Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, 710032, People's Republic of China. Electronic address:

Cancer-associated fibroblasts (CAFs) play a pivotal role in regulating tumour progression. Therefore, understanding how CAFs communicate with hepatocellular carcinoma (HCC) is crucial for HCC therapy. Recently, exosomes have been considered an important "messenger" between cells. In this study, we performed microRNA (miRNA) sequencing of exosomes derived from CAFs and corresponding para-cancer fibroblasts (PAFs) of HCC patients. We found a significant reduction in the miR-320a level in CAF-derived exosomes. Using exogenous miRNAs, we demonstrated that stromal cells could transfer miRNA to HCC cells. In vitro and in vivo studies further revealed that miR-320a could function as an antitumour miRNA by binding to its direct downstream target PBX3 to suppress HCC cell proliferation, migration and metastasis. The miR-320a-PBX3 pathway inhibited tumour progression by suppressing the activation of the MAPK pathway, which could induce the epithelial-mesenchymal transition and upregulate cyclin-dependent kinase 2 (CDK2) and MMP2 expression to promote cell proliferation and metastasis. In xenograft experiments involving CAFs mixed with MHCC97-H cells, miR-320a overexpression in CAFs could inhibit tumourigenesis. Therefore, these data suggest that CAF-mediated HCC tumour progression is partially related to the loss of antitumour miR-320a in the exosomes of CAFs and that promoting the transfer of stromal cell-derived miR-320a might be a potential treatment option to overcome HCC progression.
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http://dx.doi.org/10.1016/j.canlet.2017.03.004DOI Listing
July 2017

FOXM1 regulates glycolysis in hepatocellular carcinoma by transactivating glucose transporter 1 expression.

Oncol Rep 2017 Apr 22;37(4):2261-2269. Epub 2017 Feb 22.

Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.

The Forkhead box M1 (FOXM1) transcription factor plays crucial roles in the initiation and progression of various malignancies, including hepatocellular carcinoma (HCC). However, the mechanism by which FOXM1 regulates cancer metabolism remains unclear. In the present study, overexpression and RNA interference (RNAi) approaches were used to investigate the role of FOXM1 in the regulation of glycolysis in vitro. Luciferase reporter assays were used to explore the transcriptional regulation of the glucose transporter 1 (GLUT1) promoter by FOXM1. Then, immunohistochemical staining was used to examine the expression of FOXM1 and GLUT1 in 100 paired HCC and adjacent non-cancerous liver tissues. Chi-square test and logistic regression analysis were performed to evaluate the association between FOXM1 and GLUT1 expression with clinicopathological characteristics. Our data showed that FOXM1 promoted glycolysis in the HCC cells. FOXM1 knockdown significantly reduced the expression of GLUT1 among key glycolysis-related molecules in the different HCC cell lines. Glucose uptake and lactate production assay showed that FOXM1 positively regulated glycolysis based on GLUT1 expression. Moreover, FOXM1 overexpression increased and knockdown decreased GLUT1 expression. Luciferase reporter assays showed that the -206 to -199 bp region of the GLUT1 promoter is important for FOXM1 to enhance GLUT1 promoter activity. The results of the IHC analysis showed that the protein expression of FOXM1 and GLUT1 was closely related to the tumor histological grade and TNM stage. In addition, GLUT1 expression was also related to microvascular invasion. In conclusion, overexpression of FOXM1 and GLUT1 may play critical roles in HCC. FOXM1 promotes HCC glycolysis by transactivating GLUT1 expression.
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http://dx.doi.org/10.3892/or.2017.5472DOI Listing
April 2017

MicroRNA-200a Suppresses Cell Invasion and Migration by Directly Targeting GAB1 in Hepatocellular Carcinoma.

Oncol Res 2017 01;25(1):1-10

MicroRNA-200a (miR-200a) is frequently downregulated in most cancer types and plays an important role in carcinogenesis and cancer progression. In this study, we determined that miR-200a was downregulated in hepatocellular carcinoma (HCC) tissues and cell lines, consistent with the results of our previous study. Because a previous study suggested that downregulation of miR-200a is correlated with HCC metastasis, we aimed to elucidate the mechanism underlying the role of miR-200a in metastasis in HCC. Here we observed that overexpression of miR-200a resulted in suppression of HCC metastatic ability, including HCC cell migration, invasion, and metastasis, in vitro and in vivo. Furthermore, bioinformatics and luciferase reporter assays indicated that GAB1 is a direct target of miR-200a. Inhibition of GAB1 resulted in substantially decreased cell invasion and migration similar to that observed with overexpression of miR-200a in HCC cell lines, whereas restoration of GAB1 partially rescued the inhibitory effects of miR-200a. Taken together, these data provide novel information for comprehending the tumor-suppressive role of miR-200a in HCC pathogenesis through inhibition of GAB1 translation.
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http://dx.doi.org/10.3727/096504016X14685034103798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840785PMC
January 2017

Circular RNA Expression Profile of Pancreatic Ductal Adenocarcinoma Revealed by Microarray.

Cell Physiol Biochem 2016 19;40(6):1334-1344. Epub 2016 Dec 19.

Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, China.

Background/aims: Circular RNAs (circRNAs) are a special novel type of a stable, diverse and conserved noncoding RNA in mammalian cells. Particularly in cancer, circRNAs have been reported to be widely involved in the physiological/pathological process of life. However, it is unclear whether circRNAs are specifically involved in pancreatic ductal adenocarcinoma (PDAC).

Methods: We investigated the expression profile of circRNAs in six PDAC cancer samples and paired adjacent normal tissues using microarray. A high-throughput circRNA microarray was used to identify dysregulated circular RNAs in six PDAC patients. Bioinformatic analyses were applied to study these differentially expressed circRNAs. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to confirm these results.

Results: We revealed and confirmed that a number of circRNAs were dysregulated, which suggests a potential role in pancreatic cancer.

Conclusions: this study demonstrates that clusters of circRNAs are aberrantly expressed in PDAC compared with normal samples and provides new potential targets for the future treatment of PDAC and novel insights into PDAC biology.
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http://dx.doi.org/10.1159/000453186DOI Listing
February 2017

MicroRNA-548a-5p promotes proliferation and inhibits apoptosis in hepatocellular carcinoma cells by targeting Tg737.

World J Gastroenterol 2016 Jun;22(23):5364-73

Ge Zhao, Qi-Ke Huang, Meng Pu, Wei Sun, Zhuo-Chao Zhang, Kai-Shan Tao, Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.

Aim: To investigate whether Tg737 is regulated by microRNA-548a-5p (miR-548a-5p), and correlates with hepatocellular carcinoma (HCC) cell proliferation and apoptosis.

Methods: Assays of loss of function of Tg737 were performed by the colony formation assay, CCK assay and cell cycle assay in HCC cell lines. The interaction between miR-548a-5p and its downstream target, Tg737, was evaluated by a dual-luciferase reporter assay and quantitative real-time polymerase chain reaction. Tg737 was then up-regulated in HCC cells to evaluate its effect on miR-548a-5p regulation. HepG2 cells stably overexpressing miR-548a-5p or miR-control were also subcutaneously inoculated into nude mice to evaluate the effect of miR-548a-5p up-regulation on in vivo tumor growth. As the final step, the effect of miR-548a-5p on the apoptosis induced by cisplatin was evaluated by flow cytometry.

Results: Down-regulation of Tg737, which is a target gene of miR-548a-5p, accelerated HCC cell proliferation, and miR-548a-5p promoted HCC cell proliferation in vitro and in vivo. Like the down-regulation of Tg737, overexpression of miR-548a-5p in HCC cell lines promoted cell proliferation, increased colony forming ability and hampered cell apoptosis. In addition, miR-548a-5p overexpression increased HCC cell growth in vivo. MiR-548a-5p down-regulated Tg737 expression through direct contact with its 3' untranslated region (UTR), and miR-548a-5p expression was negatively correlated with Tg737 levels in HCC specimens. Restoring Tg737 (without the 3'UTR) significantly hampered miR-548a-5p induced cell proliferation, and rescued the miR-548a-5p induced cell proliferation inhibition and apoptosis induced by cisplatin.

Conclusion: MiR-548a-5p negatively regulates the tumor inhibitor gene Tg737 and promotes tumorigenesis in vitro and in vivo, indicating its potential as a novel therapeutic target for HCC.
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http://dx.doi.org/10.3748/wjg.v22.i23.5364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910657PMC
June 2016

Pig BMSCs Transfected with Human TFPI Combat Species Incompatibility and Regulate the Human TF Pathway in Vitro and in a Rodent Model.

Cell Physiol Biochem 2015 4;36(1):233-49. Epub 2015 May 4.

Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, People's Republic of China.

Background: The activation of tissue factor (TF) is one of the major reasons for coagulation dysregulation after pig-to-primate xenotransplantation. Tissue factor pathway inhibitor (TFPI) is the most important inhibitor of TF. Studies have demonstrated species incompatibility between pig TFPI and human TF.

Methods: A pig-to-macaque heterotopic auxiliary liver transplantation model was established to determine the origin of activated TF. Chimeric proteins of human and pig TFPI were constructed to assess the role of Kunitz domains in species incompatibility. Immortalised pig bone marrow mesenchymal stem cells transfected with human TFPI were tested for their ability to inhibit clotting in vitro.

Results: TF from recipient was activated early after liver xenotransplantation. Pig TFPI Kunitz domain 2 bound human FXa, but Kunitz domain 1 did not effectively inhibit human TF/FVIIa. Immortalised pig bone marrow mesenchymal cells (BMSCs) transfected with human TFPI showed a prolonged recalcification time in vitro and in a rodent model.

Conclusion: Recipient TF is relevant to dysregulated coagulation after xenotransplantation. Kunitz domain 1 plays the most important role in species incompatibility between pig TFPI and human TF, and clotting can be inhibited by human TFPI-transfected pig BMSCs. Our study shows a possible way to resolve the incompatibility of pig TFPI.
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http://dx.doi.org/10.1159/000374067DOI Listing
February 2016
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