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1.  Intramacrophage Infection Reinforces the Virulence of Edwardsiella tarda 
Journal of Bacteriology  2016;198(10):1534-1542.
ABSTRACT
Edwardsiella tarda is an important pathogenic bacterium that can replicate in macrophages. However, how the intramacrophage infection process affects the virulence of this bacterium is essentially unknown. Here, we show that E. tarda replicates and induces a caspase-1-dependent cell pyroptosis in a murine macrophage model. Via pyroptosis, intracellular E. tarda escapes to the extracellular milieu, forming a unique bacterial population. Being different from the bacteria cultured alone, this unique population possesses a reprogrammed transcriptional profile, particularly with upregulated type III secretion system (T3SS)/T6SS cluster genes. Subsequent studies revealed that the macrophage-released population gains enhanced infectivity for host epithelial cells and increases resistance to multiple host defenses and hence displays significantly promoted virulence in vivo. Further studies indicated that T3SS is essentially required for the macrophage infection process, while T6SS contributes to infection-induced bacterial virulence. Altogether, this work demonstrates that E. tarda can utilize macrophages as a niche for virulence priming and for spreading infection, suggesting a positive role for intramacrophage infection in bacterial pathogenesis.
IMPORTANCE Many pathogens can replicate in macrophages, which is crucial for their pathogenesis. To survive in the macrophage cell, pathogens are likely to require fitness genes to counteract multiple host-killing mechanisms. Here, Edwardsiella tarda is proved to exit from macrophages during infection. This macrophage-released population displays a reprogrammed transcriptional profile with significantly upregulated type III secretion system (T3SS)/T6SS-related genes. Furthermore, both enhanced infectivity in epithelial cells and activated resistance to complex host defenses were conferred on this macrophage-primed population, which consequently promoted the full virulence of E. tarda in vivo. Our work provides evidence that E. tarda can utilize macrophages as a niche for virulence priming and for spreading infection, highlighting the importance of the intramacrophage infection cycle for the pathogenesis of E. tarda.
doi:10.1128/JB.00978-15
PMCID: PMC4859603  PMID: 26953340
2.  Hyaluronic acid and oxidized regenerated cellulose prevent adhesion reformation after adhesiolysis in rat models 
Postsurgical adhesion formation is the most common complication in abdominal and pelvic surgery. Adhesiolysis is the most commonly applied treatment for adhesion formation but is often followed by adhesion reformation. Therefore, an efficient strategy should be adopted to solve these problems. This study aimed to explore whether hyaluronic acid and oxidized regenerated cellulose (ORC) could prevent adhesion formation and reformation. Thirty female Sprague Dawley rats were randomly divided into three groups (n=10 each) and subjected to different treatments during the first and second surgery. The control group was treated with isotonic sodium chloride, the ORC group was treated with ORC (1.5×1 cm), and the medical sodium hyaluronate (MSH) group was treated with 1% MSH (0.5 mL). At 2 weeks after the first surgery, adhesion scores in the MSH group (1.90±0.99) and the ORC group (1.40±0.97) were significantly lower than those in the control group (3.00±0.82) (P=0.005). Similarly, 2 weeks after the second surgery, adhesion scores in the MSH group (2.00±0.82) and the ORC group (1.50±1.27) were significantly lower than those in the control group (3.50±0.53) (P=0.001). In addition, body weights in the MSH group and the ORC group did not change significantly, whereas the control group showed a consistent decrease in body weight during the experiment. Histological examination revealed that inflammatory infiltration was involved in both adhesion formation and reformation. In conclusion, hyaluronic acid and ORC were both efficient in reducing adhesion formation and reformation in the rat model.
doi:10.2147/DDDT.S103824
PMCID: PMC5087760  PMID: 27822014
hyaluronic acid; oxidized regenerated cellulose; adhesion formation; adhesion reformation; rat model
3.  sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance 
Nature  2016;532(7598):250-254.
Cancer is a disease of aging, and aged cancer patients have a poorer prognosis. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumor progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression1–4 we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. We find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signaling cascade in melanoma cells that results in a decrease in β-catenin and MITF, and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to ROS-induced DNA damage, rendering them more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumor progression, offering new paradigms for the design of therapy for the elderly.
doi:10.1038/nature17392
PMCID: PMC4833579  PMID: 27042933
4.  Knockdown of poc1b causes abnormal photoreceptor sensory cilium and vision impairment in zebrafish 
Proteomic analysis of the mouse photoreceptor sensory cilium identified a set of cilia proteins, including Poc1 centriolar protein b (Poc1b). Previous functional studies in human cells and zebrafish embryos implicated that Poc1b plays important roles in centriole duplication and length control, as well as ciliogenesis. To study the function of Poc1b in photoreceptor sensory cilia and other primary cilia, we expressed a tagged recombinant Poc1b protein in cultured renal epithelial cells and rat retina. Poc1b was localized to the centrioles and spindle bundles during cell cycle progression, and to the basal body of photoreceptor sensory cilia. A morpholino knockdown and complementation assay of poc1b in zebrafish showed that loss of poc1b led to a range of morphological anomalies of cilia commonly associated with human ciliopathies. In the retina, the development of retinal laminae was significantly delayed and the length of photoreceptor outer segments was shortened. Visual behavior studies revealed impaired visual function in the poc1b morphants. In addition, ciliopathy-associated developmental defects, such as small eyes, curved body axis, heart defects, and shortened cilia in Kupffer's vesicle, were observed as well. These data suggest that poc1b is required for normal development and ciliogenesis of retinal photoreceptor sensory cilia and other cilia. Furthermore, this conclusion is supported by recent findings that mutations in POC1B gene have been identified in patients with inherited retinal dystrophy and syndromic retinal ciliopathy.
doi:10.1016/j.bbrc.2015.06.083
PMCID: PMC4601574  PMID: 26188096
poc1b; Photoreceptor sensory cilia (PSC); Basal body; Ciliopathy; Zebrafish; Inherited retinal degenerations (IRDs)
5.  Three-dimensional bulk electronic structure of the Kondo lattice CeIn3 revealed by photoemission 
Scientific Reports  2016;6:33613.
We show the three-dimensional electronic structure of the Kondo lattice CeIn3 using soft x-ray angle resolved photoemission spectroscopy in the paramagnetic state. For the first time, we have directly observed the three-dimensional topology of the Fermi surface of CeIn3 by photoemission. The Fermi surface has a complicated hole pocket centred at the Γ-Z line and an elliptical electron pocket centred at the R point of the Brillouin zone. Polarization and photon-energy dependent photoemission results both indicate the nearly localized nature of the 4f electrons in CeIn3, consistent with the theoretical prediction by means of the combination of density functional theory and single-site dynamical mean-field theory. Those results illustrate that the f electrons of CeIn3, which is the parent material of CeMIn5 compounds, are closer to the localized description than the layered CeMIn5 compounds.
doi:10.1038/srep33613
PMCID: PMC5027528  PMID: 27641364
6.  Culex pipiens quinquefasciatus: a potential vector to transmit Zika virus 
Zika virus (ZIKV) has become a threat to global health since the outbreak in Brazil in 2015. Although ZIKV is generally considered an Aedes-transmitted pathogen, new evidence has shown that parts of the virus closely resemble Culex-transmitted viruses. Therefore, it is important to evaluate the competence of Culex species for ZIKV to understand their potential as vectors. In this study, female Culex pipiens quinquefasciatus were orally exposed to ZIKV. Mosquito midguts, salivary glands and ovaries were tested for ZIKV to measure infection and dissemination at 2, 4, 6, 8, 12, 16 and 18 days post exposure (pe). In addition, saliva was collected from mosquitoes after infection and infant mice were bitten by infected mosquitoes to measure the transmission ability of Cx. p. quinquefasciatus. The results showed that the peak time of virus appearance in the salivary glands was day 8 pe, with 90% infection rate and an estimated virus titer of 3.92±0.49 lg RNA copies/mL. Eight of the nine infant mice had positive brains after being bitten by infected mosquitoes, which meant that Cx. p. quinquefasciatus could be infected with and transmit ZIKV following oral infection. These laboratory results clearly demonstrate the potential role of Cx. p. quinquefasciatus as a vector of ZIKV in China. Because there are quite different vector management strategies required to control Aedes (Stegomyia) species and Cx. p. quinquefasciatus, an integrated approach may be required should a Zika epidemic occur.
doi:10.1038/emi.2016.102
PMCID: PMC5113053  PMID: 27599470
Culex pipiens quinquefasciatus; transmission; vector competence; Zika virus (ZIKV)
7.  Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors 
The Journal of Clinical Investigation  null;126(5):1834-1856.
Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi.
doi:10.1172/JCI82661
PMCID: PMC4855947  PMID: 27043285
8.  Molecular characterization of woodchuck IFI16 and AIM2 and their expression in woodchucks infected with woodchuck hepatitis virus (WHV) 
Scientific Reports  2016;6:28776.
IFI16 and AIM2 are important DNA sensors in antiviral immunity. To characterize these two molecules in a woodchuck model, which is widely used to study hepatitis B virus (HBV) infection, we cloned and analyzed the complete coding sequences (CDSs) of woodchuck IFI16 and AIM2, and found that AIM2 was highly conserved in mammals, whereas the degree of sequence identity between woodchuck IFI16 and its mammalian orthologues was low. IFI16 and IFN-β were upregulated following VACV ds 70 mer transfection, while AIM2 and IL-1β were upregulated following poly (dA:dT) transfection, both in vitro and in vivo; IFI16-targeted siRNA decreased the transcription of IFI16 and IFN-β stimulated by VACV ds 70 mer, and AIM2 siRNA interference downregulated AIM2 and IL-1β transcripts stimulated by poly (dA:dT), in vitro, suggesting that woodchuck IFI16 and AIM2 may play pivotal roles in the DNA-mediated induction of IFN-β and IL-1β, respectively. IFI16 and AIM2 transcripts were upregulated in the liver and spleen following acute WHV infection, while IFI16 was downregulated in the liver following chronic infection, implying that IFI16 and AIM2 may be involved in WHV infection. These data provide the basis for the study of IFI16- and AIM2-mediated innate immunity using the woodchuck model.
doi:10.1038/srep28776
PMCID: PMC4926060  PMID: 27354260
9.  The P72R polymorphism of p53 predisposes to obesity and metabolic dysfunction 
Cell reports  2016;14(10):2413-2425.
SUMMARY
p53 is well known for its tumor suppressor role, but this protein also has a poorly understood role in the regulation of metabolism. Human studies have implicated a common polymorphism at codon 72 of p53 in diabetic and pre-diabetic phenotypes. To understand this role, we utilized a humanized mouse model of the p53 codon 72 variants and monitored these mice following challenge with a high fat diet (HFD). Mice with the arginine 72 (R72) variant of p53 developed more severe obesity and glucose intolerance on a HFD, compared to mice with the proline 72 variant (P72). R72 mice developed insulin resistance, islet hypertrophy, increased infiltration of immune cells, and fatty liver disease. Gene expression analyses and studies with small molecule inhibitors indicate that the p53 target genes Tnf and Npc1l1 underlie this phenotype. These results shed light on the role of p53 in obesity, metabolism and inflammation.
doi:10.1016/j.celrep.2016.02.037
PMCID: PMC4926645  PMID: 26947067
p53; obesity; diabetes; islet hypertrophy; NAFLD; lipid metabolism; inflammation; Pck1; Ccl2; Tnf; Npc1l1
10.  Differential Expression of protocadherin-19, protocadherin-17 and cadherin-6 in Adult Zebrafish Brain 
The Journal of comparative neurology  2015;523(9):1419-1442.
Cell adhesion molecule cadherins play important roles in both development and maintenance of adult structures. Most studies on cadherin expression have been carried out in developing organisms, but information on cadherin distribution in adult vertebrate brains is limited. In this study, we used in situ hybridization to examine mRNA expression of three cadherins, protocadherin-19, protocadherin-17 and cadherin-6 in adult zebrafish brain. Each cadherin exhibits a distinct expression pattern in the fish brain, with protocadherin-19 and protocadherin-17 showing much wider and stronger expression than that of cadherin-6. Both protocadherin-19 and protocadherin-17 expressing cells occur throughout the brain with strong expression in the ventromedial telencephalon, periventricular regions of the thalamus and anterior hypothalamus, stratum periventriculare of the optic tectum, dorsal tegmental nucleus, granular regions of the cerebellar body and valvula, and superficial layers of the facial and vagal lobes. Numerous sensory structures (e.g. auditory, gustatory, lateral line, olfactory and visual nuclei) and motor nuclei (e.g. oculomotor, trochlear, trigeminal motor, abducens and vagal motor nuclei) contain protocadherin-19 and/or protocadherin-17 expressing cell. Expression of these two protocadherins is similar in the ventromedial telencephalon, thalamus, hypothalamus, facial and vagal lobes, but substantially different in the dorsolateral telencephalon, intermediate layers of the optic tectum, and cerebellar valvula. In contrast to the two protocadherins, cadherin-6 expression is much weaker and limited in the adult fish brain.
doi:10.1002/cne.23746
PMCID: PMC4412781  PMID: 25612302
cell adhesion molecules; cerebellum; motor nuclei; sensory systems; RRID: 2DB-GENO-030619-2; RRID: AB_514497; Roche Cat. # 11093274910; RRID: AB_193290; RRID: XM_684743; RRID: BC_129243
11.  Critical Buckling Pressure in Mouse Carotid Arteries with Altered Elastic Fibers 
Arteries can buckle axially under applied critical buckling pressure due to a mechanical instability. Buckling can cause arterial tortuosity leading to flow irregularities and stroke. Genetic mutations in elastic fiber proteins are associated with arterial tortuosity in humans and mice, and may be the result of alterations in critical buckling pressure. Hence, the objective of this study is to investigate how genetic defects in elastic fibers affect buckling pressure. We use mouse models of human disease with reduced amounts of elastin (Eln+/−) and with defects in elastic fiber assembly due to the absence of fibulin-5 (Fbln5−/−). We find that Eln+/− arteries have reduced buckling pressure compared to their wild-type controls. Fbln5−/− arteries have similar buckling pressure to wild-type at low axial stretch, but increased buckling pressure at high stretch. We fit material parameters to mechanical test data for Eln+/−, Fbln5−/− and wild-type arteries using Fung and four-fiber strain energy functions. Fitted parameters are used to predict theoretical buckling pressure based on equilibrium of an inflated, buckled, thick-walled cylinder. In general, the theoretical predictions underestimate the buckling pressure at low axial stretch and overestimate the buckling pressure at high stretch. The theoretical predictions with both models replicate the increased buckling pressure at high stretch for Fbln5−/− arteries, but the four-fiber model predictions best match the experimental trends in buckling pressure changes with axial stretch. This study provides experimental and theoretical methods for further investigating the influence of genetic mutations in elastic fibers on buckling behavior and the development of arterial tortuosity.
doi:10.1016/j.jmbbm.2015.02.013
PMCID: PMC4395556  PMID: 25771258
elastin; fibulin-5; mechanics; tortuosity
12.  Subanesthetic isoflurane relieves zymosan-induced neutrophil inflammatory response by targeting NMDA glutamate receptor and Toll-like receptor 2 signaling 
Oncotarget  2016;7(22):31772-31789.
Neutrophil release of NO/ONOO− induces endothelial cell barrier dysfunction in inflammatory acute lung injury (ALI). Previous studies using zymosan-triggered inflammation and ALI model revealed that zymosan promotes inducible NO synthase (iNOS) expression in neutrophils, and that isoflurane inhibits zymosan-induced oxidative stress and iNOS biosynthesis. However, the underlying mechanisms remain largely unknown. We found here that in zymosan-primed neutrophils, iNOS is transcriptionally activated by NF-κB, whose nuclear translocation is triggered by excessive reactive oxygen species (ROS) and consequently activated p38 MAPK. ROS production is attributed to zymosan-initiated Toll-like receptor 2 (TLR2) signaling, in which the adaptor MyD88 recruits and activates c-Src, and c-Src activates NADPH oxidase to generate ROS. Subanesthetic isoflurane counteracts the aforementioned zymosan-induced signaling by targeting N-methyl-D-aspartic acid (NMDA) glutamate receptor and thereby suppressing calcium influx and c-Src activation. Whereas iNOS accelerates NO/ONOO− production in neutrophils which eventually promote protein leak from pulmonary microvascular endothelial cells (PMVEC), isoflurane reduced NO/ONOO− release from zymosan-treated neutrophils, and thus relieves trans- PMVEC protein leak. This study provides novel insights into the roles of neutrophils and the underlying mechanisms in zymosan-induced ALI, and has implications for the therapeutic potential of subanesthetic isoflurane in attenuating inflammatory responses causing lung endothelial cell damage.
doi:10.18632/oncotarget.9091
PMCID: PMC5077975  PMID: 27144523
isoflurane; zymosan; neutrophil; NMDA receptor; TLR2; Immunology and Microbiology Section; Immune response; Immunity
13.  Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma 
Oncotarget  2016;7(21):31191-31203.
Ubiquitin-specific protease 22 (USP22) aberrance has been implicated in several malignancies; however, whether USP22 plays a role in anaplastic thyroid carcinoma (ATC) remains unclear. Here, we report that USP22 expression is highly elevated in ATC tissues, which positively correlated with tumor size, extracapsular invasion, clinical stages, and poor prognosis of ATC patients. In vitro assays showed that USP22 depletion suppressed ATC cell survival and proliferation by decreasing Rb phosphorylation and cyclin D2, inactivating Akt, and simultaneously upregulating Rb; USP22 silencing restrained cell migration and invasion by inhibiting epithelial-mesenchymal transition; USP22 knockdown promoted mitochondrion- mediated and caspase-dependent apoptosis by upregulating Bax and Bid and promoting caspase-3 activation. Consistent with in vitro findings, downregulation of USP22 in ATC cells impeded tumor growth and lung metastasis in vivo. These results raise the applicability for USP22 as a useful predictor of ATC prognosis and a potential therapeutic target for ATC.
doi:10.18632/oncotarget.9098
PMCID: PMC5058749  PMID: 27145278
anaplastic thyroid carcinoma; ubiquitin-specific protease 22; proliferation; invasion; apoptosis
14.  Caspase-11 requires the pannexin-1 channel and the purinergic P2X7 pore to mediate pyroptosis and endotoxic shock 
Immunity  2015;43(5):923-932.
SUMMARY
The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel and ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by LPS transfection or treatment with cholera toxin B and LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11−/−, Panx1−/− or P2x7−/− mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome.
doi:10.1016/j.immuni.2015.10.009
PMCID: PMC4795157  PMID: 26572062
15.  Targeted T cell Therapy in Stage IV Breast Cancer: A Phase I Clinical Trial 
PURPOSE
This study reports a phase I immunotherapy (IT) trial in 23 women with metastatic breast cancer consisting of eight infusions of anti-CD3 × anti-HER2 bispecific antibody (HER2Bi) armed anti-CD3 activated T cells (ATC) in combination with low dose interleukin 2 (IL-2) and granulocyte-macrophage-colony stimulating factor to determine safety, maximum tolerated dose (MTD), technical feasibility, T cell trafficking, immune responses, time to progression, and overall survival (OS).
EXPERIMENTAL DESIGN
ATC were expanded from leukapheresis product using IL-2 and anti-CD3 monoclonal antibody and armed with HER2Bi. In 3+3 dose escalation design, groups of 3 patients received 5, 10, 20, or 40 × 109 armed ATC (aATC) per infusion.
RESULTS
There were no dose limiting toxicities and the MTD was not defined. It was technically feasible to grow 160 × 109 ATC from a single leukapheresis. aATC persisted in the blood for weeks and trafficked to tumors. Infusions of aATC induced anti-breast cancer responses and increases in immunokines. At 14.5 weeks after enrollment, 13 of 22 (59.1%) evaluable patients had stable disease and 9 of 22 (40.9%) had progressive disease. The median OS was 36.2 months for all patients, 57.4 months for HER2 3+ patients, and 27.4 months for HER2 0–2+ patients.
CONCLUSIONS
Targeting HER2 positive and negative tumors with aATC infusions induced anti-tumor responses, increases in Th1 cytokines and IL-12 serum levels that suggest that aATC infusions vaccinated patients against their own tumors. These results provide a strong rationale for conducting phase II trials.
doi:10.1158/1078-0432.CCR-14-2280
PMCID: PMC4433762  PMID: 25688159
Bispecific antibody; activated T cells; Immunotherapy; Stage IV Breast Cancer
16.  MicroRNA-146a inhibits cell migration and invasion by targeting RhoA in breast cancer 
Oncology Reports  2016;36(1):189-196.
MicroRNAs (miRNAs) function as genetic modulators that regulate gene expression and are involved in a wide range of biological roles, including tumor cell migration and invasion. In the present study, we demonstrated that the migration and invasion activity in MDA-MB-231 breast cancer cells could be directly influenced by altering miR-146a expression. The expression of RhoA and miR-146a in the breast cancer cells showed an inverse correlation. Upregulation of miR-146a in the MDA-MB-231 breast cancer cells by transfection of miR-146a mimics resulted in decreased RhoA protein levels. Conversely, downregulation of miR-146a by transfection of miR-146a inhibitor resulted in increased RhoA protein levels. To confirm the fact that RhoA is a potential target of miR-146a, luciferase reporter containing the RhoA 3′ untranslated region (3′UTR) was constructed. The results demonstrated that the luciferase reporter activity was reduced after overexpression of miR-146a. Moreover, the luciferase reporter which was constructed with the RhoA 3′UTR mutant did not show significantly altered luciferase reporter activity. Furthermore, after treatment with the RhoA inhibitor exoenzyme C3 transferase protein, the migratory capacity of the MDA-MB-231 cells was not significantly altered even though the amount of miR-146a was changed. Our results indicate that miR-146a functions as a tumor suppressor in breast cancer cells. Downregulation of the expression of miR-146a increased the migration of MDA-MB-231 cells, due to the upregulation of RhoA expression.
doi:10.3892/or.2016.4788
PMCID: PMC4899025  PMID: 27175941
microRNA; miR-146a; breast cancer; migration; RhoA
17.  Are tuberculosis patients adherent to prescribed treatments in China? Results of a prospective cohort study 
Background
Tuberculosis (TB) patients face numerous difficulties adhering to the long-term, rigorous TB treatment regimen. Findings on TB patients’ treatment adherence vary across existing literature and official reports. The present study attempted to determine the actual treatment adherence of new TB patients and to identify factors leading to non-adherence.
Methods
A prospective cohort of 481 newly confirmed TB patients from three counties in western China were enrolled during June to December 2012 and was followed until June 2013. Patients who missed at least one dose of drugs or one follow-up re-examination during the treatment course were deemed as non-adherent. Influencing factors were identified using a logistic regression model.
Results
A total of 173 (36.0 %) patients experienced non-adherence and the loss to follow-up cases reached 136 (28.2 %). Only 13.9 % of patients took drugs under direct observation, and 60.5 % of patients were supervised by phone calls. Factor analyses suggested that patients who were observed by family members (OR:5.54, 95 % CI:2.87–10.69) and paying monthly service expenses above 450 RMB (OR:2.08, 95 % CI:1.35–3.19) were more likely to be non-adherent, while supervision by home visit (OR:0.06, 95 % CI:0.01–0.28) and phone calls (OR:0.27, 95 % CI:0.17–0.44) were protective factors.
Conclusions
Despite recent efforts, a large proportion of newly confirmed TB patients could not adhere to standard TB treatment, and patients’ lost to follow-up was still a serious problem. Poor treatment supervision and heavy financial burden might be the main causes for non-adherence. More needs to be done to enhance treatment supervision policies and financial supports to both health providers and TB patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s40249-016-0134-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s40249-016-0134-9
PMCID: PMC4857377  PMID: 27146470
Tuberculosis; Adherence; Prospective cohort study; China
18.  Enhanced itch elicited by capsaicin in a chronic itch model 
Molecular Pain  2016;12:1744806916645349.
Chronic itch (pruritus) is an important clinical problem. However, the underlying molecular basis has yet to be understood. The Transient Receptor Potential Vanilloid 1 channel is a heat-sensitive cation channel expressed in primary sensory neurons and involved in both thermosensation and pain, but its role in chronic itch remains elusive. Here, we for the first time revealed an increased innervation density of Transient Receptor Potential Vanilloid 1-expressing sensory fibers in the skin afflicted with chronic itch. Further analysis indicated that this phenomenon is due to an expansion of Transient Receptor Potential Vanilloid 1-expressing sensory neurons under chronic itch conditions. As a functional correlates of this neuronal expansion, we observed an enhanced neuronal responsiveness to capsaicin under the dry skin conditions. Importantly, the neuronal hypersensitivity to capsaicin results in itch, rather than pain sensation, suggesting that the up-regulated Transient Receptor Potential Vanilloid 1 underlies the pain-to-itch switch under chronic itchy conditions. The study shows that there are different mechanisms of chronic pain and itching, and Transient Receptor Potential Vanilloid 1 plays an important role in chronic itch.
doi:10.1177/1744806916645349
PMCID: PMC4956172  PMID: 27118771
Chronic itch; pain; Transient Receptor Potential Vanilloid 1; calcium imaging
19.  BRAF Inhibition Stimulates Melanoma-Associated Macrophages to Drive Tumor Growth 
Purpose
To investigate the roles of melanoma-associated macrophages in melanoma resistance to BRAF inhibitors (BRAFi).
Experimental Design
An in vitro macrophage and melanoma cell co-culture system was used to investigate whether macrophages play a role in melanoma resistance to BRAFi. The effects of macrophages in tumor resistance were examined by proliferation assay, cell death assay and western blot analyses. Furthermore, two mouse preclinical models were used to validate whether targeting macrophages can increase the anti-tumor activity of BRAFi. Finally, the number of macrophages in melanoma tissues was examined by immunohistochemistry.
Results
We demonstrate that in BRAF mutant melanomas, BRAFi paradoxically activate the MAPK pathway in macrophages to produce VEGF, which reactivates the MAPK pathway and stimulates cell growth in melanoma cells. Blocking the MAPK pathway or VEGF signaling, then reverses macrophage-mediated resistance. Targeting macrophages increases the anti-tumor activity of BRAFi in mouse and human tumor models. The presence of macrophages in melanomas predicts early relapse after therapy.
Conclusions
Our findings demonstrate that macrophages play a critical role in melanoma resistance to BRAFi, suggesting that targeting macrophages will benefit patients with BRAF mutant melanoma.
doi:10.1158/1078-0432.CCR-14-1554
PMCID: PMC4383683  PMID: 25617424
20.  Panel-based Genetic Diagnostic Testing for Inherited Eye Diseases is Highly Accurate and Reproducible and More Sensitive for Variant Detection Than Exome Sequencing 
Purpose
Next-generation sequencing (NGS) based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques have not been fully defined with regard to test accuracy and reproducibility.
Methods
We developed a targeted enrichment and NGS approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy and glaucoma. In preparation for providing this Genetic Eye Disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, reproducibility as well as the clinical sensitivity of the test.
Results
The GEDi test is highly reproducible and accurate, with sensitivity and specificity for single nucleotide variant detection of 97.9% and 100%, respectively. The sensitivity for variant detection was notably better than the 88.3% achieved by whole exome sequencing (WES) using the same metrics, due to better coverage of targeted genes in the GEDi test compared to commercially available exome capture sets. Prospective testing of 192 patients with IRDs indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%.
Conclusion
The data suggest that based on quantified performance metrics, selective targeted enrichment is preferable to WES for genetic diagnostic testing.
doi:10.1038/gim.2014.172
PMCID: PMC4572572  PMID: 25412400
Genetic diagnostic testing; next-generation sequencing; sensitivity; specificity; reproducibility
21.  Resilience in rural left-behind middle school students in Yunyang county of the Three Gorges area in China: a prospective cohort study 
BMC Psychiatry  2016;16:77.
Background
The number of left-behind children in China is gradually increasing. This study aimed to understand the mental health status and changes in resilience of rural, left-behind middle school students in Yunyang County of the Three Gorges area in China.
Methods
A prospective cohort study, including two follow-up surveys, with a frequency of once every 6 months was conducted among middle school students in Yunyang County. A self-designed questionnaire was used to obtain socio-demographic factors of participants, the Mental Health Test (MHT) scale was used to assess their mental health statuses, and resilience levels were collected using Resilience Youth Development Module (RYDM) scale at baseline and at the first and second follow-up investigations.
Results
Of the 1401 students who completed the baseline survey, 1322 students were eligible for the cohort study, of whom 1160 were investigated in the first follow-up survey. Ultimately, 1101 students completed the 1-year cohort. The detection rate of mental health problems for middle school students in the rural Three Gorges area was 5.64 %, and there was no significant difference between left-behind students (LBS) and non-left-behind students (NLBS) (χ2 = 1.056, P = 0.304). The detection rates of medium resilience rose gradually (Z = 4.185, P = 0.000), while that of high resilience declined gradually (Z =−4.192, P = 0.000) in the baseline, first and second follow-up investigations. There was no significant difference between LBS and NLBS in resilience level (P > 0.05). The average RYDM scores were 2.990, 2.926, 2.904 among LBS in the baseline, first and second follow-up investigation, respectively, and the effect of time on the average RYDM scores was significant (F = 14.873, P = 0.000). The average MHT scores in LBS were 41.54, 39.79, 38.84 in the baseline, first and second follow-up investigations, respectively, and the detection rates of students who had psychological problems increased gradually (Z = 4.651, P = 0.000). The simple correlation coefficients between the RYDM and MHT scores were−0.227,−0.158, and−0.204 in the baseline, first and second follow-up survey, respectively (P = 0.000).
Conclusions
The detection rate of mental health problems for middle school students in the rural Three Gorges area is relatively low and most of the LBS in area have medium or high resilience. The mental health status of LBS positively correlated with their resilience levels. Resilience declined gradually as time went on, but further studies with longer follow-up durations are needed to confirm the variation of resilience.
doi:10.1186/s12888-016-0781-1
PMCID: PMC4804612  PMID: 27004715
Resilience; Left-behind children; Middle school students
22.  Medical school curriculum characteristics associated with intentions and frequency of tobacco dependence treatment among 3rd year U.S. medical students 
Preventive medicine  2015;72:56-63.
Objective
Physicians play a critical role in addressing tobacco dependence, yet report limited training. Tobacco dependence treatment curricula for medical students could improve performance in this area. This study identified student and medical school tobacco treatment curricula characteristics associated with intentions and use of the 5As for tobacco treatment among 3rd year U.S. medical students.
Methods
Third year medical students (N=1065, 49.3% male) from 10 U.S. medical schools completed a survey in 2009-2010 assessing student characteristics, including demographics, tobacco treatment knowledge, and self-efficacy. Tobacco curricula characteristics assessed included amount and type of classroom instruction, frequency of tobacco treatment observation, instruction, and perception of preceptors as role models.
Results
Greater tobacco treatment knowledge, self-efficacy, and curriculum-specific variables were associated with 5A intentions, while younger age, tobacco treatment self-efficacy, intentions, and each curriculum-specific variable was associated with greater 5A behaviors. When controlling for important student variables, greater frequency of receiving 5A instruction (OR = 1.07; 95%CI 1.01-1.12) and perception of preceptors as excellent role models in tobacco treatment (OR = 1.35; 95%CI 1.04-1.75) were significant curriculum predictors of 5A intentions. Greater 5A instruction (B = .06 (.03); p< .05) and observation of tobacco treatment (B= .35 (.02); p< .001) were significant curriculum predictors of greater 5A behaviors.
Conclusions
Greater exposure to tobacco treatment teaching during medical school is associated with both greater intentions to use and practice tobacco 5As. Clerkship preceptors, or those physicians who provide training to medical students, may be particularly influential when they personally model and instruct students in tobacco dependence treatment.
doi:10.1016/j.ypmed.2014.12.035
PMCID: PMC4562320  PMID: 25572623
tobacco dependence treatment; medical school curriculum and education; medical students
23.  Genome of Cnaphalocrocis medinalis Granulovirus, the First Crambidae-Infecting Betabaculovirus Isolated from Rice Leaffolder to Sequenced 
PLoS ONE  2016;11(2):e0147882.
Cnaphalocrocis medinalis is a major pest of rice in South and South-East Asia. Insecticides are the major means farmers use for management. A naturally occurring baculovirus, C. medinalis granulovirus (CnmeGV), has been isolated from the larvae and this has the potential for use as microbial agent. Here, we described the complete genome sequence of CnmeGV and compared it to other baculovirus genomes. The genome of CnmeGV is 112,060 base pairs in length, has a G+C content of 35.2%. It contains 133 putative open reading frames (ORFs) of at least 150 nucleotides. A hundred and one (101) of these ORFs are homologous to other baculovirus genes including 37 baculovirus core genes. Thirty-two (32) ORFs are unique to CnmeGV with no homologues detected in the GeneBank and 53 tandem repeats (TRs) with sequence length from 25 to 551 nt intersperse throughout the genome of CnmeGV. Six (6) homologous regions (hrs) were identified interspersed throughout the genome. Hr2 contains 11 imperfect palindromes and a high content of AT sequence (about 73%). The unique ORF28 contains a coiled-coil region and a zinc finger-like domain of 4–50 residues specialized by two C2C2 zinc finger motifs that putatively bound two atoms of zinc. ORF21 encoding a chit-1 protein suggesting a horizontal gene transfer from alphabaculovirus. The putative protein presents two carbohydrate-binding module family 14 (CBM_14) domains rather than other homologues detected from betabaculovirus that only contains one chit-binding region. Gene synteny maps showed the colinearity of sequenced betabaculovirus. Phylogenetic analysis indicated that CnmeGV grouped in the betabaculovirus, with a close relation to AdorGV. The cladogram obtained in this work grouped the 17 complete GV genomes in one monophyletic clade. CnmeGV represents a new crambidae host-isolated virus species from the genus Betabaculovirus and is most closely relative of AdorGV. The analyses and information derived from this study will provide a better understanding of the pathological symptoms caused by this virus and its potential use as a microbial pesticide.
doi:10.1371/journal.pone.0147882
PMCID: PMC4746121  PMID: 26848752
24.  In Vivo Programmed Gene Expression Based on Artificial Quorum Networks 
Applied and Environmental Microbiology  2015;81(15):4984-4992.
The quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of the Vibrio fischeri luxI-luxR quorum sensing system. In order to achieve in vivo programmed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter ParaBAD, into the QS system. In vitro expression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density. In vivo expression assays confirmed that the araQS system presented an in vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applications in vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuated Edwardsiella tarda strain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expression in vivo and might have potential uses, including, but not limited to, bacterial vector vaccines.
doi:10.1128/AEM.01113-15
PMCID: PMC4495192  PMID: 25979894
25.  A new target ligand Ser–Glu for PEPT1-overexpressing cancer imaging 
Nanoparticles functionalized with active target ligands have been widely used for tumor-specific diagnosis and therapy. The target ligands include antibodies, peptides, proteins, small molecules, and nucleic acid aptamers. Here, we utilize dipeptide Ser–Glu (DIP) as a new ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for pancreatic cancer target imaging. We demonstrate that in the first step, Ser–Glu-conjugated NPs (NPs-DIP) efficiently bind to AsPC-1 and in the following NPs-DIP are internalized into AsPC-1 in vitro. The peptide transporter 1 inhibition experiment reveals that the targeting effects mainly depend on the specific binding of DIP to peptide transporter 1, which is remarkably upregulated in pancreatic cancer cells compared with varied normal cells. Furthermore, NPs-DIP specifically accumulate in the site of pancreatic tumor xenograft and are further internalized into the tumor cells in vivo after intravenous administration, indicating that DIP successfully enhanced nanoparticles internalization efficacy into tumor cells in vivo. This work establishes Ser–Glu to be a new tumor-targeting ligand and provides a promising tool for future tumor diagnostic or therapeutic applications.
doi:10.2147/IJN.S97207
PMCID: PMC4714743  PMID: 26811678
imaging; pancreatic cancer; PEPT1 transporter; Ser–Glu; target ligand

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