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1.  TOLLIP, MUC5B, and the Response to N-Acetylcysteine among Individuals with Idiopathic Pulmonary Fibrosis 
Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology. The genes TOLLIP and MUC5B play important roles in lung host defense, which is an immune process influenced by oxidative signaling. Whether polymorphisms in TOLLIP and MUC5B modify the effect of immunosuppressive and antioxidant therapy in individuals with IPF is unknown.
Objectives: To determine whether single-nucleotide polymorphisms (SNPs) within TOLLIP and MUC5B modify the effect of interventions in subjects participating in the Evaluating the Effectiveness of Prednisone, Azathioprine, and N-Acetylcysteine in Patients with Idiopathic Pulmonary Fibrosis (PANTHER-IPF) clinical trial.
Methods: SNPs within TOLLIP (rs5743890/rs5743894/rs5743854/rs3750920) and MUC5B (rs35705950) were genotyped. Interaction modeling was conducted with multivariable Cox regression followed by genotype-stratified survival analysis using a composite endpoint of death, transplantation, hospitalization, or a decline of ≥10% in FVC.
Measurements and Main Results: Significant interaction was observed between N-acetylcysteine (NAC) therapy and rs3750920 within TOLLIP (Pinteraction = 0.001). After stratifying by rs3750920 genotype, NAC therapy was associated with a significant reduction in composite endpoint risk (hazard ratio, 0.14; 95% confidence interval, 0.02–0.83; P = 0.03) in those with a TT genotype, but a nonsignificant increase in composite endpoint risk (hazard ratio, 3.23; 95% confidence interval, 0.79–13.16; P = 0.10) was seen in those with a CC genotype. These findings were then replicated in an independent IPF cohort.
Conclusions: NAC may be an efficacious therapy for individuals with IPF with an rs3750920 (TOLLIP) TT genotype, but it was associated with a trend toward harm in those with a CC genotype. A genotype-stratified prospective clinical trial should be conducted before any recommendation regarding the use of off-label NAC to treat IPF.
PMCID: PMC4731723  PMID: 26331942
IPF; pharmacogenetics; N-acetylcysteine; drug–gene interaction; host defense
2.  Functional Characterization of Triclosan-Resistant Enoyl-acyl-carrier Protein Reductase (FabV) in Pseudomonas aeruginosa 
Pseudomonas aeruginosa is extremely resistant to triclosan. Previous studies have shown that P. aeruginosa encodes a triclosan-resistant enoyl-acyl-carrier protein reductase (ENR), FabV, and that deletion of fabV causes P. aeruginosa to be extremely sensitive to triclosan. In this report, we complemented a P. aeruginosa fabV deletion strain with several triclosan-resistant ENR encoding genes, including Vibrio cholerae fabV, Bacillus subtilis fabL and Enterococcus faecalis fabK. All complemented strains restored triclosan resistance to the level of the wild-type strain, which confirmed that triclosan-resistant ENR allows P. aeruginosa to be extremely resistant to triclosan. Moreover, fabV exhibits pleiotropic effects. Deletion of fabV led P. aeruginosa to show attenuated swarming motility, decreased rhamnolipid, pyoverdine and acyl-homoserine lactones (AHLs) production. Complementation of the fabV mutant with any one ENR encoding gene could restore these features to some extent, in comparison with the wild-type strain. Furthermore, we found that addition of exogenous AHLs could restore the fabV mutant strain to swarm on semisolid plates and to produce more virulence factors than the fabV mutant strain. These findings indicate that deletion of fabV reduced the activity of ENR in P. aeruginosa, decreased fatty acid synthesis, and subsequently depressed the production of AHLs and other virulence factors, which finally may led to a reduction in the pathogenicity of P. aeruginosa. Therefore, fabV should be an ideal target for the control of P. aeruginosa infectivity.
PMCID: PMC5126088  PMID: 27965638
Pseudomonas aeruginosa; enoyl-acyl-carrier protein reductase; triclosan; acylhomoserine lactones; swarming
3.  p53 signaling modulation of cell cycle arrest and viral replication in porcine circovirus type 2 infection cells 
Veterinary Research  2016;47:120.
Porcine circovirus type 2 (PCV2) is a ubiquitous pathogen in the swine industry worldwide. Previous studies have shown that PCV2 infection induces host cell apoptosis through up-regulation of p53. To further identify the regulatory roles of p53 signaling in the process of PCV2 infection, we established p53 gene knockout PK15 cell lines using the genomic editor tool CRISPR/Cas9, and further investigated the roles of p53 in modulating the cell cycle and viral replication in this study. The results show that PCV2 infection induced obvious S phase accumulation in wild-type PK15 cells and a compromised S phase accumulation in the p53 gene mutation cells (813PK15p53m/m), but did not induce obvious S phase accumulation in the p53 gene knockout cells (148PK15p53−/−) compared with the respective mock infection. PCV2 infection activated p53 signaling, up-regulated the expression of p21, Cyclin E, and down-regulated Cyclin A, CDK2. In p53 deficient cells, however, PCV2-induced changes in Cyclin A, CDK2, and Cyclin E were efficiently reversed to the basal levels. Detection of PCV2 replication showed decreased viral ORF1 genomic DNA in p53 deficient cells (148PK15p533−/−) and p53 mutated cells (813PK15p53m/m) compared with p53 wild-type cells after different synchronization treatment. Furthermore, PCV2 viral genomic DNA and Cap protein levels were higher in the cells released from S phase synchronized cells than in the cells released from the G0/G1 phase or G2/M phase-synchronized, or asynchronous cells after 18 h post-infection. Taken together, this study demonstrates that PCV2 infection induces S phase accumulation to favor viral replication in host cells through activation of the p53 pathway.
PMCID: PMC5129207  PMID: 27899159
4.  Activation of Wnt/β-catenin Signaling Blocks Monocyte-Macrophage Differentiation Through Antagonizing PU.1-Targeted Gene Transcription 
Leukemia  2016;30(10):2106-2109.
PMCID: PMC5053841  PMID: 27211263
Acute myeloid leukemia; the Wnt/β-catenin signaling pathway; PU.1; Egr2; monocyte-macrophage differentiation
5.  Novel H7N2 and H5N6 Avian Influenza A Viruses in Sentinel Chickens: A Sentinel Chicken Surveillance Study 
In 2014, a sentinel chicken surveillance for avian influenza viruses was conducted in aquatic bird habitat near Wuxi City, Jiangsu Province, China. Two H7N2, one H5N6, and two H9N2 viruses were isolated. Sequence analysis revealed that the H7N2 virus is a novel reassortant of H7N9 and H9N2 viruses and H5N6 virus is a reassortant of H5N1 clade 2.3.4 and H6N6 viruses. Substitutions V186 and L226 (H3 numbering) in the hemagglutinin (HA) gene protein was found in two H7N2 viruses but not in the H5N6 virus. Two A138 and A160 mutations were identified in the HA gene protein of all three viruses but a P128 mutation was only observed in the H5N6 virus. A deletion of 3 and 11 amino acids in the neuraminidase stalk region was found in two H7N2 and H5N6 viruses, respectively. Moreover, a mutation of N31 in M2 protein was observed in both two H7N2 viruses. High similarity of these isolated viruses to viruses previously identified among poultry and humans, suggests that peridomestic aquatic birds may play a role in sustaining novel virus transmission. Therefore, continued surveillance is needed to monitor these avian influenza viruses in wild bird and domestic poultry that may pose a threat to poultry and human health.
PMCID: PMC5110548  PMID: 27899915
avian influenza A virus; H7N2 virus; H9N2 virus; H5N6 virus; sentinel chicken; transmission
6.  Identification of novel and differentially expressed MicroRNAs in goat enzootic nasal adenocarcinoma 
BMC Genomics  2016;17:896.
MicroRNAs (miRNAs) post-transcriptionally regulate a variety of genes involved in eukaryotic cell growth, development, metabolism and other biological processes, and numerous miRNAs are implicated in the initiation and progression of cancer. Enzootic nasal adenocarcinoma (ENA), an epithelial tumor induced in goats and sheep by enzootic nasal tumor virus (ENTV), is a chronic, progressive, contact transmitted disease.
In this work, small RNA Illumina high-throughput sequencing was used to construct a goat nasal miRNA library. This study aimed to identify novel and differentially expressed miRNAs in the tumor and para-carcinoma nasal tissues of Nanjiang yellow goats with ENA.
Four hundred six known miRNAs and 29 novel miRNAs were identified. A total of 116 miRNAs were significantly differentially expressed in para-carcinoma nasal tissues and ENA (54 downregulated; 60 upregulated; two only expressed in control group); Target gene prediction and functional analysis revealed that 6176 non-redundancy target genes, 1792 significant GO and 97 significant KEGG pathway for 121 miRNAs (116 significant expression miRNAs and five star sequence) were predicted. GO and KEGG pathway analysis revealed the majority of target genes in ENA are involved in cell proliferation, signal transduction and other processes associated with cancer.
This is the first large-scale identification of miRNAs in Capra hircus ENA and provides a theoretical basis for investigating the complicated miRNA-mediated regulatory networks involved in the pathogenesis and progression of ENA.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-3238-5) contains supplementary material, which is available to authorized users.
PMCID: PMC5101819  PMID: 27825300
MicroRNA; Enzootic nasal adenocarcinoma; Illumina high-throughput sequencing; Gene ontology; KEGG pathway
7.  Long-term survival analysis in combined transarterial embolization and stereotactic body radiation therapy versus stereotactic body radiation monotherapy for unresectable hepatocellular carcinoma >5 cm 
BMC Cancer  2016;16:834.
The survival following transarterial chemoembolization (TACE) alone is still low in unresectable hepatocellular carcinoma (HCC) with almost patients developing disease progression after treatment. There is need to investigate additional therapeutic options that would intensify the initial response to TACE. The present study was to retrospectively compare the outcome and evaluate the prognostic factors of stereotactic body radiation therapy (SBRT) alone or as an adjunct to transarterial embolization (TAE) or TACE in the treatment of HCC >5 cm.
From January 2011 to April 2015, 77 patients received SBRT followed by TAE or TACE (TAE/TACE + SBRT group) and 50 patients received SBRT alone (SBRT group). The dose of SBRT was 30–50 Gy which was prescribed in 3–5 fractions. Eligibility criteria were: a longest tumor diameter >5.0 cm and Child-Turcotte-Pugh (CTP) Class A or B. Exclusion criteria included tumor thrombus, lymph node involvement and extrahepatic metastasis.
The median follow-up period was 20.5 months. Median tumor size was 8.5 cm (range, 5.1–21.0 cm). Median overall survival (OS) in the TAE/TACE + SBRT group was 42.0 months versus 21.0 months in the SBRT group. The 1-, 3- and 5-year OS was 75.5, 50.8, and 46.9 % in the TAE/TACE + SBRT group and was 62.4, 32.9, and 32.9 % in the SBRT group, respectively (P = 0.047). The 1-, 3- and 5-year distant metastasis-free survival (DMFS) was 66.3, 44.3, and 40.6 % in the TAE/TACE + SBRT group and was 56.8, 26.1, and 17.4 % in the SBRT group, respectively (P = 0.049). The progression-free survival (PFS) and local relapse-free survival (LRFS) were not significantly different between the two groups. In the entire patient population, a biologically effective dose (BED10) ≥100 Gy and an equivalent dose in 2 Gy fractions (EQD2) ≥74 Gy were significant prognostic factors for OS, PFS, LRFS and DMFS.
SBRT combined with TAE/TACE may be an effective complementary treatment approach for HCC >5 cm in diameter. BED10 ≥100 Gy and EQD2 ≥74 Gy should receive more attention when the SBRT plan is designed.
PMCID: PMC5093984  PMID: 27809890
Hepatocellular carcinoma; HCC; TAE/TACE; Stereotactic body radiation therapy; SBRT
8.  The Kynurenine Pathway of Tryptophan Catabolism and AIDS-associated Kaposi's Sarcoma in Africa 
Other than Kaposi's sarcoma (KS)-associated herpesvirus and CD4+ T cell lymphopenia, the mechanisms responsible for KS in the context of HIV are poorly understood. One recently explored pathway of HIV pathogenesis involves induction of the enzyme indoleamine 2,3 dioxygenase-1 (IDO), which catabolizes tryptophan into kynurenine and several other immunologically active metabolites that suppress T cell proliferation. We investigated the role of IDO in the development of KS in HIV disease.
In a case-control study among untreated HIV-infected Ugandans, cases were adults with KS and controls were without KS. IDO activity was assessed by the ratio of plasma kynurenine to tryptophan levels (KT ratio), measured by liquid chromatography tandem mass spectrometry.
We studied 631 HIV-infected subjects: 222 KS cases and 409 controls. Non-KS controls had a higher median plasma KT ratio (130, IQR: 90 to190 nM/μM) than cases (110, IQR: 90 to 150 nM/μM) (p = 0.004). After adjustment for age, sex, CD4 count and plasma HIV RNA level, subjects with the highest (fourth quartile) plasma KT ratios had a 59% reduction (95% CI: 27% to 77%) in the odds of KS compared to those with the lowest (first quartile) levels. KS was also independently associated with lower CD4+ count, higher plasma HIV RNA, and men.
Among HIV-infected individuals, greater activity of the kynurenine pathway of tryptophan catabolism, as evidenced by higher levels of plasma KT ratio, was associated with lower occurrence of KS. Some consequences of immune activation in HIV infection might actually suppress certain cancers.
PMCID: PMC4607630  PMID: 26181812
tryptophan; kynurenine; indoleamine 2,3-dioxygenase-1; HIV; Kaposi's sarcoma; plasma HIV RNA; Africa
9.  Protective Effects of Hydrogen Sulfide in the Ageing Kidney 
Aims. The study aimed to examine whether hydrogen sulfide (H2S) generation changed in the kidney of the ageing mouse and its relationship with impaired kidney function. Results. H2S levels in the plasma, urine, and kidney decreased significantly in ageing mice. The expression of two known H2S-producing enzymes in kidney, cystathionine γ-lyase (CSE) and cystathionine-β-synthase (CBS), decreased significantly during ageing. Chronic H2S donor (NaHS, 50 μmol/kg/day, 10 weeks) treatment could alleviate oxidative stress levels and renal tubular interstitial collagen deposition. These protective effects may relate to transcription factor Nrf2 activation and antioxidant proteins such as HO-1, SIRT1, SOD1, and SOD2 expression upregulation in the ageing kidney after NaHS treatment. Furthermore, the expression of H2S-producing enzymes changed with exogenous H2S administration and contributed to elevated H2S levels in the ageing kidney. Conclusions. Endogenous hydrogen sulfide production in the ageing kidney is insufficient. Exogenous H2S can partially rescue ageing-related kidney dysfunction by reducing oxidative stress, decreasing collagen deposition, and enhancing Nrf2 nuclear translocation. Recovery of endogenous hydrogen sulfide production may also contribute to the beneficial effects of NaHS treatment.
PMCID: PMC5108860  PMID: 27882191
10.  Complete Genome Sequence of IME207, a Novel Bacteriophage Which Can Lyse Multidrug-Resistant Klebsiella pneumoniae and Salmonella 
Genome Announcements  2016;4(5):e01015-16.
A novel lytic Salmonella bacteriophage was isolated by using Klebsiella pneumoniae as host cells. The phage’s genome was determined to be 47,564 bp and has the highest similarity to Salmonella phage E1 and Salmonella phage 64795_sal3, with coverages of 61% and 56%, respectively. Here, we announce the phage’s complete genome.
PMCID: PMC5084854  PMID: 27789630
11.  A TolC-Like Protein of Actinobacillus pleuropneumoniae Is Involved in Antibiotic Resistance and Biofilm Formation 
Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a significant disease that causes serious economic losses to the swine industry worldwide. Persistent infections caused by bacterial biofilms are recalcitrant to treat because of the particular drug resistance of biofilm-dwelling cells. TolC, a key component of multidrug efflux pumps, are responsible for multidrug resistance (MDR) in many Gram-negative bacteria. In this study, we identified two TolC-like proteins, TolC1 and TolC2, in A. pleuropneumoniae. Deletion of tolC1, but not tolC2, caused a significant reduction in biofilm formation, as well as increased drug sensitivity of both planktonic and biofilm cells. The genetic-complementation of the tolC1 mutation restored the competent biofilm and drug resistance. Besides, biofilm formation was inhibited and drug sensitivity was increased by the addition of phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux pump inhibitor (EPI), suggesting a role for EPI in antibacterial strategies toward drug tolerance of A. pleuropneumoniae. Taken together, TolC1 is required for biofilm formation and is a part of the MDR machinery of both planktonic and biofilm cells, which could supplement therapeutic strategies for resistant bacteria and biofilm-related infections of A. pleuropneumoniae clinical isolate SC1516.
PMCID: PMC5075564  PMID: 27822201
TolC; biofilm formation; multidrug resistance; PAβN; Actinobacillus pleuropneumoniae
12.  Aptamer-based microchip electrophoresis assays for amplification detection of carcinoembryonia antigen 
Carcinoembryonic antigen (CEA) as one of the most widely used tumor marker is used in the clinical diagnosis of colorectal, pancreatic, gastric, and cervical carcinomas. We developed an aptamer-based microchip electrophoresis assay technique for assaying CEA in human serum for cancer diagnosis.
The magnetic beads (MBs) are employed as carriers of double strand DNA that is formed by an aptamer of target and a complementary DNA of aptamer. After the aptamer in MB-dsDNA conjugate binds with target, the complementary DNA was released from MB-dsDNA conjugate. The released complementary DNA hybridizes with a fluorescein amidite (FAM) labeled DNA, and forms DNA duplex, which triggers the selective cleavage of FAM labeled DNA by nicking endonuclease Nb.BbvCI, and generating FAM labeled DNA segment. The released complementary DNA hybridizes with another FAM labeled DNA, resulting in a continuous cleavage of FAM labeled DNA, and the generation of large numbers of FAM labeled DNA segments. In MCE laser induced fluorescence detection (LIF), FAM labeled DNA segment is separated and detected.
The linear range for CEA was 130 pg/ml~8.0 ng/ml with a correlation coefficient of 0.9916 and a detection limit of 68 pg/ml. The CEA concentration in the serum samples from healthy subjects was found be in the range 1.3 ng/ml to 3.2 ng/ml. The CEA concentration in the samples from cancer patients was found to be >15 ng/ml.
This method may become a useful tool for rapid analysis of CEA and other tumor markers in biomedical analysis and clinical diagnosis.
PMCID: PMC4609300  PMID: 26344338
aptamer; carcinoembryonic antigen; microchip electrophoresis; cancer
13.  Angucyclines and Angucyclinones from Streptomyces sp. CB01913 Featuring C-ring Cleavage and Expansion 
Journal of natural products  2015;78(10):2471-2480.
Angucyclines and angucyclinones are aromatic polyketides with a tetracyclic benz[a]anthracene skeleton. The benz[a]anthracene scaffold is biosynthesized by type II polyketide synthases that catalyze the decarboxylative condensation of a short acyl-CoA starter and nine extender units. Angucyclines and angucyclinones, the largest group of polycyclic aromatic polyketides, achieve structural diversity via subsequent oxidation, ring cleavage, amino acid incorporation, and glycosylation. We here report the discovery of 14 angucyclinones and two angucyclines (1–16) from Streptomyces sp. CB01913, identifying 12 new compounds featuring various oxidations on rings A and C (1, 2, and 4), different sugar moieties attached to rings A and B (3 and 6), and C-ring cleavage (5 and 10–14) and expansion (8). These new structural features, highlighted by C-ring cleavage and expansion, enrich the structural diversity of angucyclines and angucyclinones. All compounds were tested for cytotoxicity and antibacterial activities, with 1, 5, 15, and 16 showing moderate activities against selected cancer cell lines or bacterial strains.
Graphical abstract
PMCID: PMC4845661  PMID: 26335269
14.  Immunoprotective Efficacy of Six In vivo-Induced Antigens against Actinobacillus pleuropneumoniae as Potential Vaccine Candidates in Murine Model 
Six in vivo-induced (IVI) antigens—RnhB, GalU, GalT, Apl_1061, Apl_1166, and HflX were selected for a vaccine trial in a mouse model. The results showed that the IgG levels in each immune group was significantly higher than that of the negative control (P < 0.001). Except rRnhB group, proliferation of splenocytes was observed in all immunized groups and a relatively higher proliferation activity was observed in rGalU and rGalT groups (P < 0.05). In the rGalT vaccinated group, the proportion of CD4+ T cells in spleen was significant higher than that of negative control (P < 0.05). Moreover, proportions of CD4+ T cells in other vaccinated groups were all up-regulated to varying degrees. Up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4) cytokines were detected. A survival rate of 87.5, 62.5, and 62.5% were obtained among rGalT, rAPL_1166, and rHflX group, respectively while the remaining three groups was only 25%. Histopathological analyses of lungs indicated that surviving animals from the vaccinated groups showed relatively normal pulmonary structure alveoli. These findings confirm that IVI antigens used as vaccine candidates provide partial protection against Actinobacillus pleuropneumoniae infection in a mouse model, which could be used as potential vaccine candidates in piglets.
PMCID: PMC5073529  PMID: 27818646
Actinobacillus pleuropneumoniae; IVI antigens; putative vaccine candidates; humoral immune response; cellular immune response
15.  SOXE neofunctionalization and elaboration of the neural crest during chordate evolution 
Scientific Reports  2016;6:34964.
During chordate evolution, two genome-wide duplications facilitated acquisition of vertebrate traits, including emergence of neural crest cells (NCCs), in which neofunctionalization of the duplicated genes are thought to have facilitated development of craniofacial structures and the peripheral nervous system. How these duplicated genes evolve and acquire the ability to specify NC and their derivatives are largely unknown. Vertebrate SoxE paralogues, most notably Sox9/10, are essential for NC induction, delamination and lineage specification. In contrast, the basal chordate, amphioxus, has a single SoxE gene and lacks NC-like cells. Here, we test the hypothesis that duplication and divergence of an ancestral SoxE gene may have facilitated elaboration of NC lineages. By using an in vivo expression assay to compare effects of AmphiSoxE and vertebrate Sox9 on NC development, we demonstrate that all SOXE proteins possess similar DNA binding and homodimerization properties and can induce NCCs. However, AmphiSOXE is less efficient than SOX9 in transactivation activity and in the ability to preferentially promote glial over neuronal fate, a difference that lies within the combined properties of amino terminal and transactivation domains. We propose that acquisition of AmphiSoxE expression in the neural plate border led to NCC emergence while duplication and divergence produced advantageous mutations in vertebrate homologues, promoting elaboration of NC traits.
PMCID: PMC5062122  PMID: 27734831
16.  CT-detected solitary thyroid calcification: an important imaging feature for papillary carcinoma 
OncoTargets and therapy  2016;9:6273-6279.
To evaluate computed tomography (CT) detection of solitary thyroid calcification for identifying thyroid papillary carcinoma and to determine whether the predictive ability changes when the size increases after enhancement.
Materials and methods
CT scans on all 96 patients with thyroid nodules who underwent both enhanced CT examination of neck and thyroidectomy from 2014 to 2016 in the Shandong Cancer Hospital affiliated to Shandong University were reviewed. The cases without calcification and the cases with peripheral calcification, multiple coarse calcifications, or punctate calcification were excluded. Imaging features, including location and size of the lesions, were reviewed on plain and contrast-enhanced CT. The patients were grouped by histological results. The comparisons were evaluated by using Fisher’s exact test and binary logistic regression.
The study population consisted of 96 patients (74 females, 22 males; mean age 49.8±11.3 years). Papillary thyroid carcinoma was observed in both solitary calcified thyroid nodules (85.4%) and solely coarse calcifications surrounded by low-density focus (58.2%). The difference was significant (P=0.006). Of 64 patients with an amplification of lesions after contrast enhancement, 58 (90.6%) were diagnosed with a malignant lesion. At the same time, of the 32 patients with no increase in size, 10 (31.2%) were diagnosed with carcinoma and 22 (68.8%) with nodular goiter. This difference was significant (P<0.001), and after binary logistic regression, increasing size was an independent risk factor for cancer.
Solitary calcified thyroid nodules detected on CT represent a high risk for papillary thyroid carcinoma, especially when the size of the lesions increases after contrast- enhanced CT.
PMCID: PMC5067013  PMID: 27785075
computed tomography; solitary calcified thyroid nodule; papillary thyroid carcinoma; size
17.  RNAi induced hepatotoxicity results from loss of the first synthesized isoform of miR-122 in mice 
Nature medicine  2016;22(5):557-562.
Small RNAs can be used to target and eliminate expression of virtually any disease causing gene or infectious virus, resulting in their pre-clinical and clinical development for treating disease1. To ensure success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity including hepatocyte turnover, loss of gene repression and lethality2 in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs; however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs, we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of liver microRNAs. Once this threshold was surpassed, shRNAs specifically reduced the initial synthesized 22-nucleotide isoform of miR-122-5p without substantially affecting other microRNAs resulting in functional de-repression of miR-122 target mRNAs. Delivery of an rAAV-shRNA vector expressing miR-122 could circumvent toxicity despite accounting for 70% of microRNAs. Toxicity was also not observed in miR-122 knockout mice regardless of the level or sequence of shRNA. Our study establishes limits to the microRNA machinery available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.
PMCID: PMC4860119  PMID: 27064447
18.  A Familial Cluster of Human Brucellosis Attributable to Contact with Imported Infected Goats in Shuyang, Jiangsu Province, China, 2013 
Brucellosis remains a serious public health issue in developing countries, including China. On August 8, 2013, four cases of brucellosis from one extended family were reported at Shuyang County, Jiangsu Province, China. Active case finding was performed to identify the source and the risk factors of the infection and to prevent additional cases. Multiple-locus variable number tandem repeat analysis (MLVA) was used for molecular subtyping analysis. Six people from two extended families met the case definition for brucellosis infection; four were blood culture positive for Brucella melitensis biotype 3. Four additional family members were found seropositive by using a serological test. Isolates from the four patients were indistinguishable by MLVA profiling, displaying a unique type for Jiangsu Province. Field epidemiological data combined with MLVA genotyping supported a common source of the isolates from the different patients. We recommend stronger reinforcement measures for animal quarantine practices, enhanced cooperation with veterinary service organizations, and implementation of measures that strengthen public education on brucellosis to prevent further human outbreaks in Jiangsu Province.
PMCID: PMC4596595  PMID: 26149866
19.  Bacteroides are associated with GALT iNKT cell function and reduction of microbial translocation in HIV-1 infection 
Mucosal immunology  2016;10.1038/mi.2016.34.
Invariant natural killer T (iNKT) cells are innate-like T cells that respond to lipid antigens presented by CD1d. These immunoregulatory cells have the capacity for rapid cytokine release after antigen recognition and are essential for the activation of multiple arms of the immune response. HIV-1 infection is associated with iNKT cell depletion in the peripheral blood; however, their role in the gastrointestinal-associated lymphoid tissue (GALT) is less well studied. Our results show that iNKT cells are found at a higher frequency in GALT compared to blood, particularly in HIV-1 elite controllers. The capacity of iNKT cells to produce IL-4 and IL-10 in the GALT was associated with less immune activation and lower markers of microbial translocation, while Treg frequency showed positive associations with immune activation. We hypothesized that the composition of the microbiota would influence iNKT cell frequency and function. We found positive associations between the abundance of several Bacteroides species and iNKT cell frequency and their capacity to produce IL-4 in the GALT but not in the blood. Overall, our results are consistent with the hypothesis that GALT iNKT cells, influenced by certain bacterial species, may play a key role in regulating immune activation in HIV-1 infection.
PMCID: PMC5053825  PMID: 27049061
20.  Evidence of a Synergistic Effect of Acupoint Combination: A Resting-State Functional Magnetic Resonance Imaging Study 
Objective: This study aimed to find evidence of a synergistic effect of acupoint combinations by analyzing different brain regions activated after acupuncture at different acupoint combinations.
Methods: A total of 57 healthy subjects were randomly distributed into three groups: LR3 plus KI3 acupoints, LR3 plus sham acupoint, or LR3 alone. They underwent a magnetic resonance imaging scan before and after acupuncture. The amplitude of low-frequency fluctuation (ALFF) and regional homogeneity (ReHo) values of different brain regions were analyzed to observe changes in brain function.
Results: ALFF and ReHo produced an activated area in the cerebellum posterior lobe after acupuncture at LR3 plus KI3 acupoints versus LR3 alone. ALFF and ReHo revealed altered activity in Brodmann area 10 (BA10), BA18, and brainstem pons after acupuncture at LR3 plus sham acupoint compared with at LR3 alone. A comparison of acupuncture at LR3 plus KI3 acupoints with LR3 plus sham acupoint demonstrated an increase in BA6 of ALFF and a downregulation of ReHo.
Conclusions: The increased number of brain regions with altered brain activity after acupuncture at acupoint combinations versus a single acupoint are evidence of the synergistic effect of acupoint combinations. BA6 was significantly activated after acupuncture at LR3 plus KI3 acupoints compared with at LR3 plus sham acupoint, suggesting that BA6 is the specific region of synergistic effect of acupoint combinations of LR3 plus KI3 acupoints. Affected brain regions were different between acupuncture at LR3 plus sham acupoint and LR3 alone, which indicates that the sham acupoint may have some psychological effect. However, the specific mechanism of acupoint combinations requires further research.
PMCID: PMC5067799  PMID: 27548054
acupuncture; acupoints combination; MRI; synergistic effect
21.  Effects of MicroRNA-23a on Differentiation and Gene Expression Profiles in 3T3-L1 Adipocytes 
Genes  2016;7(10):92.
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate growth, development, and programmed death of cells. A newly-published study has shown that miRNA-23a could regulate 3T3-L1 adipocyte differentiation. Here, we identified miRNA-23a as a negative regulator of 3T3-L1 adipocyte differentiation again. Over-expression of miRNA-23a inhibited differentiation and decreased lipogenesis as well as down-regulated mRNA and protein expression of both peroxisome proliferator-activated receptor (PPAR) γ and fatty acid binding protein (FABP) 4, whereas knock down of miRNA-23a showed the opposite effects on differentiation as well as increasing the number of apoptotic cells. Additionally, digital gene expression profiling sequencing (DGE-Seq) was used to assay changes in gene expression profiles following alterations in the level of miR-23a. In total, over-expression or knock down of miRNA-23a significantly changed the expression of 313 and 425 genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these genes were mainly involved in the stress response, immune system, metabolism, cell cycle, among other pathways. Additionally, the signal transducer and activator of transcription 1 (Stat1) was shown to be a target of miRNA-23a by computational and dual-luciferase reporter assays that indicated Janus Kinase (Jak)-Stat signal pathway was implicated in regulating adipogenesis mediated by miRNA-23a in adipocytes.
PMCID: PMC5083931  PMID: 27783036
microRNA-23a; adipocytes; differentiation; DGE-Seq; differentially expressed genes
22.  Absence of TolC Impairs Biofilm Formation in Actinobacillus pleuropneumoniae by Reducing Initial Attachment 
PLoS ONE  2016;11(9):e0163364.
Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a major cause of economic loss in swine industry worldwide. TolC, the key component of multidrug efflux pumps and type I secretion systems, has been well-studied as an exit duct for numerous substances in many Gram-negative bacteria. By contrast, little is known on the role of TolC in biofilm formation. In this study, a ΔtolC mutant was used to examine the importance of TolC in biofilm formation of A. pleuropneumoniae. Surface attachment assays demonstrated the essential role of TolC in initial attachment of biofilm cells. The loss of TolC function altered surface hydrophobicity, and resulted in greatly reduced autoaggregation in ΔtolC. Using both enzymatic treatments and confocal microscopy, biofilm composition and architecture were characterized. When compared against the wild-type strain, the poly-β-1, 6-N-acetyl-D-glucosamine (PGA), an important biofilm matrix component of A. pleuropneumoniae, was significantly reduced at the initial attachment stage in ΔtolC. These results were confirmed by mRNA level using quantitative RT-PCR. Additionally, defective secretion systems in ΔtolC may also contribute to the deficiency in biofilm formation. Taken together, the current study demonstrated the importance of TolC in the initial biofilm formation stage in A. pleuropneumoniae. These findings could have important clinical implications in developing new treatments against biofilm-related infections by A. pleuropneumoniae.
PMCID: PMC5040401  PMID: 27681876
23.  Laparoscopic Transcystic Common Bile Duct Exploration: Advantages over Laparoscopic Choledochotomy 
PLoS ONE  2016;11(9):e0162885.
The ideal treatment for choledocholithiasis should be simple, readily available, reliable, minimally invasive and cost-effective for patients. We performed this study to compare the benefits and drawbacks of different laparoscopic approaches (transcystic and choledochotomy) for removal of common bile duct stones.
A systematic search was implemented for relevant literature using Cochrane, PubMed, Ovid Medline, EMBASE and Wanfang databases. Both the fixed-effects and random-effects models were used to calculate the odds ratio (OR) or the mean difference (MD) with 95% confidence interval (CI) for this study.
The meta-analysis included 18 trials involving 2,782 patients. There were no statistically significant differences between laparoscopic choledochotomy for common bile duct exploration (LCCBDE) (n = 1,222) and laparoscopic transcystic common bile duct exploration (LTCBDE) (n = 1,560) regarding stone clearance (OR 0.73, 95% CI 0.50–1.07; P = 0.11), conversion to other procedures (OR 0.62, 95% CI 0.21–1.79; P = 0.38), total morbidity (OR 1.65, 95% CI 0.92–2.96; P = 0.09), operative time (MD 12.34, 95% CI −0.10–24.78; P = 0.05), and blood loss (MD 1.95, 95% CI −9.56–13.46; P = 0.74). However, the LTCBDE group showed significantly better results for biliary morbidity (OR 4.25, 95% CI 2.30–7.85; P<0.001), hospital stay (MD 2.52, 95% CI 1.29–3.75; P<0.001), and hospital expenses (MD 0.30, 95% CI 0.23–0.37; P<0.001) than the LCCBDE group.
LTCBDE is safer than LCCBDE, and is the ideal treatment for common bile duct stones.
PMCID: PMC5036868  PMID: 27668730
24.  Complete Genome Sequence of a New Enterococcus faecalis Bacteriophage, vB_EfaS_IME197 
Genome Announcements  2016;4(5):e00827-16.
We report here the whole-genome sequence of a new Enterococcus faecalis phage, vB_EfaS_IME197, which has a linear double-stranded DNA genome of 41,307 bp with 34% G+C content. We describe the main features of the genome of vB_EfaS_IME197.
PMCID: PMC5026427  PMID: 27634987
25.  Radiation Changes the Metabolic Profiling of Melanoma Cell Line B16 
PLoS ONE  2016;11(9):e0162917.
Radiation therapy can be an effective way to kill cancer cells using ionizing radiation, but some tumors are resistant to radiation therapy and the underlying mechanism still remains elusive. It is therefore necessary to establish an appropriate working model to study and monitor radiation-mediated cancer therapy. In response to cellular stress, the metabolome is the integrated profiling of changes in all metabolites in cells, which can be used to investigate radiation tolerance mechanisms and identify targets for cancer radiation sensibilization. In this study, using 1H nuclear magnetic resonance for untargeted metabolic profiling in radiation-tolerant mouse melanoma cell line B16, we comprehensively investigated changes in metabolites and metabolic network in B16 cells in response to radiation. Principal component analysis and partial least squares discriminant analysis indicated the difference in cellular metabolites between the untreated cells and X-ray radiated cells. In radiated cells, the content of alanine, glutamate, glycine and choline was increased, while the content of leucine, lactate, creatine and creatine phosphate was decreased. Enrichment analysis of metabolic pathway showed that the changes in metabolites were related to multiple metabolic pathways including the metabolism of glycine, arginine, taurine, glycolysis, and gluconeogenesis. Taken together, with cellular metabolome study followed by bioinformatic analysis to profile specific metabolic pathways in response to radiation, we deepened our understanding of radiation-resistant mechanisms and radiation sensibilization in cancer, which may further provide a theoretical and practical basis for personalized cancer therapy.
PMCID: PMC5025142  PMID: 27631970

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