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1.  si-RNA-Mediated Silencing of ADRBK1 Gene Attenuates Breast Cancer Cell Proliferation 
Breast cancer is the most prominent cause of cancer-related deaths among women worldwide. It has been found that genetic mutations play distinct roles in the onset and progression of breast cancer. Androgenic, beta, receptor kinase 1 (ADRBK1) has been reported to possess oncogenic characteristics vital for cancer cell viability. This study was designed to investigate the effects of small interference RNA (si-RNA)–mediated ADRBK1 knockdown on breast cancer cell growth in vitro. High-expression levels of ADRBK1 were observed in all tested breast cancer cell lines (MDA-MB-231, MCF-7, T-47D, and BT-474). ADRBK1 si-RNA was delivered to breast cancer cells using lentivirus delivery system. Depletion of ADRBK1 significantly attenuated the cell viability and colony-formation ability. Flow cytometry analysis further demonstrated that ADRBK1 silencing led to MDA-MB-231 cell arrest in the G0/G1 phase. Collectively, these results indicate that knockdown of ADRBK1 gene has detrimental effects on breast cancer cell growth, which may be a potential therapeutic approach for the treatment of breast cancer.
PMCID: PMC4186765  PMID: 25279970
ADRBK1; breast cancer; cell growth; si-RNA
2.  An integrated method for quantifying root architecture of field-grown maize 
Annals of Botany  2014;114(4):841-851.
Background and Aims
A number of techniques have recently been developed for studying the root system architecture (RSA) of seedlings grown in various media. In contrast, methods for sampling and analysis of the RSA of field-grown plants, particularly for details of the lateral root components, are generally inadequate.
An integrated methodology was developed that includes a custom-made root-core sampling system for extracting intact root systems of individual maize plants, a combination of proprietary software and a novel program used for collecting individual RSA information, and software for visualizing the measured individual nodal root architecture.
Key Results
Example experiments show that large root cores can be sampled, and topological and geometrical structure of field-grown maize root systems can be quantified and reconstructed using this method. Second- and higher order laterals are found to contribute substantially to total root number and length. The length of laterals of distinct orders varies significantly. Abundant higher order laterals can arise from a single first-order lateral, and they concentrate in the proximal axile branching zone.
The new method allows more meaningful sampling than conventional methods because of its easily opened, wide corer and sampling machinery, and effective analysis of RSA using the software. This provides a novel technique for quantifying RSA of field-grown maize and also provides a unique evaluation of the contribution of lateral roots. The method also offers valuable potential for parameterization of root architectural models.
PMCID: PMC4156117  PMID: 24532646
Branching order; lateral roots; root system architecture; topological structure; root architectural model; maize; Zea mays
3.  The global effect of heat on gene expression in cultured bovine mammary epithelial cells 
Cell Stress & Chaperones  2014;20(2):381-389.
Heat stress (HS) in hot climates is a major cause that strongly negatively affects milk yield in dairy cattle, leading to immeasurable economic loss. The heat stress response of bovine mammary epithelial cells (BMECs) is one component of the acute systemic response to HS. Gene networks of BMECs respond to environmental heat loads with both intra- and extracellular signals that coordinate cellular and whole-animal metabolism. Our experimental objective was to characterize the direct effects of heat stress on the cultured bovine mammary epithelial cells by microarray analyses. The data identified 2716 differentially expressed genes in 43,000 transcripts which were changed significantly between heat-stressed and normal bovine mammary epithelial cells (fold change ≥2, P ≤ 0.001). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these differentially expressed genes are involved in different pathways that regulate cytoskeleton, cell cycle, and stress response processes. Our study provides an overview of gene expression profile and the interaction between gene expression and heat stress, which will lead to further understanding of the potential effects of heat stress on bovine mammary glands.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-014-0559-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4326376  PMID: 25536930
Bovine mammary epithelial cells; Microarray analysis; Heat stress
4.  Nanoparticle mediated chemotherapy of hormone refractory prostate cancer with a novel combi-molecule 
In previous study, we synthesized a novel combi-molecule, JDF-12, with superior cytotoxicity against prostate cancer cells, but it has a poor stability in liquid after preparation with traditional method and is susceptible to hydrolysis and binding to organs highly expressing epidermal growth factor receptor (EGFR), resulting in side effects. In this study, the nanotechnology was employed to prepare JDF-12 aiming to increase its anti-tumor effect and reduce its systemic side effects. The JDF-12 loaded nanoparticles were formulated with biocompatible and biodegradable poly (D,L-lactic-co-glycolic acid)-block-poly(ethyleneglycol) (PLGA-b-PEG) copolymer and surface functionalized with a single-chain antibody that recognizes the extracellular domain of prostate stem cell antigen (PSCA), enabling a controlled release, “stealth” property, and cell-specific targeting. The targeted nanoparticles exhibited a sustained drug release in vitro and were specifically endocytosed by prostate cancer cells though the receptor-mediated endocytosis resulting in enhanced cellular toxicity in vitro. Moreover, a better outcome with reduced drug toxicity was observed in a PC3M xenograft animal model after treatment with these nanoparticles. Our results demonstrate the feasibility of nanoparticle-based technology in the development of pharmaceutically suboptimal chemotherapeutics.
PMCID: PMC4568799  PMID: 26396674
Prostate cancer; epidermal growth factor receptor; nanoparticle
5.  Insulin therapy and the risk of colorectal cancer in patients with type 2 diabetes: a meta-analysis of observational studies 
Several epidemiological studies have reported inconsistent associations between insulin therapy and the risk of colorectal cancer (CRC) in patients with type 2 diabetes mellitus. We performed this meta-analysis of observational studies to evaluate the effect of insulin therapy on the risk of CRC.
We carried out a systematic search of PubMed, Embase and the Cochrane Library Central database between January 1966 and August 2013. Fixed-effects and random-effects models were used to estimate the pooled relative risk (RR) and corresponding 95% confidence interval (CI).
A total of 12 epidemiological studies were included in the present meta-analysis, involving a total of 7947 CRC cases and 491 384 participants. There was significant heterogeneity among the studies, but no publication bias. Insulin therapy significantly increased the risk of CRC [RR = 1.69, 95% CI (1.25, 2.27)]. When the various studies were stratified by study design, we found that insulin use was associated with a statistically significant 115% higher risk of CRC among case–control studies [RR = 2.15, 95% CI (1.41, 3.26)], but not among cohort studies [RR = 1.25, 95% CI (0.95, 1.65)]. Furthermore, a significant association was noted among studies conducted in USA [RR = 1.73, 95% CI (1.15, 2.60)] and Asia [RR = 2.55, 95% CI (2.14, 3.04)], but not in Europe [RR = 1.20, 95% CI (0.92, 1.57)].
The present meta-analysis suggests that insulin therapy may increase the risk of CRC. More prospective cohort studies with longer follow-up durations are warranted to confirm this association. Furthermore, future studies should report results stratified by gender and race and should adjust the results by more confounders.
PMCID: PMC4137822  PMID: 25099257
colorectal cancer; epidemiology; insulin; meta-analysis
6.  SHP2E76K mutant promotes lung tumorigenesis in transgenic mice 
Carcinogenesis  2014;35(8):1717-1725.
Tet-inducible transgenic mice of a cancer-associated SHP2E76K mutant were generated and used to study SHP2E76K in lung tumorigenesis. Data showed that it promoted lung tumorigenesis. Tumors regressed after dox withdrawal. Moreover, SHP2E76K autoregulated tyrosine phosphorylation of its docking protein Gab1.
Lung cancer is a major disease carrying heterogeneous molecular lesions and many of them remain to be analyzed functionally in vivo. Gain-of-function (GOF) SHP2 (PTPN11) mutations have been found in various types of human cancer, including lung cancer. However, the role of activating SHP2 mutants in lung cancer has not been established. We generated transgenic mice containing a doxycycline (Dox)-inducible activating SHP2 mutant (tetO-SHP2E76K) and analyzed the role of SHP2E76K in lung tumorigenesis in the Clara cell secretory protein (CCSP)-reverse tetracycline transactivator (rtTA)/tetO-SHP2E76K bitransgenic mice. SHP2E76K activated Erk1/Erk2 (Erk1/2) and Src, and upregulated c-Myc and Mdm2 in the lungs of bitransgenic mice. Atypical adenomatous hyperplasia and small adenomas were observed in CCSP-rtTA/tetO-SHP2E76K bitransgenic mice induced with Dox for 2–6 months and progressed to larger adenoma and adenocarcinoma by 9 months. Dox withdrawal from bitransgenic mice bearing magnetic resonance imaging-detectable lung tumors resulted in tumor regression. These results show that the activating SHP2 mutant promotes lung tumorigenesis and that the SHP2 mutant is required for tumor maintenance in this mouse model of non-small cell lung cancer. SHP2E76K was associated with Gab1 in the lung of transgenic mice. Elevated pGab1 was observed in the lung of Dox-induced CCSP-rtTA/tetO-SHP2E76K mice and in cell lines expressing SHP2E76K, indicating that the activating SHP2 mutant autoregulates tyrosine phosphorylation of its own docking protein. Gab1 tyrosine phosphorylation is sensitive to inhibition by the Src inhibitor dasatinib in GOF SHP2-mutant-expressing cells, suggesting that Src family kinases are involved in SHP2 mutant-induced Gab1 tyrosine phosphorylation.
PMCID: PMC4123642  PMID: 24480804
7.  Anti-Human H1N1pdm09 and swine H1N1 Virus Antibodies among Swine Workers in Guangdong Province, China 
Scientific Reports  2015;5:12507.
To assess the potential transmission for zoonotic influenza, sero-antibodies against two kinds of influenza viruses—classical swine H1N1 and human H1N1pdm09 virus were detected in persons whose profession involved contact with swine in Guangdong province, China. Compared to the non-exposed control group, a significantly higher proportion of subjects with occupational contact to pigs exhibited positive seroreaction against the classical H1N1 SIV. Participants aged 26–50 years were at high risk of classic swine H1N1 infections. Seropositive rate to 2009 pandemic H1N1 virus among swine workers was similar with controls. The major impact of age was apparent for younger populations. Our present study has documented evidence for swine influenza virus infection among persons with occupational swine exposures. The differences of seroreactivity for the two tested influenza subtypes emphasize the necessity of regular surveillance both in pigs and human.
PMCID: PMC4513349  PMID: 26205221
8.  Pyrosequencing analysis for mutations in embB codon306 among clinical mycobacterium tuberculosis isolates from Qingdao, China 
In this study, our objectives was to analyze the molecular characteristics of mutation at embB codon306 in Mycobacterium tuberculosis in Qingdao by pyrosequencing technology, and to assess the value of embB codon306 used as a molecular marker to diagnose multidrug resistant (MDR) TB strains. Pyrosequencing was used to detect mutations at embB codon306 among M. tuberculosis isolates from tuberculosis (TB) patients in Qingdao. The correlation between embB306 mutation and MDR phenotype was evaluated by comparing with conventional drug susceptibility testing results. 60.9% of MDR strains and 15.2% of non-MDR strains carried embB306 mutation, respectively. The percentage of MDR-TB harboring embB306 mutation was significantly higher than that of non- MDR-TB (χ2=15.09, P < 0.01). EmbB306 mutation serving as a marker to diagnose MDR-TB comparing with the traditional susceptibility test, the specificity, sensitivity and accuracy were 85%, 61%, and 77%, respectively. EmbB306 mutation is the main mechanism of TB resistance to multidrug in Qingdao, showing an association with the MDR. Pyrosequencing should be a good diagnostic tool for MDR-TB in Qingdao.
PMCID: PMC4565318  PMID: 26379935
Mycobacterium tuberculosis; embB; multidrug resistance; pyrosequencing
9.  Family Clusters of Avian Influenza A H7N9 Virus Infection in Guangdong Province, China 
Since its first identification, the epizootic avian influenza A H7N9 virus has continued to cause infections in China. Two waves were observed during this outbreak. No cases were reported from Guangdong Province during the first wave, but this province became one of the prime outbreak sites during the second wave. In order to identify the transmission potential of this continuously evolving infectious virus, our research group monitored all clusters of H7N9 infections during the second wave of the epidemic in Guangdong Province. Epidemiological, clinical, and virological data on these patients were collected and analyzed. Three family clusters including six cases of H7N9 infection were recorded. The virus caused severe disease in two adult patients but only mild symptoms for all four pediatric patients. All patients reported direct poultry or poultry market exposure history. Relevant environment samples collected according to their reported exposures tested H7N9 positive. Virus isolates from patients in the same cluster shared high sequence similarities. In conclusion, although continually evolving, the currently circulating H7N9 viruses in Guangdong Province have not yet demonstrated the capacity for efficient and sustained person-to-person transmission.
PMCID: PMC4290927  PMID: 25339399
10.  Whole-genome sequencing to detect recent transmission of Mycobacterium tuberculosis in settings with a high burden of tuberculosis 
Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been used to trace the transmission of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Previously published studies using WGS were conducted in developed countries with a low TB burden. We sought to evaluate the relative usefulness of traditional VNTR and SNP typing methods, WGS and epidemiological investigations to study the recent transmission of M. tuberculosis in a high TB burden country. We conducted epidemiological investigations of 42 TB patients whose M. tuberculosis isolates were classified into three clusters based on variable-number tandem repeat (VNTR) typing. We applied WGS to 32 (76.2%) of the 42 strains and calculated the pairwise genomic distances between strains within each cluster. Eighteen (56.3%) of the 32 strains had genomic differences ≥100 SNPs with every other strain, suggesting that direct transmission did not likely occurred. Ten strains were grouped into four WGS-based clusters with genomic distances ≤5 SNPs within each cluster, and confirmed epidemiological links were identified in two of these clusters. Our results indicate that WGS provides reliable resolution for tracing the transmission of M. tuberculosis in high TB burden settings. The high resolution of WGS is particularly useful to confirm or exclude the possibility of direct transmission events defined by traditional typing methods.
PMCID: PMC4409578  PMID: 24888866
Tuberculosis; Transmission; Whole genome sequencing; Genotyping; Contact tracing
11.  α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties 
PLoS ONE  2015;10(6):e0130572.
The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer’s disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.
PMCID: PMC4472343  PMID: 26086615
12.  Profiling and initial validation of urinary microRNAs as biomarkers in IgA nephropathy 
PeerJ  2015;3:e990.
Background. MicroRNAs (miRNAs) have been found in virtually all body fluids and used successfully as biomarkers for various diseases. Evidence indicates that miRNAs have important roles in IgA nephropathy (IgAN), a major cause of renal failure. In this study, we looked for differentially expressed miRNAs in IgAN and further evaluated the correlations between candidate miRNAs and the severity of IgAN.
Methods. Microarray and RT-qRCR (real-time quantitative polymerase chain reaction) were sequentially used to screen and further verify miRNA expression profiles in urinary sediments of IgAN patients in two independent cohorts. The screening cohort consisted of 32 urine samples from 18 patients with IgAN, 4 patients with MN (membranous nephropathy), 4 patients with MCD (minimal changes disease) and 6 healthy subjects; the validation cohort consisted of 102 IgAN patients, 41 MN patients, 27 MCD patients and 34 healthy subjects. The renal pathological lesions of patients with IgAN were evaluated according to Lee’s grading system and Oxford classification.
Results. At the screening phase, significance analysis of microarrays analysis showed that no miRNA was differentially expressed in the IgAN group compared to all control groups. But IgAN grade I–II and III subgroups (according to Lee’s grading system) shared dysregulation of two miRNAs (miR-3613-3p and miR-4668-5p). At the validation phase, RT-qPCR results showed that urinary level of miR-3613-3p was significantly lower in IgAN than that in MN, MCD and healthy controls (0.47, 0.44 and 0.24 folds, respectively, all P < 0.01 by Mann–Whitney U test); urinary level of miR-4668-5p was also significantly lower in IgAN than that in healthy controls (0.49 fold, P < 0.01). Significant correlations were found between urinary levels of miR-3613-3p with 24-hour urinary protein excretion (Spearman r = 0.50, P = 0.034), eGFR (estimated glomerular filtration rate) (r = − 0.48, P = 0.043) and Lee’s grades (r = 0.57, P = 0.014). Similarly, miR-4668-5p was significantly correlated with eGFR (r = − 0.50, P = 0.034) and Lee’s grades (r = 0.57, P = 0.013). For segmental glomerulosclerosis according to Oxford classification, patients scored as S0 had significantly lower levels of urinary miR-3613-3p and miR-4668-5p than those scored as S1 (0.41 and 0.43 folds, respectively, all P < 0.05).
Conclusions. The expression profile of miRNAs was significantly altered in urinary sediments from patients with IgAN. Urinary expression of miR-3613-3p was down-regulated in patients with IgAN. Moreover, urinary levels of both miR-3613-3p and miR-4668-5p were correlated with disease severity. Further studies are needed to explore the roles of miR-3613-3p and miR-4668-5p in the pathogenesis and progression of IgA nephropathy.
PMCID: PMC4458130  PMID: 26056621
MicroRNA; Microarray; Biomarkers; IgA nephropathy
13.  StarScan: a web server for scanning small RNA targets from degradome sequencing data 
Nucleic Acids Research  2015;43(Web Server issue):W480-W486.
Endogenous small non-coding RNAs (sRNAs), including microRNAs, PIWI-interacting RNAs and small interfering RNAs, play important gene regulatory roles in animals and plants by pairing to the protein-coding and non-coding transcripts. However, computationally assigning these various sRNAs to their regulatory target genes remains technically challenging. Recently, a high-throughput degradome sequencing method was applied to identify biologically relevant sRNA cleavage sites. In this study, an integrated web-based tool, StarScan (sRNA target Scan), was developed for scanning sRNA targets using degradome sequencing data from 20 species. Given a sRNA sequence from plants or animals, our web server performs an ultrafast and exhaustive search for potential sRNA–target interactions in annotated and unannotated genomic regions. The interactions between small RNAs and target transcripts were further evaluated using a novel tool, alignScore. A novel tool, degradomeBinomTest, was developed to quantify the abundance of degradome fragments located at the 9–11th nucleotide from the sRNA 5′ end. This is the first web server for discovering potential sRNA-mediated RNA cleavage events in plants and animals, which affords mechanistic insights into the regulatory roles of sRNAs. The StarScan web server is available at
PMCID: PMC4489260  PMID: 25990732
14.  Effects of artemether on the proliferation, apoptosis, and differentiation of keratinocytes: potential application for psoriasis treatment 
Artemether exhibits diverse pharmacological effects and has multiple applications. This study aimed to investigate its antiproliferative and apoptogenic effects on HaCaT cells and keratinocyte differentiation-inducing activity in vivo. WST-8 analysis demonstrated that Artemether can inhibit the proliferation of cultured HaCaT cells in a time- and dose-dependent manner. Annexin V/PI dual staining and JC-1 staining further revealed that Artemether can dose-dependently augment HaCaT apoptosis. To investigate the keratinocyte differentiation-inducing activity of Artemether, it was prepared as topical creams at concentrations of 1%, 3%, and 5%. During the 4 weeks of topical treatment, no evidence of irritation was observed in the mouse tail test. Artemether cream dose-dependently increased the degree of orthokeratosis and the relative epidermal thickness of mouse tail skin, indicative of the keratinocyte differentiation-inducing activity. Taking the in vitro and in vivo findings together, the present study suggests that Artemether may be a promising antipsoriatic agent worthy of further investigation.
PMCID: PMC4509189  PMID: 26221244
Artemether; psoriasis; antipsoriatic; keratinocyte; HaCaT cells; proliferation; apoptosis; differentiation; mouse tail test
15.  Environmental Sampling for Avian Influenza A(H7N9) in Live-Poultry Markets in Guangdong, China 
PLoS ONE  2015;10(5):e0126335.
To provide an increased understanding of avian influenza A(H7N9) activity in live-poultry market in space and time and hence improve H7N9 epidemic control, an ongoing environmental sampling program in multiple live-poultry markets across Guangdong, China was conducted during March 2013–June 2014.
A total of 625 live-poultry markets throughout 21 prefecture areas took part in the study. A total of 10 environmental sites in markets for sampling were identified to represent 4 different poultry-related activity areas. At least 10 environmental samples were collected from each market every month. The real time RT-PCR was performed to detect the avian influenza A(H7N9) virus. Field survey was conducted to investigate the sanitation status of live-poultry markets.
There were 109 human infections with H7N9 avian influenza in Guangdong, of which 37 (34%) died. A total of 18741 environmental swabs were collected and subjected to real-time RT-PCR test, of which 905(4.83%) were found positive for H7N9 virus. There were 201 (32.16%) markets affected by H7N9 in 16 prefecture areas. The detection of H7N9 virus in markets spiked in winter months. 63.33% markets (38/60) had no physical segregation for poultry holding, slaughter or sale zones. Closing live-poultry market significantly decreased the H7N9 detection rate from 14.83% (112/755) to 1.67% (5/300).
This study indicates the importance of live-poultry market surveillance based on environmental sampling for H7N9 Avian Influenza control. Improving live-poultry market management and sanitation and changing consumer practices are critical to reduce the risk of H7N9 infection.
PMCID: PMC4416787  PMID: 25933138
16.  Prognostic Nomogram for Thoracic Esophageal Squamous Cell Carcinoma after Radical Esophagectomy 
PLoS ONE  2015;10(4):e0124437.
Nomogram has demonstrated its capability in individualized estimates of survival in diverse cancers. Here we retrospectively investigated 1195 patients with esophageal squamous-cell carcinoma (ESCC) who underwent radical esophagectomy at Zhejiang Cancer Hospital in Hangzhou, China. We randomly assigned two-thirds of the patients to a training cohort (n = 797) and one-third to a validation cohort (n = 398). Cox proportional hazards regression analyses were performed using the training cohort, and a nomogram was developed for predicting 3-year and 5-year overall survival rates. Multivariate analysis identified tumor length, surgical approach, number of examined lymph node, number of positive lymph node, extent of positive lymph node, grade, and depth of invasion as independent risk factors for survival. The discriminative ability of the nomogram was externally determined using the validation cohort, showing that the nomogram exhibited a sufficient level of discrimination according to the C-index (0.715, 95% CI 0.671–0.759). The C-index of the nomogram was significantly higher than that of the sixth edition (0.664, P-value<0.0001) and the seventh edition (0.696, P-value<0.0003) of the TNM classification. This study developed the first nomogram for ESCC, which can be applied in daily clinical practice for individualized survival prediction.
PMCID: PMC4404051  PMID: 25893524
17.  Mixed infections of Mycobacterium tuberculosis in tuberculosis patients in Shanghai, China 
We applied a 7 loci Variable-Number-Tandem-Repeats (VNTR-7) analysis method to identify mixed infections of Mycobacterium tuberculosis and to estimate the rate of mixed infections among pulmonary tuberculosis patients in Shanghai, China. We validated the VNTR-7 method and used it to genotype an isolate from each of the 249 from pulmonary tuberculosis patients reported from the Songjiang and Chongming districts in Shanghai during 2006. We identified 14 patients with mixed infections, and the estimated rate of mixed infections was 5.6% (14/249) (95% CI 3.1%–9.2%). Mixed infections were observed more frequently among tuberculosis patients undergoing retreatment (15.6%) than among new cases (4.1%) (p < 0.05), and among tuberculosis patients whose disease was caused by non-Beijing genotype strains (12.5%) versus Beijing genotype strains (3.5%) (p < 0.05). The VNTR-7 method is a highly sensitive, practical tool with relatively high discriminatory power, making it useful for studying mixed infections.
PMCID: PMC4391511  PMID: 18424179
Tuberculosis; Mixed infections; VNTR
18.  Next-generation sequencing reveals the biological significance of the N2,3-ethenoguanine lesion in vivo 
Nucleic Acids Research  2015;43(11):5489-5500.
Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N2,3-ethenoguanine (N2,3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2′-fluoro-2′-deoxyribose analog of N2,3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N2,3-εG and its isomer 1,N2-ethenoguanine (1,N2-εG) were evaluated in various repair and replication backgrounds. We found that N2,3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N2-εG induces various substitutions and frameshifts. We also found that N2,3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N2,3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.
PMCID: PMC4477646  PMID: 25837992
19.  Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome 
PLoS Genetics  2015;11(3):e1005097.
Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.
Author Summary
Mitochondrial respiratory chain (MRC) disease represents a large and heterogeneous group of energy deficiency disorders. Here we report three mutations in NARS2, a mitochondrial asparaginyl-tRNA synthetase, associated with non-syndromic hearing loss (NSHL) and Leigh syndrome in two independent families. Located in the predicted catalytic domain of the protein, missense mutation p.(Val213Phe) results in NSHL (DFNB94) while compound heterozygous mutation (p.Tyr323*; p.Asn381Ser) is leading to Leigh syndrome with auditory neuropathy. In vivo analysis deemed p.Tyr323* mutant protein to be unstable. Co-immunoprecipitation assays show that p.Asn381Ser mutant disrupts the dimerization ability of NARS2. Leigh syndrome patient fibroblasts exhibit a decreased steady-state level of mt-tRNAAsn. In addition, in these cells, the mitochondrial respiratory chain is deficient, including significantly decreased oxygen consumption rates and electron transport chain activities. These functions can be partially restored with over-expression of wild-type NARS2 but not with p.Val213Phe mutant protein. Our study provides new insights into the genes that are necessary for the function of brain and inner ear sensory cells in humans.
PMCID: PMC4373692  PMID: 25807530
20.  Optimization and Decision Science 
The Scientific World Journal  2015;2015:159169.
PMCID: PMC4390113  PMID: 25884019
21.  Inhibition of Shp2 suppresses mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcinoma 
Oncotarget  2015;6(8):6191-6202.
Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma.
PMCID: PMC4467431  PMID: 25730908
Shp2; EGFR; phosphatase; transgenic mice; lung cancer
22.  Fatty acid composition of developing tree peony (Paeonia section Moutan DC.) seeds and transcriptome analysis during seed development 
BMC Genomics  2015;16(1):208.
Tree peony (Paeonia section Moutan DC.) is known for its excellent ornamental and medicinal values. In 2011, seeds from P. ostii have been identified as novel resource of α-linolenic acid (ALA) for seed oil production and development in China. However, the molecular mechanism on biosynthesis of unsaturated fatty acids in tree peony seeds remains unknown. Therefore, transcriptome data is needed to better understand the underlying mechanisms.
In this study, lipid accumulation contents were measured using GC-MS methods across developing tree peony seeds, which exhibited an extraordinary ALA content (49.3%) in P. ostii mature seeds. Transcriptome analysis was performed using Illumina sequencing platform. A total of 144 million 100-bp paired-end reads were generated from six libraries, which identified 175,874 contigs. In the KEGG Orthology enrichment of differentially expressed genes, lipid metabolism pathways were highly represented categories. Using this data we identified 388 unigenes that may be involved in de novo fatty acid and triacylglycerol biosynthesis. In particular, three unigenes (SAD, FAD2 and FAD8) encoding fatty acid desaturase with high expression levels in the fast oil accumulation stage compared with the initial stage of seed development were identified.
This study provides the first comprehensive genomic resources characterizing tree peony seeds gene expression at the transcriptional level. These data lay the foundation for further understanding of molecular mechanism responsible for lipid biosynthesis and the high unsaturated fatty acids (especially ALA) accumulation. Meanwhile, it provides theoretical base for potential oilseed application in the respect of n-6 to n-3 ratio for human diets and future regulation of target healthy components of oils.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1429-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4404109  PMID: 25887415
Paeoniaceae; Tree peony; Transcriptome; α-linolenic acid; Omega-3 fatty acid; Triacylglycerols
23.  Phylogeny of Salix subgenus Salix s.l. (Salicaceae): delimitation, biogeography, and reticulate evolution 
The taxonomy and systematics of Salix subgenus Salix s.l. is difficult. The reliability and evolutionary implications of two important morphological characters (number of stamens, and morphology of bud scales) used in subgeneric classification within Salix remain untested, and a disjunct Old–New World distribution pattern of a main clade of subgenus Salix s.l., revealed by a previous study, lacks a reasonable explanation. To study these questions, we conducted phylogenetic analyses based on 4,688 bp of sequence data from four plastid (rbcL, trnD–T, matK, and atpB–rbcL) and two nuclear markers (ETS and ITS) covering all subgenera of Salix, and all sections of subgenus Salix s.l.
Subgenus Salix came out as para- or polyphyletic in both nrDNA and plastid trees. The plastid phylogeny successfully resolved relationships among the major clades of Salix, but resolution within subgenus Salix s.l. remained low. Nevertheless, three monophyletic groups were identifiable in subgenus Salix s.l.: the ‘main clade’ of subgenus Salix s.l., with New and Old World species being reciprocally monophyletic; the section Triandroides clade; and the subgenus Pleuradenia clade. While nrDNA regions showed higher resolution within subgenus Salix s.l., they failed to resolve subgeneric relationships. Extensive, statistically significant gene-tree incongruence was detected across nrDNA–plastid as well as nrDNA ETS–ITS phylogenies, suggesting reticulate evolution or hybridization within the group. The results were supported by network analyses. Ancestral-state reconstructions indicated that multiple stamens and free bud scales represent the plesiomorphic states within Salix, and that several significant shifts in stamen number and bud scale morphology have occurred.
Subgenus Salix s.l. is not monophyletic, and the evolutionary history of the subgenus has involved multiple reticulation events that may mainly be due to hybridization. The delimitation of subgenus Salix s.l. should be redefined by excluding section Triandrae and subgenus Pleuradenia from it. The evolutionary lability of bud-scale morphology and stamen number means that these characters are unreliable bases for classification. The disjunct Old–New World distribution of subgenus Salix s.l. appears to be linked to the profound climatic cooling during the Tertiary, which cut off gene exchange between New and Old World lineages.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-015-0311-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4357182  PMID: 25886526
Salix subgenus Salix; Phylogeny; Biogeography; Reticulate evolution; Character evolution
24.  Upregulation of heat shock protein 32 with hemin alleviates acute heat-induced hepatic injury in mice 
Cell Stress & Chaperones  2014;19(5):675-683.
Heat shock protein 32 (HSP32) is a stress response protein that can be induced by heat stress in the liver, and its induction can act as an important cellular defence mechanism against heat-induced liver injury. To investigate the functional role of HSP32 in protecting liver tissue against heat stress in mice and the mechanism by which it achieves this protective effect, HSP32 expression and carbon monoxide (CO) contents in a model of mice subjected to acute, transient heat exposure were examined. Furthermore, functional and histological parameters of liver damage and the possible involvement of oxidative stress to induce oxidative deterioration of liver functions and caspase-3 expression were also investigated in this study. We found that heat treatment of mice produced severe hepatic injury, whereas upregulation of HSP32 with hemin pretreatment prevented mice from liver damage. In contrast, addition of Sn-protoporphyrin (SnPP) to inhibit HSP32 expression completely reversed its hepatoprotective effect. It is concluded that upregulation of HSP32 by hemin could alleviate acute heat-induced hepatocellular damage in mice, and its by-product CO seems to play a more important role in hepatoprotective mechanism.
PMCID: PMC4147065  PMID: 24473736
HSP32; Liver injury; Acute heat stress; Caspase-3; Hemin
25.  Altered Molecular Expression of the TLR4/NF-κB Signaling Pathway in Mammary Tissue of Chinese Holstein Cattle with Mastitis 
PLoS ONE  2015;10(2):e0118458.
Toll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor κB (NF-κB) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differential gene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 79 genes associated with the TLR4/NF-κB signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4, MyD88, IL-6, and IL-10, were up-regulated, while, CD14, TNF-α, MD-2, IL-β, NF-κB, and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland. In conclusion, our study provides new evidence for better understanding the differential expression and mechanisms of the TLR4 /NF-κB signaling pathway in Chinese Holstein cattle with mastitis.
PMCID: PMC4338248  PMID: 25706977

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