Search

Background Despite extensive molecular epidemiological studies, the prevalence and characteristics of Mouse Mammary Tumor Virus-Like Virus (MMTV-LV) in Chinese women breast cancer are still unclear. Besides, the prevalence of MMTV-LV in women breast cancer tissue varies in different countries and its dependent factors remain inconclusive. Methods In the first part of the study, a case-control study was performed. 119 breast cancer samples (84 from Northern China and 35 from Southern China) and 50 breast fibroadenoma specimens were collected from Chinese women patients. MMTV-like env sequence and the homology to MMTV env gene were analysed by semi-nested polymerase chain reaction (PCR). We also explored the association of MMTV-LV prevalence with sample sources (Southern and Northern China) and patients’ clinicopathological characteristics. To investigate the dependent factors of the prevalence of MMTV-LV in breast cancer worldwide, a meta-analysis was conducted in the second part of the study. Results We found that the prevalence of MMTV-LV was much higher in breast cancer tissues (17.65%) than that in breast fibroadenoma specimens (4.00%) (P P P Conclusion The distribution of house mice may be a crucial environmental factor that explains the geographic differences in human breast cancer incidence. Our findings may provide a potential avenue of prevention, diagnosis and treatment for breast cancer.

HeLa cells are a commonly used cell line in many biological research areas. They are not picky for culture medium and proliferate rapidly. HeLa cells are a notorious source of cell cross‐contamination and have been found to be able to contaminate a wide range of cell lines in cell culture. In this study, we reported a simple and efficient method for detecting the presence of HeLa cell contamination in cell culture. HPV‐18 was used as a biomarker. The cell culture supernatant was used directly as the template for nested PCR without extracting nucleic acid. By PCR amplification of the cell culture supernatant with the designed primers, we were able to detect the presence of HeLa cells in the culture. The sensitivity of this method can reach 1%, which is 10‐fold higher than Short tandem repeat sequence (STR) profiling. This simple, rapid, and “noninvasive” quality checking method should find applications in routine cell culture practice.

Endometriosis is a common gynecological condition with unclear pathogenesis. Although a dysregulated lncRNA expression profile has been speculated, very few studies have addressed this hypothesis. We determined the differential lncRNA and mRNA expression patterns between endometriosis and control tissues, and between eutopic and normal endometrium in the proliferative phase, using RNA sequencing. The potential targets of lncRNA were predicted on the basis of cis and trans action, and lncRNAs were functionally annotated in relation to their co-expressed mRNAs. Dysregulated lncRNAs and mRNAs were screened relative to the biological features of endometriosis, and the five filtered lncRNAs were validated using qRT-PCR. A total of 9924 novel lncRNA transcripts were identified, and 86 lncRNAs and 1228 mRNAs were differentially expressed between the endometriosis and control groups. GO and KEGG pathway analysis showed that the differentially expressed lncRNAs were enriched in the biological processes and signaling pathways involved in endometriosis. A coding-noncoding gene (CNC) co-expression network was constructed using the dysregulated lncRNAs and their co-expressed mRNAs to simulate the complex intergenic interactions. This study is the first to use sequencing technology to elucidate the differentially lncRNA expression profiles of eutopic and normal endometrium in the proliferative phase of endometriosis. The dysregulated lncRNAs can potentially be novel diagnostic biomarkers and therapeutic targets of endometriosis.

Neutrophils play a critical role in host defense against Pseudomonas aeruginosa infection. Mechanisms underlying the negative regulation of neutrophil function in bacterial clearance remain incompletely defined. Here, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of P. aeruginosa clearance by neutrophils. PTP1B-deficient neutrophils display greatly enhanced bacterial phagocytosis and killing, which are accompanied by increased Toll-like receptor 4 (TLR4) signaling activation and nitric oxide (NO) production following P. aeruginosa infection. Interestingly, PTP1B deficiency mainly upregulates the production of IL-6 and IFN-β, leads to enhanced TLR4-dependent STAT1 activation and iNOS expression by neutrophils following P. aeruginosa infection. Further studies reveal that PTP1B and STAT1 are physically associated. These findings demonstrate a negative regulatory mechanism in neutrophil underlying the elimination of P. aeruginosa infection though a PTP1B-STAT1 interaction.

The aim of this study was to investigate the hepatotoxic effect and its underlying mechanism of aloe emodin (AE). AE was docked with the targets of NF-kappa B inflammatory pathway and P53 apoptosis pathway respectively by using molecular docking technique. To verify the results of molecular docking and further investigate the hepatotoxicity mechanism of AE, the zebrafish Tg (fabp10: EGFP) was used as an animal model in vivo. The pathological sections of zebrafish liver were analyzed to observe the histopathological changes and Sudan black B was used to study whether there were inflammatory reactions in zebrafish liver or not. Then TdT-mediated dUTP Nick-End Labeling (TUNEL) was used to detect the apoptotic signal of zebrafish liver cells, finally the mRNA expression levels as well as the protein expression levels of the targets in NF-kappa B and P53 pathways in zebrafish were measured by quantitative Real-Time PCR (qRT-PCR) and western blot. Molecular docking results showed that AE could successfully dock with all the targets of NF-kappa B and P53 pathways, and the docking scores of most of the targets were equal to or higher than that of the corresponding ligands. Pathological sections showed AE could cause zebrafish liver lesions and the result of Sudan black B staining revealed that AE blackened the liver of zebrafish with Sudan black B. Then TUNEL assay showed that a large number of dense apoptotic signals were observed in AE group, mainly distributed in the liver and yolk sac of zebrafish. The results of qRT-PCR and western blot showed that AE increased the mRNA and protein expression levels of pro-inflammatory and pro-apoptotic targets in NF-kappa B and P53 pathways. AE could activate the NF-kappa B inflammatory pathway and the P53 apoptosis pathway, and its hepatotoxic mechanism was related to activation of NF-.B-P53 inflammation-apoptosis pathways.

Diatom test is the most commonly used method to diagnose drowning in forensic laboratories. However, microscopic examination and identification of diatom frustules is time-consuming and requires taxonomic expertise. At present, the identification of drowning is still a challenge in forensic casework. In this study, we developed a novel diatom microarray based on the detection of specific 18S rRNA gene fragments of diatom species. The array covers 169 diatom species which were documented as commonly found in a wide range of fresh waters in China. Diatom arrays were prepared from species specific oligonucleotide probes targeting to variable regions of the 18S rRNA gene. We also developed an auxiliary sample preparation method for isolation of diatom DNA from tissues, which enabled detection of diatom species in real forensic samples as well as environmental waters. We applied the diatom arrays to analyze six drowned cases and eight environmental samples. The diatom arrays showed much better sensitivity and more consistent results than those of the conventional SEM methods. We discovered major discrepancies between results generated by the diatom arrays and the routinely used SEM based diatom tests. We verified the results of our diatom arrays by species specific PCR and Sanger sequencing and found that the currently used SEM diatom test method has a serious deficiency in sensitivity due to high loss rate of frustules in the sample preparation procedure. We anticipate that the application of diatom arrays will transform current forensic practice of diagnosing drowning deaths.

The interaction between bone marrow stromal cells and leukemia cells is critical for the persistence and progression of leukemia, and this interaction may account for residual disease. However, the link between leukemia cells and their environment is still poorly understood. In our study, runt‑related transcription factor 3 (RUNX3) was identified as a novel target gene affected by As2O3 and involved in mesenchymal stem cell (MSC)‑mediated protection of leukemia cells from As2O3‑induced apoptosis. We observed induction of RUNX3 expression and the translocation of RUNX3 into the nucleus after As2O3 treatment in leukemia cells. In K562 chronic myeloid leukemia cells, downregulation of endogenous RUNX3 compromised As2O3‑induced growth inhibition, cell cycle arrest, and apoptosis. In the presence of MSC, As2O3‑induced expression of RUNX3 was reduced significantly and this reduction was modulated by CXCL12/CXCR4 signaling. Furthermore, overexpression of RUNX3 restored, at least in part, the sensitivity of leukemic cells to As2O3. We conclude that RUNX3 plays an important role in As2O3‑induced cellular responses and allows cells to overcome MSC‑mediated drug resistance. Therefore, RUNX3 is a promising target for therapeutic approaches to overcome MSC‑mediated drug resistance.

Carica papaya L. is an important economic crop worldwide and is used as a model plant for sex-determination research. To study the different flower sex types, we screened sex-related genes using alternative splicing sequences (AS-seqs) from a transcriptome database of the three flower sex types, i.e., males, females, and hermaphrodites, established at 28 days before flowering using 15 bacterial artificial chromosomes (BACs) of C. papaya L. After screening, the cDNA regions of the three sex-related loci, including short vegetative phase-like (CpSVPL), the chromatin assembly factor 1 subunit A-like (CpCAF1AL), and the somatic embryogenesis receptor kinase (CpSERK), which contained eight sex-related single-nucleotide polymorphisms (SNPs) from the different sex types of C. papaya L., were genotyped using high-resolution melting (HRM). The three loci were examined regarding the profiles of the third whorl, as described below. CpSVPL, which had one SNP associated with the three sex genotypes, was highly expressed in the male and female sterile flowers (abnormal hermaphrodite flowers) that lacked the fourth whorl structure. CpCAF1AL, which had three SNPs associated with the male genotype, was highly expressed in male and normal hermaphrodite flowers, and had no AS-seqs, whereas it exhibited low expression and an AS-seqs in intron 11 in abnormal hermaphrodite flowers. Conversely, carpellate flowers (abnormal hermaphrodite flowers) showed low expression of CpSVPL and AS-seqs in introns 5, 6, and 7 of CpSERK, which contained four SNPs associated with the female genotype. Specifically, the CpSERK and CpCAF1AL loci exhibited no AS-seq expression in the third whorl of the male and normal hermaphrodite flowers, respectively, and variance in the AS-seq expression of all other types of flowers. Functional mapping of the third whorl of normal hermaphrodites indicated no AS-seq expression in CpSERK, low CpSVPL expression, and, for CpCAF1AL, high expression and no AS-seq expression on XYh-type chromosomes.

Gender is one of the factors influencing the intestinal microbial composition in mammals, but whether fish also have gender-specific intestinal microbial patterns remains unknown. In this decade, endocrine disrupting chemicals in surface and ground water of many areas and increasing observation of freshwater male fish displaying female sexual characteristics have been reported. Here we identified the difference in intestinal microbiota between male and female zebrafish, and revealed the influence of endocrine disrupting chemicals on zebrafish intestinal microbiota by using high-throughput sequencing. The results indicated that Fusobacteria, Bacteroidetes and Proteobacteria were dominant in the gut of zebrafish and there were no obvious gender-specific intestinal microbial patterns. Two endocrine disrupting chemicals, Estradiol (E2) and Bisphenol A (BPA), were selected to treat male zebrafish for 5 weeks. E2 and BPA increased vitellogenin expression in the liver of male zebrafish and altered the intestinal microbial composition with the abundance of the phylum CKC4 increased significantly. Our results suggested that because of the developmental character and living environment, gender did not influence the assembly of intestinal microbiota in zebrafish as it does in mammals, but exposure extra to endocrine disrupting chemicals disturbed the intestinal microbial composition, which may be related to changes in host physiological metabolism.

p53 is one of the most widely investigated molecular markers in bladder cancer and its polymorphisms have been related to individual cancer risks. The objective of this study was to explore the association of p53 codon 72 polymorphism with susceptibility and clinicopathologic characteristics of bladder cancer in a Chinese population.We investigated the impact of p53 codon 72 polymorphism in a hospital-based case-control study of bladder cancer. We tested peripheral blood samples from 120 patients with bladder cancer and 120 healthy individuals of similar age and from the same geographical region. The polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism assay.There was an association between smoking status and bladder cancer (odds ratio [OR] = 2.25; 95% confidence interval [95% CI] = 1.31, 3.87; p = 0.003). Patients with bladder cancer had a significantly lower frequency of Arg/Arg (OR = 0.53; 95% CI = 0.31, 0.89; p = 0.02) and Arg allele (OR = 0.66; 95% CI = 0.45, 0.95; p = 0.03) than controls. Patients with invasive bladder cancer had a significantly lower frequency of Arg/Arg (OR = 0.29; 95% CI = 0.10, 0.88; p = 0.03) than those with superficial bladder cancer. When stratifying by the grade and histological type of bladder cancer, we found no statistical association.These data suggest that the p53 codon 72 Arg/Arg genotype and Arg allele are associated with a lower risk of bladder cancer in Chinese population.

The ferric enterobactin receptor CfrA not only is responsible for high-affinity iron acquisition in Campylobacter jejuni but also is essential for C. jejuni colonization in animal intestines. In this study, we determined the feasibility of targeting the iron-regulated outer membrane protein CfrA for immune protection against Campylobacter colonization. Alignment of complete CfrA sequences from 15 Campylobacter isolates showed that the levels of amino acid identity for CfrA range from 89% to 98%. Immunoblotting analysis using CfrA-specific antibodies demonstrated that CfrA was dramatically induced under iron-restricted conditions and was widespread and produced in 32 Campylobacter primary strains from various sources and from geographically diverse areas. The immunoblotting survey results were highly correlated with the results of an enterobactin growth promotion assay and a PCR analysis using cfrA -specific primers. Inactivation of the cfrA gene also impaired norepinephrine-mediated growth promotion, suggesting that CfrA is required for C. jejuni to sense intestinal stress hormones during colonization. Complementation of the cfrA mutant with a wild-type cfrA allele in trans fully restored the production and function of CfrA. A growth assay using purified anti-CfrA immunoglobulin G demonstrated that specific CfrA antibodies could block the function of CfrA, which diminished ferric enterobactin-mediated growth promotion under iron-restricted conditions. The inhibitory effect of CfrA antibodies was dose dependent. Immunoblotting analysis also indicated that CfrA was expressed and immunogenic in chickens experimentally infected with C. jejuni . Amino acid substitution mutagenesis demonstrated that R327, a basic amino acid that is highly conserved in CfrA, plays a critical role in ferric enterobactin acquisition in C. jejuni. Together, these findings strongly suggest that CfrA is a promising vaccine candidate for preventing and controlling Campylobacter infection in humans and animal reservoirs.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a major role in integrin signaling pathways. Although cardiovascular defects were observed in FAK total KO mice, the embryonic lethality prevented investigation of FAK function in the hearts of adult animals. To circumvent these problems, we created mice in which FAK is selectively inactivated in cardiomyocytes (CFKO mice). We found that CFKO mice develop eccentric cardiac hypertrophy (normal LV wall thickness and increased left chamber dimension) upon stimulation with angiotensin II or pressure overload by transverse aortic constriction as measured by echocardiography. We also found increased heart/body weight ratios, elevated markers of cardiac hypertrophy, multifocal interstitial fibrosis, and increased collagen I and VI expression in CFKO mice compared with control littermates. Spontaneous cardiac chamber dilation and increased expression of hypertrophy markers were found in the older CFKO mice. Analysis of cardiomyocytes isolated from CFKO mice showed increased length but not width. The myocardium of CFKO mice exhibited disorganized myofibrils with increased nonmyofibrillar space filled with swelled mitochondria. Last, decreased tyrosine phosphorylation of FAK substrates p130Cas and paxillin were observed in CFKO mice compared with the control littermates. Together, these results provide strong evidence for a role of FAK in the regulation of heart hypertrophy in vivo.

Summary Mast cells have recently been found to be a major player in the host defence against bacterial infection through secretion of potent mediators. Identification of bacteria-induced mast cell mediators and intracellular signalling molecules involved during bacterial infection remains a major area of investigation. Recently we found an active interaction between mast cells and Pseudomonas aeruginosa bacteria. To further characterize specific genes in mast cells modulated by P. aeruginosa, we used a new approach for the study of mast cell–bacteria interaction; the suppression subtractive hybridization (SSH). SSH approach does not require a prerequisite knowledge of target genes and does not rely on the availability of the assay reagents for the specific genes. Using SSH, 94 clones were randomly selected from the subtracted cDNA library for differential screening leading to the identification of 14 P. aeruginosa–up-regulated transcripts. Sequence analysis revealed that expression of IL-1, IL-8 and CCL4 was increased by human mast cells after P. aeruginosa infection. Increased production of IL-1, IL-8 and CCL4 was confirmed at the protein levels. In addition, sequence analysis of the clones also suggests that ribosomal protein S3 and cytochrome b as well as additional 4 uncharacterized genes may potentially be involved in P. aeruginosa pathogenesis. Thus, SSH is an effective approach by identifying potential molecular targets for the study of mechanisms involved in P. aeruginosa and mast cell interaction.

Background An animal model for HBV that more closely approximates the disease in humans is needed. The tree shrew (Tupaia belangeri) is closely related to primates and susceptible to HBV. We previously established that neonatal tree shrews can be persistently infected with HBV in vivo, and here present a six year follow-up histopathological study of these animals. Methods Group A consists of six tree shrews with persistent HBV infection, group B consists of three tree shrews with suspected persistent HBV infection, while group C consists of four tree shrews free of HBV infection. Serum and liver tissues samples were collected periodically from all animals. HBV antigen and HBV antibodies were detected by ELISA and/or TRFIA. HBV DNA in serum and in liver biopsies was measured by FQ-PCR. Liver biopsies were applied for general histopathologic observation and scoring, immunohistochemical detections of HBsAg and HBcAg, and ultrastructural observation with electron microscope technique. Results Hydropic, fatty and eosinophilic degeneration of hepatocytes, lymphocytic infiltration and hyperplasia of small bile ducts in the portal area were observed in group A. One animal infected with HBV for over six years showed multiple necrotic areas which had fused to form bridging necrosis and fibrosis, and megalocytosis. The hepatic histopathological scores of group A were higher than those of group B and C. The histopathological score correlated positively with the duration of infection. Conclusions Hepatic histopathological changes observed in chronically HBV-infected tree shrews are similar to those observed in HBV-infected humans. The tree shrew may represent a novel animal model for HBV infection.

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130Cas and its subsequent binding to several SH2 domains, which depended on both the p130Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130Cas further increased cell migration on FN and coexpression of the p130Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.

UNLABELLED PREMISE OF THE STUDY Microsatellite loci were developed in Launaea arborescens, an endangered and medicinal Asteraceae species in North Africa, for further investigation of its conservation genetics. • METHODS AND RESULTS We isolated and characterized 10 polymorphic and nine monomorphic microsatellite loci from L. arborescens using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). For the 10 polymorphic loci, the number of alleles detected per locus varied from two to six, and the observed and expected heterozygosities ranged from 0.000 to 0.833 and 0.059 to 0.713, respectively. • CONCLUSIONS The polymorphic markers provide a useful tool for conservation genetics studies of L. arborescens, including analysis of mating system, estimating gene flow, and identifying discrete genetic units within the species.

Background and aim The sensitivity and specificity of biomarkers and scoring systems used for predicting fatality of severe sepsis patients remain unsatisfactory. This study aimed to determine the prognostic value of circulating plasma DNA levels in severe septic patients presenting at the Emergency Department (ED). Methods Sixty-seven consecutive patients with severe sepsis and 33 controls were evaluated. Plasma DNA levels were estimated by real-time quantitative polymerase chain reaction assay using primers for the human β-hemoglobin and ND2 gene. The patients’ clinical and laboratory data on admission were analyzed. Results The median plasma nuclear and mitochondria DNA levels for severe septic patients on admission were significantly higher than those of the controls. The mean plasma nuclear DNA level on admission correlated with lactate concentration (γ = 0.36, p = 0.003) and plasma mitochondrial DNA on admission (γ = 0.708, p 0.001). Significant prognostic factors for fatality included mechanical ventilation within the first 24 hours (p = 0.013), mean sequential organ failure assessment (SOFA) score on admission (p = 0.04), serum lactate (p < 0.001), and both plasma nuclear and mitochondrial DNA on admission (p < 0.001). Plasma mitochondrial DNA was an independent predictor of fatality by stepwise logistic regression such that an increase by one ng/mL in level would increase fatality rate by 0.7%. Conclusion Plasma DNA has potential use for predicting outcome in septic patients arriving at the emergency room. Plasma mitochondrial DNA level on admission is a more powerful predictor than lactate concentration or SOFA scores on admission.

To assess the relationship between folate biomarkers levels and IGF2 imprinting status among second trimester pregnant Chinese women.Three hundred women in their second trimester were screened by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for analysis of IGF2 imprinting status. Statistical differences of folate biomarkers levels were calculated in the groups with different imprinting status.Of the 300 women analyzed, 133 (44.33%) cases were homozygous allele with A, 3 (1.00%) cases were homozygous allele with G, while 164 (54.67%) cases were heterozygous with A and G, qualifying them for analysis of loss of imprinting (LOI). Among the 164 cases undergoing LOI analysis, 44 (26.83%) were IGF2 LOI cases, while 120 (73.17%) were IGF2 retention of imprinting (ROI) ones. The mean level of serum folate, vitamin B12 and tHcy was 28.46 +/- 10.74 ng/mL, 380.20 +/- 206.13 pg/mL, 14.24 +/- 6.34 micromol/L among women with IGF2 ROI, and 30.89 +/- 9.97 ng/mL, 394.28 +/- 195.92 pg/ mL, and 13.12 +/- 6.23 micromol/L among women with IGF2 LOI, respectively.No significant difference of folate biomarkers levels was observed between IGF2 ROI and IGF2 LOI groups.

CmeABC, a multidrug efflux system in Campylobacter jejuni, plays an important role in the resistance to different antimicrobials and toxic compounds. Although this efflux system has been well characterized in C. jejuni and to a less extent in C. coli, it is unknown if CmeABC homologs are functional in other Campylobacter spp. In this study, the cmeABC homologs were identified and functionally characterized in five Campylobacter species including C. jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus. Our results indicated that cmeABC is present in all five Campylobacter spp. and the genomic organization of this efflux operon is similar among the Campylobacter spp. Insertional mutagenesis of cmeB increased the susceptibilities of all the five Campylobacter spp. to structurally diverse antimicrobials. Together, these results indicated that the CmeABC efflux system is conserved at both the genomic and functional levels in all five Campylobacter spp. examined in this study, further highlighting the significant role of CmeABC in Campylobacter pathobiology.

To observe the hepatitis B virus (HBV) replication in the tree shrews that were inoculated with HBV at neonatal period.Six new-born tree shrews were inoculated with human HBV positive serum. Blood samples and liver biopsies were collected at different time points after inoculation. The HBV infection markers were tested by nested polymerase chain reaction (nPCR), fluorescence quantitative polymerase chain reaction (FQ-PCR), Southern blot, ELISA and immunohistochemistry staining. The liver tissues were observed under electron and light microscope.48 weeks after inoculation, HBV DNA and HBV cccDNA were detected in the serum and liver samples of three animals (number 1, 2 and 6) by nPCR. The copy-numbers of HBV DNA detected by FQ-PCR in their serum and liver samples were 103 and-104/ml respectively,and the total DNA in 1microg liver tissue was 107-108. Southern blot indicated that HBV replication intermediates such as HBV cccDNA and HBV ssDNA was detectable in liver tissues. HBsAg was detected by ELISA, and immunohistochemical staining showed a gradual increase of HBsAg-positive liver cells. High copy number of HBV DNA and suspected HBV EM particles could be detected in the liver samples from one of the three animals that have survived more than 2 years after inoculation. The other three animals showed low HBV DNA copy number, and the rest of the signs of HBV infection were negative or transiently positive.Neonatal tree shrews can be infected with human HBV. HBV can replicate inside the liver cells of tree shrew.

A study was conducted to determine if homologous integrons occurred in Escherichia coli and Salmonella spp. within livestock production sites in the United States and Thailand suggesting transfer of genetic resistance elements between those organisms. Fecal samples were collected via rectal swabs from live swine in the United States and Thailand, and cloacal swabs from live chickens in Thailand, and killed chickens at a U.S. abattoir. Isolates were derived only from farms harboring both Salmonella and E. coli, resulting in the inclusion of 571 E. coli and 98 Salmonella isolates derived from both livestock species in the United States and Thailand. Class 1 integron variable regions were detected using polymerase chain reaction targeting 5' and 3' conserved sequences. When integron-positive E. coli and Salmonella from the same farm had identical amplicon patterns, polymerase chain reaction products were sequenced to determine homology. Nine integron amplicons, with sizes ranging from 0.5 to 2.5 kb, were observed in bacterial isolates, and we found a single swine farm in Thailand from which identical amplicons were observed in both E. coli and Salmonella. Sequence analysis revealed a 1.0 kb amplicon common to both bacteria contained an aadA1 gene cassette encoding aminoglycoside 3'-adenyltransferase, conferring resistance to streptomycin and spectinomycin. A 2.0 kb amplicon was also found in both types of bacteria from that farm, containing an aadA5 gene encoding aminoglycoside 3'-adenyltransferase, an additional reading frame, orfD, with unknown function, and a dfrA17 gene encoding dihydrofolate reductase, conferring resistance to trimethoprim. Further analyses determined the amplicons were contained on plasmid DNA in both E. coli and Salmonella, and a plasmid of similar size was identified in both species and was found to harbor the class 1 integron. Our results indicate that while in most cases, integrons of coexisting E. coli and Salmonella differed, identical integron amplicons were found in those species from a single swine farm in Thailand, suggesting horizontal transfer between these two organisms may have occurred on-farm.

The aim of this study was to identify genetic loci required for polymyxin (PM) resistance in Campylobacter jejuni using an efficient in vivo random mutagenesis system. PM has been widely used as a model peptide to examine mechanisms of bacterial resistance to antimicrobial peptides (AMPs), the major effectors of host innate immunity and also candidates for a new generation of antibiotics. In this study, a commercially available transposon mutagenesis approach (EZ-Tn5KAN-2Transposome; Epicentre, Madison, WI) was evaluated and used to systematically identify Campylobacter mutants with increased susceptibility to PM. This simple, yet efficient, transposon mutagenesis approach identified 12 mutants representing seven different genes of C. jejuni 81-176 involved in acquired PM resistance. Backcrossing of the transposon mutations into the parent strain confirmed that the PM-sensitive phenotype in each mutant was linked to the gene with a specific transposon insertion. The genes are identified as being involved in the synthesis of cell-surface carbohydrates, modification of intracellular targets, signal transduction, and modulation of transmembrane potential. The mutant with the highest susceptibility to PM contains a transposon insertion in a putative galU gene that is essential for production of uridine diphosphate glucose (UDP)-glucose, a precursor required for lipooligosaccharide (LOS) synthesis. LOS analysis by tricine SDSPAGE showed significant truncation of the LOS core structure in the galU mutant. Susceptibility assays also indicated that GalU contributed C. jejuni resistance to some natural AMPs. Complementation of the galU mutant in trans fully restored LOS synthesis and resistance to the levels of the parent strain. Together, these results define seven C. jejuni genetic loci that will be useful for characterizing the molecular basis of Campylobacter resistance to PM and natural AMPs, and also highlight the usefulness of the in vivo mutagenesis approach for systematic characterization of functionally important Campylobacter genes.

Tetracyclines and bacitracin are used extensively as a growth promotant and a therapeutic, respectively in livestock. In this study, we test whether there is an interaction between bacitracin and tetracycline that can select for an increase in microbial tetracycline sensitivity, using Escherichia coli and Salmonella as a model. We found via in vitro studies that bacitracin at sublethal concentrations preferentially selects for outgrowth of tetracycline-sensitive bacteria from among a population of tetracycline-resistant and tetracycline-sensitive E. coli and Salmonella strains. Most of the bacterial strains employed in our study were shown by PCR to possess the tetracycline resistance gene tet(A) or tet(C), either of whose activity is associated with tetracycline efflux. Bacitracin could be substituted for the lipophylic chelator, fusaric acid, used as a positive selective agent for tetracycline sensitivity. Together, these observations suggest that bacitracin-mediated selection for tetracycline sensitivity alters the function of tetracycline efflux proteins. Using bacitracin and tetracycline simultaneously may improve the effectiveness of the antibiotics, while decreasing the risk of selecting for a population of tetracycline-resistant microorganisms.

To identify the mayven-interacting proteins and study the effect of mayven on multiple sclerosis pathogenesis.The full length and four different fragments of mayven gene were amplified by polymerase chain reaction (PCR) with specific primers and were cloned into the yeast expression vector pDBLeu. Then, the recombinant plasmids were transformed into MAV203 yeast strain. The autonomous activation of their expression products was detected.The yeast expression vectors of mayven, which include a full-length and four different fragments, were constructed successfully. Full length P1, fragments P3 and P4 have no effect on the expression of HIS3 and LacZ gene, but fragments P7 and P8 do. The C-terminal of Mayven gene may contain a transcription activation domain.Full length P1, fragments P3 and P4 of the mayven gene can be used to screen the mayven-interacting proteins, but whether Mayven has transcriptional activation activity need to be studied.

Alpha-fetoprotein (AFP) is a marker often used to detect carcinoma cells in peripheral blood of patients with hepatocellular carcinoma (HCC). This study was to explore the correlation of perioperative AFP expression in peripheral blood to recurrence of HCC.Peripheral blood samples from 56 HCC patients, obtained perioperatively, were tested for the presence of AFP mRNA by nested reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan MGB probe real-time quantitative PCR. The peripheral blood samples from 15 patients with benign liver masses and cirrhosis, and 30 healthy volunteers were used as control.The positive rate of AFP mRNA in preoperative peripheral blood was significantly higher in HCC patients than in patients with benign liver masses and cirrhosis (42.9% vs. 13.3%, P=0.035) and healthy volunteers (42.9% vs. 10.0%,P=0.002). The expression of AFP mRNA in preoperative peripheral blood was significantly related to tumor vascular invasion, either observed macroscopically (P=0.029) or microscopically (P0.001). The 1-, 2-, and 3-year overall and disease-free survival rates were significantly higher in AFP-negative patients than in AFP-positive patients (P=0.003, P=0.039). Cox multivariate analysis showed that preoperative expression of AFP mRNA in peripheral blood was an independent factor for predicting recurrence (P=0.018). The positive rate of AFP mRNA in postoperative peripheral blood samples from HCC patients was 37.5%. The presence of AFP mRNA in postoperative peripheral blood was not related to any clinicopathologic factors, AFP mRNA levels in preoperative peripheral blood, and prognosis of the HCC patients.The presence of AFP mRNA in preoperative peripheral blood is related to tumor invasion of HCC, and may be a predictor maker of tumor recurrence.

We previously reported the selective killing of HER2-positive tumor cells by a class of immunoproapoptotic proteins containing single-chain antibody, translocation domain of Pseudomonas exotoxin A (domain II; PEA II), and constitutively active human apoptotic molecules. In this study, a novel class of antitumor immunoproapoptotic proteins was explored to mediate tumor-specific apoptosis both in vitro and in vivo. Three furin cleavage sequences, including a synthetic polyarginine tract, and two furin cleavable sequences from PEA and diphtheria toxin were respectively used to replace PEA II in the previously constructed immunoproapoptotic protein. When produced and secreted by the genetically modified Jurkat cells, the novel targeted proapoptotic proteins selectively bound to HER2, which is often overexpressed on tumor cell surface. Followed by receptor-mediated endocytosis and furin cleavage in the endosome, the recombinant proteins could translocate into the cytosol, leading to irreversible cell death. Moreover, delivery of these proteins by either i.m. plasmid injection or i.v. injection of plasmid-expressing Jurkat cells led to tumor regression and prolonged animal survival in a nude mouse xenograft tumor model, indicating in vivo antitumor activity of the recombinant proteins. We conclude that the new class of immunoproapoptotic proteins show comparable activity with PEA II–containing counterpart and provide an attractive therapeutic alternative as they contain much less exogenous fragments. [Cancer Res 2007;67(24):11830–39]

To express the TSO45W-4BX of Taenia solium in combination with CD58 as a molecular adjuvant for improving the protective efficacy of the TSO45W-4BX recombinant vaccine.TSO45W-4BX and porcine CD58 genes were amplified by PCR, using recombinant plasmids pGEM-4B and pGEM-CD58 as template respectively. The CD58 fragment was inserted into the recombinant plasmid pGEX-4T-1 with directly ligated TSO45W-4BX. The transformant was induced with IPTG and followed by identifying the integrity of the recombinant containing TS045W-4BX and porcine CD58 with PCR and sequencing. The products were analyzed by SDS-PAGE and Western blotting.The expression products of Mr 69,000 GST-4BX/CD58 and Mr 41,000 GST-4BX were present mainly in the form of inclusion bodies and soluble substance respectively, and both were recognized by sera of cysticercosis patients.The TSO45W-4BX co-expressed with porcine CD58 conserves its immune reactivity.

AIM: To probe into the genetic susceptibility of HLA-DRB1 alleles to esophageal carcinoma in Han Chinese in Hubei Province. METHODS: HLA-DRB1 allele polymorphisms were typed by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 42 unrelated patients with esophageal cancer and 136 unrelated normal control subjects and the associated HLA-DRB1 allele was measured by nucleotide sequence analysis with PCR.SAS software was used in statistics. RESULTS: Allele frequency (AF) of HLA-DRB1*0901 was significantly higher in esophageal carcinoma patients than that in the normal controls (0.2500 vs0.1397, P=0.028, the odds ratio 2.053, etiologic fraction 0.1282). After analyzed the allele nucleotide sequence of HLA-DRB1*0901 which approachs to the corresponded exon 2 sequence of the allele in genebank. There was no association between patients and controls in the rested HLA-DRB1 alleles. CONCLUSION: HLA-DRB1*0901 allele is more common in the patients with esophageal carcinoma than in the healthy controls, which is positively associated with the patients of Hubei Han Chinese. Individuals carrying HLA-DRB1*0901 may be susceptible to esophageal carcinoma.