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Objective: We investigated the epidemiological characteristics of drug resistance and virulence factors of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from paediatric patients in Shanghai. Methods: CRKP strains were consecutively collected between January and December in 2018. Antimicrobial susceptibility was determined by VITEK 2 compact. Polymerase chain reaction (PCR) was used to analyse drug resistance determinants, virulence genes and plasmid types. wzi sequencing and multilocus sequence typing was used to determine clonal relatedness. Results: Among 172 CRKP strains, blaKPC-2 and blaNDM-5 were the predominant carbapenemase genes. Compared with NDM-5, KPC-2 producers showed higher resistance rates to fluoroquinolones and aminoglycosides. The majority of KPC-2 producers belonged to KL64-ST11 background, while NDM-5 producers were mainly identified as KL62-ST48. Plasmid typing shown that IncF and IncFIB were the most prevalent plasmids in KPC-2 producers and IncX3 was widely spread in NDM-5-KP. Thirty-seven isolates carried various hypervirulence genes and the profiles of these genes showed high diversity. Conclusions: The predominant carbapenemase of CRKP strains from paediatric patients in Shanghai were KPC-2 and NDM-5. KL47-ST11 KPC-2-KP and KL62-ST48 NDM-5-KP were representative clonal lineages. Although not prevalent, hypervirulence associated genes have begun to spread. Active long-term surveillance should be performed in both drug resistance characteristics and virulence factors. Keywords: Klebsiella pneumoniae, Carbapenemase, Virulence, Epidemiology, Paediatric

This study investigated the antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) isolated from cases of subclinical bovine mastitis in China, as well as resistance mechanisms and virulence genes encoding adhesins and toxins. We determined antimicrobial susceptibility using the disk diffusion method, and analyzed resistance, adhesin, and toxin genes using PCR. We confirmed MRSA in 73 of 498 (14.7%) Staph. aureus isolates recovered from subclinical mastitic milk samples. All isolates were positive for mecA. The MRSA isolates showed high resistance to penicillin (100.0%), gentamicin (100.0%), and tetracycline (98.6%). All MRSA isolates harbored resistance genes blaZ (penicillin), aacA/aphD (gentamicin), and tetM (alone or in combination with tetK, tetracycline). Moreover, all isolates carried the adhesin genes fnbpA, clfA, clfB, cna, sdrE, and map/eap, and most carried sdrC (98.6%), sdrD (95.9%), bbp (94.5%), and ebpS (80.8%). The toxin genes seh, hla, and hld were present in all isolates, and most isolates carried sea (71.2%), seg (84.9%), sei (82.2%), lukE-lukD (97.3%), and hlg (72.6%). These findings of high-level resistance to antimicrobials commonly used in dairy cattle should lead to calls for antibiogram analysis before antimicrobial therapy. The high frequency of adhesin and toxin genes in MRSA indicates their potential virulence in bovine mastitis in China.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and has poor outcomes. However, the potential molecular biological process underpinning the occurrence and development of HCC is still largely unknown. The purpose of this study was to identify the core genes related to HCC and explore their potential molecular events using bioinformatics methods. HCC-related expression profiles GSE25097 and GSE84005 were selected from the Gene Expression Omnibus (GEO) database, and the differentially expressed genes (DEGs) between 306 HCC tissues and 281 corresponding noncancerous tissues were identified using GEO2R online tools. The protein-protein interaction network (PPIN) was constructed and visualized using the STRING database. Gene Ontology (GO) and KEGG pathway enrichment analyses of the DEGs were carried out using DAVID 6.8 and KOBAS 3.0. Additionally, module analysis and centrality parameter analysis were performed by Cytoscape. The expression differences of key genes in normal hepatocyte cells and HCC cells were verified by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR). Additionally, survival analysis of key genes was performed by GEPIA. Our results showed that a total of 291 DEGs were identified including 99 upregulated genes and 192 downregulated genes. Our results showed that the PPIN of HCC was made up of 287 nodes and 2527 edges. GO analysis showed that these genes were mainly enriched in the molecular function of protein binding. Additionally, KEGG pathway analysis also revealed that DEGs were mainly involved in the metabolic, cell cycle, and chemical carcinogenesis pathways. Interestingly, a significant module with high centrality features including 10 key genes was found. Among these, CDK1, NDC80, HMMR, CDKN3, and PTTG1, which were only upregulated in HCC patients, have attracted much attention. Furthermore, qRT-PCR also confirmed the upregulation of these five key genes in the normal human hepatocyte cell line (HL-7702) and HCC cell lines (SMMC-7721, MHCC-97L, and MHCC-97H); patients with upregulated expression of these five key genes had significantly poorer survival and prognosis. CDK1, NDC80, HMMR, CDKN3, and PTTG1 can be used as molecular markers for HCC. This finding provides potential strategies for clinical diagnosis, accurate treatment, and prognosis analysis of liver cancer.

To evaluate the performance of polymerase chain reaction (PCR)-free whole genome sequencing (WGS) for clinical diagnosis, and thereby revealing how experimental parameters affect variant detection.Five NA12878 samples were sequenced using MGISEQ-2000. NA12878 samples underwent WGS with differing deoxyribonucleic acid (DNA) input and library preparation protocol (PCR-based vs PCR-free protocols for library preparation). The depth of coverage and genotype quality of each sample were compared. The performance of each sample was measured for sensitivity, coverage of depth and breadth of coverage of disease-related genes, and copy number variants. We also developed a systematic WGS pipeline (PCR-free) for the analysis of 11 clinical cases.In general, NA12878-2 (PCR-free WGS) showed better depth of coverage and genotype quality distribution than NA12878-1 (PCR-based WGS). With a mean depth of ∼40×, the sensitivity of homozygous and heterozygous single nucleotide polymorphisms (SNPs) of NA12878-2 showed higher sensitivity (>99.77% and >99.82%) than NA12878-1, and positive predictive value exceeded 99.98% and 99.07%. The sensitivity and positive predictive value of homozygous and heterozygous indels for NA12878-2 (PCR-free WGS) showed great improvement than NA128878-1. The breadths of coverage for disease-related genes and copy number variants are slightly better for samples with PCR-free library preparation protocol than the sample with PCR-based library preparation protocol. DNA input also influences the performance of variant detection in samples with PCR-free WGS. All the 19 previously confirmed variants in 11 clinical cases were successfully detected by our WGS pipeline (PCR free).Different experimental parameters may affect variant detection for clinical WGS. Clinical scientists should know the range of sensitivity of variants for different methods of WGS, which would be useful when interpreting and delivering clinical reports.

Peculiar findings of orofacial actinomycosis mimicking the clinical appearance of a tumor of the upper gingiva are reported. An 83-year-old man with bleeding of the gingiva visited our hospital. The clinical diagnosis was a benign gingival tumor, and the lesion was surgically removed. Histologically, the excised specimens showed an ulcerative granuloma lesion covered by bacterial colonies consisting of club-shaped filaments. DNA samples were extracted from paraffin sections and examined by polymerase chain reaction (PCR) for Actinomyces species. The PCR products examined by direct DNA sequencing demonstrated the presence of Actinomyces israelii. Finally, a pathological diagnosis was made of a pyogenic granuloma associated with actinomycosis. The PCR method aided the early and exact diagnosis of the paraffin-embedded sample of oral mucosal infectious diseases including actinomycosis. Keywords: Actinomyces israelii, actinomycosis, DNA sequence, nested PCR, pyogenic granuloma

Background Mycoplasma-related vaginitis gradually has been growing as a threat in adults-genitourinary infection contributes to funisitis, spontaneous abortion, and low birth weight. Until now, use of loop-mediated isothermal amplification (LAMP) to detect Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), or Mycoplasma genitalium (MG) has been reported by some researchers. However, previous studies focused on purified DNA as the template for LAMP assay, which is usually extracted via commercial kit. Methods We developed a LAMP assay for rapid detection of UU, MH, and MG genital mycoplasmas using a simple boiling method for DNA extraction, in a cohort of pregnant women with mycoplasma-related vaginitis. We monitored amplicons with the naked eye using SYBR Green I. Results The cohort in our study showed a prevalence of 22.6% in pregnant women, as detected by UU-LAMP assay. Compared to the polymerase chain reaction (PCR) test with purified DNA, the sensitivity of the UU-LAMP in clinical specimens with crude DNA was 87.5% (95% confidence interval [CI], 64.6%->99.9). For crude DNA specimens, UU-LAMP was more sensitive and reliable than PCR, with a higher agreement rate (96.8%) and Youden index value (0.88). Conclusions As a point-of-care test, LAMP is a useful, specific, and efficient way to detect genital mycoplasmas in resource-limited settings, especially for crude DNA.

Supplemental Digital Content is available in the text, The aim of this study was toexplore the long non-coding RNA (lncRNA) expression pattern of non-small cell lung cancer (NSCLC) on a genome-wide scale and investigate their potential biological function in NSCLC. LncRNAs were investigated in 6 pairs of NSCLC and matched adjacent non-tumor lung tissues (NTL) by microarray. A validation cohort was obtained from The Cancer Genome Atlas (TCGA) database and the effect of LINC01614 on diagnosis and prognosis in NSCLC was analyzed. Gene set enrichment analysis (GSEA) was used to predict the potential molecular mechanism of LINC01614, one identified lncRNA. A total of 1392 differentially expressed lncRNAs were identified. LINC01614 was the most aberrantly expressed lncRNA in NSCLC compared with NTL. We confirmed the significantly upregulated LINC01614 in NSCLC patients from TCGA database. Furthermore, in TCGA database, LINC01614 was significantly upregulated in both adenocarcinoma and squamous cell carcinoma. And high expression of LINC01614 indicated poor overall survival of NSCLC patients. A sensitivity of 93% was calculated conditional on a high specificity of 95% for the discrimination of NSCLC tissues from normal tissues. Furthermore, the expression levels of LINC01614 were associated with the stage of tumor, but had no relationship with age and sex. Additionally, GSEA found that LINC01614 might be involved in TGF-β-, P53-, IGF-IR-mediated, Wnt and RTK/Ras/MAPK signaling pathways. lncRNAs may play key roles in the development of NSCLC. LINC01614 is the most aberrantly expressed lncRNA in NSCLC tissues in our experiment and is also significantly differentially expressed in NSCLC patients from TCGA database. LINC01614 could be a prognostic indicator and has the potential to be a diagnostic biomarker of NSCLC.

PhoH is a host-derived auxiliary metabolic gene that can be used as a new biomarker for surveying phage diversity in marine and paddy waters. However, the applicability of this gene in other environments has not been addressed. In this paper, we surveyed the phoH gene in four wetland sediments in northeast China. DNA was extracted directly from sediments and used for PCR amplification with the degenerate primers vPhoHf and vPhoHr. In total, 44 and 58 phoH sequences were identified as belonging to bacteria and phages, respectively, suggesting that this primer set is not highly specific to the phage phoH gene. A BLASTp search showed that the 58 phage phoH sequences had the highest identity to the known viral sequences, ranging from 48% to 100%. Phylogenetic analysis showed that all phage sequences from wetlands distributed into the previously designated Groups 2, 3, 4 and 6. In addition, two new subgroups, Groups 2c and 4c, which contained sequences exclusively from wetlands, were detected in this study. Nonmetric multidimensional scaling analysis showed that the phage phoH assemblage from a coastal wetland was similar to that in marine environments, while the phage phoH assemblage from a lake wetland was similar to that in paddy waters. These findings indicated that different types of wetlands had distinct phage phoH compositions.

Extensive insecticide use has led to the resistance of mosquitoes to these insecticides, posing a major barrier to mosquito control. Previous Solexa high-throughput sequencing ofCulex pipiens pallensin the laboratory has revealed that the abundance of a novel microRNA (miRNA), miR-13664, was higher in a deltamethrin-sensitive (DS) strain than a deltamethrin-resistant (DR) strain. Real-time quantitative PCR revealed that the miR-13664 transcript level was lower in the DR strain than in the DS strain. MiR-13664 oversupply in the DR strain increased the susceptibility of these mosquitoes to deltamethrin, whereas inhibition of miR-13664 made the DS strain more resistant to deltamethrin. Results of bioinformatic analysis, quantitative reverse-transcriptase polymerase chain reaction, luciferase assay and miR mimic/inhibitor microinjection revealedCpCYP314A1to be a target of miR-13664. In addition, downregulation ofCpCYP314A1expression in the DR strain reduced the resistance of mosquitoes to deltamethrin. Taken together, our results indicate that miR-13664 could regulate deltamethrin resistance by interacting withCpCYP314A1, providing new insights into mosquito resistance mechanisms.

The mosquito, Culex pipiens pallens (L.), is an important vector of encephalitis and filariasis in northern China. The control of these mosquitoes occurs primarily via the use of pyrethroid insecticides, such as deltamethrin. The widespread and improper application of pyrethroid has resulted in the evolution of pyrethroid resistance amongst many mosquito populations, including Cx. pipiens pallens. Previous studies using high-throughput transcriptome sequencing have identified that the venom allergen 5 gene is differentially expressed between deltamethrin-susceptible and deltamethrin-resistant Cx. pipiens pallens. In this study, quantitative real-time polymerase chain reaction analyses revealed that venom allergen 5 was significantly overexpressed in adult females of both deltamethrin-resistant laboratory populations and two field populations. The transcriptional level of venom allergen 5 in the laboratory populations was elevated as the levels of deltamethrin resistance increased. Full-length cDNAs of the venom allergen 5 gene were cloned from Cx. pipiens pallens, and contained an open reading frame of 765 bp, encoding a protein with 254 amino acids. The deduced amino acid sequence shared 100% identity with the ortholog in Culex quinquefasciatus Say. The overexpression of venom allergen 5 decreased the susceptibility of mosquito cells to deltamethrin, while knockdown of this gene by RNAi increased the susceptibility of mosquitoes to deltamethrin. This study provides the first evidence of the association between the venom allergen 5 gene and deltamethrin resistance in mosquitoes.

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8 DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense., Author Summary TLR signaling induces expression of key inflammatory cytokines and pro-survival factors that facilitate control of microbial infection. TLR signaling can also engage cell death pathways through activation of enzymes known as caspases. Caspase-8 activates apoptosis in response to infection by pathogens that interfere with NF-κB signaling, including Yersinia, but has also recently been linked to control of inflammatory gene expression. Pathogenic Yersinia can cause severe disease ranging from gastroenteritis to plague. While caspase-8 mediates cell death in response to Yersinia infection as well as other signals, its precise role in gene expression and host defense during in vivo infection is unknown. Here, we show that caspase-8 activity promotes cell-intrinsic cytokine expression, independent of its role in cell death in response to Yersinia infection. Our studies further demonstrate that caspase-8 enzymatic activity plays a previously undescribed role in ensuring optimal TLR-induced gene expression by innate cells during bacterial infection. This work sheds new light on mechanisms that regulate essential innate anti-bacterial immune defense.

The epididymis and efferent ducts play major roles in sperm maturation, transport, concentration and storage by reabsorbing water, ions and proteins produced from seminiferous tubules. Gpr48-null male mice demonstrate reproductive tract defects and infertility. In the present study, we found that estrogen receptor alpha (ERalpha) was dramatically reduced in the epididymis and efferent ducts in Gpr48-null male mice. We further revealed that ERalpha could be upregulated by Gpr48 activation via the cAMP/PKA signaling pathway. Moreover, we identified a cAMP responsive element (Cre) motif located at -1307 to -1300 bp in the ERalpha promoter that is able to interact with Cre binding protein (Creb). In conclusion, Gpr48 participates in the development of the male epididymis and efferent ducts through regulation of ERalpha expression via the cAMP/PKA signaling pathway.

The progression of colorectal cancer (CRC) is a multistep process and metastatic CRC is always incurable; consequently, CRC is the leading cause of cancer-related deaths. There is therefore an urgent need for identifying useful biomarkers with enough sensitivity and specificity to detect this disease at early stages, which will significantly reduce the mortality for this malignancy. In this study, we performed an integrating analysis of different RNA-Seq data sets to find new candidate biomarkers for diagnosis, prognosis and as therapeutic targets for this malignancy, as well as to elucidate the molecular mechanisms of CRC carcinogenesis. We identified 883 differentially expressed genes (DEGs) across the studies between CRC and normal control (NC) tissues by combining five RNA-Seq data sets. Gene function analysis revealed high correlation with carcinogenesis. The top 10 most significantly DEGs were further evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in both rectal cancer (RC) and colon cancer (CC), and the results matched well with integrating data, suggesting that the method of integrating analysis of different RNA-seq data sets is acceptable. Therefore, integrating analysis of different RNA-seq data sets may be a useful way to overcome the limitation of small sample size in a single RNA-seq study. In addition, our study showed that some genes, such as SIM2, ADAMTS6, FOXD4L4 and DNAH5, may have an important role in the development of CRC, which could be applied for diagnosis, prognosis and as therapy for this malignancy. Our findings would also help to understand the pathology of CRC.

Background Targeted next-generation sequencing (NGS) is a cost-effective approach for rapid and accurate detection of genetic mutations in patients with suspected genetic disorders, which can facilitate effective diagnosis. Methodology/Principal Findings We designed a capture array to mainly capture all the coding sequence (CDS) of 2,181 genes associated with 561 Mendelian diseases and conducted NGS to detect mutations. The accuracy of NGS was 99.95%, which was obtained by comparing the genotypes of selected loci between our method and SNP Array in four samples from normal human adults. We also tested the stability of the method using a sample from normal human adults. The results showed that an average of 97.79% and 96.72% of single-nucleotide variants (SNVs) in the sample could be detected stably in a batch and different batches respectively. In addition, the method could detect various types of mutations. Some disease-causing mutations were detected in 69 clinical cases, including 62 SNVs, 14 insertions and deletions (Indels), 1 copy number variant (CNV), 1 microdeletion and 2 microduplications of chromosomes, of which 35 mutations were novel. Mutations were confirmed by Sanger sequencing or real-time polymerase chain reaction (PCR). Conclusions/Significance Results of the evaluation showed that targeted NGS enabled to detect disease-causing mutations with high accuracy, stability, speed and throughput. Thus, the technology can be used for the clinical diagnosis of 561 Mendelian diseases.

To elucidate the role of hepatitis G virus (HGV) infection in chronic non-A-E hepatitis and sequence the partial NS5 genome of HGV isolated from the serum of a Chinese patient with chronic non-A-E hepatitisSerum samples of patients with chronic non-A-E hepatitis were collected and total nucleic acids were extracted and subjected to reverse transcriptase-nested-polymerase chain reaction (RT-nested-PCR) using primers from the putative NS5 region of HGV genome. Then, 994bp cDNA was prepared from the positive serum, purified with electrophoresis of polyacrylamide gels, and directly sequenced using the dideoxy-mediated chain-termination method.HGV-RNA was detected in 1 of the 35 patients with chronic non-A-E hepatitis. Compared with the 2 HGV isolates (PNF2161 and R10291) obtained from American patients, the HGV NS5 gene of this Beijing isolate (HG-G) showed homology of 88.0% and 89.2% respectively. On the other hand, in comparison with the West African isolate (GBV-C), the Beijing isolate showed homology of 93.5%. The patient showed persistent increase of alanine transaminase, but normal levels were achieved after interferon therapy with persistent positive HGV RNA.HGV is one of the causes of chronic non-A-E hepatitis, but it may not be a very important cause. The nucleotide sequence of partial NS5 gene of HG-G was found to be highly homologous to the West Africa isolate.

In the present study, we studied the relationship between plasma cell-free DNA and the effect of preoperative chemoradiotherapy in patients with rectal cancer. The concentration, KRAS mutation and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status of cell-free DNA were measured by using polymerase chain reaction (PCR) analyses. The response to chemoradiotherapy was assessed using tumor regression grading (TRG) scores. The cell-free DNA concentrations in patients with rectal cancer (n=34) were significantly higher compared to healthy controls (n=10). The 400-base pair (bp) DNA concentration, 400-/100-bp DNA ratio decreased significantly after chemoradiotherapy in the good response group. The incidence of KRAS mutation decreased significantly after chemoradiotherapy in both good and poor response groups. Higher MGMT promoter methylation status at baseline DNA was associated with a better tumor response. Therefore, cell-free DNA detection may be useful in evaluating the effect of preoperative chemoradiotherapy in patients with rectal cancer.

Background - Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings - Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance - In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.

The study was performed to assess the potential role of GSTM1, GSTT1 and GSTP1 polymorphisms in the risk of nasopharyngeal cancer in Chinese population.We collected 182 cases undergoing pathologic examination and 366 controls from the affiliated hospital of Medical College of Qingdao University from April 2006 to July 2010. Genotyping was based upon duplex polymerase-chain-reactions with the PCR- CTPP method.More smokers were found in NPC patients than controls, and a higher IgA/VCA+. Individuals carrying null GSTM1 and GSTT1 had 1.76 and 2.01 fold risk of NPC when compared with non-null genotypes, respectively. A non-significant increase risk of NPC was found in individuals with 1b/1b genotype when compared with 1a/1a genotype (OR=1.32, 95%CI=0.60-2.94). When compared with non-null GSTM1 and GSTT1 genotypes, the combination of null/null GSTM1 and GSTT1 genotypes showed moderate increased risk of NPC (OR=3.03, 95% CI=1.74-5.08).Our study provides evidence that genetic deletion of GSTM1 and GSTT1 may contribute to increased susceptibility to NPC in Chinese population, while GSTP1 may not. Our findings provide information relevant to the prevention of NPC.

Pseudomonas aeruginosa is a major cause of blindness and visual impairment in the United States and worldwide. Using a murine model of keratitis in which abraded corneas are infected with P. aeruginosa parent and ΔfliC (aflagellar) strains 19660 and PAO1, we found that F4/80+ macrophages were the predominant cell type in the cornea expressing TLR2, TLR4, and TLR5. Depletion of macrophages and dendritic cells using transgenic Mafia mice, in which Fas ligand is selectively activated in these cells, resulted in diminished cytokine production and cellular infiltration to the corneal stroma and unimpaired bacterial growth. TLR4−/− mice showed a similar phenotype postinfection with ΔfliC strains, whereas TLR4/5−/− mice were susceptible to corneal infection with parent strains. Bone marrow-derived macrophages stimulated with ΔfliC bacteria induced Toll/IL-1R intracellular domain (TIR)-containing adaptor inducing IFN-β (TRIF)-dependent phosphorylation of IFN regulatory factor 3 in addition to TIR-containing adaptor protein/MyD88-dependent phosphorylation of IκB and nuclear translocation of the p65 subunit of NFκB. Furthermore, TRIF−/− mice showed a similar phenotype as TLR4−/− mice in regulating only ΔfliC bacteria, whereas MyD88−/− mice were unable to clear parent or ΔfliC bacteria. Finally, IL-1R1−/− and IL-1α/β−/− mice were highly susceptible to infection. Taken together, these findings indicate that P. aeruginosa activates TLR4/5 on resident corneal macrophages, which signal through TRIF and TIR-containing adaptor protein/MyD88 pathways, leading to NF-κB translocation to the nucleus, transcription of CXCL1 and other CXC chemokines, recruitment of neutrophils to the corneal stroma, and subsequent bacterial killing and tissue damage. IL-1α and IL-1β are also produced, which activate an IL-1R1/MyD88-positive feedback loop in macrophages and IL-1R on other resident cells in the cornea.

Pyrethroid insecticides have been extensively used in China and worldwide for public health pest control. Accurate resistance monitoring is essential to guide the rational use of insecticides and resistance management. Here we examined the nucleotide diversity of the para-sodium channel gene, which confers knockdown resistance (kdr) in Culex pipiens pallens mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations. We developed and validated allele-specific PCR and the real-time TaqMan methods for resistance diagnosis. The real-time TaqMan method is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity. Significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014S mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance monitoring in natural Cx. pipiens pallens populations in the East China region. The laboratory selection experiment found that L1014F mutation frequency, but not L1014S mutation, responded to deltamethrin selection, suggesting that the L1014F mutation is the key mutation conferring resistance to deltamethrin. High L1014F mutation frequency detected in six populations of Cx. pipens pallens suggests high prevalence of pyrethroid resistance in Eastern China, calling for further surveys to map the resistance in China and for investigating alternative mosquito control strategies.

Background The microbiota of an animal's intestinal tract plays important roles in the animal's overall health, productivity and well-being. There is still a scarcity of information on the microbial diversity in the gut of livestock species such as cattle. The primary reason for this lack of data relates to the expense of methods needed to generate such data. Here we have utilized a bacterial tag-encoded FLX 16s rDNA amplicon pyrosequencing (bTEFAP) approach that is able to perform diversity analyses of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. We have evaluated the microbiome from the feces of 20 commercial, lactating dairy cows. Results Ubiquitous bacteria detected from the cattle feces included Clostridium, Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophage, Anaerotruncus, and Acidaminococcus spp. Foodborne pathogenic bacteria were detected in several of the cattle, a total of 4 cows were found to be positive for Salmonella spp (tentative enterica) and 6 cows were positive for Campylobacter spp. (tentative lanienae). Conclusion Using bTEFAP we have examined the microbiota in the feces of cattle. As these methods continue to mature we will better understand the ecology of the major populations of bacteria the lower intestinal tract. This in turn will allow for a better understanding of ways in which the intestinal microbiome contributes to animal health, productivity and wellbeing.

BACKGROUND: Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections. METHODS AND FINDINGS: Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp. CONCLUSIONS: In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is definitely warranted and needed in order to prove whether the FEPs concept is a viable hypothesis. The findings here also suggest that traditional culturing methods may be extremely biased as a diagnostic tool as they select for easily cultured organisms such as Staphylococcus aureus and against difficult to culture bacteria such as anaerobes. While PCR methods also have bias, further work is now needed in comparing traditional culture results to high-resolution molecular diagnostic methods such as bTEFAP.

To accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed.Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients.Of 29 live-born recipients, 19 had the presence of human CD45(+) cells in peripheral blood (PB) detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues (i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin) examined at the time of tissue collection, as confirmed by detecting human beta2-microglobulin expression using immunohistochemistry. In this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CD45-positive human cells in chimeric spleen and thymus of recipients.Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future. The potential for new advances in our better understanding the living biological systems in human provided by investigators in humanized animals will remain promising.

To analyze the correlation of hepatitis B virus (HBV) DNA with the serological markers of HB in the serum of chronic HB patients after treatment PCR method and to analyze the status of these markers and the multiplication of virus.Peripheral blood samples were collected from 480 chronic HB patients, aged 15 - 50, who had been treated by anti-nucleotide drugs or traditional Chinese herbs and showed normal ALT/AST. Both COBAS AMPLICOR HBV MONIORTM kit (internal-standard PCR method) and Light Cycler real time fluorescent quantitative PCR instrument (external-quantitative standard PCR method) were used to measure the HBV DNAS level. 42 of the 312 patients with the HBV DNA level lower than the minimum test limit measured by COBAS AMPLICOR HBV MONIORTM kit and HBeAg positive (4 S/CO) underwent microparticle enzyme immunoassay (MEIA) to test the HBsAg, anti-HBs, HBeAg, anti-HBe, and HBcIgG.Seven of the 42 patients with HBV DNA negative measured by COBAS AMPLICOR HBV MONIORTM kit lower then the minimum test limit were shown as HBV DNA positive by Light Cycler real time fluorescent quantitative PCR. The 42 patients were HBsAg (+), anti-HBs (-), HBeAg (+), anti-HBe (-), and anti-HBcIgG (-), with an average HBeAg level of 42.26 S/CO and a positive HBeAg rate of 13.46%.HBeAg positivity does not necessarily means an active multiplication of HBV. The changes of the serological markers of HBV may be not consistent with that of HBV DNA. It is more objective to undergo both internal-standard and external-quantitative standard methods.

Neprilysin has been reported to be a major beta-amyloid peptide (Ab)-degrading enzyme. The decreased expression and activity of it may contribute to the development of Alzheimer’s disease by promoting the accumulation of Ab. We used denaturing high-performance liquid chromatography to screen the neprilysin gene (NEP) for single nucleotide polymorphisms (SNPs) in 257 Chinese sporadic Alzheimer’s disease patients and 242 cognitive normal controls. As a result, eight novel and one known SNP were identified. Three of them, � 204GfiC in the promoter region, IVS17� 294CfiT, and IVS22þ36CfiA showed a significant association with Alzheimer’s disease (p ¼ .006, .017, and .003, respectively). Subsequent haplotype analysis provided further evidence of the association (global p , .0001 for the three SNPs mentioned above, and global p , .01 for the eight SNPs with rare allele frequency .1%). These findings indicate that genetic variations within or extremely close to NEP might influence the susceptibility to Alzheimer’s disease in Chinese persons.

One of the characteristics of hepatocellular carcinoma (HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1, 20), and it has been identified as a "hotspot" mutation in heptocellular carcinomas occurring in populations exposed to aflatoxin and with high prevalence of hepatitis B virus carriers (2,3,9, 10,16,24). We evaluated in this paper whether this "hotspot" mutation could be detected in cell-free DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker.We collected blood samples from 25 hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 microl of plasma from each sample. The 249(Ser) p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products.We found in exon 7 of p53 gene G-T transversion at the third base of codon 249 resulting 249(Arg) - 249(Ser) mutation in 10/25 (40 %) hepatocellular carcinoma cases, 4/20 (20 %) cirrhotics, and 2/30 (7 %) healthy controls. The adjusted odds ratio for having the mutation was 22.1(95 % CI, 3.2-91.7) for HCC cases compared to controls.These data show that the 249(Ser) p53 mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency, in plasma DNA of Qidong cirrhotics and healthy controls; We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249(Ser) p53 mutation should be developed to a new early diagnostic marker for HCC.

To develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers.The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction (PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis (CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 μm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of 114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma (HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis.CGE allowed the separation of PCR products differing in size3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls (47.1% vs 28.1%, P0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC (95%CI: 1.723-5.122, P0.001), large deletion (99 bp) did not show any association with HCC (P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9% (8/9) had deletions 2-5 years prior to HCC, while only 44.4%4 (4/9) contained such deletions 6-9 years prior to HCC.CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.

Wei Liu1,*, Jun-yu Peng2,*, Fen Li1, Hong-yan Sun3, Ying Ding1, Jing He1, Yi Liu11College of Veterinary Medicine, Hunan Agricultural University, Changsha; 2NeX Eco-Agriculture Technologies, Inc, Chenzhou, Hunan; 3Key Laboratory of Applied Marine Biology of Guangdong Province and Chinese Academy of Sciences, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, People's Republic of China*These authors contributed equally to this workBackground: In this study, methods for the in vitro microculture of Histomonas meleagridis were defined to lay the foundation for further study of its diagnosis, life cycle, and pathogenic mechanism.Methods: After serial dilutions of H. meleagridis, a single parasite was taken and cultured at 40°C. Its proliferation was checked by microscopy, and an 18S rRNA gene fragment of H. meleagridis was amplified using specific primers (Hmf/Hmr) and sequenced. The sequences were assembled manually and aligned using the Clustal X 1.81 program.Results: Two tubes showed proliferation of parasites and, after infection with parasites from these tubes, acute histomoniasis was rapidly induced in turkeys. 18S rRNA gene fragments of the two strains were 492 and 495 base pairs in length, respectively, and the sequence homologies with known H. meleagridis sequences were both 99.4%. Sequence homology between these two strains was 99.6%, and the variation between the two sequences and Trichomonas was more than 20%.Conclusion: We conclude that microculture can produce high-purity and highly pathogenic H. meleagridis. Keywords: Histomonas meleagridis, microculture technology, polymerase chain reaction, identification&nbsp

Background Schistosomiasis is a parasitic disease infecting more than 200 million people in the world. Although chemotherapy targeting on killing schistosomes is one of the main strategies in the disease control, there are few effective ways of dealing with liver fibrosis caused by the parasite infection in the chronic and advanced stages of schistosomiasis. For this reason, new strategies and prospective drugs, which exert antifibrotic effects, are urgently required. Methods and findings The antifibrotic effects of praziquantel were assessed in the murine models of schistosomiasis japonica. Murine fibrosis models were established by cutaneous infection with 14 ± 2 Schistosoma japonicum cercariae. Then, the mice of both chronic (8 weeks post-infection) and advanced (15 weeks post-infection) schistosomiasis were treated by gavage of praziquantel (250 mg/kg, once daily for 3 days) to eliminate worms, and followed by praziquantel anti-fibrosis treatment (300 mg/kg, twice daily for 30 days). The fibrosis-related parameters assessed were areas of collagen deposition, content of hydroxyproline and mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-β, MMP9, TIMP1, IL-4, IL-10, IL-13 and IFN-γ of liver. Spleen weight index, alanine aminotransferase activity and liver portal venous pressure were also measured. The results showed that anti-fibrosis treatment improved liver fibrosis, splenomegaly, hepatic function, as well as liver portal hypertension. In order to confirm the anti-fibrotic properties of praziquantel, we established a CCL(4)-induced model and revealed that CCL(4)-induced liver fibrosis was inhibited by PZQ treatment for 30 days. Furthermore, we analyzed the effects of praziquantel on mouse primary hepatic stellate cells (HSCs). It is indicated that mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-β, MMP9 and TIMP1 of HSCs were all inhibited after praziquantel anti-parasite treatments. Conclusions The significant amelioration of hepatic fibrosis by praziquantel treatment validates it as a promising drug of anti-fibrosis and offers potential of a new chemotherapy for hepatic fibrosis resulting from schistosomiasis.