Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.     

A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever in Central Africa

Deep sequencing was used to discover a novel rhabdovirus (Bas-Congo virus, or BASV) associated with a 2009 outbreak of 3 human cases of acute hemorrhagic fever in Mangala village, Democratic Republic of Congo (DRC), Africa. The cases, presenting over a 3-week period, were characterized by abrupt disease onset, high fever, mucosal hemorrhage, and, in two patients, death within 3 days. BASV was detected in an acute serum sample from the lone survivor at a concentration of 1.09×10⁶ RNA copies/mL, and 98.2% of the genome was subsequently de novo assembled from ∼140 million sequence reads. Phylogenetic analysis revealed that BASV is highly divergent and shares less than 34% amino acid identity with any other rhabdovirus. High convalescent neutralizing antibody titers of >1∶1000 were detected in the survivor and an asymptomatic nurse directly caring for him, both of whom were health care workers, suggesting the potential for human-to-human transmission of BASV. The natural animal reservoir host or arthropod vector and precise mode of transmission for the virus remain unclear. BASV is an emerging human pathogen associated with acute hemorrhagic fever in Africa.     

Incidence of Hemorrhagic Disease in White-Tailed Deer Is Associated with Winter and Summer Climatic Conditions

Hemorrhagic disease (HD) is an important vector-borne disease of white-tailed deer (Odocoileus virginianus). The objective of this study was to determine whether temperature and precipitation were associated with a measure of annual incidence of HD in white-tailed deer from Virginia. The annual percentages of deer with hoof wall growth interruptions (a clinical sign of HD) from four climate divisions in the HD endemic area of Virginia recorded during 1993–2006 were used as indicators of annual HD incidence. Pearson’s correlation coefficients between these indicators of incidence and average temperature (°F) or total precipitation (in.) for each month, as well as for winter (January–February), early summer (June–July), and late summer/fall (August–September–October) seasons were calculated. Strong direct correlations between the measure of annual HD incidence and average temperature for winter (r = 0.39, P = 0.003, n = 57), early summer (r = 0.51, P < 0.0001, n = 57), and late summer/fall (r = 0.42, P = 0.001, n = 57) were evident. There also was a strong inverse correlation between the measured annual HD incidence and June precipitation (r = −0.44, P = 0.0006, n = 57). Poisson regression models of seasonal temperatures and June precipitation to annual percentage of deer with hoof wall growth interruptions were developed. Based on Akaike’s Information Criterion with small sample size correction (AICc), the global model was selected as the top model. Higher winter and summer temperatures may increase vector capacity and competence, and lower precipitation in June may create favorable breeding sites for midges.     

Necrotizing lesions in the intestine, gizzard, and liver in captive capercaillies (Tetrao urogallus) associated with Clostridium perfringens.

During the period from 1982 to 1991, 863 captive and 32 wild capercaillies (Tetrao urogallus) were necropsied. The most common cause of death in captive capercaillies was necrotizing enteritis, diagnosed in 110 (13%) birds. Of these, 31 (28%) birds also had necrotizing lesions in the liver. Necrotizing gastritis occurred in 29 birds, two of which had concurrent necrotizing enteritis. In the capercaillies with necrotizing enteritis, Clostridium perfringens type A was isolated more frequently and in larger numbers than in birds which died from other causes. Thus, Clostridium perfringens type A may be of etiological importance in necrotizing enteritis. Necrotizing enteritis was not diagnosed in wild capercaillies.     

Immunological Characterization of the Neurotoxin Produced by Clostridium botulinum Type A Associated with Infant Botulism in Japan

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino-terminal half (H-1 fragment) of the heavy chain. This may support the hypothesis that the H-1 fragment functions in the transport of the light chain in the target cell.     

Insulin Treatment Is Associated with Increased Mortality in Patients with COVID-19 and Type 2 Diabetes

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Ongoing Spillover of Hantaan and Gou Hantaviruses from Rodents Is Associated with Hemorrhagic Fever with Renal Syndrome (HFRS) in China

Background

Longquan City, Zhejiang province, China, has been seriously affected by hemorrhagic fever with renal syndrome (HFRS) since the first cases were registered in 1974. To understand the epidemiology and emergence of HFRS in Longquan, which may be indicative of large parts of rural China, we studied long-term incidence patterns and performed a molecular epidemiological investigation of the causative hantaviruses in human and rodent populations.

Method/Principal Findings

During 1974–2011, 1866 cases of HFRS were recorded in Longquan, including 20 deaths. In 2011, the incidence of HFRS remained high, with 19.61 cases/100,000 population, despite the onset of vaccination in 1997. During 1974–1998, HFRS cases in Longquan occurred mainly in winter, while in the past decade the peak of HFRS has shifted to the spring. Notably, the concurrent prevalence of rodent-borne hantaviruses in the region was also high. Phylogenetic analyses of viral sequences recovered from rodents in Longquan revealed the presence of novel genetic variants of Gou virus (GOUV) in Rattus sp. rats and Hantaan virus (HTNV) in the stripe field mice, respectively. Strikingly, viral sequences sampled from infected humans were very closely related to those from rodents.

Conclusions/Significance

HFRS represents an important public health problem in Longquan even after years of preventive measures. Our data suggest that continual spillover of the novel genetic variant of GOUV and the new genetic lineage of HTNV are responsible for the high prevalence of HFRS in humans. In addition, this is the first report of GOUV associated with human HFRS cases, and our data suggest that GOUV is now the major cause of HFRS in this region.     

Constitutively Elevated Blood Serotonin Is Associated with Bone Loss and Type 2 Diabetes in Rats

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)₂D₃ decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.     

Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells

Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin–Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes.     

Pentraxin 3 (PTX3) Is Associated with Severe Sepsis and Fatal Disease in Emergency Room Patients with Suspected Infection: A Prospective Cohort Study

Background

Early diagnostic and prognostic stratification of patients with suspected infection is a difficult clinical challenge. We studied plasma pentraxin 3 (PTX3) upon admission to the emergency department in patients with suspected infection.

Methods

The study comprised 537 emergency room patients with suspected infection: 59 with no systemic inflammatory response syndrome (SIRS) and without bacterial infection (group 1), 67 with bacterial infection without SIRS (group 2), 54 with SIRS without bacterial infection (group 3), 308 with sepsis (SIRS and bacterial infection) without organ failure (group 4) and 49 with severe sepsis (group 5). Plasma PTX3 was measured on admission using a commercial solid-phase enzyme-linked immunosorbent assay (ELISA).

Results

The median PTX3 levels in groups 1–5 were 2.6 ng/ml, 4.4 ng/ml, 5.0 ng/ml, 6.1 ng/ml and 16.7 ng/ml, respectively (p<0.001). The median PTX3 concentration was higher in severe sepsis patients compared to others (16.7 vs. 4.9 ng/ml, p<0.001) and in non-survivors (day 28 case fatality) compared to survivors (14.1 vs. 5.1 ng/ml, p<0.001). A high PTX3 level predicted the need for ICU stay (p<0.001) and hypotension (p<0.001). AUC^ROC in the prediction of severe sepsis was 0.73 (95% CI 0.66–0.81, p<0.001) and 0.69 in case fatality (95% CI 0.58–0.79, p<0.001). PTX3 at a cut-off level for 14.1 ng/ml (optimal cut-off value for severe sepsis) showed 63% sensitivity and 80% specificity. At a cut-off level 7.7 ng/ml (optimal cut-off value for case fatality) showed 70% sensitivity and 63% specificity in predicting case fatality on day 28.In multivariate models, high PTX3 remained an independent predictor of severe sepsis and case fatality after adjusting for potential confounders.

Conclusions

A high PTX3 level on hospital admission predicts severe sepsis and case fatality in patients with suspected infection.     

The SOS Response Master Regulator LexA Is Associated with Sporulation,Motility and Biofilm Formation in Clostridium difficile

The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925"}}R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.     

A Single Nucleotide Polymorphism within DUSP9 Is Associated with Susceptibility to Type 2 Diabetes in a Japanese Population

Aims

The DUSP9 locus on chromosome X was identified as a susceptibility locus for type 2 diabetes in a meta-analysis of European genome-wide association studies (GWAS), and GWAS in South Asian populations identified 6 additional single nucleotide polymorphism (SNP) loci for type 2 diabetes. However, the association of these loci with type 2 diabetes have not been examined in the Japanese. We performed a replication study to investigate the association of these 7 susceptibility loci with type 2 diabetes in the Japanese population.

Methods

We genotyped 11,319 Japanese participants (8,318 with type 2 diabetes and 3,001 controls) for each of the 7 SNPs–rs5945326 near DUSP9, rs3923113 near GRB14, rs16861329 in ST6GAL1, rs1802295 in VPS26A, rs7178572 in HMG20A, rs2028299 near AP3S2, and rs4812829 in HNF4A–and examined the association of each of these 7 SNPs with type 2 diabetes by using logistic regression analysis.

Results

All SNPs had the same direction of effect (odds ratio [OR]>1.0) as in the original reports. One SNP, rs5945326 near DUSP9, was significantly associated with type 2 diabetes at a genome-wide significance level (p = 2.21×10⁻⁸; OR 1.39, 95% confidence interval [CI]: 1.24−1.56). The 6 SNPs derived from South Asian GWAS were not significantly associated with type 2 diabetes in the Japanese population by themselves (p≥0.007). However, a genetic risk score constructed from 6 South Asian GWAS derived SNPs was significantly associated with Japanese type 2 diabetes (p = 8.69×10⁻⁴, OR  = 1.06. 95% CI; 1.03−1.10).

Conclusions/interpretation

These results indicate that the DUSP9 locus is a common susceptibility locus for type 2 diabetes across different ethnicities, and 6 loci identified in South Asian GWAS also have significant effect on susceptibility to Japanese type 2 diabetes.     

Recombination Is Associated with an Outbreak of Novel Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Viruses in China

In 2009 to 2010, there was a marked increase in the number of infections with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in China. Through phylogenetic analysis, we show that viruses from this outbreak originated from a single recombination event, illustrating the potential importance of this process for disease emergence.     

Isolation of Nontoxigenic Variants Associated with Enhanced Sporulation and Alteration in the Cell Wall from Clostridium botulinum Type A 190L by Treatment with Detergents

Nontoxigenic variants were isolated from Clostridium botulinum type A strain 190L after treatment with detergents such as deoxycholate, sodium dodecyl sulfate, Tween 80 and Brij-58. Deoxycholate was most effective for obtaining the variants. The variants exhibited a markedly increased frequency of sporulation compared with the oligosporogenic parent strain. The cell wall of the parent strain was composed of an outer layer and an inner layer, whereas that of the variants lost the outer layer. After treatment with mitomycin C the parent strain was subjected to lysis and produced bacteriophages with a hexagonal head and a contractible tail, while the nontoxigenic variants did not yield bacteriophages or phage-like structures. There appears to be a close relationship among the toxigenic and sporogenic properties, formation of the outer cell wall layer and lysogeny.     

Presence of CHD1L Over-Expression Is Associated with Aggressive Tumor Biology and Is a Novel Prognostic Biomarker for Patient Survival in Human Breast Cancer

Background

The chromodomain helicase/adenosine triphosphatase DNA binding protein 1–like gene (CHD1L) is a recently identified oncogene localized at 1q21. CHD1L protein over-expression in primary hepatocellular carcinoma is correlated with enhanced apoptosis inhibition, reduced chemosensitivity and shortened patient survival. However, CHD1L protein status or mRNA expression in breast cancer and its clinical significance remain obscure.

Material and Methods

In this study, immunohistochemical staining for CHD1L expression was performed on tissue microarrays containing 179 primary invasive breast cancers and 65 matched normal breast tissue specimens. Clinico-pathological features were collected and compared between different CHD1L statuses. Kaplan-Meier curves were applied to estimate disease-free survival (DFS) and overall survival (OS). Cox regression was used to identify independent prognostic factors. Also, quantitative real-time polymerase chain reaction (QRT-PCR) was employed to evaluate the mRNA level expression of CHD1L in six breast cancer cell lines.

Results

Presence of CHD1L over-expression was observed in 87 of the 179 patients (48.6%), which associated with a younger age (P = 0.011), higher grade (P = 0.004), higher Ki-67 index (P = 0.018) and HER2 over-expression/amplification (P = 0.037). After a median follow-up of 55 months, patients with presence of CHD1L over-expression had significantly poorer DFS (82.6% Vs 76.3%, P = 0.035), but not OS (87.0% Vs 94.9%, P = 0.439). In multivariate analysis, CHD1L status (HR = 2.169, [95%CI, 1.029–4.573], P = 0.042), triple negative subtype (HR = 2.809, [95%CI 1.086–7.264], P = 0.033) and HER2 positive subtype (HR = 5.221, [95%CI 1.788–15.240], P = 0.002) were identified as independent prognostic factors for DFS. In vitro study indicated that relative mRNA expression level of CHD1L was higher in breast cancer cell lines, especially in MDA-MB-231 and LM2-4175, when compared to normal breast epithelial cell line.

Conclusions

Presence of CHD1L over-expression is probably associated with aggressive tumor biology in breast cancer. CHD1L status might be a novel prognostic biomarker for patients with breast cancer.     

Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24

Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP) located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.     

Nuclear S100A7 Is Associated with Poor Prognosis in Head and Neck Cancer

Background

Tissue proteomic analysis of head and neck squamous cell carcinoma (HNSCC) and normal oral mucosa using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and liquid chromatography-mass spectrometry, led to the identification of a panel of biomarkers including S100A7. In the multi-step process of head and neck tumorigenesis, the presence of dysplastic areas in the epithelium is proposed to be associated with a likely progression to cancer; however there are no established biomarkers to predict their potential of malignant transformation. This study aimed to determine the clinical significance of S100A7 overexpression in HNSCC.

Methodology

Immunohistochemical analysis of S100A7 expression in HNSCC (100 cases), oral lesions (166 cases) and 100 histologically normal tissues was carried out and correlated with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. Overexpression of S100A7 protein was significant in oral lesions (squamous cell hyperplasia/dysplasia) and sustained in HNSCC in comparison with oral normal mucosa (p_trend<0.001). Significant increase in nuclear S100A7 was observed in HNSCC as compared to dysplastic lesions (p = 0.005) and associated with well differentiated squamous cell carcinoma (p = 0.031). Notably, nuclear accumulation of S100A7 also emerged as an independent predictor of reduced disease free survival (p = 0.006, Hazard ratio (HR = 7.6), 95% CI = 1.3−5.1) in multivariate analysis underscoring its relevance as a poor prognosticator of HNSCC patients.

Conclusions

Our study demonstrated nuclear accumulation of S100A7 may serve as predictor of poor prognosis in HNSCC patients. Further, increased nuclear accumulation of S100A7 in HNSCC as compared to dysplastic lesions warrants a large-scale longitudinal study of patients with dysplasia to evaluate its potential as a determinant of increased risk of transformation of oral premalignant lesions.