Challenges for standardization of Clostridium difficile typing methods

Typing of Clostridium difficile facilitates understanding of the epidemiology of the infection. Some evaluations have shown that certain strain types (for example, ribotype 027) are more virulent than others and are associated with worse clinical outcomes. Although restriction endonuclease analysis (REA) and pulsed-field gel electrophoresis have been widely used in the past, PCR ribotyping is the current method of choice for typing of C. difficile. However, global standardization of ribotyping results is urgently needed. Whole-genome sequencing of C. difficile has the potential to provide even greater epidemiologic information than ribotyping

Miniature Schnauzers under primary veterinary care in the UK in 2013: demography, mortality and disorders

Individual dog breeds are often reported as predisposed to specific breed-related disorders but reliable epidemiological data on disease prevalence are sparse. The Miniature Schnauzer in the UK is a popular small breed dog that is often considered as relatively healthy and long-lived, but is this really true? This study aimed to use data from the VetCompass™ Programme at the Royal Veterinary College to characterise the demography, mortality and common disorders of the general population of Miniature Schnauzers under veterinary care in the UK

Persistent DNA strand breaks induce a CAF-like phenotype in normal fibroblasts.

Cancer-associated fibroblasts (CAFs) are an emerging target for cancer therapy as they promote tumour growth and metastatic potential. However, CAF targeting is complicated by the lack of knowledge-based strategies aiming to selectively eliminate these cells. There is a growing body of evidence suggesting that a pro-inflammatory microenvironment (e.g. ROS and cytokines) promotes CAF formation during tumorigenesis, although the exact mechanisms involved remain unclear. In this study, we reveal that a prolonged pro-inflammatory stimulation causes a de facto deficiency in base excision repair, generating unrepaired DNA strand breaks and thereby triggering an ATF4-dependent reprogramming of normal fibroblasts into CAF-like cells. Based on the phenotype of in vitro-generated CAFs, we demonstrate that midostaurin, a clinically relevant compound, selectively eliminates CAF-like cells deficient in base excision repair and prevents their stimulatory role in cancer cell growth and migration

Invasion of Wolbachia into Anopheles and Other Insect Germlines in an Ex vivo Organ Culture System

The common bacterial endosymbiont Wolbachia manipulates its host's reproduction to promote its own maternal transmission, and can interfere with pathogen development in many insects making it an attractive agent for the control of arthropod-borne disease. However, many important species, including Anopheles mosquitoes, are uninfected. Wolbachia can be artificially transferred between insects in the laboratory but this can be a laborious and sometimes fruitless process. We used a simple ex vivo culturing technique to assess the suitability of Wolbachia-host germline associations. Wolbachia infects the dissected germline tissue of multiple insect species when the host tissue and bacteria are cultured together. Ovary and testis infection occurs in a density-dependent manner. Wolbachia strains are more capable of invading the germline of their native or closely related rather than divergent hosts. The ability of Wolbachia to associate with the germline of novel hosts is crucial for the development of stably-transinfected insect lines. Rapid assessment of the suitability of a strain-host combination prior to transinfection may dictate use of a particular Wolbachia strain. Furthermore, the cultured germline tissues of two major Anopheline vectors of Plasmodium parasites are susceptible to Wolbachia infection. This finding further enhances the prospect of using Wolbachia for the biological control of malaria

Viral Paratransgenesis in the Malaria Vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae

Labrador retrievers under primary veterinary care in the UK: demography, mortality and disorders

Abstract Background Labrador retrievers are reportedly predisposed to many disorders but accurate prevalence information relating to the general population are lacking. This study aimed to describe demography, mortality and commonly recorded diseases in Labrador retrievers under UK veterinary care. Methods The VetCompass™ programme collects electronic patient record data on dogs attending UK primary-care veterinary practices. Demographic analysis covered all33,320 Labrador retrievers in the VetCompass™ database under veterinary care during 2013 while disorder and mortality data were extracted from a random sample of 2074 (6.2%) of these dogs. Results Of the Labrador retrievers with information available, 15,427 (46.4%) were female and 15,252 (53.6%) were male. Females were more likely to be neutered than males (59.7% versus 54.8%, P <  0.001). The overall mean adult bodyweight was 33.0 kg (SD 6.1). Adult males were heavier (35.2 kg, SD 5.9 kg) than adult females (30.4 kg, SD 5.2 kg) (P <  0.001). The median longevity of Labrador retrievers overall was 12.0 years (IQR 9.9–13.8, range 0.0–16.0). The most common recorded colours were black (44.6%), yellow (27.8%) and liver/chocolate (reported from hereon as chocolate) (23.8%). The median longevity of non-chocolate coloured dogs (n = 139, 12.1 years, IQR 10.2–13.9, range 0.0–16.0) was longer than for chocolate coloured animals (n = 34, 10.7 years, IQR 9.0–12.4, range 3.8–15.5) (P = 0.028). Of a random sample of 2074 (6.2%) Labrador retrievers under care in 2013 that had full disorder data extracted, 1277 (61.6%) had at least one disorder recorded. The total number of dogs who died at any date during the study was 176. The most prevalent disorders recorded were otitis externa (n = 215, prevalence 10.4%, 95% CI: 9.1–11.8), overweight/obesity (183, 8.8%, 95% CI: 7.6–10.1) and degenerative joint disease (115, 5.5%, 95% CI: 4.6–6.6). Overweight/obesity was not statistically significantly associated with neutering in females (8.3% of entire versus 12.5% of neutered, P = 0.065) but was associated with neutering in males (4.1% of entire versus 11.4% of neutered, P < 0.001). The prevalence of otitis externa in black dogs was 12.8%, in yellow dogs it was 17.0% but, in chocolate dogs, it rose to 23.4% (P < 0.001). Similarly, the prevalence of pyo-traumatic dermatitis in black dogs was 1.1%, in yellow dogs it was 1.6% but in chocolate dogs it rose to 4.0% (P = 0.011). Conclusions The current study assists prioritisation of health issues within Labrador retrievers. The most common disorders were overweight/obesity, otitis externa and degenerative joint disease. Males were significantly heavier females. These results can alert prospective owners to potential health issues and inform breed-specific wellness checks

Potent Antimalarial 2-Pyrazolyl Quinolone bc1 (Qi) Inhibitors with Improved Drug-like Properties

A series of 2-pyrazolyl quinolones has been designed and synthesized in 5-7 steps to optimize for both in vitro antimalarial potency and various in vitro drug metabolism and pharmacokinetics (DMPK) features. The most potent compounds display no cross-resistance with multidrug resistant parasite strains (W2) compared to drug sensitive strains (3D7), with IC50 (concentration of drug required to achieve half maximal growth suppression) values in the range of 15-33 nM. Furthermore, members of the series retain moderate activity against the atovaquone-resistant parasite isolate (TM90C2B). The described 2-pyrazoyl series displays improved DMPK properties, including improved aqueous solubility compared to previously reported quinolone series and acceptable safety margin through in vitro cytotoxicity assessment. The 2-pyrazolyl quinolones are believed to bind to the ubiquinone-reducing Qi site of the parasite bc1 complex, which is supported by crystallographic studies of bovine cytochrome bc1 complex

Choriocarcinoma in a 73-year-old woman: a case report and review of the literature

<p>Abstract</p> <p>Introduction</p> <p>Choriocarcinoma is a highly malignant tumor of trophoblastic origin. Most cases present within one year of the antecedent pregnancy (molar or non-molar). However, very rarely, choriocarcinoma can develop from germ cells or from dedifferentiation of endometrial carcinoma into choriocarcinoma. This article concerns a case of choriocarcinoma developing 38 years after the patient's last pregnancy and 23 years after menopause.</p> <p>Case presentation</p> <p>A 73-year-old African-American woman presented with a three-week history of vaginal bleeding. A vaginal mass was seen on pelvic examination. Ultrasonography showed a thickened complex endometrial echo. Her β-human chorionic gonadotrophin level was found to be elevated (2,704,040 mIU/mL). Vaginal and uterine biopsies were suggestive of choriocarcinoma. Immunohistochemistry tests were positive for β-human chorionic gonadotrophin as well as cytokeratin and negative for octamer binding transcription factor 3/4 and α-fetoprotein, supporting the diagnosis of choriocarcinoma. A combination of etoposide, methotrexate, and dactinomycin, followed by cyclophosphamide and vincristine (the so-called EMA/CO regimen) was initiated. After seven cycles of chemotherapy, her β-human chorionic gonadotrophin level dropped below 5 mIU/mL. Our patient is being followed up at our oncology institute.</p> <p>Conclusions</p> <p>We report an extremely rare case of choriocarcinoma arising 23 years after menopause. A postmenopausal woman presenting with vaginal bleed from a mass and β-human chorionic gonadotrophin elevation should be evaluated by immunohistochemical analysis to rule out the possibilities of a germ cell origin of the tumor or dedifferentiation of an epithelial tumor. Absence of octamer binding transcription factor 3/4, α-fetoprotein and CD-30 staining helps in exclusion of most germ cell tumors. DNA polymorphism studies can be used to differentiate between gestational and non-gestational tumor origin. These require fresh tissue samples and are time consuming. Finally, the effective first-line therapy for β-human chorionic gonadotrophin-producing high-risk gestational as well as non-gestational trophoblastic tumors is combination chemotherapy (the EMA/CO regimen). Therefore, treatment should be commenced when a potential diagnosis of metastatic trophoblastic tumor is being considered.</p

Apoptosis assays with lymphoma cell lines: problems and pitfalls

Much attention has been focused on the manner in which tumour cells die after treatment with cytotoxic agents. The basic question is whether cells die via apoptosis or via direct damage from the toxic agent. Various assays have been used to make this distinction. However, we show herein that some of the widely used assays for apoptosis do not in fact distinguish between apoptosis and other forms of cell death. More specifically: (1) A sub-G1 DNA content, identified by propidium iodide staining, does not distinguish between apoptotic and necrotic cells; (2) loss of mitochondrial membrane potential does not distinguish between apoptotic and necrotic cells, unless combined with an assay for an intact cell membrane; (3) subcellular fragments that arise from dead cells or from apoptotic bodies can interfere with some assays for apoptosis such as annexin V staining, as they may be close to the size of intact cells, making it difficult to decide where to set the size threshold; (4) irradiated cells display a large increase in nonspecific Ab binding. This may be partly due to an increase in cell size, but, regardless of the cause, it can lead to a mistaken conclusion that there is an increase in a particular antigen if appropriate control reagents are not tested; and (5) experiments utilising Ab crosslinking have neglected the role of cell aggregation, which can cause multiple problems including death from mechanical stress when cells are handled. Consideration of these factors will improve our ability to determine the mode of cell death

Clostridium difficile Infections amongst Patients with Haematological Malignancies: A Data Linkage Study

OBJECTIVES: Identify risk factors for Clostridium difficile infection (CDI) and assess CDI outcomes among Australian patients with a haematological malignancy. METHODS: A retrospective cohort study involving all patients admitted to hospitals in Western Australia with a haematological malignancy from July 2011 to June 2012. Hospital admission data were linked with all hospital investigated CDI case data. Potential risk factors were assessed by logistic regression. The risk of death within 60 and 90 days of CDI was assessed by Cox Proportional Hazards regression. RESULTS: There were 2085 patients of whom 65 had at least one CDI. Twenty percent of CDI cases were either community-acquired, indeterminate source or had only single-day admissions in the 28 days prior to CDI. Using logistic regression, having acute lymphocytic leukaemia, neutropenia and having had bacterial pneumonia or another bacterial infection were associated with CDI. CDI was associated with an increased risk of death within 60 and 90 days post CDI, but only two deaths had CDI recorded as an antecedent factor. Ribotyping information was available for 33 of the 65 CDIs. There were 19 different ribotypes identified. CONCLUSIONS: Neutropenia was strongly associated with CDI. While having CDI is a risk factor for death, in many cases it may not be a direct contributor to death but may reflect patients having higher morbidity. A wide variety of C. difficile ribotypes were found and community-acquired infection may be under-estimated in these patients