Cytopenia induction by 5-fluorouracil identifies thrombopoietic mutants in sensitized ENU mutagenesis screens

The ability of random mutagenesis techniques to annotate the mammalian genome can be hampered due to genetic redundancy and compensatory pathways that mask heterozygous mutations under homeostatic conditions. The objective of this study was to devise a pharmacologically sensitized screen using the chemotherapeutic drug, 5-fluorouracil (5FU), to induce cytopenia. 5FU dose was optimized in the 129/SvImJ, C57BL/6J, BALB/cJ, and C3H/HeJ strains of laboratory mice. N-ethyl-N-nitrosourea (ENU) mutagenesis was performed on 129/SvImJ males and phenotypic variants were identified by backcrossing on to the C57BL/6J background. G1 animals were challenged with 100 μg/g 5FU and phenodeviants with altered platelet recovery were monitored. Of 546 G1 animals tested, 15 phenodeviants were identified that displayed increased baseline platelet number, a platelet overshoot, or delayed platelet recovery, thereby demonstrating the utility of this approach for uncovering mutations in megakaryocyte and platelet development. Four G1 mice were selected for further analysis. The phenotypes were heritable in all four strains and genetic mapping identified a chromosome location in two of the three G2 lines tested. In conclusion, our group has developed a sensitized random mutagenesis screen utilizing 5FU and has shown that the strain combination of 129/SvImJ × C57BL/6J is robust for identification of founder lines with defects in megakaryocyte and platelet development.     

Tonic regulation of vascular permeability

Our major theme is that the layered structure of the endothelial barrier requires continuous activation of signalling pathways regulated by sphingosine‐1‐phosphate (S1P) and intracellular cAMP. These pathways modulate the adherens junction, continuity of tight junction strands, and the balance of synthesis and degradation of glycocalyx components. We evaluate recent evidence that baseline permeability is maintained by constant activity of mechanisms involving the small GTPases Rap1 and Rac1. In the basal state, the barrier is compromised when activities of the small GTPases are reduced by low S1P supply or delivery. With inflammatory stimulus, increased permeability can be understood in part as the action of signalling to reduce Rap1 and Rac1 activation. With the hypothesis that microvessel permeability and selectivity under both normal and inflammatory conditions are regulated by mechanisms that are continuously active, it follows that when S1P or intracellular cAMP are elevated at the time of inflammatory stimulus, they can buffer changes induced by inflammatory agents and maintain normal barrier stability. When endothelium is exposed to inflammatory conditions and subsequently exposed to elevated S1P or intracellular cAMP, the same processes restore the functional barrier by first re‐establishing the adherens junction, then modulating tight junctions and glycocalyx. In more extreme inflammatory conditions, loss of the inhibitory actions of Rac1‐dependent mechanisms may promote expression of more inflammatory endothelial phenotypes by contributing to the up‐regulation of RhoA‐dependent contractile mechanisms and the sustained loss of surface glycocalyx allowing access of inflammatory cells to the endothelium.     

Single incision laparoscopic adjustable gastric band: technique,feasibility, safety and learning curve

Introduction

Single incision laparoscopic surgery (SILS) is established in many procedures but not in bariatric surgery. One explanation may be that SILS is technically demanding in morbidly obese patients. This report describes our technique and experience with single incision laparoscopic adjustable gastric banding (SILAGB).

Methods

Prospective data collection was performed on consecutive obese patients who underwent SILAGB between November 2009 and February 2011. A single 3cm transverse incision in the right upper quadrant was used for a Covidien SILS^™ multichannel access port. The technique is described with a standard pars flaccida approach and the ‘tips and tricks’ needed for a wide range of candidates using standard laparoscopic equipment.

Results

A total of 29 patients (27 female) with a median body mass index of 41kg/m² (range: 35–52kg/m²) and median age of 44 years (range: 22–57 years) underwent SILAGB. There were no ‘conversions’ to a standard laparoscopic technique. Two cases required the addition of one single 5mm port. The only complications were two postoperative wound infections (one with a port site infection requiring replacement of the port) and one faulty band requiring replacement. There were therefore two returns to theatre and no 30-day deaths. All patients were discharged on the first postoperative day. In this series, operative times reduced significantly to be comparable with the conventional laparoscopic approach.

Conclusions

SILAGB is safe and feasible in the morbidly obese. Proficiency in this technique using conventional laparoscopic equipment can be achieved with a short learning curve.     

Thoracic surgical management of colorectal lung metastases: a questionnaire survey of members of the Society for Cardiothoracic Surgery in great Britain and Ireland

Introduction

Distant metastases to liver and lung are not uncommon in colorectal cancer. Resection of metastases is accepted widely as the standard of care. However, there is no firm evidence base for this. This questionnaire survey was carried out to assess the current practice preferences of cardiothoracic surgeons in Great Britain and Ireland.

Methods

An online questionnaire survey was emailed to cardiothoracic surgeons in Great Britain and Ireland. The survey was live for 12 weeks. Responses were collated with SurveyMonkey^®.

Results

Overall, there were 75 respondents. The majority (83%) indicated thoracic surgery as a specialist interest. Almost all (99%) used thoracic computed tomography (CT) for staging; 70% added liver CT and 51% added pelvic CT. Fluorodeoxyglucose positron emission tomography was used by 86%. The most frequent indication for pulmonary resection (97%) was solitary lung metastasis without extrathoracic disease. Video assisted thoracoscopic surgery (VATS) was used by 85%. In addition, thoracotomy was used by 96%. A third (33%) used radiofrequency ablation. Synchronous liver and lung resection was contraindicated for 83% of respondents. Over three-quarters (77%) thought that scientific equipoise exists presently for lung resection for colorectal lung metastases but only 21% supported a moratorium on this type of surgery until further evidence becomes available.

Conclusions

The results confirm that the majority of respondents use conventional cross-sectional imaging and either VATS or formal thoracotomy for resection. The results emphasise the continuing need for formal randomised trials to provide evidence of any survival benefit from pulmonary metastasectomy for colorectal lung metastases.     

Evidence That Alpha-9 Human Papillomavirus Infections are a Major Etiologic Factor for Oropharyngeal Carcinoma in Black South Africans

Human papillomavirus (HPV) infection, most commonly genotype 16 of the alpha-9 family, is implicated in the etiology of a subset of oropharyngeal squamous cell carcinomas (OPSC) worldwide. Data are scarce regarding OPSC in South Africans, and three prior studies suggest no significant etiologic role for HPV. We aimed to investigate for evidence of HPV etiology in OPSCs from black South Africans by polymerase chain reaction (PCR) methodologies with determination of HPV subtype by sequencing, in situ hybridization (ISH), and p16^INK4a immunohistochemistry (IHC), as a surrogate marker for an HPV-driven tumor. It was hypothesized that HPV-driven tumors would be positive by PCR plus IHC and/or ISH whereas OPSCs with HPV background infections (HPV-passenger) would be positive by PCR alone. Formalin-fixed, paraffin-embedded tissues from 51 OPSCs collected between 2005 and 2010 from 41 patients were analyzed for HPV by GP5+6+ PCR (targeting the HPV L1 region), pU-1M/pU-2R PCR (targeting the HPV E6/E7 region) and HPV-31 specific PCR (targeting the E5 region), chromogenic ISH, and p16^INK4a IHC. All cases positive by PCR were subject to sequencing to determine HPV genotype. The patient mean age was 58.0 years and 88 % were male. Of the 51 evaluable tumors, 48 (94.1 %) were positive for HPV DNA by PCR: 25 (49.1 %) met criteria for an HPV-driven tumor, 23 (45.1 %) for HPV-passenger, and 3 (5.9 %) were HPV-unrelated. Sequencing of the PCR-positive cases revealed the following genotypes: combined HPV-16 and 31 (41.7 %), HPV-31 (25.0 %), HPV-16 (22.9 %), combined HPV-16 and 18 (6.3 %), and a single case each of HPV 18 and HPV 33. Studies via ISH were negative in all cases. In accordance with worldwide trends but contrary to prior South African data, HPV likely plays an etiologic role in a significant subset (at least 49.1 %) of OPSC in black South Africans. We found that the alpha-9 HPV family, particularly HPV-16 and 31 either in combination or separately, to predominate in our sample tumors. The use of multiple PCR primers increased sensitivity of viral detection, and a HPV-31 specific primer confirmed the presence of this genotype in many samples. Further studies including HPV E6/E7 mRNA assays are needed to better elucidate the pathogenic role of HPV in black South African OPSCs.     

Clinical characteristics and treatment of malignant brainstem gliomas in elderly patients

Adult brainstem gliomas (BSG) are uncommon tumors that constitute only 2% of all brain tumors. Due to its rare occurrence in the elderly (60 years and older), there is no literature discussing the natural history, prognosis, and best treatment strategy for malignant BSG in this population to our knowledge. We report seven elderly patients with malignant BSG and propose treatment strategies to manage these aggressive tumors. The median age at onset in this cohort was 65 years, with the majority of patients being male (71.4%) and Caucasian (85.7%). The median duration of symptoms prior to presentation was 0.5 months, with the most common symptoms being facial weakness, blurry vision, headache, and extremity weakness. Tumors were most commonly located in the pons (85.7%), with one tumor being located in the tectal plate. Five of seven (71.4%) patients underwent biopsies, with two patients undergoing partial resections. Following tissue diagnosis, patients received radiation therapy and concurrent temozolomide, followed by additional chemotherapeutics upon progression. Side effects as a result of treatment were seen in three patients and all involved reversible hematological complications such as neutropenia and thrombopenia. The median time to progression was 6.7 months and the median overall survival was 13.5 months. While malignant BSG in elderly patients are aggressive gliomas with an overall poor prognosis, these patients are able to safely undergo aggressive chemoradiotherapy, resulting in improved survival. Resection may be considered for select patients in which the tumor is mostly exophytic, near the brainstem surface, and easily accessible.     

Pilot Randomized Controlled Trial of the WHO Caregiver Skills Training in Public Health Services in Italy

Journal of Autism and Developmental Disorders - Parents of children with ASD (N?=?86; mean age 44.8 months; 67 boys) were randomized to either WHO Caregiver Skills Training...