The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains

T-Bet and Eomes Regulate the Balance between the Effector/Central Memory T Cells versus Memory Stem Like T Cells

Memory T cells are composed of effector, central, and memory stem cells. Previous studies have implicated that both T-bet and Eomes are involved in the generation of effector and central memory CD8 T cells. The exact role of these transcription factors in shaping the memory T cell pool is not well understood, particularly with memory stem T cells. Here, we demonstrate that both T-bet or Eomes are required for elimination of established tumors by adoptively transferred CD8 T cells. We also examined the role of T-bet and Eomes in the generation of tumor-specific memory T cell subsets upon adoptive transfer. We showed that combined T-bet and Eomes deficiency resulted in a severe reduction in the number of effector/central memory T cells but an increase in the percentage of CD62LhighCD44low Sca-1+ T cells which were similar to the phenotype of memory stem T cells. Despite preserving large numbers of phenotypic memory stem T cells, the lack of both of T-bet and Eomes resulted in a profound defect in antitumor memory responses, suggesting T-bet and Eomes are crucial for the antitumor function of these memory T cells. Our study establishes that T-bet and Eomes cooperate to promote the phenotype of effector/central memory CD8 T cell versus that of memory stem like T cells. © 2013 Li et al

How Do Police Respond to Stalking? An Examination of the Risk Management Strategies and Tactics Used in a Specialized Anti-Stalking Law Enforcement Unit

How do police respond to and manage complaints of stalking? To answer this question, we conducted a 3-phase study. First, we reviewed the literature to identify risk management tactics used to combat stalking. Second, we asked a group of police officers to review those tactics for completeness and group them into categories reflecting more general risk management strategies. The result was 22 categories of strategies. Finally, we used qualitative methods to evaluate the files of 32 cases referred to the specialized anti-stalking unit of a metropolitan police department. We coded specific risk management tactics and strategies used by police. Results indicated that a median number of 19 specific tactics from 7 general strategies were used to manage risk. Also, the implementation of strategies and tactics reflected specific characteristics of the cases (e.g., perpetrator risk factors, victim vulnerability factors), suggesting that the risk management decisions made by police were indeed strategic in nature. Qualitative analyses indicated that some of the strategies and tactics were more effective than others. We discuss how these findings can be used to understand and use stalking risk management more generally, as well as improve research on the efficacy of risk assessment and management for stalking

Muscle-specific overexpression of AdipoR1 or AdipoR2 gives rise to common and discrete local effects whilst AdipoR2 promotes additional systemic effects

Hypoadiponectinemia and adiponectin resistance are implicated in the aetiology of obesity-related cardiometabolic disorders, hence represent a potential therapeutic axis. Here we characterised the effects of in vivo electrotransfer-mediated overexpression of the adiponectin receptors, AdipoR1 or AdipoR2, into tibialis anterior muscle (TAM) of lean or obese mice. In lean mice, TAM-specific overexpression of AdipoR1 (TAMR1) or AdipoR2 (TAMR2) increased phosphorylation of AMPK, AKT and ERK and expression of the insulin responsive glucose transporter glut4. In contrast, only TAMR2 increased pparα and a target gene acox1. These effects were decreased in obese mice despite no reduction in circulating adiponectin levels. TAMR2 also increased expression of adipoQ in TAM of lean and obese mice. Furthermore, in obese mice TAMR2 promoted systemic effects including; decreased weight gain; reduced epididymal fat mass and inflammation; increased epididymal adipoQ expression; increased circulating adiponectin. Collectively, these results demonstrate that AdipoR1 and AdipoR2 exhibit overlapping and distinct effects in skeletal muscle consistent with enhanced adiponectin sensitivity but these appear insufficient to ameliorate established obesity-induced adiponectin resistance. We also identify systemic effects upon TAMR2 in obese mice and postulate these are mediated by altered myokine production. Further studies are warranted to investigate this possibility which may reveal novel therapeutic approaches

Prognostic Value of Body Mass Index on Short-Term and Long-Term Outcome after Resection of Esophageal Cancer

Introduction: Cachexia and obesity have been suggested to be risk factors for postoperative complications. However, high body mass index (BMI) might result in a higher R0-resection rate because of the presence of more fatty tissue surrounding the tumor. The purpose of this study was to investigate whether BMI is of prognostic value with regard to short-term and long-term outcome in patients who undergo esophagectomy for cancer. Methods: In 556 patients who underwent esophagectomy (1991-2007), clinical and pathological outcome were compared between different BMI classes (underweight, normal weight, overweight, obesity). Results: Overall morbidity, mortality, and reoperation rate did not differ in underweight and obese patients. However, severe complications seemed to occur more often in obese patients (p = 0.06), and the risk for anastomotic leakage increased with higher BMI (12.5% in underweight patients compared with 27.6% in obese patients, p = 0.04). Histopathological assessment showed comparable pTNM stages, although an advanced pT stage was seen more often in patients with low/normal BMI (p = 0.02). A linear association between BMI and R0-resection rate was detected (p = 0.02): 60% in underweight patients compared with 81% in obese patients. However, unlike pT-stage (p < 0.001), BMI was not an independent predictor for R0 resection (p = 0.12). There was no significant difference in overall or disease-free 5-year survival between the BMI classes (p = 0.25 and p = 0.6, respectively). Conclusions: BMI is not of prognostic value with regard to short-term and long-term outcome in patients who undergo esophagectomy for cancer and is not an independent predictor for radical R0 resection. Patients oncologically eligible for esophagectomy should not be denied surgery on the basis of their BMI class

Mitochondrial complex 1 activity measured by spectrophotometry is reduced across all brain regions in ageing and more specifically in neurodegeneration

Mitochondrial function, in particular complex 1 of the electron transport chain (ETC), has been shown to decrease during normal ageing and in neurodegenerative disease. However, there is some debate concerning which area of the brain has the greatest complex 1 activity. It is important to identify the pattern of activity in order to be able to gauge the effect of age or disease related changes. We determined complex 1 activity spectrophotometrically in the cortex, brainstem and cerebellum of middle aged mice (70–71 weeks), a cerebellar ataxic neurodegeneration model (pcd5J) and young wild type controls. We share our updated protocol on the measurements of complex1 activity and find that mitochondrial fractions isolated from frozen tissues can be measured for robust activity. We show that complex 1 activity is clearly highest in the cortex when compared with brainstem and cerebellum (p<0.003). Cerebellum and brainstem mitochondria exhibit similar levels of complex 1 activity in wild type brains. In the aged brain we see similar levels of complex 1 activity in all three-brain regions. The specific activity of complex 1 measured in the aged cortex is significantly decreased when compared with controls (p<0.0001). Both the cerebellum and brainstem mitochondria also show significantly reduced activity with ageing (p<0.05). The mouse model of ataxia predictably has a lower complex 1 activity in the cerebellum, and although reductions are measured in the cortex and brain stem, the remaining activity is higher than in the aged brains. We present clear evidence that complex 1 activity decreases across the brain with age and much more specifically in the cerebellum of the pcd5j mouse. Mitochondrial impairment can be a region specific phenomenon in disease, but in ageing appears to affect the entire brain, abolishing the pattern of higher activity in cortical regions

Prediction of postoperative hepatic insufficiency by liver stiffness measurement (FibroScan®) before curative resection of hepatocellular carcinoma: a pilot study

BACKGROUND: Liver stiffness measurement (LSM) using transient elastography (FibroScan((R))) reflects the degree of hepatic fibrosis. This prospective study investigated how well LSM predicts the development of hepatic insufficiency after curative liver resection surgery for hepatocellular carcinoma. METHODS: The study enrolled 72 consecutive patients who underwent a preoperative LSM to assess the degree of liver fibrosis followed by curative liver resection surgery for hepatocellular carcinoma between July 2006 and December 2007. The primary end point was the development of hepatic insufficiency. RESULTS: The mean age of the patients was 54.9 years. Twenty patients (27.7%) had chronic hepatitis and 52 (72.3%) had cirrhosis (44 and 8 patients showed Child-Pugh class A and B, respectively). The mean LSM was 17.1 kPa. Twelve patients (16.6%) had segmentectomy only, 16 patients (22.2%) had bisegmentectomy, and 44 patients (61.2%) had lobectomy. Nine patients (12.5%) had stage I tumor, 56 (77.7%) had stage II, and 7 (9.8%) had stage III. Univariate and subsequent multivariate analyses revealed that preoperative LSM was the only independent risk factor for predicting the development of postoperative hepatic insufficiency (cutoff, 25.6 kPa; P = 0.001; relative risk, 19.14; 95% confidence interval, 2.71-135.36). CONCLUSIONS: LSM is potentially useful to predict the development of postoperative hepatic insufficiency in patients with hepatocellular carcinoma undergoing curative liver resection surgery.ope

Valgus and varus deformity after wide-local excision, brachytherapy and external beam irradiation in two children with lower extremity synovial cell sarcoma: case report

BACKGROUND: Limb-salvage is a primary objective in the management of extremity soft-tissue sarcoma in adults and children. Wide-local excision combined with radiation therapy is effective in achieving local tumor control with acceptable morbidity and good functional outcomes for most patients. CASE PRESENTATION: Two cases of deformity after wide-local excision, brachytherapy and external beam irradiation for lower-extremity synovial cell sarcoma are presented and discussed to highlight contributing factors, time course of radiation effects and orthopedic management. In an effort to spare normal tissues from the long-term effects of radiation therapy, more focal irradiation techniques have been applied to patients with musculoskeletal tumors including brachytherapy and conformal radiation therapy. As illustrated in this report, the use of these techniques results in the asymmetric irradiation of growth plates and contributes to the development of valgus or varus deformity and leg-length discrepancies. CONCLUSIONS: Despite good functional outcomes, progressive deformity in both patients required epiphysiodesis more than 3 years after initial management. There is a dearth of information related to the effects of radiation therapy on the musculoskeletal system in children. Because limb-sparing approaches are to be highlighted in the next generation of cooperative group protocols for children with musculoskeletal tumors, documentation of the effects of surgery and radiation therapy will lead to improved decision making in the selection of the best treatment approach and in the follow-up of these patients

C2-phytoceramide perturbs lipid rafts and cell integrity in Saccharomyces cerevisiae in a sterol-dependent manner

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.This work was supported by Fundacao para a Ciencia e Tecnologia through projects PTDC/BIA-BCM/69448/2006 and PEst-C/BIA/UI4050/2011, and fellowships to A. P. (SFRH/BPD/65003) and F. A. (SFRH/BD/80934/2011), as well as by FEDER through POFC - COMPETE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript