Identification and Characterization of σS, a Novel Component of the Staphylococcus aureus Stress and Virulence Responses

S. aureus is a highly successful pathogen that is speculated to be the most common cause of human disease. The progression of disease in S. aureus is subject to multi-factorial regulation, in response to the environments encountered during growth. This adaptive nature is thought to be central to pathogenesis, and is the result of multiple regulatory mechanisms employed in gene regulation. In this work we describe the existence of a novel S. aureus regulator, an as yet uncharacterized ECF-sigma factor (σS), that appears to be an important component of the stress and pathogenic responses of this organism. Using biochemical approaches we have shown that σS is able to associates with core-RNAP, and initiate transcription from its own coding region. Using a mutant strain we determined that σS is important for S. aureus survival during starvation, extended exposure to elevated growth temperatures, and Triton X-100 induced lysis. Coculture studies reveal that a σS mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth (7 days), or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS. Assays performed hourly for 72h revealed expression in typically background ranges. Analysis of a potential anti-sigma factor, encoded downstream of sigS, revealed it to have no obvious role in the upregulation of sigS expression. Using a murine model of septic arthritis, sigS-mutant infected animals lost significantly less weight, developed septic arthritis at significantly lower levels, and had increased survival rates. Studies of mounted immune responses reveal that sigS-mutant infected animals had significantly lower levels of IL-6, indicating only a weak immunological response. Finally, strains of S. aureus lacking sigS were far less able to undergo systemic dissemination, as determined by bacterial loads in the kidneys of infected animals. These results establish that σS is an important component in S. aureus fitness, and in its adaptation to stress. Additionally it appears to have a significant role in its pathogenic nature, and likely represents a key component in the S. aureus regulatory network

A Note on Flux Induced Superpotentials in String Theory

Non-vanishing fluxes in M-theory and string theory compactifications induce a superpotential in the lower dimensional theory. Gukov has conjectured the explicit form of this superpotential. We check this conjecture for the heterotic string compactified on a Calabi-Yau three-fold as well as for warped M-theory compactifications on Spin(7) holonomy manifolds, by performing a Kaluza-Klein reduction.Comment: 19 pages, no figure

PKCθ Regulates T Cell Motility via Ezrin-Radixin-Moesin Localization to the Uropod

Cell motility is a fundamental process crucial for function in many cell types, including T cells. T cell motility is critical for T cell-mediated immune responses, including initiation, activation, and effector function. While many extracellular receptors and cytoskeletal regulators have been shown to control T cell migration, relatively few signaling mediators have been identified that can modulate T cell motility. In this study, we find a previously unknown role for PKCθ in regulating T cell migration to lymph nodes. PKCθ localizes to the migrating T cell uropod and regulates localization of the MTOC, CD43 and ERM proteins to the uropod. Furthermore, PKCθ-deficient T cells are less responsive to chemokine induced migration and are defective in migration to lymph nodes. Our results reveal a novel role for PKCθ in regulating T cell migration and demonstrate that PKCθ signals downstream of CCR7 to regulate protein localization and uropod formation.</p

A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo

Pseudomonas aeruginosa Adaptation to Lungs of Cystic Fibrosis Patients Leads to Lowered Resistance to Phage and Protist Enemies

Pathogenic life styles can lead to highly specialized interactions with host species, potentially resulting in fitness trade-offs in other ecological contexts. Here we studied how adaptation of the environmentally transmitted bacterial pathogen, Pseudomonas aeruginosa, to cystic fibrosis (CF) patients affects its survival in the presence of natural phage (14/1, ΦKZ, PNM and PT7) and protist (Tetrahymena thermophila and Acanthamoebae polyphaga) enemies. We found that most of the bacteria isolated from relatively recently intermittently colonised patients (1-25 months), were innately phage-resistant and highly toxic for protists. In contrast, bacteria isolated from long time chronically infected patients (2-23 years), were less efficient in both resisting phages and killing protists. Moreover, chronic isolates showed reduced killing of wax moth larvae (Galleria mellonella) probably due to weaker in vitro growth and protease expression. These results suggest that P. aeruginosa long-term adaptation to CF-lungs could trade off with its survival in aquatic environmental reservoirs in the presence of microbial enemies, while lowered virulence could reduce pathogen opportunities to infect insect vectors; factors that are both likely to result in poorer environmental transmission. From an applied perspective, phage therapy could be useful against chronic P. aeruginosa lung infections that are often characterized by multidrug resistance: chronic isolates were least resistant to phages and their poor growth will likely slow down the emergence of beneficial resistance mutations

A hypothetico-deductive approach to assessing the social function of chemical signalling in a non-territorial solitary carnivore

The function of chemical signalling in non-territorial solitary carnivores is still relatively unclear. Studies on territorial solitary and social carnivores have highlighted odour capability and utility, however the social function of chemical signalling in wild carnivore populations operating dominance hierarchy social systems has received little attention. We monitored scent marking and investigatory behaviour of wild brown bears Ursus arctos, to test multiple hypotheses relating to the social function of chemical signalling. Camera traps were stationed facing bear ‘marking trees’ to document behaviour by different age sex classes in different seasons. We found evidence to support the hypothesis that adult males utilise chemical signalling to communicate dominance to other males throughout the non-denning period. Adult females did not appear to utilise marking trees to advertise oestrous state during the breeding season. The function of marking by subadult bears is somewhat unclear, but may be related to the behaviour of adult males. Subadults investigated trees more often than they scent marked during the breeding season, which could be a result of an increased risk from adult males. Females with young showed an increase in marking and investigation of trees outside of the breeding season. We propose the hypothesis that females engage their dependent young with marking trees from a young age, at a relatively ‘safe’ time of year. Memory, experience, and learning at a young age, may all contribute towards odour capabilities in adult bears

The presentation, diagnosis and management of non-traumatic wrist pain: an evaluation of current practice in secondary care in the UK NHS

AbstractObjectivesThe study aims were to assess the burden of non-traumatic wrist pain in terms of numbers of referrals to secondary care, and to characterise how patients present, are diagnosed and are managed in secondary care in the United Kingdom National Health Service.MethodsTen consecutive patients presenting with non-traumatic wrist pain were identified retrospectively at each of 16 participating hospitals and data was extracted for twelve months following the initial referral.ResultsThe 160 patients consisted of 100 females and 60 males with a median age of 49, accounting for approximately 13% of all new hand/wrist referrals. The dominant wrist was affected in 60% of cases and the mean symptom duration was 13.3 months. Diagnoses were grouped into: osteoarthritis (OA) (31%), tendinopathy (13%), ganglion (14%), ulnar sided pain (17%) and other (25%). The OA group was significantly older than other groups, while other groups contained a predominance of females.The non-surgical interventions in decreasing frequency of usage were: steroid injections (39%), physiotherapy (32%), splint (31%) and analgesics (12%). Of those who underwent surgery, all patients had previously received non-surgical treatment, however 42% had undergone only one non-surgical intervention.ConclusionNon-traumatic wrist pain represents a significant burden to secondary care both in terms of new patient referrals and in terms of investigation, follow up and treatment. Those presenting with osteoarthritis are more likely to be older and male, while those presenting with other diagnoses are more likely to be younger and female

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors

Several 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity

Do Web-Based Interventions Improve Well-Being in Type 2 Diabetes? A Systematic Review and Meta-Analysis

BACKGROUND: Poor diabetes self-care can have a negative impact on psychological well-being and quality of life. Given the scarcity of traditional psychological support and the barriers to uptake of and attendance at face-to-face education programs, Web-based interventions are becoming a popular approach to provide an additional platform for psychological support in long-term conditions. However, there is limited evidence to assess the effect of Web-based psychological support in people with type 2 diabetes. OBJECTIVE: This systematic review is the first review to critically appraise and quantify the evidence on the effect of Web-based interventions that aim to improve well-being in people with type 2 diabetes. METHODS: Searches were carried out in the following electronic databases: MEDLINE, EMBASE, CINAHL, PsycINFO, and Cochrane Library. Reference lists were hand-searched. A meta-analysis was conducted for depression and distress outcomes. RESULTS: A total of 16 randomized controlled studies met the inclusion criteria for the systematic review and 9 were included in the meta-analyses. Theories were applied to the majority of the interventions. The most common behavior change techniques were "General information" and "Tracking/monitoring." Interventions with a duration of 2-6 months providing professional-led support with asynchronous and synchronous communication appeared to be associated with significant well-being outcomes. The pooled mean (95% confidence interval) difference between the intervention and control arms at follow-up on depression score was -0.31 (-0.73 to 0.11). The pooled mean difference on distress scores at follow-up was -0.11 (-0.38 to 0.16). No significant improvements in depression (P=.15) or distress (P=.43) were found following meta-analyses. CONCLUSIONS: While the meta-analyses demonstrated nonsignificant results for depression and distress scores, this review has shown that there is a potential for Web-based interventions to improve well-being outcomes in type 2 diabetes. Further research is required to confirm the findings of this review