The Human Fungal Pathogen Cryptococcus neoformans Escapes Macrophages by a Phagosome Emptying Mechanism That Is Inhibited by Arp2/3 Complex-Mediated Actin Polymerisation

The lysis of infected cells by disease-causing microorganisms is an efficient but risky strategy for disseminated infection, as it exposes the pathogen to the full repertoire of the host's immune system. Cryptococcus neoformans is a widespread fungal pathogen that causes a fatal meningitis in HIV and other immunocompromised patients. Following intracellular growth, cryptococci are able to escape their host cells by a non-lytic expulsive mechanism that may contribute to the invasion of the central nervous system. Non-lytic escape is also exhibited by some bacterial pathogens and is likely to facilitate long-term avoidance of the host immune system during latency. Here we show that phagosomes containing intracellular cryptococci undergo repeated cycles of actin polymerisation. These actin ‘flashes’ occur in both murine and human macrophages and are dependent on classical WASP-Arp2/3 complex mediated actin filament nucleation. Three dimensional confocal imaging time lapse revealed that such flashes are highly dynamic actin cages that form around the phagosome. Using fluorescent dextran as a phagosome membrane integrity probe, we find that the non-lytic expulsion of Cryptococcus occurs through fusion of the phagosome and plasma membranes and that, prior to expulsion, 95% of phagosomes become permeabilised, an event that is immediately followed by an actin flash. By using pharmacological agents to modulate both actin dynamics and upstream signalling events, we show that flash occurrence is inversely related to cryptococcal expulsion, suggesting that flashes may act to temporarily inhibit expulsion from infected phagocytes. In conclusion, our data reveal the existence of a novel actin-dependent process on phagosomes containing cryptococci that acts as a potential block to expulsion of Cryptococcus and may have significant implications for the dissemination of, and CNS invasion by, this organism.\ud \u

Six-week high-intensity exercise program for middle-aged patients with knee osteoarthritis: a randomized controlled trial [ISRCTN20244858]

BACKGROUND: Studies on exercise in knee osteoarthritis (OA) have focused on elderly subjects. Subjects in this study were middle-aged with symptomatic and definite radiographic knee osteoarthritis. The aim was to test the effects of a short-term, high-intensity exercise program on self-reported pain, function and quality of life. METHODS: Patients aged 36–65, with OA grade III (Kellgren & Lawrence) were recruited. They had been referred for radiographic examination due to knee pain and had no history of major knee injury. They were randomized to a twice weekly supervised one hour exercise intervention for six weeks, or to a non-intervention control group. Exercise was performed at ≥ 60% of maximum heart rate (HR max). The primary outcome measure was the Knee injury and Osteoarthritis Outcome Score (KOOS). Follow-up occurred at 6 weeks and 6 months. RESULTS: Sixty-one subjects (mean age 56 (SD 6), 51 % women, mean BMI 29.5 (SD 4.8)) were randomly assigned to intervention (n = 30) or control group (n = 31). No significant differences in the KOOS subscales assessing pain, other symptoms, or function in daily life or in sport and recreation were seen at any time point between exercisers and controls. In the exercise group, an improvement was seen at 6 weeks in the KOOS subscale quality of life compared to the control group (mean change 4.0 vs. -0.7, p = 0.05). The difference between groups was still persistent at 6 months (p = 0.02). CONCLUSION: A six-week high-intensive exercise program had no effect on pain or function in middle-aged patients with moderate to severe radiographic knee OA. Some effect was seen on quality of life in the exercise group compared to the control group

The attitudes, beliefs and behaviours of GPs regarding exercise for chronic knee pain: a systematic review

<p>Abstract</p> <p>Background</p> <p>Joint pain, specifically chronic knee pain (CKP), is a frequent cause of chronic pain and limitation of function and mobility among older adults. Multiple evidence-based guidelines recommend exercise as a first-line treatment for all patients with CKP or knee osteoarthritis (KOA), yet healthcare practitioners' attitudes and beliefs may limit their implementation. This systematic review aims to identify the attitudes, beliefs and behaviours of General Practitioners (GPs) regarding the use of exercise for CKP/KOA.</p> <p>Methods</p> <p>We searched four electronic databases between inception and January 2008, using subject headings to identify studies examining the attitudes, beliefs or behaviours of GPs regarding the use of exercise for the treatment of CKP/KOA in adults aged over 45 years in primary care. Studies referring to patellofemoral pain syndrome or CKP secondary to other causes or that occurring in a prosthetic joint were excluded. Once inclusion and exclusion criteria were applied, study data were extracted and summarised. Study quality was independently reviewed using two assessment tools.</p> <p>Results</p> <p>From 2135 potentially relevant articles, 20 were suitable for inclusion. A variety of study methodologies and approaches to measuring attitudes beliefs and behaviours were used among the studies. Quality assessment revealed good reporting of study objective, type, outcome factors and, generally, the sampling frame. However, criticisms included use of small sample sizes, low response rates and under-reporting of non-responder factors. Although 99% of GPs agreed that exercise should be used for CKP/KOA and reported ever providing advice or referring to a physiotherapist, up to 29% believed that rest was the optimum management approach. The frequency of actual provision of exercise advice or physiotherapy referral was lower. Estimates of provision of exercise advice and physiotherapy referral were generally higher for vignette-based studies (exercise advice 9%-89%; physiotherapy referral 44%-77%) than reviews of actual practice (exercise advice 5%-52%; physiotherapy referral 13-63%). <it>A</it><it>dvice to exercise </it>and exercise <it>prescription </it>were not clearly differentiated.</p> <p>Conclusions</p> <p>Attitudes and beliefs of GPs towards exercise for CKP/KOA vary widely and exercise appears to be underused in the management of CKP/KOA. Limitations of the evidence base include the paucity of studies directly examining attitudes of GPs, poor methodological quality, limited generalisability of results and ambiguity concerning GPs' expected roles. Further investigation is required of the roles of GPs in using exercise as first-line management of CKP/KOA.</p

Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control.

N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia

Supreme activity of gramicidin S against resistant, persistent and biofilm cells of staphylococci and enterococci.

Three promising antibacterial peptides were studied with regard to their ability to inhibit the growth and kill the cells of clinical strains of Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. The multifunctional gramicidin S (GS) was the most potent, compared to the membranotropic temporin L (TL), being more effective than the innate-defence regulator IDR-1018 (IDR). These activities, compared across 16 strains as minimal bactericidal and minimal inhibitory concentrations (MIC), are independent of bacterial resistance pattern, phenotype variations and/or biofilm-forming potency. For S. aureus strains, complete killing is accomplished by all peptides at 5 × MIC. For E. faecalis strains, only GS exhibits a rapid bactericidal effect at 5 × MIC, while TL and IDR require higher concentrations. The biofilm-preventing activities of all peptides against the six strains with the largest biofilm biomass were compared. GS demonstrates the lowest minimal biofilm inhibiting concentrations, whereas TL and IDR are consistently less effective. In mature biofilms, only GS completely kills the cells of all studied strains. We compare the physicochemical properties, membranolytic activities, model pharmacokinetics and eukaryotic toxicities of the peptides and explain the bactericidal, antipersister and antibiofilm activities of GS by its elevated stability, pronounced cell-penetration ability and effective utilization of multiple modes of antibacterial action

A far-ultraviolet-driven photoevaporation flow observed in a protoplanetary disk.

Most low-mass stars form in stellar clusters that also contain massive stars, which are sources of far-ultraviolet (FUV) radiation. Theoretical models predict that this FUV radiation produces photodissociation regions (PDRs) on the surfaces of protoplanetary disks around low-mass stars, which affects planet formation within the disks. We report James Webb Space Telescope and Atacama Large Millimeter Array observations of a FUV-irradiated protoplanetary disk in the Orion Nebula. Emission lines are detected from the PDR; modeling their kinematics and excitation allowed us to constrain the physical conditions within the gas. We quantified the mass-loss rate induced by the FUV irradiation and found that it is sufficient to remove gas from the disk in less than a million years. This is rapid enough to affect giant planet formation in the disk