The anti-mitotic agents PTC-028 and PTC596 display potent activity in pre-clinical models of multiple myeloma but challenge the role of BMI-1 as an essential tumour gene

Future progress in the treatment of multiple myeloma (MM) requires both the characterisation of key drivers of the disease and novel, innovative approaches to tackle these vulnerabilities. The present study focussed on the pre-clinical evaluation of a novel drug class, BMI-1 modulators, in MM. We demonstrate potent activity of PTC-028 and PTC596 in a comprehensive set of in vitro and in vivo models, including models of drug resistance and stromal support. Treatment of MM cells with PTC-028 and PTC596 downregulated BMI-1 protein levels, which was found to correlate with drug activity. Surprisingly, BMI-1 was dispensable for the activity of BMI-1 modulators and MM cell growth. Our data rather point to mitotic arrest accompanied by myeloid cell leukaemia-1 (MCL-1) loss as key anti-MM mechanisms and reveal impaired MYC and AKT signalling activity due to BMI-1 modulator treatment. Moreover, we observed a complete eradication of MM after PTC596 treatment in the 5TGM.1 in vivo model and define epigenetic compounds and B cell leukaemia/lymphoma 2 homology domain 3 (BH3) mimetics as promising combination partners. These results bring into question the postulated role of BMI-1 as an essential MM gene and confirm BMI-1 modulators as potent anti-mitotic agents with encouraging pre-clinical activity that supports their rapid translation into clinical trials.     

Disinvestment and reinvestment in respiratory care: use of programme budgeting and marginal analysis in north Wales,UK

BackgroundRespiratory care is a large cost driver for Betsi Cadwaladr University Health Board, the largest health board in Wales. Since health boards face increasing pressure to deliver services that are value for money, a programme budgeting marginal analysis (PBMA) of respiratory care was commissioned.MethodsA research group with health economics, clinical, National Health Service (NHS) finance, and pharmacy prescribing expertise was established to gather evidence on the current respiratory care pathway in north Wales and to recommend where expenditure within the service should be focussed using PBMA. A PBMA panel was established consisting of managers and directors of medicines' management, therapies, finance, planning, and health-care professionals. The review identified a budget of £86·9 million spent on respiratory care in 2012–13 for a population of 700 000. With an agreed list of criteria developed by the research team and the panel, electronic voting was used to establish criteria for decision making and vote on candidates to disinvest and reinvest in. The panel was also asked to make a set of recommendations about the allocation of funding within the respiratory service. Evidence was provided in a booklet of the potential for cost savings (from NHS finance staff) and health benefits (from a rapid review of the evidence) of the proposed recommendations to equip the panel with the notion of opportunity cost when making their investment and disinvestment decisions.Findings13 candidates were discussed; they were individual proposals for resource reallocation, ranging from specific interventions to cross agency partnership. After extensive discussion facilitated by a chairperson, four candidates received recommendations to disinvest, seven to invest, and two to maintain current activity. The panel were also able to rank the candidates in order of highest priority for the health board to lowest priority.InterpretationThis exercise demonstrates the potential for health boards to use evidence-based approaches to reach potentially controversial disinvestment and reinvestment decisions. This exercise, though in its early stages, could offer further support for changes in respiratory care implementation in north Wales, if recommendations were to be implemented. PBMA also has wider implications across health agencies in the UK for resource allocation decisions, such as diabetes care.FundingNone.     

ParP prevents dissociation of CheA from chemotactic signaling arrays and tethers them to a polar anchor

Bacterial chemotaxis proteins are organized into ordered arrays. In peritrichous organisms, such as Escherichia coli, stochastic assembly processes are thought to account for the placement of chemotaxis arrays, which are nonuniformly distributed. In contrast, we previously found that chemotactic signaling arrays in polarly flagellated vibrios are uniformly polar and that array localization is dependent on the ParA-like ATPase ParC. However, the processes that enable ParC to facilitate array localization have not been described. Here, we show that a previously uncharacterized protein, ParP, interacts with ParC and that ParP is integral to array localization in Vibrio parahaemolyticus. ParC’s principal contribution to chemotaxis appears to be via positioning of ParP. Once recruited to the pole by ParC, ParP sequesters arrays at this site by capturing and preventing the dissociation of chemotactic signaling protein (CheA). Notably, ParP also stabilizes chemotactic protein complexes in the absence of ParC, indicating that some of its activity is independent of this interaction partner. ParP recruits CheA via CheA’s localization and inheritance domain, a region found only in polarly flagellated organisms that encode ParP, ParC, and CheA. Thus, a tripartite (ParC–ParP–CheA) interaction network enables the polar localization and sequestration of chemotaxis arrays in polarly flagellated organisms. Localization and sequestration of chemotaxis clusters adjacent to the flagella—to which the chemotactic signal is transmitted—facilitates proper chemotaxis as well as accurate inheritance of these macromolecular machines.Motile bacteria use chemotaxis to survey their environments and navigate in response to them. In particular, chemotactic sensing and response systems enable bacteria to recognize chemical gradients in their surroundings and to direct themselves toward favorable environments and away from detrimental ones. Chemotactic behavior is mediated by two-component signal-transduction pathways that detect such gradients and transmit this information via a phosphorelay to the bacterial flagella. Unfavorable gradients, i.e., a decrease in attractants or an increase in repellants, generate a signal to change flagellar rotation, which results in a change in swimming direction and a net movement toward more favorable conditions. This ability to react to changes in the external milieu can be essential for viability and competitiveness (reviewed in ref. 1).Chemoeffectors typically are detected by transmembrane chemosensory proteins termed “methyl-accepting chemotaxis proteins” (MCPs). These receptors interact with a histidine kinase, CheA, whose interaction with MCPs is stabilized by an adaptor protein, CheW. Upon recognition of a decrease in attractant or increase in repellant, the MCPs induce CheA autophosphorylation. Phosphorylated CheA transfers its phosphate group to the response regulator CheY, which diffuses freely in the cytoplasm. Accumulation and binding of phosphorylated CheY to the flagellar switch protein FliM increases the probability of a change in flagellar motor rotation, resulting in movement toward more favorable conditions (Fig. S1A) (1).Chemosensory proteins are found universally in large macromolecular clusters known as “chemotactic signaling arrays” (2–5). MCPs, together with CheA and CheW, form the sensory core of these clusters. Interactions between the cytoplasmic signaling domains of the receptors are sufficient for the formation of receptor arrays (6), although the formation is enhanced in the presence of CheW or CheA (6–8). Recent studies have suggested that array formation in Escherichia coli is a stochastic process in which individual receptors are inserted randomly in the membrane, where they diffuse freely and either join existing arrays or nucleate new ones (9). This process results in a nonuniform distribution of signaling arrays at cell poles and randomly along the cell length (10). In other organisms chemosensory arrays are localized only to the cell poles (11–14); however, the mechanisms behind polar localization are incompletely understood.We recently reported that the principal chemotaxis proteins in Vibrio cholerae, the Gram-negative bacterium responsible for the diarrheal disease cholera, display a markedly different distribution from that observed in E. coli (15). Newborn cells contain a single focus of chemotaxis proteins at their old pole. Subsequently, as cells mature, a second focus develops at the new pole, resulting in bipolar localization. Uni-and bipolar targeting of these chemotaxis proteins is dependent on ParC, a representative of a distinct family of ParA-like ATPases encoded within chemotaxis operons of many polar-flagellated bacteria. Like the chemotaxis signaling proteins, ParC exhibits a cell cycle-dependent unipolar/bipolar distribution (15). The mechanisms that enable ParC to control the localization of chemotactic signaling arrays have not been described. Targeting of ParC to the pole is dependent on the polar determinant, HubP, although HubP and ParC do not interact directly (16).ParA-like ATPases are responsible for the spatiotemporal positioning of several additional intracellular processes, including positioning of chromosomes and plasmids and identification of the cell division plane (reviewed in refs. 17 and 18). ParA-like proteins modulate the localization of cytosolic chemotaxis clusters and carboxysomes in Rhodobacter sphaeroides (19) and Synechococcus elongatus (20), respectively. The localization and activity of ParA-like proteins typically is controlled by ATP binding and hydrolysis, because ATP binding governs their capacity to oligomerize and to interact with other proteins (reviewed in refs. 17 and 18). Notably, many ParA-like proteins are encoded adjacent to genes encoding interacting proteins that also are required for their function (e.g., ParB). Like ParA proteins, these partners typically display a restricted and dynamic distribution within the cell. In general, the role of the partner protein is to act as a bridge between ParA and its target and to regulate the enzyme’s ATPase activity.Here, we investigated the function of an unannotated open reading frame, now designated parP, which is encoded downstream of parC. ParP proved to be a ParC partner protein, and, like ParC, ParP colocalizes with polar chemotactic signaling arrays in Vibrio parahaemolyticus. Positioning of ParP is dependent on ParC, and localization of ParP appears to be ParC’s principal function. ParP promotes array stability as well as positioning and retention at the pole. Following its recruitment to the pole by ParC, ParP sequesters arrays at this site by capturing and preventing the dissociation of CheA. Notably, in contrast to many other ParA-associated proteins, we found that ParP has activity that is independent of its ParA-like partner. Even in the absence of ParC, ParP prevents the dissociation of CheA from clusters of chemotaxis proteins. ParP maintains clusters of CheA by interacting with a localization and inheritance domain (LID) that is present only in CheA proteins with coresident ParP and ParC and also can be bound by ParC. Thus, our data indicate that a tripartite ParC–ParP–CheA interaction network promotes proper polar localization, sequestration, and inheritance of the macromolecular machine that is responsible for chemotactic signaling.     

Enterotoxicity of a nonribosomal peptide causes antibiotic-associated colitis

Antibiotic therapy disrupts the human intestinal microbiota. In some patients rapid overgrowth of the enteric bacterium Klebsiella oxytoca results in antibiotic-associated hemorrhagic colitis (AAHC). We isolated and identified a toxin produced by K. oxytoca as the pyrrolobenzodiazepine tilivalline and demonstrated its causative action in the pathogenesis of colitis in an animal model. Tilivalline induced apoptosis in cultured human cells in vitro and disrupted epithelial barrier function, consistent with the mucosal damage associated with colitis observed in human AAHC and the corresponding animal model. Our findings reveal the presence of pyrrolobenzodiazepines in the intestinal microbiota and provide a mechanism for colitis caused by a resident pathobiont. The data link pyrrolobenzodiazepines to human disease and identify tilivalline as a target for diagnosis and neutralizing strategies in prevention and treatment of colitis.The human digestive tract houses several hundred microbial species (1). This complex community, known collectively as the intestinal microbiota, is important for human digestive physiology, immune function, and protection from pathogens (2). In healthy humans homeostasis between the microbiota and the host is maintained. Perturbed homeostasis, a state known as dysbiosis, is defined by shifts in bacterial abundances, their altered local distribution, and modified metabolic activities. Infections or nutritional or therapeutic stressors can induce dysbiosis and alter the integrity of the mucosal barrier. The pathogenesis of many disorders has been associated with dysbiotic changes in the intestinal microbiota, including metabolic diseases, cancer, allergies, and afflictions of the bowel, liver, and lung (3–6). The underlying mechanisms are poorly understood; however, recent work is beginning to show that microbial metabolites are important mediators of complex interactions between the enteric microbiota and the host. Thus, bacterial metabolites produced in a dysbiotic intestinal environment together with host factors may be important determinants in the pathogenesis of disease (7, 8).Many drug therapies, and especially antibiotics, disrupt the human intestinal microbiota. Up to 30% of patients taking antibiotics experience diarrhea (9). With the exception of infection with toxin-producing Clostridium difficile, the underlying causes for the vast majority of diarrhea or colitis cases after antibiotic therapy are not known. A link to antibiotic-driven changes in microbial abundances and overgrowth of certain bacterial species is observed; however, the pathophysiological mechanisms remain to be determined (10). The expanding species are often not pathogens (11, 12) and in the majority of cases no single organism seems to explain either the presence or absence of disease.In contrast to this general view, antibiotic-associated hemorrhagic colitis (AAHC) is a disease associated with antibiotic-driven enterobacterial overgrowth, which is dominated by a single organism, Klebsiella oxytoca. This bacterium is a resident of the gut in 2–10% of healthy individuals (13–15). In AAHC, a brief therapy with penicillins triggers dysbiosis with sudden onset of bloody diarrhea and abdominal cramps (14). K. oxytoca constitutively expresses resistance to amino- and carboxypenicillins (16). Antibiotic clearance of a niche in the colon facilitates overgrowth of K. oxytoca (10⁷ cfu/g feces during acute phases of AAHC compared with 10² cfu/g feces in healthy subjects) (15). Our earlier work showed that the dysbiotic population shift dominated by K. oxytoca causes colitis (14), thereby identifying the resident organism as a pathobiont (17).Here we aimed to characterize the pathogenicity of K. oxytoca in colitis. We identified the nonribosomal peptide tilivalline from K. oxytoca and demonstrate its causative action in an animal model of AAHC. Unlike most known enterotoxins, tilivalline is not a protein but a pyrrolobenzodiazepine metabolite not previously linked with the intestinal microbiota and human disease. Our findings define a mechanism whereby antibiotic-driven enterobacterial overgrowth and concomitant production of the small molecule enterotoxin ultimately leads to colitis.     

The usage of multidisciplinary physical therapies at the Rio de Janeiro 2016 Olympic Summer Games: an observational study

BackgroundThis observational research study analyses the uptake of physical therapies treatments in the Polyclinic during the Rio 2016 Olympic Games.ObjectiveTo describe the usage of physical therapies services – physical therapy, osteopath, chiropractic, and sports massage – by athletes and non-athletes and across different sports.MethodsThe multidisciplinary team of physical therapies recorded treatment modalities, information on provider discipline and reason for attendance, in an Electronic Medical Record system throughout the 32 days of operation of the Olympic Polyclinic. Cold-therapy total immersion ice baths (TIIB) were provided as part of the services, but were reported and analysed separately.ResultsThere were 4993 encounters (4038 athletes, 955 non-athlete encounters). 1395 athletes (12.4% of all athletes) and 393 non-athletes sought treatment. For all four provider disciplines, in addition to TIIB, the primary reason for athlete attendance was for recovery (52% of all encounters), followed by injury treatment (30%), and maintenance (16%). Athletes reported “injury” as the main reason for physical therapy (92% of all encounters, 2.8 encounters per athlete), chiropractic (94%, 1.9) and osteopathy (91%, 1.8) visits. Almost all TIIB visits were used for recovery (98% of all TIIB encounters; 2.1 encounters per athlete). Athletes from handball (37% of all handball athletes), followed by judo (22%), and athletics (21%), presented the largest user groups.ConclusionThis Olympic Polyclinic study evaluates the physical therapies’ activity, and athlete's reason for use of the multidisciplinary physical therapies team, including total immersion ice bath provision. These results emphasise the importance of a multidisciplinary approach.