Enhanced heterogeneity of rpoB in Mycobacterium tuberculosis found at low pH.

OBJECTIVES: The aim of this study was to gain an insight into the molecular mechanisms of the evolution of rifampicin resistance in response to controlled changes in the environment. METHODS: We determined the proportion of rpoB mutants in the chemostat culture and characterized the sequence of mutations found in the rifampicin resistance-determining region of rpoB in a steady-state chemostat at pH 7.0 and 6.2. RESULTS: The overall proportion of rpoB mutants of strain H37Rv remained constant for 37 days at pH 7.0, ranging between 3.6 x 10(-8) and 8.9 x 10(-8); however, the spectrum of mutations varied. The most commonly detected mutation, serine to leucine mutation at codon 531 (S531L), increased from 40% to 89%, while other mutations (S531W, H526Y, H526D, H526R, S522L and D516V) decreased over the 37 day sampling period. Changing the pH from 7.0 to 6.2 did not significantly alter the overall proportion of mutants, but resulted in a decrease in the percentage of strains harbouring S531L (from 89% to 50%) accompanied by an increase in the range of different mutations from 4 to 12. CONCLUSIONS: The data confirm that the fitness of strains with the S531L mutation is greater than that of strains containing other mutations. We also conclude that at low pH the environment is permissive for a wider spectrum of mutations, which may provide opportunities for a successful mutant to survive

StereoTactic radiotherapy for wet Age-Related macular degeneration (STAR):Study protocol for a randomized controlled clinical trial

BACKGROUND: The standard of care for neovascular age-related macular degeneration (nAMD) involves ongoing intravitreal injections of anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF). The most commonly used anti-VEGF drugs are ranibizumab, bevacizumab and aflibercept. The main objective of the STAR trial is to determine if stereotactic radiotherapy can reduce the number of anti-VEGF injections that patients with nAMD require. METHODS/DESIGN: STAR is a multicentre, double-masked, randomised, sham-controlled clinical trial. It evaluates a new device (manufactured by Oraya, Newark, CA, USA) designed to deliver stereotactic radiotherapy (SRT) to nAMD lesions. The trial enrols participants with chronic, active nAMD. Participants receive a single SRT treatment (16 Gy or sham) with a concomitant baseline intravitreal injection of 0.5 mg ranibizumab. Thereafter, they attend every month for 24 months, and ranibizumab is administered at the visit if retreatment criteria are met. The primary outcome is the number of pro re nata ranibizumab injections during the first 24 months. Secondary outcomes include visual acuity, lesion morphology, quality of life and safety. Additional visits occur at 36 and 48 months to inspect for radiation retinopathy. The target sample size of 411 participants (randomised 2:1 in favour of radiation) is designed to detect a reduction of 2.5 injections against ranibizumab monotherapy, at 90% power, and a significance level (alpha) of 0.025 (one-sided two-sample t test). This gives 97% power to detect non-inferiority of visual acuity at a five-letter margin. The primary analyses will be by intention to treat. DISCUSSION: The safety and efficacy outcomes will help determine the role of SRT in the management of chronic, active nAMD. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number: ISRCTN12884465. Registered on 28 November 2014. ClinicalTrials.gov: NCT02243878. Registered on 17 September 2014

Cognitive behavioural therapy for adults with dissociative seizures (CODES): a pragmatic, multicentre, randomised controlled trial.

BACKGROUND: Dissociative seizures are paroxysmal events resembling epilepsy or syncope with characteristic features that allow them to be distinguished from other medical conditions. We aimed to compare the effectiveness of cognitive behavioural therapy (CBT) plus standardised medical care with standardised medical care alone for the reduction of dissociative seizure frequency. METHODS: In this pragmatic, parallel-arm, multicentre randomised controlled trial, we initially recruited participants at 27 neurology or epilepsy services in England, Scotland, and Wales. Adults (≥18 years) who had dissociative seizures in the previous 8 weeks and no epileptic seizures in the previous 12 months were subsequently randomly assigned (1:1) from 17 liaison or neuropsychiatry services following psychiatric assessment, to receive standardised medical care or CBT plus standardised medical care, using a web-based system. Randomisation was stratified by neuropsychiatry or liaison psychiatry recruitment site. The trial manager, chief investigator, all treating clinicians, and patients were aware of treatment allocation, but outcome data collectors and trial statisticians were unaware of treatment allocation. Patients were followed up 6 months and 12 months after randomisation. The primary outcome was monthly dissociative seizure frequency (ie, frequency in the previous 4 weeks) assessed at 12 months. Secondary outcomes assessed at 12 months were: seizure severity (intensity) and bothersomeness; longest period of seizure freedom in the previous 6 months; complete seizure freedom in the previous 3 months; a greater than 50% reduction in seizure frequency relative to baseline; changes in dissociative seizures (rated by others); health-related quality of life; psychosocial functioning; psychiatric symptoms, psychological distress, and somatic symptom burden; and clinical impression of improvement and satisfaction. p values and statistical significance for outcomes were reported without correction for multiple comparisons as per our protocol. Primary and secondary outcomes were assessed in the intention-to-treat population with multiple imputation for missing observations. This trial is registered with the International Standard Randomised Controlled Trial registry, ISRCTN05681227, and ClinicalTrials.gov, NCT02325544. FINDINGS: Between Jan 16, 2015, and May 31, 2017, we randomly assigned 368 patients to receive CBT plus standardised medical care (n=186) or standardised medical care alone (n=182); of whom 313 had primary outcome data at 12 months (156 [84%] of 186 patients in the CBT plus standardised medical care group and 157 [86%] of 182 patients in the standardised medical care group). At 12 months, no significant difference in monthly dissociative seizure frequency was identified between the groups (median 4 seizures [IQR 0-20] in the CBT plus standardised medical care group vs 7 seizures [1-35] in the standardised medical care group; estimated incidence rate ratio [IRR] 0·78 [95% CI 0·56-1·09]; p=0·144). Dissociative seizures were rated as less bothersome in the CBT plus standardised medical care group than the standardised medical care group (estimated mean difference -0·53 [95% CI -0·97 to -0·08]; p=0·020). The CBT plus standardised medical care group had a longer period of dissociative seizure freedom in the previous 6 months (estimated IRR 1·64 [95% CI 1·22 to 2·20]; p=0·001), reported better health-related quality of life on the EuroQoL-5 Dimensions-5 Level Health Today visual analogue scale (estimated mean difference 6·16 [95% CI 1·48 to 10·84]; p=0·010), less impairment in psychosocial functioning on the Work and Social Adjustment Scale (estimated mean difference -4·12 [95% CI -6·35 to -1·89]; p<0·001), less overall psychological distress than the standardised medical care group on the Clinical Outcomes in Routine Evaluation-10 scale (estimated mean difference -1·65 [95% CI -2·96 to -0·35]; p=0·013), and fewer somatic symptoms on the modified Patient Health Questionnaire-15 scale (estimated mean difference -1·67 [95% CI -2·90 to -0·44]; p=0·008). Clinical improvement at 12 months was greater in the CBT plus standardised medical care group than the standardised medical care alone group as reported by patients (estimated mean difference 0·66 [95% CI 0·26 to 1·04]; p=0·001) and by clinicians (estimated mean difference 0·47 [95% CI 0·21 to 0·73]; p<0·001), and the CBT plus standardised medical care group had greater satisfaction with treatment than did the standardised medical care group (estimated mean difference 0·90 [95% CI 0·48 to 1·31]; p<0·001). No significant differences in patient-reported seizure severity (estimated mean difference -0·11 [95% CI -0·50 to 0·29]; p=0·593) or seizure freedom in the last 3 months of the study (estimated odds ratio [OR] 1·77 [95% CI 0·93 to 3·37]; p=0·083) were identified between the groups. Furthermore, no significant differences were identified in the proportion of patients who had a more than 50% reduction in dissociative seizure frequency compared with baseline (OR 1·27 [95% CI 0·80 to 2·02]; p=0·313). Additionally, the 12-item Short Form survey-version 2 scores (estimated mean difference for the Physical Component Summary score 1·78 [95% CI -0·37 to 3·92]; p=0·105; estimated mean difference for the Mental Component Summary score 2·22 [95% CI -0·30 to 4·75]; p=0·084), the Generalised Anxiety Disorder-7 scale score (estimated mean difference -1·09 [95% CI -2·27 to 0·09]; p=0·069), and the Patient Health Questionnaire-9 scale depression score (estimated mean difference -1·10 [95% CI -2·41 to 0·21]; p=0·099) did not differ significantly between groups. Changes in dissociative seizures (rated by others) could not be assessed due to insufficient data. During the 12-month period, the number of adverse events was similar between the groups: 57 (31%) of 186 participants in the CBT plus standardised medical care group reported 97 adverse events and 53 (29%) of 182 participants in the standardised medical care group reported 79 adverse events. INTERPRETATION: CBT plus standardised medical care had no statistically significant advantage compared with standardised medical care alone for the reduction of monthly seizures. However, improvements were observed in a number of clinically relevant secondary outcomes following CBT plus standardised medical care when compared with standardised medical care alone. Thus, adults with dissociative seizures might benefit from the addition of dissociative seizure-specific CBT to specialist care from neurologists and psychiatrists. Future work is needed to identify patients who would benefit most from a dissociative seizure-specific CBT approach. FUNDING: National Institute for Health Research, Health Technology Assessment programme

A definition of flare in low back pain (LBP): A multiphase process involving perspectives of individuals with LBP and expert consensus

Low back pain (LBP) varies over time. Consumers, clinicians and researchers use various terms to describe fluctuations of LBP symptoms. Although "flare" is commonly used to describe symptom fluctuation, there is no consensus on how it is defined. This study aimed to obtain consensus for a LBP flare definition using a mixed-method approach. Step 1 involved derivation of a preliminary candidate flare definition based on thematic analysis of consumers' views in consultation with an expert consumer writer. In Step 2, a workshop was conducted to incorporate perspectives of LBP experts into the preliminary flare definition, which resulted in two alternative LBP flare definitions. Step 3 refined the definition using a two-round Delphi consensus process with experts in musculoskeletal conditions. The definition favoured by experts was further tested with individuals with LBP in Step 4, using the definition in three scenarios. This multiphase study produced a LBP flare definition that distinguishes it from other LBP fluctuations, represents views of consumers, involves expert consensus, and is understandable by consumers in clinical and research contexts: "A flare-up is a worsening of your condition that lasts from hours to weeks that is difficult to tolerate and generally impacts your usual activities and/or emotions". Perspective: A multiphase processes produced a low back pain (LBP) flare definition that distinguishes it from other LBP fluctuations, involves expert consensus and represents consumers' views

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway(1,2). Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis

Adaptative Potential of the Lactococcus Lactis IL594 Strain Encoded in Its 7 Plasmids

The extrachromosomal gene pool plays a significant role both in evolution and in the environmental adaptation of bacteria. The L. lactis subsp. lactis IL594 strain contains seven plasmids, named pIL1 to pIL7, and is the parental strain of the plasmid-free L. lactis IL1403, which is one of the best characterized lactococcal strains of LAB. Complete nucleotide sequences of pIL1 (6,382 bp), pIL2 (8,277 bp), pIL3 (19,244 bp), pIL4 (48,979), pIL5 (23,395), pIL6 (28,435 bp) and pIL7 (28,546) were established and deposited in the generally accessible database (GeneBank). Nine highly homologous repB-containing replicons, belonging to the lactococcal theta-type replicons, have been identified on the seven plasmids. Moreover, a putative region involved in conjugative plasmid mobilization was found on four plasmids, through identification of the presence of mob genes and/or oriT sequences. Detailed bioinformatic analysis of the plasmid nucleotide sequences provided new insight into the repertoire of plasmid-encoded functions in L. lactis, and indicated that plasmid genes from IL594 strain can be important for L. lactis adaptation to specific environmental conditions (e.g. genes coding for proteins involved in DNA repair or cold shock response) as well as for technological processes (e.g. genes encoding citrate and lactose utilization, oligopeptide transport, restriction-modification system). Moreover, global gene analysis indicated cooperation between plasmid- and chromosome-encoded metabolic pathways

Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): Survival results from an adaptive, multiarm, multistage, platform randomised controlled trial

BACKGROUND Long-term hormone therapy has been the standard of care for advanced prostate cancer since the 1940s. STAMPEDE is a randomised controlled trial using a multiarm, multistage platform design. It recruits men with high-risk, locally advanced, metastatic or recurrent prostate cancer who are starting first-line long-term hormone therapy. We report primary survival results for three research comparisons testing the addition of zoledronic acid, docetaxel, or their combination to standard of care versus standard of care alone. METHODS Standard of care was hormone therapy for at least 2 years; radiotherapy was encouraged for men with N0M0 disease to November, 2011, then mandated; radiotherapy was optional for men with node-positive non-metastatic (N+M0) disease. Stratified randomisation (via minimisation) allocated men 2:1:1:1 to standard of care only (SOC-only; control), standard of care plus zoledronic acid (SOC + ZA), standard of care plus docetaxel (SOC + Doc), or standard of care with both zoledronic acid and docetaxel (SOC + ZA + Doc). Zoledronic acid (4 mg) was given for six 3-weekly cycles, then 4-weekly until 2 years, and docetaxel (75 mg/m(2)) for six 3-weekly cycles with prednisolone 10 mg daily. There was no blinding to treatment allocation. The primary outcome measure was overall survival. Pairwise comparisons of research versus control had 90% power at 2·5% one-sided α for hazard ratio (HR) 0·75, requiring roughly 400 control arm deaths. Statistical analyses were undertaken with standard log-rank-type methods for time-to-event data, with hazard ratios (HRs) and 95% CIs derived from adjusted Cox models. This trial is registered at ClinicalTrials.gov (NCT00268476) and ControlledTrials.com (ISRCTN78818544). FINDINGS 2962 men were randomly assigned to four groups between Oct 5, 2005, and March 31, 2013. Median age was 65 years (IQR 60-71). 1817 (61%) men had M+ disease, 448 (15%) had N+/X M0, and 697 (24%) had N0M0. 165 (6%) men were previously treated with local therapy, and median prostate-specific antigen was 65 ng/mL (IQR 23-184). Median follow-up was 43 months (IQR 30-60). There were 415 deaths in the control group (347 [84%] prostate cancer). Median overall survival was 71 months (IQR 32 to not reached) for SOC-only, not reached (32 to not reached) for SOC + ZA (HR 0·94, 95% CI 0·79-1·11; p=0·450), 81 months (41 to not reached) for SOC + Doc (0·78, 0·66-0·93; p=0·006), and 76 months (39 to not reached) for SOC + ZA + Doc (0·82, 0·69-0·97; p=0·022). There was no evidence of heterogeneity in treatment effect (for any of the treatments) across prespecified subsets. Grade 3-5 adverse events were reported for 399 (32%) patients receiving SOC, 197 (32%) receiving SOC + ZA, 288 (52%) receiving SOC + Doc, and 269 (52%) receiving SOC + ZA + Doc. INTERPRETATION Zoledronic acid showed no evidence of survival improvement and should not be part of standard of care for this population. Docetaxel chemotherapy, given at the time of long-term hormone therapy initiation, showed evidence of improved survival accompanied by an increase in adverse events. Docetaxel treatment should become part of standard of care for adequately fit men commencing long-term hormone therapy. FUNDING Cancer Research UK, Medical Research Council, Novartis, Sanofi-Aventis, Pfizer, Janssen, Astellas, NIHR Clinical Research Network, Swiss Group for Clinical Cancer Research

Abiraterone acetate plus prednisolone for metastatic patients starting hormone therapy: 5-year follow-up results from the STAMPEDE randomised trial (NCT00268476)

Abiraterone acetate plus prednisolone (AAP) previously demonstrated improved survival in STAMPEDE, a multiarm, multistage platform trial in men starting long-term hormone therapy for prostate cancer. This long-term analysis in metastatic patients was planned for 3 years after the first results. Standard-of-care (SOC) was androgen deprivation therapy. The comparison randomised patients 1:1 to SOC-alone with or without daily abiraterone acetate 1000 mg + prednisolone 5 mg (SOC + AAP), continued until disease progression. The primary outcome measure was overall survival. Metastatic disease risk group was classified retrospectively using baseline CT and bone scans by central radiological review and pathology reports. Analyses used Cox proportional hazards and flexible parametric models, accounting for baseline stratification factors. One thousand and three patients were contemporaneously randomised (November 2011 to January 2014): median age 67 years; 94% newly-diagnosed; metastatic disease risk group: 48% high, 44% low, 8% unassessable; median PSA 97 ng/mL. At 6.1 years median follow-up, 329 SOC-alone deaths (118 low-risk, 178 high-risk) and 244 SOC + AAP deaths (75 low-risk, 145 high-risk) were reported. Adjusted HR = 0.60 (95% CI: 0.50-0.71; P = 0.31 × 10−9) favoured SOC + AAP, with 5-years survival improved from 41% SOC-alone to 60% SOC + AAP. This was similar in low-risk (HR = 0.55; 95% CI: 0.41-0.76) and high-risk (HR = 0.54; 95% CI: 0.43-0.69) patients. Median and current maximum time on SOC + AAP was 2.4 and 8.1 years. Toxicity at 4 years postrandomisation was similar, with 16% patients in each group reporting grade 3 or higher toxicity. A sustained and substantial improvement in overall survival of all metastatic prostate cancer patients was achieved with SOC + abiraterone acetate + prednisolone, irrespective of metastatic disease risk group

Mechanisms of sporadic cerebral small vessel disease:insights from neuroimaging

The term “cerebral small vessel disease” (SVD) describes a range of neuroimaging, pathological and associated clinical features. The latter range from none, to discrete focal neurological symptoms (stroke), to insidious global neurological dysfunction and dementia. The public health burden is considerable. The pathogenesis is largely unknown. Although associated with vascular risk factors, and generally considered to result from an intrinsic cerebral arteriolar occlusive disease, the pathological processes leading to the arteriolar disease, how these result in brain disease, how SVD lesions contribute to neurological or cognitive symptoms and the relationship to risk factors, have been the subject of much speculation. Pathology often reflects end-stage disease making determination of the earliest stages difficult. Neuroimaging provides considerable insights: the small vessels are not easily seen themselves, but the effects of their malfunction on the brain can be tracked on detailed brain imaging. We review the growing evidence for the most likely mechanisms