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Metastasis remains the leading cause of cancer deaths worldwide and lung cancer, known for its highly metastatic progression, remains among the most lethal of malignancies. Lung cancer metastasis can selectively spread to multiple different organs, however the genetic and molecular drivers for this process are still poorly understood. Understanding the heterogeneous genomic profile of lung cancer metastases is considered key in identifying therapeutic targets that prevent its spread. Research has identified the key source for metastasis being clusters of cells rather than individual cancer cells. These clusters, known as metastatic cancer cell clusters (MCCCs) have been shown to be 100-fold more tumorigenic than individual cancer cells. Unfortunately, access to these primary drivers of metastases remains difficult and has limited our understanding of their molecular and genomic profiles. Strong evidence in the literature suggests that differentially regulated biological pathways in MCCCs can provide new therapeutic drug targets to help combat cancer metastases. In order to expand research into MCCCs and their role in metastasis, we demonstrate a novel, proof of principle technology, to capture MCCCs directly from patients' whole blood. Our platform can be readily tuned for different solid tumor types by combining a biomimicry-based margination effect coupled with immunoaffinity to isolate MCCCs. Adopting a selective capture approach based on overexpressed CD44 in MCCCs provides a methodology that preferentially isolates them from whole blood. Furthermore, we demonstrate a high capture efficiency of more than 90% when spiking MCCC-like model cell clusters into whole blood. Characterization of the captured MCCCs from lung cancer patients by immunofluorescence staining and genomic analyses, suggests highly differential morphologies and genomic profiles. This study lays the foundation to identify potential drug targets thus unlocking a new area of anti-metastatic therapeutics.

Thousands of offshore oil and gas platforms have been installed throughout the world's oceans and more structures are being installed as part of the transition to renewable energy. These structures increase the availability of ecological niches by providing hard substrate in midwater and complex 3D habitat on the seafloor. This can lead to 'hotspots' of biodiversity, or increased densities of flora and fauna, which potentially spill over into the local area. However, the distances over which these higher densities extend (the 'range of influence') can be highly variable. Fish aggregate at such structures, but the range of influence and any implications for wider fish populations, are unclear. We investigated the relationship between fish and platform areal densities using high resolution fisheries acoustic data. Data were collected in the waters surrounding the vessel exclusions zones around 16 oil and gas platforms in the North Sea, and throughout the wider area. We estimated densities of schooling fish using echo-integration, and densities of non-schooling fish using echo-counting. At 10 platforms, non-schooling fish densities were elevated near the platform relative to background levels in the equivalent wider area. The range of influence, defined here as the range to which fish densities were elevated above background, varied from 0.8 to 23 km. In areas of high platform density, fish schools were encountered more often, and non-schooling fish densities were higher, when controlling for other sources of environmental variation. This is the first time such long-range effects have been identified; previously, ranges of influence have been reported in the order of just 10s-100s of metres. These findings suggest that the environmental impact of these structures may extend further than previously thought, which may be relevant in the context of upcoming management decisions around the decommissioning of these structures.

IntroductionNot all patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection develop symptomatic coronavirus disease 2019 (COVID-19), making it challenging to assess the burden of COVID-19-related hospitalizations and mortality. We aimed to determine the proportion, resource utilization, and outcomes of SARS-CoV-2 positive patients admitted for COVID-19, and assess the impact of using the Center for Disease Control's (CDC) discharge diagnosis-based algorithm and the Massachusetts state department's drug administration-based classification system on identifying admissions for COVID-19.MethodsIn this retrospective cohort study, we enrolled consecutive SARS-CoV-2 positive patients admitted to one of five hospitals in British Columbia between December 19, 2021 and May 31,2022. We completed medical record reviews, and classified hospitalizations as being primarily for COVID-19 or with incidental SARS-CoV-2 infection. We applied the CDC algorithm and the Massachusetts classification to estimate the difference in hospital days, intensive care unit (ICU) days and in-hospital mortality and calculated sensitivity and specificity.ResultsOf 42,505 Emergency Department patients, 1,651 were admitted and tested positive for SARS-CoV-2, with 858 (52.0%, 95% CI 49.6-54.4) admitted for COVID-19. Patients hospitalized for COVID-19 required ICU admission (14.0% versus 8.2%, pConclusionHalf of SARS-CoV-2 hospitalizations were for COVID-19 during the Omicron wave. The CDC algorithm was more specific and sensitive than the Massachusetts classification, but underestimated the burden of COVID-19 admissions.Trial registrationClinicaltrials.gov, NCT04702945.

An ecologically valid, reliable and sensitive method of quantifying punch force variables would be useful for coaches and practitioners monitoring combat-specific performance. The present study utilised a vertically mounted force plate to quantify the peak punch force and rate of force development (RFD) of amateur boxers. Ten male senior elite amateur boxers performed maximal jab, cross, and hook punches across two separate days. The force plate showed excellent within-day and good-to-excellent between-day reliability for peak punch impact force and RFD (ICC 0.89-0.99). The CV% for all punch force variables were similar on day 1 (3-9%) and day 2 (4-10%). Standard error of measurement (SEM) and smallest worthwhile changes (SWC) revealed the force plate can detect small-to-moderate changes in punch performance. The greatest impact forces and RFD were found in the rear hook (2624 ± 581 N, 296448 ± 101823 N.s-1), followed by the lead hook (2524 ± 532 N, 256813 ± 81735 N.s-1), cross 2425 ± 545 N, 193004 ± 62671 N.s-1) and jab (1645 ± 537 N, 116675 ± 41577 N.s-1). The vertically mounted force plate is a reliable and sensitive test of punch performance, thus may be useful in determining the efficacy of training interventions.

Data from the past 30+ years of the male boxing World Heavyweight Championship (n = 182 bouts) was obtained. The USA were the most represented and produced more champions than any other nation, followed by the UK, Ukraine and Russia. Denmark (100%), Ukraine (85.4%) and the UK (67.3%) produced the greatest ‘success rates’ in World Heavyweight Championship contests. Where possible, comparisons between bout winners and losers were also made. Winners were significantly taller (p < 0.001, d = 0.35) and had a greater reach (p = 0.003, d = 0.23) when compared to losers. Championship bouts were settled by the following methods: a form of knockout (101), points decision (57), retirement (14), draw (3), disqualification (3), technical decision (1), whilst 3 no contests were omitted from the analysis. Total punches thrown and landed, and jabs and power punches thrown and landed were consistently significantly greater (p < 0.005, d = 0.27–0.73) in winners, compared to losers. Winners were more accurate compared to their losing counterparts by ~ 8 percentage points. The data presented in the present study clearly show some anthropometric advantages of championship bout winners, compared to their unsuccessful counterparts, and that winners are more active and accurate when compared to losers. The punch output data, albeit very basic, may be useful in informing tactical strategy and preparation of heavyweight prospects. Likewise, the data in the present study may be an interesting resource for professional boxing enthusiasts. Future research should seek to replicate the analysis in the present study across other weight divisions to explore any potential differences between weight classes. Additionally, extending the analysis to female boxing may provide interesting comparisons.

Fisheries harvest strategies are formal frameworks that represent a best-practice approach for sustainable fisheries management. A key component of a harvest strategy is a ’pre-agreed rule’, known as a harvest control rule (HCR), that sets fishing opportunities, e.g. catch limits, based on an estimate of fish stock status, e.g. estimated stock biomass. The harvest strategy development process is driven by stakeholders who are required to make a range of informed decisions, including on the selection of the preferred HCR. Capacity building may be required to facilitate the stakeholder engagement, particularly regarding the technical components of harvest strategies, including HCRs. The AMPLE package for R provides three interactive apps that support capacity building and stakeholder engagement on HCRs. These apps have been used during in-country national workshops around the western and central Pacific Ocean (WCPO) to support the development of harvest strategies for the Western and Central Pacific Fisheries Commission. These apps include several novel features: they take users from a gentle introduction to how HCRs work, to using methods for testing, comparing and selecting a preferred HCR from a suite of candidates. They include an introduction to the impact of uncertainty on the performance of an HCR, introduce performance indicators and discuss methods for selecting the preferred HCR based on management objectives. As such they provide a more detailed overview of HCRs than currently existing alternatives. These apps provide an effective platform for hands-on learning and have proven to be successful at supporting capacity building on HCRs in the WCPO. For example, using them for group activities and competitions stimulated productive discussions and increased understanding. As the model fishery in AMPLE is generic and not based on a real example, the apps will also be of interest to scientists, managers and stakeholders developing harvest strategies in other regions.

ObjectiveAlthough high visit-to-visit blood pressure variability (BPV) is an independent risk factor for cardiovascular events, the frequency of high BPV is unknown. We conducted this study to define the frequency of high BPV in primary care patients, clinical correlates, and association with antihypertensive therapies.MethodsRetrospective cohort study using electronic medical record data (with previously validated case definitions based on billing codes, free text analysis of progress notes, and prescribing data) from the Canadian Primary Care Sentinel Surveillance Network of 221,803 adults with multiple clinic visits over a 2-year period. We a priori defined a standard deviation>13.0 mm Hg in visit-to-visit systolic blood pressure (SBP) as "high BPV" based on prior literature.ResultsOverall, 85,455 (38.5%) patients had hypertension (mean 6.56 visits with SBP measurement, mean SBP 134.4 with Standard Deviation [SD] 11.3, 33.2% exhibited high BPV) and 136,348 did not (mean 3.96 visits with SBP measurement, mean SBP 120.9 with SD 8.2, 16.5% had high BPV). BPV increased with age regardless of whether individuals had hypertension or not; at all ages BPV varied across antihypertensive treatment regimens and was greater in those receiving renin angiotensin blockers or beta-blockers (pConclusionsHigh visit-to-visit BPV is present in one sixth of non-hypertensive adults and one third of hypertensive individuals and is more common in those with comorbidities. The frequency of high BPV varies across antihypertensive treatment regimens.

Physical training, testing, and monitoring are three key constitutes of athlete physical performance; however, there is a currently a lack of information on the prevalence of such methods in amateur boxing. This study aimed to explore the physical preparation practices of senior elite (SEB) and senior development (SDB) amateur boxers, and to determine whether these practices were discriminated by competitor level. One hundred and one amateur boxers (SEB n = 59, SDB n = 42) were surveyed on their understanding, perceptions and application of physical training, monitoring, and testing practices. SEB were associated with strength/power training (SEB 78%, SDB 50%, P = 0.005), monitor of training intensities (SEB 68%, SDB 40%, P = 0.006), and performing regular fitness testing (SEB 76%, SDB 50%, P = 0.006), compared to SDB. Likewise, SEB were twice as likely (56%) to have their physical preparation managed by a strength and conditioning (S&C) coach or sport scientist, compared to SDB (26%; P = 0.005). For the first time, these data demonstrate the extent to which competitor level is associated with preparatory practices in amateur boxing. Cost was identified as the main barrier in implementing several forms of scientific support in SDB. These data serve as a framework to enhance preparatory practices across different competitor levels in amateur boxing. This might include boxer and coach education on the benefits to a more scientific approach, and the use of cost-effective methods to develop, monitor and assess amateur boxers physical performance. This may be of particular importance where boxers are not funded, such as the SDB in the current study. However, this work may also be used to emphasise the importance of strength/power training, physical fitness testing and monitoring at the elite level of amateur boxing.

Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel®) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments, which cover different regions of a treatment area, resulted in a sustained or increased dermal remodeling response, with many genes either differentially regulated or continuously upregulated, supporting previous observations that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of human skin activates a number of biological processes involved in wound healing and tissue regeneration.

PurposeThe Palliative Prognostic (PaP) score; Palliative Prognostic Index (PPI); Feliu Prognostic Nomogram (FPN) and Palliative Performance Scale (PPS) have all been proposed as prognostic tools for palliative cancer care. However, clinical judgement remains the principal way by which palliative care professionals determine prognoses and it is important that the performance of prognostic tools is compared against clinical predictions of survival (CPS).MethodsThis was a multi-centre, cohort validation study of prognostic tools. Study participants were adults with advanced cancer receiving palliative care, with or without capacity to consent. Key prognostic data were collected at baseline, shortly after referral to palliative care services. CPS were obtained independently from a doctor and a nurse.ResultsPrognostic data were collected on 1833 participants. All prognostic tools showed acceptable discrimination and calibration, but none showed superiority to CPS. Both PaP and CPS were equally able to accurately categorise patients according to their risk of dying within 30 days. There was no difference in performance between CPS and FPN at stratifying patients according to their risk of dying at 15, 30 or 60 days. PPI was significantly (pConclusionsAlthough four commonly used prognostic algorithms for palliative care generally showed good discrimination and calibration, none of them demonstrated superiority to CPS. Prognostic tools which are less accurate than CPS are of no clinical use. However, prognostic tools which perform similarly to CPS may have other advantages to recommend them for use in clinical practice (e.g. being more objective, more reproducible, acting as a second opinion or as an educational tool). Future studies should therefore assess the impact of prognostic tools on clinical practice and decision-making.

BackgroundPrognosis in Palliative care Study (PiPS) models predict survival probabilities in advanced cancer. PiPS-A (clinical observations only) and PiPS-B (additionally requiring blood results) consist of 14- and 56-day models (PiPS-A14; PiPS-A56; PiPS-B14; PiPS-B56) to create survival risk categories: days, weeks, months. The primary aim was to compare PIPS-B risk categories against agreed multi-professional estimates of survival (AMPES) and to validate PiPS-A and PiPS-B. Secondary aims were to assess acceptability of PiPS to patients, caregivers and health professionals (HPs).Methods and findingsA national, multi-centre, prospective, observational, cohort study with nested qualitative sub-study using interviews with patients, caregivers and HPs. Validation study participants were adults with incurable cancer; with or without capacity; recently referred to community, hospital and hospice palliative care services across England and Wales. Sub-study participants were patients, caregivers and HPs. 1833 participants were recruited. PiPS-B risk categories were as accurate as AMPES [PiPS-B accuracy (910/1484; 61%); AMPES (914/1484; 61%); p = 0.851]. PiPS-B14 discrimination (C-statistic 0.837) and PiPS-B56 (0.810) were excellent. PiPS-B14 predictions were too high in the 57-74% risk group (Calibration-in-the-large [CiL] -0.202; Calibration slope [CS] 0.840). PiPS-B56 was well-calibrated (CiL 0.152; CS 0.914). PiPS-A risk categories were less accurate than AMPES (pConclusionsPiPS-B risk categories are as accurate as AMPES made by experienced doctors and nurses. PiPS-A categories are less accurate. Patients, carers and HPs regard PiPS as potentially helpful in clinical practice.Study registrationISRCTN13688211.

BACKGROUND:Although hypertension is the most common risk factor for atrial fibrillation (AF), whether blood pressure (BP) control varies across the spectrum of stroke risk in patients with AF or by adequacy of their thromboprophylaxis management is unclear. METHODS:We examined data from the RE-LY AF registry conducted at 164 emergency departments (EDs) in 47 countries between December 2007 and October 2011. RESULTS:Of the 15,400 patients in the registry, we analyzed the 9929 (mean age 67.5 years, 51.9% men) with a prior history of AF and complete BP data. While 6508 (66.5%) AF patients had hypertension, the prevalence varied widely depending on comorbidity profiles: from 45.4% in those without other cardiovascular risk factors to 82.5% in those with AF and diabetes. Although 93.9% of AF patients with hypertension were on at least one antihypertensive agent, fewer than half had BP levels ≤ 140/90 with no difference across risk profiles: 45.9% of those with NVAF and CHADS2 scores of 1 and 45.6% of those with NVAF and CHADS2 scores of 2 or more (46.9% and 45.3% for CHA2DS2-VASc scores of 1 versus 2 or more). BP control rates were not significantly better in those NVAF patients receiving guideline concordant thromboprophylaxis management (47.2%, aOR 1.03, 95%CI 0.89-1.20) than in those not receiving guideline-concordant antithrombotic therapy (45.3%). CONCLUSIONS:Hypertension was common in patients with AF but BP control rates were sub-optimal and varied little across the spectrum of stroke risk or by adequacy of thromboprophylaxis. This highlights the need for an increased focus on total atherosclerotic risk rather than just thromboprophylaxis management in AF patients.

The pathogenesis of Salmonella Typhimurium depends on the bacterium's ability to survive and replicate within host cells. The formation and maintenance of a unique membrane-bound compartment, termed the Salmonella-containing vacuole (SCV), is essential for S. Typhimurium pathogenesis. SCV-bound S. Typhimurium induces formation of filamentous tubules that radiate outwards from the SCV, termed Salmonella-induced filaments (SIFs). SIF formation is concomitant with the onset of replication within host epithelial cells. SIF biogenesis, formation and maintenance of the SCV, and the intracellular positioning of the SCV within the host cell requires translocation of bacterial proteins (effectors) into the host cell. Effectors secreted by the type III secretion system encoded on Salmonella pathogenicity island 2 (T3SS2) function to interfere with host cellular processes and promote both intracellular survival and replication of S. Typhimurium. Seven T3SS2-secreted effectors, SifA, SopD2, PipB2, SteA, SseJ, SseF, and SseG have previously been implicated to play complementary, redundant, and/or antagonistic roles with respect to SIF biogenesis, intracellular positioning of the SCV, and SCV membrane dynamics modulation during infection. We undertook a systematic study to delineate the contribution of each effector to these processes by (i) deleting all seven of these effectors in a single S. Typhimurium strain; and (ii) deleting combinations of multiple effectors based on putative effector function. Using this deletion mutant library, we show that each of SIF biogenesis, intracellular SCV localization, intramacrophage replication, colonization, and virulence depends on the activities of multiple effectors. Together, our data demonstrates the complex interplay between these seven effectors and highlights the necessity to study T3SS2-secreted effectors as groups, rather than studies of individual effectors.

[This corrects the article DOI: 10.1371/journal.pone.0232142.].

BackgroundAutoimmune conditions (AICs) and/or their treatment may alter risk of human papilloma virus (HPV) infection and females with AICs are therefore at an increased risk of cervical dysplasia. However, inclusion of these at-risk populations in cervical cancer screening and HPV-vaccination guidelines, are mostly lacking. This study aimed to determine the prevalence of cervical dysplasia in a wide range of AICs and compare that to HIV and immunocompetent controls to support the optimisation of cervical cancer preventive health measures.MethodsData linkage was used to match cervical screening episodes to emergency department records of females with AICs or HIV to immunocompetent controls over a 14-year period. The primary outcome was histologically confirmed high-grade cervical disease. Results, measured as rates by cytology and histology classification per 1,000 females screened, were analysed per disease group, and intergroup comparisons were performed.ResultsFemales with inflammatory bowel disease (2,683), psoriatic and enteropathic arthropathies (1,848), multiple sclerosis (MS) (1,426), rheumatoid arthritis (1,246), systemic lupus erythematosus and/or mixed connective tissue disease (SLE/MCTD) (702), HIV (44), and 985,383 immunocompetent controls were included. SLE/MCTD and HIV groups had greater rates of high-grade histological and cytological abnormalities compared to controls. Increased rates of low-grade cytological abnormalities were detected in all females with AICs, with the exception of the MS group.ConclusionsFemales with SLE/MCTD or HIV have increased rates of high-grade cervical abnormalities. The increased low-grade dysplasia rate seen in most females with AICs is consistent with increased HPV infection. These findings support expansion of cervical cancer preventative programs to include these at-risk females.

BackgroundPatients with non-tuberculous mycobacteria (NTM) or Mycobacterium tuberculosis (MTB) pulmonary disease may have similar clinical presentation. The potential for misdiagnosis and inappropriate treatment exists in settings with limited testing capacity for Xpert® MTB/RIF (Xpert), phenotypic culture and NTM speciation. We describe treatment outcomes among people living with HIV (PLHIV) who received anti-tuberculosis treatment and were found to have NTM or MTB positive sputum cultures.MethodsPLHIV attending one of the 22 participating HIV clinics, who screened positive for ≥1 tuberculosis (TB) symptoms (cough, fever, night sweats, or weight loss) were asked to submit sputa for culture and speciation from August 2012 to November 2014. The national intensified TB case finding algorithms were followed: initially symptomatic patients were evaluated by testing sputum samples using a smear (smear-based TB diagnostic algorithm) and, after GeneXpert instruments were installed, by testing with Xpert (Xpert-based TB diagnostic algorithm). Within the study period, TB diagnostic algorithms used for MTB did not include screening, diagnosis, and management of NTM. Despite MTB negative culture, some symptomatic patients, including those with NTM positive culture, received empirical anti-TB treatment at the discretion of treating clinicians. Per the World Health Organization treatment outcomes classification: died, treatment failure or loss-to-follow-up were classified as unfavorable (unsuccessful) outcome; cured and treatment completed were classified as favorable (successful) outcome. Empiric treatment was defined as initiating treatment without or before receiving a test result indicating MTB. We compare treatment outcomes and characteristics among patients with NTM or MTB positive culture who received anti-TB treatment.ResultsAmong 314 PLHIV, who were found co-infected with TB, 146 cases had microbiological evidence; and for 131/146 MTB positive cultures were reported. One-hundred fifty-two of the 314 were clinically diagnosed with TB and treated empirically. Among those empirically treated for TB, 36/152 had culture results positive for NTM, and another 43/152 had culture results positive for MTB, reported after patients received empirical anti-TB treatment. Overall, MTB positive culture results were reported for 174 (131 plus 43) patients. Treatment outcomes were available for 32/36 NTM and 139/174 MTB; unfavorable outcomes were 12.5% and 8.7% for NTM and MTB, respectively, p = 0.514, respectively. For 34/36 tested NTM patients, all Xpert results indicated 'no MTB'. Among patients who initially received empiric anti-TB treatment and ultimately were found to have MTB positive culture, the unfavorable outcome was 11.8% (4/34), compared to 12.5% (4/32) of patients with NTM positive culture, Fisher's exact test p = 1.00.ConclusionsWhile the higher unfavorable outcome was non statistically significant, the impact of inappropriate treatment among NTM patients should not be overlooked. Our findings suggest that Xpert has the potential to rapidly rule-out NTM and avoid sub-optimal treatment; further research is needed to evaluate such potential.

ObjectiveCompetency based medical education (CBME) requires novel approaches to surgical education. Significant investment has been made in laparoscopic simulation, which has been shown to foster skill development prior to patient encounters. However, research suggests variable voluntary use of these resources by residents, and little is known about the motivational factors that influence their utilization. The purpose of this study was to characterize factors that motivate residents to seek laparoscopic simulation experience outside of the formal curriculum.DesignWe developed a questionnaire grounded in Expectancy Value Theory, an established psychological theory of motivation, by adapting validated measures to fit the study context. We conducted a cross sectional survey of Canadian obstetrics and gynecology residents.SettingWe invited residents enrolled in English-language obstetrics and gynecology training programs in Canada to participate.ParticipantsAll residents engaged in clinical duties during Winter 2018 were invited to complete the questionnaire. Forty-four Obstetrics and Gynecology (Ob/Gyn) residents participated in the study.ResultsResidents reported limited use of simulation resources and identified multiple barriers including lack of time, access, and supervision. They expressed concern about development of bad habits during independent practice, and simulation use was positively correlated with perceived utility. Compared to junior residents, senior residents reported greater enjoyment of laparoscopic surgery, less emotional costs, and higher self-efficacy for learning laparoscopy.ConclusionsResidents' perception of utility and barriers impede voluntary simulation use and overall use was limited. As programs undertake curricula redevelopment for CBME, mitigating barriers and improving perceived utility of laparoscopic simulation is vital to increase use and enhance skill development.

IntroductionTuberculosis (TB) remains a significant cause of morbidity and mortality in Vietnam. The current TB burden is unknown as not all individuals with TB are diagnosed, recorded and notified. The second national TB prevalence survey was conducted in 2017-2018 to assess the current burden of TB disease in the country.MethodEighty-two clusters were selected using a multistage cluster sampling design. Adult (≥15 years of age) residents having lived for 2 weeks or more in the households of the selected clusters were invited to participate in the survey. The survey participants were screened for TB by a questionnaire and digital chest X-ray after providing written informed consent. Individuals with a positive symptom screen and/or chest X-ray suggestive of TB were asked to provide sputum samples to test for Mycobacterium tuberculosis by Ziehl-Neelsen direct light microscopy, Xpert MTB/RIF G4, BACTEC MGIT960 liquid culture and Löwenstein-Jensen solid culture. Bacteriologically confirmed TB cases were defined by an expert panel following a standard decision tree.ResultOf 87,207 eligible residents, 61,763 (70.8%) participated, and 4,738 (7.7%) screened positive for TB. Among these, 221 participants were defined as bacteriologically confirmed TB cases. The estimated prevalence of bacteriologically confirmed adult pulmonary TB was 322 (95% CI: 260-399) per 100,000, and the male-to-female ratio was 4.0 (2.8-5.8, pConclusionWith highly sensitive diagnostics applied, this survey showed that the TB burden in Vietnam remains high. Half of the TB cases were not picked up by general symptom-based screening and were identified by chest X-ray only. Our results indicate that improving TB diagnostic capacity and access to care, along with reducing TB stigma, need to be top priorities for TB control and elimination in Vietnam.

Colored dissolved organic matter (CDOM), a major component of the dissolved organic carbon (DOC) pool in many lakes, is an important controlling factor in lake ecosystem functioning. Absorption coefficients at 440 nm (a440, m-1), a common measure of CDOM, exhibited strong associations with dissolved iron (Fediss) and DOC in 280 lakes of the Upper Great Lakes States (UGLS: Minnesota, Wisconsin, and Michigan), as has been found in Scandinavia and elsewhere. Linear regressions between the three variables on UGLS lake data typically yielded R2 values of 0.6-0.9, suggesting that some underlying common processes influence organic matter and Fediss. Statistical and experimental evidence, however, supports only a minor role for iron contributions to a440 in UGLS lakes. Although both DOC and Fediss were significant variables in linear and log-log regressions on a440, DOC was the stronger predictor; adding Fediss to the linear a440-DOC model improved the R2 only from 0.90 to 0.93. Furthermore, experimental additions of FeIII to colored lake waters had only small effects on a440 (average increase of 0.242 m-1 per 100 μg/L of added FeIII). For 136 visibly stained waters (with a440 > 3.0 m-1), where allochthonous DOM predominates, DOM accounted for 92.3 ± 5.0% of the measured a440 values, and Fediss accounted for the remainder. In 75% of the lakes, Fediss accounted for < 10% of a440, but contributions of 15-30% were observed for 7 river-influenced lakes. Contributions of Fediss in UGLS lakes to specific UV absorbance at 254 nm (SUVA254) generally were also low. Although Fediss accounted for 5-10% of measured SUVA254 in a few samples, on average, 98.1% of the SUVA254 signal was attributable to DOM and only 1.9% to Fediss. DOC predictions from measured a440 were nearly identical to those from a440 corrected to remove Fediss contributions. Overall, variations in Fediss in most UGLS lakes have very small effects on CDOM optical properties, such as a440 and SUVA254, and negligible effects on the accuracy of DOC estimated from a440, data for which can be obtained at broad regional scales by remote sensing methods.

Leptomeningeal metastasis remains a difficult clinical challenge. Some success has been achieved by direct administration of therapeutics into the cerebrospinal fluid (CSF) circumventing limitations imposed by the blood brain barrier. Here we investigated continuous infusion versus bolus injection of therapy into the CSF in a preclinical model of human Group 3 medulloblastoma, the molecular subgroup with the highest incidence of leptomeningeal disease. Initial tests of selected Group 3 human medulloblastoma cell lines in culture showed that D283 Med and D425 Med were resistant to cytosine arabinoside and methotrexate. D283 Med cells were also resistant to topotecan, whereas 1 μM topotecan killed over 99% of D425 Med cells. We therefore introduced D425 Med cells, modified to express firefly luciferase, into the CSF of immunodeficient mice. Mice were then treated with topotecan or saline in five groups: continuous intraventricular (IVT) topotecan via osmotic pump (5.28 μg/day), daily bolus IVT topotecan injections with a similar daily dose (6 μg/day), systemic intraperitoneal injections of a higher daily dose of topotecan (15 μg/day), daily IVT pumped saline and daily intraperitoneal injections of saline. Bioluminescence analyses revealed that both IVT topotecan treatments effectively slowed leptomeningeal tumor growth in the brains. Histological analysis showed that they were associated with localized brain necrosis, possibly due to backtracking of topotecan around the catheter. In the spines, bolus IVT topotecan showed a trend towards slower tumor growth compared to continuous (pump) IVT topotecan, as measured by bioluminescence. Both continuous and bolus topotecan IVT showed longer survival compared to other groups. Thus, both direct IVT topotecan CSF delivery methods produced better anti-medulloblastoma effect compared to systemic therapy at the dosages used here.

The cytokinin dehydrogenase gene HvCKX2.1 is the regulatory target for the most abundant heterochromatic small RNAs in drought-stressed barley caryopses. We investigated the diversity of HvCKX2.1 in 228 barley landraces and 216 wild accessions and identified 14 haplotypes, five of these with ten or more members, coding for four different protein variants. The third largest haplotype was abundant in wild accessions (51 members), but absent from the landrace collection. Protein structure predictions indicated that the amino acid substitution specific to haplotype 3 could result in a change in the functional properties of the HvCKX2.1 protein. Haplotypes 1-3 have overlapping geographical distributions in the wild population, but the average rainfall amounts at the collection sites for haplotype 3 plants are significantly higher during November to February compared to the equivalent data for plants of haplotypes 1 and 2. We argue that the likelihood that haplotype 3 plants were excluded from landraces by sampling bias that occurred when the first wild barley plants were taken into cultivation is low, and that it is reasonable to suggest that plants with haplotype 3 are absent from the crop because these plants were less suited to the artificial conditions associated with cultivation. Although the cytokinin signalling pathway influences many aspects of plant development, the identified role of HvCKX2.1 in the drought response raises the possibility that the particular aspect of cultivation that mitigated against haplotype 3 relates in some way to water utilization. Our results therefore highlight the possibility that water utilization properties should be looked on as a possible component of the suite of physiological adaptations accompanying the domestication and subsequent evolution of cultivated barley.

This paper describes a methodology that combines meta-population theory and stock assessment models to gain insights about spatial heterogeneity of the meta-population in an operational time frame. The methodology was tested with stochastic simulations for different degrees of connectivity between sub-populations and applied to two case studies, North Sea cod (Gadus morua) and Northeast Atlantic sardine (Sardina pilchardus). Considering that the biological components of a population can be partitioned into discrete spatial units, we extended this idea into a property of additivity of sub-population abundances. If the additivity results hold true for putative sub-populations, then assessment results based on sub-populations will provide information to develop and monitor the implementation of finer scale/local management. The simulation study confirmed that when sub-populations are independent and not too heterogeneous with regards to productivity, the sum of stock assessment model estimates of sub-populations' SSB is similar to the SSB estimates of the meta-population. It also showed that a strong diffusion process can be detected and that the stronger the connection between SSB and recruitment, the better the diffusion process will be detected. On the other hand it showed that weak to moderate diffusion processes are not easy to identify and large differences between sub-populations productivities may be confounded with weak diffusion processes. The application to North Sea cod and Atlantic sardine exemplified how much insight can be gained. In both cases the results obtained were sufficiently robust to support the regional analysis.

In this paper, we examine the inequality in the dynamics of the total fertility rate within 21 sub-Saharan African countries by wealth quintiles. We also examine the associated inequality within each country in the proximate determinants of fertility-marriage, contraception, and breastfeeding. Applying Bongaarts' proximate determinants of fertility framework, for 14/21 countries we analyze, we find that those in the richest wealth quintiles have had a more rapid decline in fertility rates than those in the poorest wealth quintiles. The rapid decline for those in the richest wealth quintiles is attributable to delayed marriage and modest increases in contraceptive use. Although the poorest lag in fertility decline, postpartum abstinence and breastfeeding are the most important factors for them for fertility regulation. Further encouraging maternal health programs that focus on natural methods of fertility regulation will work in favor of the poorest in sub-Saharan Africa in moving through the demographic transition.

In 2011, the Botswana National Tuberculosis Program adopted World Health Organization guidelines and introduced Xpert MTB/RIF (Xpert) assay to support intensified case finding among people living with HIV enrolling in care. An evaluation was designed to assess performance under operational conditions to inform the national Xpert scale-up.Xpert was implemented from August 2012 through November 2014 with 13 GeneXpert instruments (GeneXpert) deployed in a phased approach over nine months: nine centralized laboratory and four point-of-care (POC) peripheral clinics. Clinicians and laboratorians were trained on the four-symptom tuberculosis screening algorithm and Xpert testing. We documented our experience with staff training and GeneXpert performance. Test results were extracted from GeneXpert software; unsuccessful tests were analysed in relation to testing sites and trends over time.During 276 instrument-months of operation a total of 3,630 tests were performed, of which 3,102 (85%) were successful with interpretable results. Mycobacterium tuberculosis complex was detected for 447 (14%); of these, 36 (8%) were rifampicin resistant. Of all 3,630 Xpert tests, 528 (15%) were unsuccessful; of these 361 (68%) were classified as "error", 119 (23%) as "invalid" and 48 (9%) as "no result". The total number of recorded error codes was 385 and the most common reasons were related to sample processing (211; 55%) followed by power supply (77; 20%) and cartridge/module related (54; 14%). Cumulative incidence of unsuccessful test was similar between POC (17%, 95% CI: 11-25%) and centralized laboratory-based GeneXpert instruments (14%, 95% CI: 11-17%; p = 0.140).Xpert introduction was successful in the Botswana setting. The incidence of unsuccessful test was similar by GeneXpert location (POC vs. centralized laboratory). However, unsuccessful test incidence (15%) in our settings was higher than previously reported and was mostly related to improper sample processing. Ensuring adequate training among Xpert testing staff is essential to minimize errors.

Madagascar conducted the first two phases of a national free mass distribution campaign of long-lasting insecticidal nets (LLINs) during a political crisis in 2009 aiming to achieve coverage of two LLINs per household as part of the National Malaria Control Strategy. The campaign targeted households in 19 out of 91 total health districts.A community-based cross-sectional household survey using a three-stage cluster sample design was conducted four months post campaign to assess LLIN ownership, access and use. Multivariable logistic regression analysis was used to identify factors associated with household LLIN access and individual LLIN use.A total of 2211 households were surveyed representing 8867 people. At least one LLIN was present in 93.5% (95% confidence interval [CI], 91.6-95.5%) of households and 74.8% (95% CI, 71.0-78.6%) owned at least two LLINs. Access measured as the proportion of the population that could potentially be covered by household-owned LLINs was 77.2% (77.2% (95% CI, 72.9-81.3%) and LLIN use by all individuals was 84.2% (95% CI, 81.2-87.2%). LLIN use was associated with knowledge of insecticide treated net use to prevent malaria (OR = 3.58, 95% CI, 1.85-6.94), household ownership of more LLINs (OR 2.82, 95% CI 1.85-4.3), presence of children under five (OR = 2.05, 95% CI, 1.67-2.51), having traveled to the distribution point and receiving information about hanging a bednet (OR = 1.56, 95% CI, 1.41-1.74), and having received a post-campaign visit by a community mobilizer (OR = 1.75, 95% CI, 1.26-2.43). Lower LLIN use was associated with increasing household size (OR = 0.81 95% CI 0.77-0.85) and number of sleeping spaces (OR = 0.55, 95% CI, 0.44-0.68).A large scale free mass LLIN distribution campaign was feasible and effective at achieving high LLIN access and use in Madagascar. Campaign process indicators highlighted potential areas for strengthening implementation to optimize access and equity.

Voice change is one of the earliest features of Parkinson's disease. However, quantitative studies of vocal fold dynamics which are needed to provide insight into disease biology, aid diagnosis, or track progression, are few.We therefore quantified arytenoid cartilage movements and glottic area during repeated phonation in 15 patients with Parkinson's disease (symptom duration < 6 years) and 19 controls, with 320-slice computerised tomography (CT). We related these measures to perceptual voice evaluations and spirometry. We hypothesised that Parkinson's disease patients have a smaller inter-arytenoid distance, a preserved or larger glottic area because vocal cord bowing has previously been reported, less variability in loudness, more voice dysdiadochokinesis and breathiness and a shortened phonation time because of arytenoid hypokinesis relative to glottic area.Inter-arytenoid distance in Parkinson's disease patients was moderately smaller (Mdn = 0.106, IQR = 0.091-0.116) than in controls (Mdn = 0.132, IQR = 0.116-0.166) (W = 212, P = 0.015, r = -0.42), normalised for anatomical and other inter-subject variance, analysed with two-tailed Wilcoxon's rank sum test. This finding was confirmed in a linear mixed model analysis-Parkinson's disease significantly predicted a reduction in the dependent variable, inter-arytenoid distance (b = -0.87, SEb = 0.39, 95% CI [-1.66, -0.08], t(31) = -2.24, P = 0.032). There was no difference in glottic area. On perceptual voice evaluation, patients had more breathiness and dysdiadochokinesis, a shorter maximum phonation time, and less variability in loudness than controls. There was no difference in spirometry after adjustment for smoking history.As predicted, vocal fold adduction movements are reduced in Parkinson's disease on repeated phonation but glottic area is maintained. Some perceptual characteristics of Parkinsonian speech reflect these changes. We are the first to use 320-slice CT to study laryngeal motion. Our findings indicate how Parkinson's disease affects intrinsic laryngeal muscle position and excursion.

BACKGROUND:Non-tuberculous mycobacteria (NTM) can cause pulmonary infection and disease especially among people living with HIV (PLHIV). PLHIV with NTM disease may clinically present with one of the four symptoms consistent with tuberculosis (TB). We describe the prevalence of NTM and Mycobacterium tuberculosis complex (MTBC) isolated among PLHIV who presented for HIV care and treatment. METHODS:All PLHIV patients presenting for HIV care and treatment services at 22 clinical sites in Botswana were offered screening for TB and were recruited. Patients who had ≥1 TB symptom were asked to submit sputa for Xpert MTB/RIF and culture. Culture growth was identified as NTM and MTBC using the SD-Bioline TB Ag MPT64 Kit and Ziehl Neelsen microscopy. NTM and MTBC isolates underwent species identification by the Hain GenoType CM and AS line probe assays. RESULTS:Among 16, 259 PLHIV enrolled 3068 screened positive for at least one TB symptom. Of these, 1940 submitted ≥1 sputum specimen, 427 (22%) patients had ≥1 positive-culture result identified phenotypically for mycobacterial growth. Of these 247 and 180 patients were identified as having isolates were NTM and MTBC, respectively. Of the 247 patients identified with isolates containing NTM; 19 were later excluded as not having NTM based on additional genotypic testing. Among the remaining 408 patients 228 (56%, 95% confidence interval, 46-66%) with NTM. M. intracellulare was the most common isolated (47.8%). Other NTMs commonly associated with pulmonary disease included M. malmoense (3.9%), M. avium (2.2%), M. abscessus (0.9%) and M. kansasii (0.4%). After excluding NTM isolates that were non-speciated and M. gordonae 154 (67.5%) of the NTM isolates were potential pathogens. CONCLUSIONS:In the setting of HIV care and treatment, over-half (56%) of a positive sputum culture among PLHIV with TB symptoms was NTM. Though we were not able to distinguish in our study NTM disease and colonization, the study suggests culture and species identification for PLHIV presenting with TB symptoms remains important to facilitate NTM diagnosis and hasten time to appropriate treatment.

BACKGROUND:Little is known about potential ethnic differences in stroke incidence. We compared incidence and time trends of ischemic stroke and primary intracerebral hemorrhage in South Asian, Chinese and white persons in a population-based study. METHODS:Population based census and administrative data analysis in the provinces of Ontario and British Columbia, Canada using validated ICD 9/ICD 10 coding for acute ischemic and hemorrhagic stroke (1997-2010). RESULTS:There were 3290 South Asians, 4444 Chinese and 160944 white patients with acute ischemic stroke and 535 South Asian, 1376 Chinese and 21842 white patients with intracerebral hemorrhage. South Asians were younger than whites at onset of stroke (70 vs. 74 years for ischemic and 67 vs. 71 years for hemorrhagic stroke). Age and sex adjusted ischemic stroke incidence in 2010 was 43% lower in Chinese and 63% lower in South Asian than in White patients. Age and sex adjusted intracerebral hemorrhage incidence was 18% higher in Chinese patients, and 66% lower in South Asian relative to white patients. Stroke incidence declined in all ethnic groups (relative reduction 69% in South Asians, 25% in Chinese, and 34% in white patients for ischemic stroke and for intracerebral hemorrhage, 79% for South Asians, 51% for Chinese and 30% in white patients). CONCLUSION:Although stroke rates declined across all ethnic groups, these rates differed significantly by ethnicity. Further study is needed to understand mechanisms underlying the higher ischemic stroke incidence in white patients and intracerebral hemorrhage in Chinese patients.

Estimating fish stock status is very challenging given the many sources and high levels of uncertainty surrounding the biological processes (e.g. natural variability in the demographic rates), model selection (e.g. choosing growth or stock assessment models) and parameter estimation. Incorporating multiple sources of uncertainty in a stock assessment allows advice to better account for the risks associated with proposed management options, promoting decisions that are more robust to such uncertainty. However, a typical assessment only reports the model fit and variance of estimated parameters, thereby underreporting the overall uncertainty. Additionally, although multiple candidate models may be considered, only one is selected as the 'best' result, effectively rejecting the plausible assumptions behind the other models. We present an applied framework to integrate multiple sources of uncertainty in the stock assessment process. The first step is the generation and conditioning of a suite of stock assessment models that contain different assumptions about the stock and the fishery. The second step is the estimation of parameters, including fitting of the stock assessment models. The final step integrates across all of the results to reconcile the multi-model outcome. The framework is flexible enough to be tailored to particular stocks and fisheries and can draw on information from multiple sources to implement a broad variety of assumptions, making it applicable to stocks with varying levels of data availability The Iberian hake stock in International Council for the Exploration of the Sea (ICES) Divisions VIIIc and IXa is used to demonstrate the framework, starting from length-based stock and indices data. Process and model uncertainty are considered through the growth, natural mortality, fishing mortality, survey catchability and stock-recruitment relationship. Estimation uncertainty is included as part of the fitting process. Simple model averaging is used to integrate across the results and produce a single assessment that considers the multiple sources of uncertainty.

Members of the Inhibitor of APoptosis (IAP) protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF) superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC), which binds to selected baculovirus IAP repeat (BIR) domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP) assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR) analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki) of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α). Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to cytotoxicity mediated by TNF and LT-α.

Analysis of the mouse transcriptional response to Listeria monocytogenes infection reveals that a large set of genes are perturbed in both blood and tissue and that these transcriptional responses are enriched for pathways of the immune response. Further we identified enrichment for both type I and type II interferon (IFN) signaling molecules in the blood and tissues upon infection. Since type I IFN signaling has been reported widely to impair bacterial clearance we examined gene expression from blood and tissues of wild type (WT) and type I IFNαβ receptor-deficient (Ifnar1-/-) mice at the basal level and upon infection with L. monocytogenes. Measurement of the fold change response upon infection in the absence of type I IFN signaling demonstrated an upregulation of specific genes at day 1 post infection. A less marked reduction of the global gene expression signature in blood or tissues from infected Ifnar1-/- as compared to WT mice was observed at days 2 and 3 after infection, with marked reduction in key genes such as Oasg1 and Stat2. Moreover, on in depth analysis, changes in gene expression in uninfected mice of key IFN regulatory genes including Irf9, Irf7, Stat1 and others were identified, and although induced by an equivalent degree upon infection this resulted in significantly lower final gene expression levels upon infection of Ifnar1-/- mice. These data highlight how dysregulation of this network in the steady state and temporally upon infection may determine the outcome of this bacterial infection and how basal levels of type I IFN-inducible genes may perturb an optimal host immune response to control intracellular bacterial infections such as L. monocytogenes.

Uropathogenic Escherichia coli (UPEC) strains live as commensals in the digestive tract of the host, but they can also initiate urinary tract infections. The aim of this work was to determine how a host detects the presence of a new UPEC strain in the digestive tract. Mice were orally challenged with UPEC strains 536 and CFT073, non-pathogenic strain K12 MG1655, and ΔPAI-536, an isogenic mutant of strain 536 lacking all 7 pathogenicity islands whose virulence is drastically attenuated. Intestinal colonization was measured, and cytokine expression was determined in various organs recovered from mice after oral challenge. UPEC strain 536 efficiently colonized the mouse digestive tract, and prior Enterobacteriaceae colonization was found to impact strain 536 colonization efficiency. An innate immune response, detected as the production of TNFα, IL-6 and IL-10 cytokines, was activated in the ileum 48 hours after oral challenge with strain 536, and returned to baseline within 8 days, without a drop in fecal pathogen load. Although inflammation was detected in the ileum, histology was normal at the time of cytokine peak. Comparison of cytokine secretion 48h after oral gavage with E. coli strain 536, CFT073, MG1655 or ΔPAI-536 showed that inflammation was more pronounced with UPECs than with non-pathogenic or attenuated strains. Pathogenicity islands also seemed to be involved in host detection, as IL-6 intestinal secretion was increased after administration of E. coli strain 536, but not after administration of ΔPAI-536. In conclusion, UPEC colonization of the mouse digestive tract activates acute phase inflammatory cytokine secretion but does not trigger any pathological changes, illustrating the opportunistic nature of UPECs. This digestive tract colonization model will be useful for studying the factors controlling the switch from commensalism to pathogenicity.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a foodborne enteric pathogen and a major cause of gastroenteritis in humans. It is known that molecules derived from the human fecal microbiota downregulate S. Typhimurium virulence gene expression and induce a starvation-like response. In this study, S. Typhimurium was cultured in minimal media to mimic starvation conditions such as that experienced by S. Typhimurium in the human intestinal tract, and the pathogen's virulence in vitro and in vivo was measured. S. Typhimurium cultured in minimal media displayed a reduced ability to invade human epithelial cells in a manner that was at least partially independent of the Salmonella Pathogenicity Island 1 (SPI-1) type III secretion system. Nutrient deprivation did not, however, alter the ability of S. Typhimurium to replicate and survive inside epithelial cells. In a murine model of S. Typhimurium-induced gastroenteritis, prior cultivation in minimal media did not alter the pathogen's ability to colonize mice, nor did it affect levels of gastrointestinal inflammation. Upon examining the post-infection fecal gastrointestinal microbiota, we found that specifically in the 129Sv/ImJ murine strain S. Typhimurium cultured in minimal media induced differential microbiota compositional shifts compared to that of S. Typhimurium cultured in rich media. Together these findings demonstrate that S. Typhimurium remains a potent pathogen even in the face of nutritional deprivation, but nevertheless that nutrient deprivation encountered in this environment elicits significant changes in the bacterium genetic programme, as well as its capacity to alter host microbiota composition.

To determine the data sources and 'look back' intervals to define comorbidities.Hospital discharge abstracts database (DAD), physician claims, population registry and death registry from April 1, 1994 to March 31, 2010 in Alberta, Canada.Newly-diagnosed hypertension cases from 1997 to 2008 fiscal years were identified and followed up to 12 years. We defined comorbidities using data sources and duration of retrospective observation (6 months, 1 year, 2 years, and 3 years). The C-statistics for logistic regression and concordance index (CI) for Cox model of mortality and cardiovascular disease hospitalization were used to evaluate discrimination performance for each approach of defining comorbidities.The comorbidities prevalence became higher with a longer duration. Using DAD alone underestimated the prevalence by about 75%, compared to using both DAD and physician claims. The C-statistic and CI were highest when both DAD and physician claims were used, and model performance improved when observation duration increased from 6 months to one year or longer.The comorbidities prevalence is greatly impacted by the data source and duration of retrospective observation. A combination of DAD and physicians claims with at least one year observation duration improves predictions for cardiovascular disease and one-year mortality outcome model performance.

BACKGROUND:Sleep quality in hospitalized Canadian General Internal Medicine patients is not well characterized. Our goals were to characterize hospital sleep quality in this population and identify potentially modifiable barriers to good sleep. METHODS:GIM inpatients at a quaternary centre in Edmonton, Canada completed a survey, including the Verran-Snyder Halpern (VSH) questionnaire, to characterize the previous night's sleep within 48 hours prior to discharge. A chart review was also completed to assess comorbidities, discharge diagnoses, and pharmaceutical sleep aid use. RESULTS:Patients reported significantly worse nighttime sleep duration in hospital compared with home (mean 5.5 versus 7.0 hours per night, p < 0.0001). Sleep quality was poor, as measured by the VSH disturbance (mean 371), effectiveness (190), and supplementation (115) subscales. Variables independently associated with poorer sleep duration in multivariable regression include prior diagnosis of sleep disorder and multi-patient occupancy rooms. Age, sex, admitting diagnosis, length of stay, frequency of vital checks, and use of sleep pharmaceuticals during the index hospitalization were not associated with sleep duration. The most frequently reported reasons for poor sleep included noise (59%), nursing interruptions (30%), uncomfortable beds (18%), bright lights (16%), unfamiliar surroundings (14%), and pain (9%). CONCLUSIONS:Sleep quality for GIM inpatients is significantly worse in hospital than at home. There is a need to test non-pharmacologic interventions to address the most frequently identified factors causing poor sleep hygiene for GIM inpatients.

Background and purposeWe studied whether anticoagulant use and outcomes differed between rural versus urban Canadian non-valvular atrial fibrillation (NVAF) patients prior to the introduction of direct oral anticoagulant drugs.MethodsRetrospective cohort study of 25,284 adult Albertans with NVAF between April 1, 1999 and December 31, 2008.ResultsCompared to urban patients, rural patients were older (p = 0.0009) and had more comorbidities but lower bleeding risk at baseline. In the first year after NVAF diagnosis, urban patients were less likely to be hospitalized (aOR 0.82, 95%CI 0.77-0.89) or have an emergency department visit for any reason (aOR 0.61, 95%CI 0.56-0.66) but warfarin dispensation rates (72.2% vs 71.8% at 365 days, p = 0.98) and clinical outcomes were similar: 7.8% died in both groups, 3.2% rural vs. 2.8% urban had a stroke or systemic embolism (SSE) (aOR 0.92, 95%CI 0.77-1.11), and 6.6% vs. 5.7% (aOR 0.93, 95%CI 0.81-1.06) had a bleed. Baseline SSE risk did not impact warfarin dispensation (73.0% in those with high vs. 72.8% in those with low CHADS2 score, p = 0.85) but patients at higher baseline bleeding risk were less likely to be using warfarin (69.2% high vs. 73.6% low HASBLED score, pConclusionWarfarin use and clinical event rates did not differ between rural and urban NVAF patients in a universal access publically-funded healthcare system.

Psychologists tend to report high levels of occupational stress, with serious implications for themselves, their clients, and the discipline as a whole. Recent research suggests that self-compassion is a promising construct for psychologists in terms of its ability to promote psychological wellbeing and resilience to stress; however, the potential benefits of self-compassion are yet to be thoroughly explored amongst this occupational group. Additionally, while a growing body of research supports self-compassion as a key predictor of psychopathology, understanding of the processes by which self-compassion exerts effects on mental health outcomes is limited. Structural equation modelling (SEM) was used to test an emotion regulation model of self-compassion and stress among psychologists, including postgraduate trainees undertaking clinical work (n = 198). Self-compassion significantly negatively predicted emotion regulation difficulties and stress symptoms. Support was also found for our preliminary explanatory model of self-compassion, which demonstrates the mediating role of emotion regulation difficulties in the self-compassion-stress relationship. The final self-compassion model accounted for 26.2% of variance in stress symptoms. Implications of the findings and limitations of the study are discussed.

Administrative health data have been used in hypertension surveillance using the 1H2P method: the International Classification of Disease (ICD) hypertension diagnosis codes were recorded in at least 1 hospitalization or 2 physician claims within 2 year-period. Accumulation of false positive cases over time using the 1H2P method could result in the overestimation of hypertension prevalence. In this study, we developed and validated a new reclassification method to define hypertension cases using regularized logistic regression with the age, sex, hypertension and comorbidities in physician claims, and diagnosis of hypertension in hospital discharge data as independent variables. A Bayesian method was then used to adjust the prevalence estimated from the reclassification method. We evaluated the hypertension prevalence in data from Alberta, Canada using the currently accepted 1H2P method and these newly developed methods. The reclassification method with Bayesian adjustment produced similar prevalence estimates as the 1H2P method. This supports the continued use of the 1H2P method as a simple and practical way to conduct hypertension surveillance using administrative health data.

Restrictions on retail purchases of pseudoephedrine are one regulatory approach to reduce the social costs of methamphetamine production and use, but may impose costs on legitimate users of nasal decongestants. This is the first study to evaluate the costs of restricting access to medications on consumer welfare. Our objective was to measure the inconvenience cost consumers place on restrictions for cold medication purchases including identification requirements, purchase limits, over-the-counter availability, prescription requirements, and the active ingredient.We conducted a contingent choice experiment with Amazon Mechanical Turk workers that presented participants with randomized, hypothetical product prices and combinations of restrictions that reflect the range of public policies. We used a conditional logit model to calculate willingness-to-accept each restriction.Respondents' willingness-to-accept prescription requirements was $14.17 ($9.76-$18.58) and behind-the-counter restrictions was $9.68 ($7.03-$12.33) per box of pseudoephedrine product. Participants were willing to pay $4.09 ($1.66-$6.52) per box to purchase pseudoephedrine-based products over phenylephrine-based products.Restricting access to medicines as a means of reducing the social costs of non-medical use can imply large inconvenience costs for legitimate consumers. These results are relevant to discussions of retail access restrictions on other medications.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and "triple negative" variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not with AKT or ERK phosphorylation, consistent with the mTOR pathway being a principal target. We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.

BACKGROUND:Worsening and improving renal function during acute heart failure have been associated with adverse outcomes but few studies have considered the admission level of renal function upon which these changes are superimposed. OBJECTIVES:The objective of this study was to evaluate definitions that incorporate both admission renal function and change in renal function. METHODS:696 patients with acute heart failure with calculable eGFR were classified by admission renal function (Reduced [R, eGFR

Many quality-of-care and risk prediction metrics rely on time to first rehospitalization even though heart failure (HF) patients may undergo several repeat hospitalizations. The aim of this study is to compare repeat hospitalization models. Using a population-based cohort of 40,667 patients, we examined both HF and all cause re-hospitalizations using up to five years of follow-up. Two models were examined: the gap-time model which estimates the adjusted time between hospitalizations and a multistate model which considered patients to be in one of four states; community-dwelling, in hospital for HF, in hospital for any reason, or dead. The transition probabilities and times were then modeled using patient characteristics and number of repeat hospitalizations. We found that during the five years of follow-up roughly half of the patients returned for a subsequent hospitalization for each repeat hospitalization. Additionally, we noted that the unadjusted time between hospitalizations was reduced ∼40% between each successive hospitalization. After adjustment each additional hospitalization was associated with a 28 day (95% CI: 22-35) reduction in time spent out of hospital. A similar pattern was seen when considering the four state model. A large proportion of patients had multiple repeat hospitalizations. Extending the gap between hospitalizations should be an important goal of treatment evaluation.

The anti-diabetic drug metformin regulates T-cell responses to immune activation and is proposed to function by regulating the energy-stress-sensing adenosine-monophosphate-activated protein kinase (AMPK). However, the molecular details of how metformin controls T cell immune responses have not been studied nor is there any direct evidence that metformin acts on T cells via AMPK. Here, we report that metformin regulates cell growth and proliferation of antigen-activated T cells by modulating the metabolic reprogramming that is required for effector T cell differentiation. Metformin thus inhibits the mammalian target of rapamycin complex I signalling pathway and prevents the expression of the transcription factors c-Myc and hypoxia-inducible factor 1 alpha. However, the inhibitory effects of metformin on T cells did not depend on the expression of AMPK in T cells. Accordingly, experiments with metformin inform about the importance of metabolic reprogramming for T cell immune responses but do not inform about the importance of AMPK.

Cellular signaling pathways involving mTOR, PI3K and ERK have dominated recent studies of breast cancer biology, and inhibitors of these pathways have formed a focus of numerous clinical trials. We have chosen trametinib, a drug targeting MEK in the ERK pathway, to address two questions. Firstly, does inhibition of a signaling pathway, as measured by protein phosphorylation, predict the antiproliferative activity of trametinib? Secondly, do inhibitors of the mTOR and PI3K pathways synergize with trametinib in their effects on cell proliferation? A panel of 30 human breast cancer cell lines was chosen to include lines that could be classified according to whether they were ER and PR positive, HER2 over-expressing, and "triple negative". Everolimus (targeting mTOR), NVP-BEZ235 and GSK2126458 (both targeting PI3K/mTOR) were chosen for combination experiments. Inhibition of cell proliferation was measured by IC50 values and pathway utilization was measured by phosphorylation of signaling kinases. Overall, no correlation was found between trametinib IC50 values and inhibition of ERK signaling. Inhibition of ERK phosphorylation was observed at trametinib concentrations not affecting proliferation, and sensitivity of cell proliferation to trametinib was found in cell lines with low ERK phosphorylation. Evidence was found for synergy between trametinib and either everolimus, NVP-BEZ235 or GSK2126458, but this was cell line specific. The results have implications for the clinical application of PI3K/mTOR and MEK inhibitors.

Unlike all other New World (platyrrine) monkeys, both male and female howler monkeys (Alouatta sp.) are obligatory trichromats. In all other platyrrines, only females can be trichromats, while males are always dichromats, as determined by multiple behavioral, electrophysiological, and genetic studies. In addition to obligatory trichromacy, Alouatta has an unusual fovea, with substantially higher peak cone density in the foveal pit than every other diurnal anthropoid monkey (both platyrrhines and catarrhines) and great ape yet examined, including humans. In addition to documenting the general organization of the retinal ganglion cell layer in Alouatta, the distribution of cones is compared to retinal ganglion cells, to explore possible relationships between their atypical trichromacy and foveal specialization. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from five Alouatta caraya. Ganglion cell density peaked at 0.5 mm between the fovea and optic nerve head, reaching 40,700-45,200 cells/mm2. Displaced amacrine cell density distribution peaked between 0.5-1.75 mm from the fovea, reaching mean values between 2,050-3,100 cells/mm2. The mean number of ganglion cells was 1,133,000±79,000 cells and the mean number of displaced amacrine cells was 537,000±61,800 cells, in retinas of mean area 641±62 mm2. Ganglion cell and displaced amacrine cell density distribution in the Alouatta retina was consistent with that observed among several species of diurnal Anthropoidea, both platyrrhines and catarrhines. The principal alteration in the Alouatta retina appears not to be in the number of any retinal cell class, but rather a marked gradient in cone density within the fovea, which could potentially support high chromatic acuity in a restricted central region.

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

The transcription factor SOX2 is essential for maintaining pluripotency in a variety of stem cells. It has important functions during embryonic development, is involved in cancer stem cell maintenance, and is often deregulated in cancer. The mechanism of SOX2 regulation has yet to be clarified, but the SOX2 gene lies in an intron of a long multi-exon non-coding RNA called SOX2 overlapping transcript (SOX2OT). Here, we show that the expression of SOX2 and SOX2OT is concordant in breast cancer, differentially expressed in estrogen receptor positive and negative breast cancer samples and that both are up-regulated in suspension culture conditions that favor growth of stem cell phenotypes. Importantly, ectopic expression of SOX2OT led to an almost 20-fold increase in SOX2 expression, together with a reduced proliferation and increased breast cancer cell anchorage-independent growth. We propose that SOX2OT plays a key role in the induction and/or maintenance of SOX2 expression in breast cancer.

The concealed phase of arrhythmogenic right ventricular cardiomyopathy (ARVC) may initially manifest electrophysiologically. No studies have examined dynamic conduction/repolarization kinetics to distinguish benign right ventricular outflow tract ectopy (RVOT ectopy) from ARVC's early phase. We investigated dynamic endocardial electrophysiological changes that differentiate early ARVC disease expression from RVOT ectopy.22 ARVC (12 definite based upon family history and mutation carrier status, 10 probable) patients without right ventricular structural anomalies underwent high-density non-contact mapping of the right ventricle. These were compared to data from 14 RVOT ectopy and 12 patients with supraventricular tachycardias and normal hearts. Endocardial & surface ECG conduction and repolarization parameters were assessed during a standard S1-S2 restitution protocol.Definite ARVC without RV structural disease could not be clearly distinguished from RVOT ectopy during sinus rhythm or during steady state pacing. Delay in Activation Times at coupling intervals just above the ventricular effective refractory period (VERP) increased in definite ARVC (43 ± 20 ms) more than RVOT ectopy patients (36 ± 14 ms, p = 0.03) or Normals (25 ± 16 ms, p = 0.008) and a progressive separation of the repolarisation time curves between groups existed. Repolarization time increases in the RVOT were also greatest in ARVC (definite ARVC: 18 ± 20 ms; RVOT ectopy: 5 ± 14, Normal: 1 ± 18, p