Past progress in environmental nanoanalysis and a future trajectory for atomic mass-spectrometry methods.

The development of engineered nanotechnology has necessitated a commensurate maturation of nanoanalysis capabilities. Building off a legacy established by electron microscopy and light-scattering, environmental nanoanalysis has now benefited from ongoing advancements in instrumentation and data analysis, which enable a deeper understanding of nanomaterial properties, behavior, and impacts. Where once environmental nanoparticles and colloids were grouped into broad 'dissolved or particulate' classes that are dependent on a filter size cut-off, now size distributions of submicron particles can be separated and characterized providing a more comprehensive examination of the nanoscale. Inductively coupled plasma-quadrupole mass spectrometry (ICP-QMS), directly coupled to field flow fractionation (FFF-ICP-QMS) or operated in single particle mode (spICP-MS) have spearheaded a revolution in nanoanalysis, enabling research into nanomaterial behavior in environmental and biological systems at expected release concentrations. However, the complexity of the nanoparticle population drives a need to characterize and quantify the multi-element composition of nanoparticles, which has begun to be realized through the application of time-of-flight MS (spICP-TOFMS). Despite its relative infancy, this technique has begun to make significant strides in more fully characterizing particulate systems and expanding our understanding of nanoparticle behavior. Though there is still more work to be done with regards to improving instrumentation and data processing, it is possible we are on the cusp of a new nanoanalysis revolution, capable of broadening our understanding of the size regime between dissolved and bulk particulate compartments of the environment.

Staphylococcus aureus bacteriuria: implications and management.

Background: Staphylococcus aureus is isolated in around 0.2%-4% of positive urinary cultures, more commonly in the contexts of long-term care, urological abnormalities and procedures, male sex, older age and comorbidities. Isolation may represent contamination, colonization, urinary tract infection or bacteraemic seeding from another site, and may be linked to S. aureus bacteraemia. However, there is little guidance on investigation and management of S. aureus bacteriuria. We performed a retrospective analysis of cases in our service, including clinical characteristics, investigations and treatment. Methods: Data were collected on all urine samples taken from adult patients over a 5-year period from which S. aureus was isolated. Detailed analysis including investigations and management was conducted in those collected over a 1-year period. Results: From 511 patients, 668 urine cultures positive for S. aureus were identified; 6.5% of cases were positive for MRSA. Of 93 patients who had blood cultures taken, there were 6 cases of S. aureus bacteraemia, 4 of which were associated with urological instrumentation. Of 94 cases analysed in detail, 57% were treated with antibiotics, and 49% had repeat urine cultures. Factors associated with recurrence were urinary catheterization, urological abnormality, diabetes and inpatient status. Conclusions: Our experience does not support the routine taking of blood cultures or treatment of asymptomatic bacteriuria in well patients with S. aureus bacteriuria in this setting. However, repeat urine culture, and investigation and treatment of higher risk patients, for example, prior to bladder instrumentation, may be warranted. We propose a simple algorithm to guide clinicians.

Adult life-course trajectories of psychological distress and economic outcomes in midlife during the COVID-19 pandemic: evidence from the 1958 and 1970 British birth cohorts.

PURPOSE: Financial adversity in times of economic recession have been shown to have an unequal effect on individuals with prior mental health problems. This study investigated the relationship between mental health groupings across the adult life-course and change in financial situation and employment status during the COVID-19 pandemic, as well as the use of financial measures to mitigate the economic shock. METHODS: Using two nationally representative British birth cohorts, the National Child Development Study (1958) n = 17,415 and 1970 British Cohort Study n = 17,198, we identified 5 different life-course trajectories of psychological distress from adolescence to midlife which were similar but not identical across the two cohorts. We explored their relation to changes in financial and employment circumstances at different stages during the pandemic from May 2020 to March 2021, applying multinomial logistic regression and controlling for numerous early life covariates, including family socio-economic status (SES). In addition, we ran modified Poisson models with robust standard errors to identify whether different mental health trajectories were supported by government and used other methods to mitigate their financial situation. RESULTS: We found that the financial circumstances of pre-pandemic trajectories of psychological distress with differential onset, severity, and chronicity across the life-course were exacerbated by the COVID-19 economic shock. The 'stable-high' (persistent severe symptoms) and 'adult-onset' (symptoms developing in 30s, but later decreasing) groups were vulnerable to job loss. Compared to pre-pandemic trajectory groupings with no, minor, or psychological distress symptoms in early adulthood, the 'stable-high', 'midlife-onset' (symptoms developing in midlife), and 'adult-onset' trajectory groups were more likely to seek support from the UK governments economic response package. However, trajectories with pre-pandemic psychological distress were also at greater risk of reducing consumption, dis-saving, relying on increased financial help from family and friends, and also taking payment holidays (agreements with lenders to pause mortgage, credit card or loan payments for a set period) and borrowing. CONCLUSION: This work highlights different trajectories of pre-pandemic psychological distress, compared to groups with no symptoms were more vulnerable to pandemic-related economic shock and job loss. By adopting unsustainable mitigating measures (borrowing and payment holidays) to support their financial circumstances during COVID-19, these mental health trajectories are at even more risk of lasting adverse impacts and future economic difficulties.

A multisite evaluation of antifungal use in critical care: implications for antifungal stewardship.

Background: ICUs are settings of high antifungal consumption. There are few data on prescribing practices in ICUs to guide antifungal stewardship implementation in this setting. Methods: An antifungal therapy (AFT) service evaluation (15 May-19 November 2019) across ICUs at three London hospitals, evaluating consumption, prescribing rationale, post-prescription review, de-escalation and final invasive fungal infection (IFI) diagnostic classification. Results: Overall, 6.4% of ICU admissions (305/4781) received AFT, accounting for 11.41 days of therapy/100 occupied bed days (DOT/100 OBD). The dominant prescribing mode was empirical (41% of consumption), followed by targeted (22%), prophylaxis (18%), pre-emptive (12%) and non-invasive (7%). Echinocandins were the most commonly prescribed drug class (4.59 DOT/100 OBD). In total, 217 patients received AFT for suspected or confirmed IFI; 12%, 10% and 23% were classified as possible, probable or proven IFI, respectively. Hence, in 55%, IFI was unlikely. Proven IFI (n = 50) was mostly invasive candidiasis (92%), of which 48% had been initiated on AFT empirically before yeast identification. Where on-site (1 → 3)-ß-d-glucan (BDG) testing was available (1 day turnaround), in those with suspected but unproven invasive candidiasis, median (IQR) AFT duration was 10 (7-15) days with a positive BDG (≥80 pg/mL) versus 8 (5-9) days with a negative BDG (<80 pg/mL). Post-prescription review occurred in 79% of prescribing episodes (median time to review 1 [0-3] day). Where suspected IFI was not confirmed, 38% episodes were stopped and 4% de-escalated within 5 days. Conclusions: Achieving a better balance between promptly treating IFI patients and avoiding inappropriate antifungal prescribing in the ICU requires timely post-prescription review by specialist multidisciplinary teams and improved, evidence-based-risk prescribing strategies incorporating rapid diagnostics to guide AFT start and stop decisions.

Gut Commensal Bacteroidetes Encode a Novel Class of Vitamin B12-Binding Proteins.

Human gut commensal Bacteroidetes rely on multiple transport systems to acquire vitamin B12 and related cobamides for fitness in the gut. In addition to a set of conserved transport proteins, these systems also include a diverse repertoire of additional proteins with unknown function. Here, we report the function and structural characterization of one of these proteins, BtuH, which binds vitamin B12 directly via a C-terminal globular domain that has no known structural homologs. This protein is required for efficient B12 transport and competitive fitness in the gut, demonstrating that members of the heterogeneous suite of accessory proteins encoded in Bacteroides cobamide transport system loci can play key roles in vitamin acquisition. IMPORTANCE The gut microbiome is a complex microbial community with important impacts on human health. One of the major groups within the gut microbiome, the Bacteroidetes, rely on their ability to capture vitamin B12 and related molecules for fitness in the gut. Unlike well-studied model organisms, gut Bacteroidetes genomes often include multiple vitamin B12 transport systems with a heterogeneous set of components. The role, if any, of these components was unknown. Here, we identify new proteins that play key roles in vitamin B12 capture in these organisms. Notably, these proteins are associated with some B12 transport systems and not others (even in the same bacterial strain), suggesting that these systems may assemble into functionally distinct machines to capture vitamin B12 and related molecules.

Co-design of interventions to improve acute care in hospital: A rapid review of the literature and application of the BASE methodology, a novel system for the design of patient centered service prototypes.

Co-design in acute care is challenged by the inability of unwell patients to participate in the process and the often transient nature of acute care. We undertook a rapid review of the literature on co-design, co-production and co-creation of solutions for acute care that were developed with patients. We found limited little evidence for co-design methods in acute care. We adapted a novel design driven method (BASE methodology) that creates stakeholder groups through epistemological criteria for the rapid development of interventions for acute care. We demonstrated feasibility of the methodology in two case studies: A mHealth application with checklists for patients undergoing treatment for cancer and a patient held record for self-clerking on admission to hospital.

Patient reported outcomes after risk-reducing surgery in patients at increased risk of ovarian cancer.

OBJECTIVE: To describe the quality of life of women at an increased risk of ovarian cancer undergoing risk-reducing bilateral salpingo-oophorectomy (RRBSO). METHODS: Patients evaluated in our gynecologic oncology ambulatory practice between January 2018-December 2019 for an increased risk of ovarian cancer were included. Patients received the EORTC QLQ-C30 and PROMIS emotional and instrumental support questionnaires along with a disease-specific measure (PROM). First and last and pre- and post-surgical PROM responses in each group were compared as were PROMs between at-risk patients and patients with other ovarian diseases. RESULTS: 195 patients with an increased risk of ovarian cancer were identified, 155 completed PROMs (79.5%). BRCA1 or BRCA2 mutations were noted in 52.8%. Also included were 469 patients with benign ovarian disease and 455 with ovarian neoplasms. Seventy-two at-risk patients (46.5%) had surgery and 36 had both pre- and post-operative PROMs. Post-operatively, these patients reported significantly less tension (p = 0.011) and health-related worry (p = 0.021) but also decreased levels of health (p = 0.018) and quality of life <7d (0.001), less interest in sex (p = 0.014) and feeling less physically attractive (p = 0.046). No differences in body image or physical/sexual health were noted in at-risk patients who did not have surgery. When compared to patients with ovarian neoplasms, at-risk patients reported lower levels of disease-related life interference and treatment burden, less worry, and better overall health. CONCLUSIONS: In patients with an increased risk of ovarian cancer, RRBSO is associated with decreased health-related worry and tension, increased sexual dysfunction and poorer short-term quality of life. Patients with ovarian neoplasms suffer to a greater extent than at-risk patients and report higher levels of treatment burden and disease-related anxiety.

Ion temperature measurements from tomographic reconstruction of Doppler spectra in the presence of multi-component flow in two dimensions.

A new ion Doppler diagnostic has been constructed to measure ion temperature profiles in the presence of multi-component flow during magnetic reconnection experiments. The inversion technique and diagnostic setup are applicable to axisymmetric plasmas with two-component flow across the measurement cross section, which occurs during magnetic reconnection. The particular design discussed here is optimized for operation on the Magnetic Reconnection eXperiment (MRX) at Princeton Plasma Physics Laboratory. To prove the viability of this diagnostic for MRX and the future Facility for Laboratory Reconnection Experiments, measurements have been taken and ion temperature and perpendicular flow profiles have been obtained. The radial velocity on MRX does not contribute to the Doppler shift of the measured spectra but does contribute to the broadening of the spectra, while toroidal flow contributes to both. It is shown that neglecting the radial velocity for vR = 20 km/s leads to an error in the ion temperature inversion of 20%. Results from MRX discharges are shown, and the impact of radial velocity on ion temperature inversions is discussed.

The use of neoadjuvant chemotherapy in advanced endometrial cancer.

The objective of this retrospective cohort study was to review the use of neoadjuvant chemotherapy followed by interval cytoreductive surgery in patients presenting with advanced, unresectable endometrial cancer at two large cancer centers. Patients with advanced endometrial cancer treated with neoadjuvant chemotherapy between 2008 and 2015 were identified from an institutional database. Clinical and surgical variables were analyzed and time to recurrence and death was calculated and compared between surgical groups. Thirty-three patients were identified (mean age 64.8 (range 42-86 years)). Overall, 28% of patients had endometrioid histology, 48% serous, 4% clear cell, 4% carcinosarcoma, 12% mixed and 4% other. Ineligibility for primary surgery was due to unresectable disease (85%), comorbidities (6%) and unknown reasons (9%). All patients received neoadjuvant chemotherapy with 91% of patients receiving carboplatin and paclitaxel. On reimaging, 12% of patients had progressed, 76% had a partial response and 3% had a complete response to chemotherapy. 76% of patients underwent interval surgery, with cytoreduction to no visible residual disease achieved in 52%. Overall, 91% of patients recurred and 85% died during follow-up. Patients undergoing surgery after chemotherapy had significantly longer progression-free survival (11.53 vs. 4.99 months, p = 0.0096) and overall survival (24.13 vs. 7.04 months, p = 0.0042) when compared to patients who did not have surgery. Neoadjuvant chemotherapy is a feasible treatment option to allow for interval cytoreductive surgery in patients with advanced endometrial cancer not amenable to primary debulking. Patients who undergo surgery after chemotherapy have significantly improved progression free and overall survival.

White matter integrity after cannabidiol administration for treatment resistant epilepsy.

OBJECTIVE: The effects of individual cannabinoids on white matter integrity are unclear. Human studies have shown white matter maturation alterations in regular recreational cannabis users with the magnitude of these effects dependent on the age of exposure. However, studies have yet to determine which phytocannabinoids are most responsible for these changes. In the current study, we analyzed the effects of pharmaceutical grade cannabidiol oral solution (CBD; Epidiolex® in the U.S.; Epidyolex® in the EU; 100 mg/mL oral solution) on white matter integrity using diffusion MRI in patients with treatment resistant epilepsy (TRE). METHODS: 15 patients with TRE underwent 3 T diffusion MRI prior to receiving CBD and then again approximately 12 weeks later while on a stable dose of CBD for at least two weeks. DTI analyzes were conducted using DSI Studio and tract-based spatial statistics (TBSS). RESULTS: DTI analysis using DSI Studio showed significant increases in fractional anisotropy (FA) in the right medial lemniscus (p = 0.03), right superior cerebellar peduncle (p = 0.03) and the pontine crossing tract (p = 0.04); decreased mean diffusivity (MD) in the left uncinate fasciculus (p = 0.02) and the middle cerebellar peduncle (p = 0.04); decreased axial diffusivity (AD) in the left superior cerebellar peduncle (p = 0.05), right anterior limb of the internal capsule (p = 0.03), and right posterior limb of the internal capsule (p = 0.02); and decreased radial diffusivity (RD) in the middle cerebellar peduncle (p = 0.03) and left uncinate fasiculus (p = 0.01). The follow-up ANCOVA also yielded significant results when controlling for covariates of CBD dosage, age, sex, change in seizure frequency, and scanner type: FA increased in the pontine crossing tract (p = 0.03); RD decreased in the middle cerebellar peduncle (p = 0.04) and left uncinate fasciculus (p = 0.04). Subsequent TBSS analysis controlling for the same variables yielded no significant white matter differences between groups. CONCLUSION: These findings indicate relatively minor short-term effects of highly-purified plant-derived CBD on white matter structural integrity in patients with TRE.

Development and internal validation of clinical prediction models for outcomes of complicated intra-abdominal infection.

BACKGROUND: Complicated intra-abdominal infections (cIAIs) are associated with significant morbidity and mortality. The aim of this study was to describe the clinical characteristics of patients with cIAI in a multicentre study and to develop clinical prediction models (CPMs) to help identify patients at risk of mortality or relapse. METHODS: A multicentre observational study was conducted from August 2016 to February 2017 in the UK. Adult patients diagnosed with cIAI were included. Multivariable logistic regression was performed to develop CPMs for mortality and cIAI relapse. The c-statistic was used to test model discrimination. Model calibration was tested using calibration slopes and calibration in the large (CITL). The CPMs were then presented as point scoring systems and validated further. RESULTS: Overall, 417 patients from 31 surgical centres were included in the analysis. At 90 days after diagnosis, 17.3 per cent had a cIAI relapse and the mortality rate was 11.3 per cent. Predictors in the mortality model were age, cIAI aetiology, presence of a perforated viscus and source control procedure. Predictors of cIAI relapse included the presence of collections, outcome of initial management, and duration of antibiotic treatment. The c-statistic adjusted for model optimism was 0.79 (95 per cent c.i. 0.75 to 0.87) and 0.74 (0.73 to 0.85) for mortality and cIAI relapse CPMs. Adjusted calibration slopes were 0.88 (95 per cent c.i. 0.76 to 0.90) for the mortality model and 0.91 (0.88 to 0.94) for the relapse model; CITL was -0.19 (95 per cent c.i. -0.39 to -0.12) and - 0.01 (- 0.17 to -0.03) respectively. CONCLUSION: Relapse of infection and death after complicated intra-abdominal infections are common. Clinical prediction models were developed to identify patients at increased risk of relapse or death after treatment, these now require external validation.

Novel DNA methylation signatures of tobacco smoking with trans-ethnic effects.

BACKGROUND: Smoking remains one of the leading preventable causes of death. Smoking leaves a strong signature on the blood methylome as shown in multiple studies using the Infinium HumanMethylation450 BeadChip. Here, we explore novel blood methylation smoking signals on the Illumina MethylationEPIC BeadChip (EPIC) array, which also targets novel CpG-sites in enhancers. METHOD: A smoking-methylation meta-analysis was carried out using EPIC DNA methylation profiles in 1407 blood samples from four UK population-based cohorts, including the MRC National Survey for Health and Development (NSHD) or 1946 British birth cohort, the National Child Development Study (NCDS) or 1958 birth cohort, the 1970 British Cohort Study (BCS70), and the TwinsUK cohort (TwinsUK). The overall discovery sample included 269 current, 497 former, and 643 never smokers. Replication was pursued in 3425 trans-ethnic samples, including 2325 American Indian individuals participating in the Strong Heart Study (SHS) in 1989-1991 and 1100 African-American participants in the Genetic Epidemiology Network of Arteriopathy Study (GENOA). RESULTS: Altogether 952 CpG-sites in 500 genes were differentially methylated between smokers and never smokers after Bonferroni correction. There were 526 novel smoking-associated CpG-sites only profiled by the EPIC array, of which 486 (92%) replicated in a meta-analysis of the American Indian and African-American samples. Novel CpG sites mapped both to genes containing previously identified smoking-methylation signals and to 80 novel genes not previously linked to smoking, with the strongest novel signal in SLAMF7. Comparison of former versus never smokers identified that 37 of these sites were persistently differentially methylated after cessation, where 16 represented novel signals only profiled by the EPIC array. We observed a depletion of smoking-associated signals in CpG islands and an enrichment in enhancer regions, consistent with previous results. CONCLUSION: This study identified novel smoking-associated signals as possible biomarkers of exposure to smoking and may help improve our understanding of smoking-related disease risk.

Sex-related Differences in Stress Reactivity and Cingulum White Matter.

The prefrontal cortex and limbic system are important components of the neural circuit that underlies stress and anxiety. These brain regions are connected by white matter tracts that support neural communication including the cingulum, uncinate fasciculus, and the fornix/stria-terminalis. Determining the relationship between stress reactivity and these white matter tracts may provide new insight into factors that underlie stress susceptibility and resilience. Therefore, the present study investigated sex differences in the relationship between stress reactivity and generalized fractional anisotropy (GFA) of the white matter tracts that link the prefrontal cortex and limbic system. Diffusion weighted images were collected and deterministic tractography was completed in 104 young adults (55 men, 49 women; mean age = 18.87 SEM = 0.08). Participants also completed self-report questionnaires (e.g., Trait Anxiety) and donated saliva (later assayed for cortisol) before, during, and after the Trier Social Stress Test. Results revealed that stress reactivity (area under the curve increase in cortisol) and GFA of the cingulum bundle varied by sex. Specifically, men demonstrated greater cortisol reactivity and greater GFA within the cingulum than women. Further, an interaction between sex, stress reactivity, and cingulum GFA was observed in which men demonstrated a positive relationship while women demonstrated a negative relationship between GFA and cortisol reactivity. Finally, trait anxiety was positively associated with the GFA of the fornix/stria terminalis - the white matter pathways that connect the hippocampus/amygdala to the hypothalamus. These findings advance our understanding of factors that underlie individual differences in stress reactivity.

Role of molecular biomarker human papilloma virus (HPV) E6 oncoprotein in cervical cancer screening.

OBJECTIVE: The Onco E6™ Cervical Test, based on detection of the E6 oncoprotein of HPV 16 and 18 genotypes is evaluated as a screen for the early detection cervical neoplasia in resource-limited countries. METHODS: This prospective study from June 2018 to June 2019 evaluated 235 women aged 21-65 years, who came to Gynaecological Oncology Outpatient Department by VIA, cytology, E6 oncoprotein test and by colposcopy. Screen-positive women by any of the tests or women with suspicious findings were further evaluated by biopsy at colposcopy. The McNemar test was used to compare the performance of E6 oncoprotein test with other screening tests. RESULTS: The E6 oncoprotein positivity rate was 6.8% (n = 16) with 81.25% HPV 16 positive and 18.75% HPV 18 positive. Among VIA positive cases (n = 100), E6 oncoprotein was positive in 9% (p < .001). In histopathology confirmed chronic cervicitis, CIN I, CIN II, CIN III and invasive cervical cancer, E6 test was positive for 2.8%, 4.7%, 25%, 50% and 100% respectively. E6 oncoprotein test had the highest specificity and Positive Predictive Value (PPV; 97% and 75%) compared to VIA (42% and 18%), cytology (95% and 46%) and colposcopy (94% and 59%). Sensitivity of the E6 oncoprotein test for detection of CIN3+ was significantly higher than that of cytology (52% VS 25%) but lower than that of VIA (52% VS 74%). CONCLUSIONS: The HPV E6 oncoprotein test is highly specific and is an effective triage test to reduce colposcopy referrals for the large number of false positive test outcomes seen with VIA.

Imaging in the investigation and management of Staphylococcus aureus bacteraemia: a role for advanced imaging techniques.

Staphylococcus aureus bacteraemia (SAB) continues to affect ∼25,000 patients in the UK per year with a high crude mortality of 30% at 90 days. Prompt source control improves outcomes in sepsis and SAB and is included in sepsis guidelines. A recent clinical trial of adjunctive antibiotic treatment in SAB found that the majority of recurrences of SAB were associated with a failure of source management. In this condition, the ability to control the source of infection may be limited by the ability to detect a focus of infection. Echocardiogram is now a routinely used tool to detect such unknown foci in the form of unexpected infectious vegetations. We review the literature to explore the utility of advanced imaging techniques, such as [18F]fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) and magnetic resonance imaging (including whole-body MRI), to detect foci which may otherwise be missed. As unknown foci are associated with increased mortality, we propose that increasing the detection of foci could enable improved source control and result in improved outcomes in SAB.

A challenging case of twin pregnancy with complete hydatidiform mole and co-existing normal live fetus - A case report and review of the literature.

Hydatidiform mole coexisting with a normal live fetus in a twin pregnancy is extremely rare. Management of these cases is challenging due to the risk of severe antepartum and post-partum complications. Herein, we report the case of a 24-year-old gravida 2 para 1 who presented at 28 weeks gestation with severe preeclampsia, vulvar edema and a serum ß-HCG of 285,000 IU/mL. Ultrasonography demonstrated a single live intra-uterine pregnancy with concurrent hydatidiform mole. Conservative management with magnesium sulfate and anti-hypertensive medications was initiated however the patient developed HELLP syndrome and required urgent delivery at 33 weeks. Copious molar tissue was removed from the uterus during delivery. Four weeks post-partum, her ß-HCG had dropped to 14,000 IU/ml and continued to decline at 6 weeks (2900 IU/ml). However, at eight weeks, it increased to 3500 IU/ml and the patient was treated with nine cycles of intramuscular methotrexate. Current guidelines for management of a twin pregnancy with coexistent mole recommend close clinical monitoring if the mother and fetus are stable and urgent delivery in the setting of complications. During the postpartum period, careful follow up with clinical evaluation and serial serum ß-HCG is important for the diagnosis and treatment of persistent trophoblastic disease.

A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa.

The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa's outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic.IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

Associations of birth characteristics with perimenopausal disorders: a prospective cohort study.

Perimenopausal disorders (PDs) are prevalent and importantly affect quality of life among middle-aged women. Yet, very little is known about the developmental origins of these disorders. The objective of this study was to investigate the associations of birth characteristics with PDs. This cohort study is based on archived birth records for birth weight and gestational age, and followed prospectively in Swedish inpatient and outpatient registers for 8 years (n=3212). The main outcomes were menopausal and climacteric states (e.g. flushing, sleeplessness), perimenopausal bleeding and other PDs (e.g. atrophic vaginitis). Cox proportional hazards regression models were used to estimate the hazard ratios (HRs) for three subtypes of PDs separately. During the follow-up, 218 women had PDs, among whom 125 had menopausal and climacteric states, 61 had perimenopausal bleeding and 58 had other PDs as first recorded disorder. Birth weight was linearly associated with incidence rate of menopausal and climacteric states [HR=1.66 per 1 kg increase, 95% confidence interval (95% CI)=1.14-2.41]. Gestational age (rather than birth weight) was associated with incidence rate of other PDs (HR=0.87 per 1 week increase, 95% CI=0.79-0.95). Neither birth weight nor gestational age was associated with perimenopausal bleeding. Similar results were found after adjustment for other early-life and adult socio-demographic characteristics. This observational study provides, for the first time, evidence regarding the developmental origins of PDs. Future research is required to investigate the underlying causal mechanisms, which may shed further light on the etiology of this class of disorders.

Adjuvant bevacizumab for melanoma patients at high risk of recurrence: survival analysis of the AVAST-M trial.

Background: Bevacizumab is a recombinant humanised monoclonal antibody to vascular endothelial growth factor shown to improve survival in advanced solid cancers. We evaluated the role of adjuvant bevacizumab in melanoma patients at high risk of recurrence. Patients and methods: Patients with resected AJCC stage IIB, IIC and III cutaneous melanoma were randomised to receive either adjuvant bevacizumab (7.5 mg/kg i.v. 3 weekly for 1 year) or standard observation. The primary end point was detection of an 8% difference in 5-year overall survival (OS) rate; secondary end points included disease-free interval (DFI) and distant metastasis-free interval (DMFI). Tumour and blood were analysed for prognostic and predictive markers. Results: Patients (n=1343) recruited between 2007 and 2012 were predominantly stage III (73%), with median age 56 years (range 18-88 years). With 6.4-year median follow-up, 515 (38%) patients had died [254 (38%) bevacizumab; 261 (39%) observation]; 707 (53%) patients had disease recurrence [336 (50%) bevacizumab, 371 (55%) observation]. OS at 5 years was 64% for both groups [hazard ratio (HR) 0.98; 95% confidence interval (CI) 0.82-1.16, P = 0.78). At 5 years, 51% were disease free on bevacizumab versus 45% on observation (HR 0.85; 95% CI 0.74-0.99, P = 0.03), 58% were distant metastasis free on bevacizumab versus 54% on observation (HR 0.91; 95% CI 0.78-1.07, P = 0.25). Forty four percent of 682 melanomas assessed had a BRAFV600 mutation. In the observation arm, BRAF mutant patients had a trend towards poorer OS compared with BRAF wild-type patients (P = 0.06). BRAF mutation positivity trended towards better OS with bevacizumab (P = 0.21). Conclusions: Adjuvant bevacizumab after resection of high-risk melanoma improves DFI, but not OS. BRAF mutation status may predict for poorer OS untreated and potential benefit from bevacizumab. Clinical Trial Information: ISRCTN 81261306; EudraCT Number: 2006-005505-64.