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Objective: To identify a microbial signature for endometriosis for use as a diagnostic non-invasive biomarker., Design: Prospective cohort pilot study., Setting: Nepean Hospital and UNSW Microbiome Research Centre, Australia., Population: Sixty-four age- and sex-matched subjects (n = 19 healthy control (HC); n = 24 non-endometriosis (N-ENDO) and n = 21 confirmed endometriosis (ENDO)). All study participants, besides healthy controls, underwent laparoscopic surgical assessment for endometriosis, and histology was performed on excised lesions., Methods: Oral, stool and, vaginal samples were self-collected at a single time point for healthy controls, and preoperatively for patients undergoing laparoscopy. Samples underwent 16S rRNA amplicon sequencing, followed by bioinformatics analysis., Main Outcome Measures: Compositional differences between cohorts as identified by diversity analyses, and differentially abundant microbial taxa, as identified by LEfSE analysis., Results: The composition of the oral (adjusted p = 0.003), and stool (adjusted p = 0.042) microbiota is different between the three cohorts. Differentially abundant taxa are present within each cohort as identified by LEfSE analysis. Particularly, Fusobacterium was enriched in the oral samples of patients with moderate/severe endometriosis., Conclusions: Taxonomic and compositional differences were found between the microbiota in the mouth, gut and, vagina of patients with and without endometriosis and healthy controls. Fusobacterium was enriched in patients with moderate/severe endometriosis. Fusobacterium is noted as a key pathogen in periodontal disease, a common comorbidity in endometriosis. These findings suggest a role for the oral, stool and, vaginal microbiome in endometriosis, and present potential for microbial-based treatments and the design of a diagnostic swab., (© 2024 The Author(s). BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd.)

Importance: Extended work hours and night shifts are essential in health care, but negatively affect physician sleep, well-being, and patient care. Alternative schedules with shorter work hours and/or reduced irregularity might mitigate these issues., Objective: To compare sleep, well-being, and cognition between interns working irregular, extended shifts (call schedule), and those working a more regular schedule with restricted hours (float schedule)., Design, Setting, and Participants: In this observational longitudinal cohort study, interns in a Singapore-based teaching hospital were studied for 8 weeks from January 2022 to July 2023. Data were analyzed from July 2023 to July 2024., Exposure: Participants worked either regular approximately 10-hour workdays, interspersed with 24 hour or more overnight calls 4 to 5 times a month, or a float schedule, which included regular approximately 10-hour workdays, and 5 to 7 consecutive approximately 12-hour night shifts every 2 months. Exposure was based on departmental training and operational needs., Main Outcomes and Measures: Sleep was measured with wearable sleep trackers and an electronic diary. Day-to-day well-being and cognitive assessments were collected through a smartphone application. Assessments included the Sleep Regularity Index (SRI; determines the probability of an individual being in the same state [sleep or wake] at any 2 time points 24 hours apart, with 0 indicating highly random sleep patterns and 100 denoting perfect regularity) and Pittsburgh Sleep Quality Inventory (PSQI; scores ranges from 0 to 21, with higher scores indicating poorer sleep; a score greater than 5 suggests significant sleep difficulties)., Results: Participants (mean [SD] age, 24.7 [1.1] years; 57 female participants [59.4%]; 41 on call schedule [42.7%]; 55 on float schedule [57.3%]) provided 4808 nights of sleep (84.2%) and 3390 days (59.3%) of well-being and cognition assessments. Participants on a float schedule had higher SRI scores (mean [SD] score, 69.4 [6.16]) and had better quality sleep (PSQI mean [SD] score, 5.4 [2.3]), than participants on call schedules (SRI mean [SD] score, 56.1 [11.3]; t91 = 6.81; mean difference, 13.3; 95% CI, 9.40 to 17.22; P < .001; PSQI mean [SD] score, 6.5 [2.3]; t79 = 2.16; 95% CI, 0.09 to 2.15; P = .03). Overnight call shifts, but not night float shifts, were associated with poorer mood (-13%; β = -6.79; 95% CI, -9.32 to -4.27; P < .001), motivation (-21%; β = -10.09; 95% CI, -12.55 to -7.63; P < .001), and sleepiness ratings (29%; β = 15.96; 95% CI, 13.01 to 18.90; P < .001) and impaired vigilance (21 ms slower; β = 20.68; 95% CI, 15.89 to 25.47; P < .001) compared with regular day shifts. Night shifts with naps were associated with better vigilance (16 ms faster; β = -15.72; 95% CI, -28.27 to -3.17; P = .01) than nights without naps., Conclusions and Relevance: In this cohort study, 24-hour call schedules were associated with poorer sleep, well-being, and cognition outcomes than float schedules. Naps during night shifts benefited vigilance in both schedules.

Sequencing the tyrosine phosphoproteome using MS-based proteomics is challenging due to the low abundance of tyrosine phosphorylation in cells, a challenge compounded in scarce samples like primary cells or clinical samples. The broad-spectrum optimisation of selective triggering (BOOST) method was recently developed to increase phosphotyrosine sequencing in low protein input samples by leveraging tandem mass tags (TMT), phosphotyrosine enrichment, and a phosphotyrosine-loaded carrier channel. Here, we demonstrate the viability of BOOST in T cell receptor (TCR)-stimulated primary murine T cells by benchmarking the accuracy and precision of the BOOST method and discerning significant alterations in the phosphoproteome associated with receptor stimulation. Using 1 mg of protein input (about 20 million cells) and BOOST, we identify and precisely quantify more than 2000 unique pY sites compared to about 300 unique pY sites in non-BOOST control samples. We show that although replicate variation increases when using the BOOST method, BOOST does not jeopardise quantitative precision or the ability to determine statistical significance for peptides measured in triplicate. Many pY previously uncharacterised sites on important T cell signalling proteins are quantified using BOOST, and we identify new TCR responsive pY sites observable only with BOOST. Finally, we determine that the phase-spectrum deconvolution method on Orbitrap instruments can impair pY quantitation in BOOST experiments., (© 2024 Wiley‐VCH GmbH.)

Objective: The objective of this study is to explore the information needs related to insulin therapy in children and adolescents with type 1 diabetes mellitus (T1DM) from the children's perspectives as well as their caregivers., Design: Qualitative study; semistructured interviews. To identify emerging themes relating to information needs, open coding and thematic analysis were employed., Setting: Participants were recruited from a tertiary care children's hospital in Kuala Lumpur, Malaysia and a specialist hospital in Riyadh, Saudi Arabia., Participants: Thirty one children with a mean age of 11.5 years (SD=1.9) and their caregivers were interviewed. Seventeen participants were from Malaysia and 14 were from Saudi Arabia., Results: Four themes of information emerged from the interviews, including information related to (1) hypoglycaemia and hyperglycaemia, (2) insulin therapy, (3) injection technique and (4) other information needs pertaining to continuous glucose monitoring, access to peer groups and future advances in insulin therapy., Conclusion: This study provided valuable insights into the information needs related to T1DM and insulin therapy among children and adolescents with T1DM that should be considered by stakeholders in the development of age-appropriate education materials. Such materials will assist children and adolescents to better manage their life-long T1DM condition from adolescence until adulthood., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Vaginal estrogen is a treatment for genitourinary symptoms of menopause (GSM), which comprises vaginal atrophy and urinary dysfunction, including incontinence. Previous studies show that estrogen therapy promotes lactobacilli abundance and is associated with reduced GSM symptoms, including reduction of stress incontinence. However, detailed longitudinal studies that characterize how the microbiome changes in response to estrogen are scarce. We aimed to compare the vaginal microbiota of postmenopausal women, before and 12 weeks after vaginal estrogen cream., Methods: A total of 44 paired samples from 22 postmenopausal women with vaginal atrophy and stress incontinence were collected pre-vaginal estrogens and were compared to 12 weeks post-vaginal estrogen. Microbiota was characterized by 16S rRNA amplicon sequencing and biodiversity was investigated by comparing the alpha- and beta-diversity and potential markers were identified using differential abundance analysis., Results: Vaginal estrogen treatment was associated with a reduction in vaginal pH and corresponded with a significant reduction in alpha diversity of the microbiota. Healthy vaginal community state type was associated with lower mean pH 4.89 (SD = 0.6), in contrast to dysbiotic state which had a higher mean pH 6.4 (SD = 0.74). Women with lactobacilli dominant community pre-treatment, showed stable microbiota and minimal change in their pH. Women with lactobacilli deficient microbiome pre-treatment improved markedly ( p = 0.004) with decrease in pH -1.31 and change to heathier community state types., Conclusion: In postmenopausal women with stress incontinence, vaginal estrogen promotes Lactobacillus and Bifidobacterium growth and lowers vaginal pH. Maximum response is seen in those with a dysbiotic vaginal microbiota pre-treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Moore, Ognenovska, Chua, Chen, Hicks, El-Assaad, te West and El-Omar.)

Background: Alzheimer's disease (AD) and vascular dementia (VaD) are two of the commonest causes of dementia in the elderly. Of the myriad biomolecules implicated in dementia pathogenesis, sphingolipids have attracted relatively scant research attention despite their known involvement in multiple pathophysiological processes. The potential utility of peripheral sphingolipids as biomarkers in dementia cohorts with high concomitance of cerebrovascular diseases is also unclear., Methods: Using a lipidomics platform, we performed a case-control study of plasma sphingolipids in a prospectively assessed cohort of 526 participants (non-cognitively impaired, NCI = 93, cognitively impaired = 217, AD = 166, VaD = 50) using a lipidomics platform., Results: Distinct patterns of sphingolipid alterations were found in AD and VaD, namely an upregulation of d18:1 species in AD compared to downregulation of d16:1 species in VaD. In particular, GM3 d18:1/16:0 and GM3 d18:1/24:1 showed the strongest positive associations with AD. Furthermore, evaluation of sphingolipids panels showed specific combinations with higher sensitivity and specificity for classification of AD (Cer d16:1/24:0. Cer d18:1/16:0, GM3 d16:1/22:0, GM3 d18:1/16:0, SM d16:1/22:0, HexCer d18:1/18:0) and VAD (Cer d16:1/24:0, Cer d18:1/16:0, Hex2Cer d16:1/16:0, HexCer d18:1/18:0, SM d16:1/16:0, SM d16:1/20:0, SM d18:2/22:0) compared to NCI., Conclusions: AD and VaD are associated with distinct changes of plasma sphingolipids, warranting further studies into underlying pathophysiological mechanisms and assessments of their potential utility as dementia biomarkers and therapeutic targets., (© 2023. The Author(s).)

Study Objectives: Country or regional differences in sleep duration are well-known, but few large-scale studies have specifically evaluated sleep variability, either across the work week, or in terms of differences in weekday and weekend sleep., Methods: Sleep measures, obtained over 50 million night's sleep from ∼220,000 wearable device users in 35 countries, were analysed. Each person contributed an average of ∼242 nights of data. Multiple regression was used to assess the impact country of residence had on sleep duration, timing, efficiency, weekday sleep variability, weekend sleep extension and social jetlag., Results: Nocturnal sleep was shorter and had a later onset in Asia than other regions. Despite this, sleep efficiency was lower and weekday sleep variability was higher. Weekend sleep extension was longer in Europe and the USA than in Asia, and was only partially related to weekday sleep duration. There were also cross-country differences in social jetlag although the regional differences were less distinct than for weekend sleep extension., Conclusions: In addition to regional differences in sleep duration, cross-country differences in sleep variability and weekend sleep extension suggest that using the latter as an indicator of sleep debt may need to be reconsidered. In countries exhibiting both short sleep and high weekday sleep variability, a culturally different means of coping with inadequate sleep is likely. Country or region differences in culture, particularly those related to work, merit closer examination as factors influencing the variability in normative sleep patterns around the world., Competing Interests: Declaration of competing interest M.K. and I.A. work for Oura Health Oy, which provided the data for this study. M.W.L.C. has received research funding for performing performance evaluation of Oura Rings. A.R.W. and X.Y.C. declare no competing financial interests. Neither the company nor its employees influenced the scientific integrity of the work., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The microbiome has emerged as a key determinant of human health and reproduction, with recent evidence suggesting a dysbiotic microbiome is implicated in adverse perinatal health outcomes. The existing research has been limited by the sample collection and timing, cohort design, sample design, and lack of data on the preconception microbiome. This prospective, longitudinal cohort study will recruit 2000 Australian women, in order to fully explore the role of the microbiome in the development of adverse perinatal outcomes. Participants are enrolled for a maximum of 7 years, from 1 year preconception, through to 5 years postpartum. Assessment occurs every three months until pregnancy occurs, then during Trimester 1 (5 + 0-12 + 6 weeks gestation), Trimester 2 (20 + 0-24 + 6 weeks gestation), Trimester 3 (32 + 0-36 + 6 weeks gestation), and postpartum at 1 week, 2 months, 6 months, and then annually from 1 to 5 years. At each assessment, maternal participants self-collect oral, skin, vaginal, urine, and stool samples. Oral, skin, urine, and stool samples will be collected from children. Blood samples will be obtained from maternal participants who can access a study collection center. The measurements taken will include anthropometric, blood pressure, heart rate, and serum hormonal and metabolic parameters. Validated self-report questionnaires will be administered to assess diet, physical activity, mental health, and child developmental milestones. Medications, medical, surgical, obstetric history, the impact of COVID-19, living environments, and pregnancy and child health outcomes will be recorded. Multiomic bioinformatic and statistical analyses will assess the association between participants who developed high-risk and low-risk pregnancies, adverse postnatal conditions, and/or childhood disease, and their microbiome for the different sample types.

Dietary intake during pregnancy may influence the antenatal microbiome, which is proposed to impact maternal and infant health during the pregnancy and beyond. The aim of this sub-study was to examine associations between dietary intake and microbiota diversity during pregnancy using whole metagenomic sequencing and examine associations in low-risk versus high-risk pregnancies, as well as complicated versus uncomplicated pregnancies. Pregnancy data were analysed from women participating in the MUMS cohort study in Sydney, Australia (women followed from trimester 1 of pregnancy to 1-year postpartum), who had dietary intake data at either trimester 1 or 3, assessed using the Australian Eating Survey, and a matched stool sample ( n = 86). Correlations of microbial alpha diversity with dietary intake data were determined using the repeated-measures correlation, rmcorr, in R. In the combined cohort, no associations were found between diet quality or diet composition and microbial alpha diversity or beta diversity. However, trends in our analysis suggested that dietary intake of specific macro- and micronutrients may influence microbial diversity differently, depending on particular pregnancy conditions. Our findings suggest that dietary intake during pregnancy may have a variable influence on the maternal microbiota, unique to the individual maternal pregnancy phenotype. More research is needed to disentangle these associations.

In recent decades, mass spectrometry-based lipidomics has provided a fertile environment for scientific investigations of biochemical and mechanistic processes in biological systems. Notably, this approach has been used to characterize physiological and pathological processes relevant to the central nervous system by identifying changes in the sphingolipid content in the brain, cerebral spinal fluid, and blood plasma. However, despite a preponderance of studies identifying correlations between specific lipids and disease progression, this powerful resource has not yet substantively translated into clinically useful diagnostic assays. Part of this gap may be explained by insufficient depth of the lipidomic profiles in many studies, by lab-to-lab inconsistencies in methodology, and a lack of absolute quantification. These issues limit the identification of specific molecular species and the harmonization of results across independent studies. In this chapter, we contextualize these issues with recent reports identifying correlations between brain lipids and neurological diseases, and we describe the workflow our group has optimized for analysis of the blood plasma sphingolipidome, adapted to the characterization of the human brain tissue., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The microbiome has been implicated in the development of metabolic conditions which occur at high rates in people with schizophrenia and related psychoses. This exploratory proof-of-concept study aimed to: (i) characterize the gut microbiota in antipsychotic naïve or quasi-naïve people with first-episode psychosis, and people with established schizophrenia receiving clozapine therapy; (ii) test for microbiome changes following a lifestyle intervention which included diet and exercise education and physical activity. Participants were recruited from the Eastern Suburbs Mental Health Service, Sydney, Australia. Anthropometric, lifestyle and gut microbiota data were collected at baseline and following a 12-week lifestyle intervention. Stool samples underwent 16S rRNA sequencing to analyse microbiota diversity and composition. Seventeen people with established schizophrenia and five people with first-episode psychosis were recruited and matched with 22 age-sex, BMI and ethnicity matched controls from a concurrent study for baseline comparisons. There was no difference in α-diversity between groups at baseline, but microbial composition differed by 21 taxa between the established schizophrenia group and controls. In people with established illness pre-post comparison of α-diversity showed significant increases after the 12-week lifestyle intervention. This pilot study adds to the current literature that detail compositional differences in the gut microbiota of people with schizophrenia compared to those without mental illness and suggests that lifestyle interventions may increase gut microbial diversity in patients with established illness. These results show that microbiome studies are feasible in patients with established schizophrenia and larger studies are warranted to validate microbial signatures and understand the relevance of lifestyle change in the development of metabolic conditions in this population.

Intravenous cannulation is experientially traumatic to children. To minimize this, EMLA ® is applied on the would-be-cannulated area before IV cannula insertion. However, the time to achieve its maximum efficacy may be affected due to incomplete cutaneous absorption and the duration of application. The latter may be a limiting factor in a busy healthcare facility. The usage of dissolvable maltose microneedles may circumvent this problem by introducing micropores that will facilitate EMLA ® absorption. A randomized phase II cross-over trial will be conducted to compare the Visual Analogue Scale (VAS) pain scores and skin conductance algesimeter index between 4 different interventions (1 fingertip unit (FTU) of EMLA ® with microneedle patch for 30 min before cannulation; 0.5 FTU of EMLA ® with microneedle patch for 30 min; 1 FTU of EMLA ® with microneedle for 15 min; 1 FTU of EMLA ® with sham patch for 30 min). A total of 26 pediatric patients with thalassemia aged between 6 and 18 years old and requiring blood transfusion will be recruited in this trial. During the visits, the VAS scores and skin conductance algesimeter index at venous cannulation will be obtained using the VAS rulers and PainMonitor™ machine, respectively. The trial will commence in August 2021 and is anticipated to end by August 2022.

Excessive antibiotic consumption is still common among critically ill patients admitted to intensive care units (ICU), especially during the coronavirus disease 2019 (COVID-19) period. Moreover, information regarding antimicrobial consumption among ICUs in South-East Asia remains scarce and limited. This study aims to determine antibiotics utilization in ICUs by measuring antibiotics consumption over the past six years (2016−2021) and specifically evaluating carbapenems prescribed in a COVID-19 ICU and a general intensive care unit (GICU) during the second year of the COVID-19 pandemic. (2) Methods: This is a retrospective cross-sectional observational analysis of antibiotics consumption and carbapenems prescriptions. Antibiotic utilization data were estimated using the WHO Defined Daily Doses (DDD). Carbapenems prescription information was extracted from the audits conducted by ward pharmacists. Patients who were prescribed carbapenems during their admission to COVID-19 ICU and GICU were included. Patients who passed away before being reviewed by the pharmacists were excluded. (3) Results: In general, antibiotics consumption increased markedly in the year 2021 when compared to previous years. Majority of carbapenems were prescribed empirically (86.8%). Comparing COVID-19 ICU and GICU, the reasons for empirical carbapenems therapy in COVID-19 ICU was predominantly for therapy escalation (64.7% COVID-19 ICU vs. 34% GICU, p < 0.001), whereas empirical prescription in GICU was for coverage of extended-spectrum beta-lactamases (ESBL) gram-negative bacteria (GNB) (45.3% GICU vs. 22.4% COVID-19 ICU, p = 0.005). Despite microbiological evidence, the empirical carbapenems were continued for a median (interquartile range (IQR)) of seven (5−8) days. This implies the need for a rapid diagnostic assay on direct specimens, together with comprehensive antimicrobial stewardship (AMS) discourse with intensivists to address this issue., Competing Interests: The authors declare no conflict of interest.

Spent coffee grounds (SCG) disposal is an environmental problem. These residues from coffee brewing and instant coffee production have potential to produce novel alcoholic beverages. SCG valorization through sequential alcoholic and malolactic fermentation was investigated using a yeast, Lachancea thermotolerans Concerto and a lactic acid bacterium (LAB), Oenococcus oeni Lalvin 31 in this study. Our results showed that sequential inoculation prevented early death of yeast confronted when simultaneous inoculation was adopted, allowing for growth and persistence of both yeast and LAB till the end of fermentation. Adequate ethanol production (4.91 ± 0.13 %, v/v) with low residual sugar content was also attained. In addition, relatively lower levels of acetic, lactic, and succinic acids were produced by sequential inoculation than that of simultaneous inoculation. Furthermore, SCG hydrolysates fermented via sequential inoculation had the widest variety of volatiles (e.g. esters and ketones). Overall, our results indicated that sequential inoculation of L. thermotolerans and O. oeni in SCG hydrolysates might be a way to develop novel beverages with pleasant flavor profiles., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

The essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ-ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C-terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C-terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation in polymer-guided interactions., (© 2022 The Protein Society.)

Study Objectives: COVID-19 lockdowns drastically affected sleep, physical activity, and wellbeing. We studied how these behaviors evolved during reopening the possible contributions of continued working from home and smartphone usage., Methods: Participants (N = 198) were studied through the lockdown and subsequent reopening period, using a wearable sleep/activity tracker, smartphone-delivered ecological momentary assessment (EMA), and passive smartphone usage tracking. Work/study location was obtained through daily EMA ascertainment., Results: Upon reopening, earlier, shorter sleep and increased physical activity were observed, alongside increased self-rated stress and poorer evening mood ratings. These reopening changes were affected by post-lockdown work arrangements and patterns of smartphone usage. Individuals who returned to work or school in-person tended toward larger shifts to earlier sleep and wake timings. Returning to in-person work/school also correlated with more physical activity. Contrary to expectation, there was no decrease in objectively measured smartphone usage after reopening. A cluster analysis showed that persons with relatively heavier smartphone use prior to bedtime had later sleep timings and lower physical activity., Conclusions: These observations indicate that the reopening after lockdown was accompanied by earlier sleep timing, increased physical activity, and altered mental wellbeing. Moreover, these changes were affected by work/study arrangements and smartphone usage patterns., (© Sleep Research Society 2021. Published by Oxford University Press on behalf of the Sleep Research Society.)

Inflammation is usually a tightly regulated process whose termination by mediators including Annexin A1 (AnxA1) results in the resolution of inflammatory responses. In neurodegenerative dementias, chronic neuroinflammation, along with accumulation of aggregated β-amyloid (Aβ) peptides and apoptosis, has long been recognized to be a pathological hallmark; but it is unclear whether a failure of inflammation resolution contributes to this pathophysiological process. In this study, we measured AnxA1 immunoreactivities in postmortem neocortex (Brodmann areas BA9 and BA40) of well characterized Alzheimer's disease (AD), Parkinson disease dementia (PDD) and dementia with Lewy bodies (DLB) patients as well as aged controls. Inactive cleaved AnxA1 was found to be elevated in AD and DLB in BA40. Levels of cleaved AnxA1 also positively correlated with amyloidogenic brain Aβ, anti-inflammatory markers such as IL10 and IL13, as well as with the pro-apoptotic marker cleaved caspase-3 in BA40. Our findings suggest that elevated cleaved AnxA1 in neurodegenerative dementias may reflect a failure of inflammation resolution in certain regions of the diseased brain, and also support a mechanistic link between AnxA1 and amyloid pathology, neuroinflammation and apoptosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Activation of the T cell receptor (TCR) leads to a network of early signaling predominantly orchestrated by tyrosine phosphorylation in T cells. The TCR is commonly activated using soluble anti-TCR antibodies, but this approach is not antigen-specific. Alternatively, activating the TCR using specific antigens of a range of binding affinities in the form of a peptide-major histocompatibility complex (pMHC) is presumed to be more physiological. However, due to the lack of wide-scale phosphotyrosine (pTyr) proteomic studies directly comparing anti-TCR antibodies and pMHC, a comprehensive definition of these activated states remains enigmatic. Elucidation of the tyrosine phosphoproteome using quantitative pTyr proteomics enables a better understanding of the unique features of these activating agents and the role of ligand binding affinity on signaling. Here, we apply the recently established Broad-spectrum Optimization Of Selective Triggering (BOOST) to examine perturbations in tyrosine phosphorylation of human TCR triggered by anti-TCR antibodies and pMHC. Our data reveal that high-affinity ovalbumin (OVA) pMHC activation of the human TCR triggers a largely similar, albeit potentially stronger, pTyr-mediated signaling regulatory axis compared to the anti-TCR antibody. The signaling output resulting from OVA pMHC variants correlates well with their weaker affinities, enabling affinity-tunable control of signaling strength. Collectively, we provide a framework for applying BOOST to compare pTyr-mediated signaling pathways of human T cells activated in an antigen-independent and antigen-specific manner.

Using polysomnography over multiple weeks to characterize an individual's habitual sleep behavior while accurate, is difficult to upscale. As an alternative, we integrated sleep measurements from a consumer sleep-tracker, smartphone-based ecological momentary assessment, and user-phone interactions in 198 participants for 2 months. User retention averaged >80% for all three modalities. Agreement in bed and wake time estimates across modalities was high (rho = 0.81-0.92) and were adrift of one another for an average of 4 min, providing redundant sleep measurement. On the ~23% of nights where discrepancies between modalities exceeded 1 h, k-means clustering revealed three patterns, each consistently expressed within a given individual. The three corresponding groups that emerged differed systematically in age, sleep timing, time in bed, and peri-sleep phone usage. Hence, contrary to being problematic, discrepant data across measurement modalities facilitated the identification of stable interindividual differences in sleep behavior, underscoring its utility to characterizing population sleep and peri-sleep behavior.

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected., (© 2021 Fisheries Society of the British Isles.)

Sphingosine 1-phosphates (S1Ps) are bioactive lipids that mediate a diverse range of effects through the activation of cognate receptors, S1P 1 -S1P 5 . Scrutiny of S1P-regulated pathways over the past three decades has identified important and occasionally counteracting functions in the brain and cerebrovascular system. For example, while S1P 1 and S1P 3 mediate proinflammatory effects on glial cells and directly promote endothelial cell barrier integrity, S1P 2 is anti-inflammatory but disrupts barrier integrity. Cumulatively, there is significant preclinical evidence implicating critical roles for this pathway in regulating processes that drive cerebrovascular disease and vascular dementia, both being part of the continuum of vascular cognitive impairment (VCI). This is supported by clinical studies that have identified correlations between alterations of S1P and cognitive deficits. We review studies which proposed and evaluated potential mechanisms by which such alterations contribute to pathological S1P signaling that leads to VCI-associated chronic neuroinflammation and neurodegeneration. Notably, S1P receptors have divergent but overlapping expression patterns and demonstrate complex interactions. Therefore, the net effect produced by S1P represents the cumulative contributions of S1P receptors acting additively, synergistically, or antagonistically on the neural, vascular, and immune cells of the brain. Ultimately, an optimized therapeutic strategy that targets S1P signaling will have to consider these complex interactions.

While Lck has been widely recognized to play a pivotal role in the initiation of the T cell receptor (TCR) signaling pathway, an understanding of the precise regulation of Lck in T cells upon TCR activation remains elusive. Investigation of protein-protein interaction (PPI) using proximity labeling techniques such as TurboID has the potential to provide valuable molecular insights into Lck regulatory networks. By expressing Lck-TurboID in Jurkat T cells, we have uncovered a dynamic, short-range Lck protein interaction network upon 30 min of TCR stimulation. In this novel application of TurboID, we detected 27 early signaling-induced Lck-proximal interactors in living T cells, including known and novel Lck interactors, validating the discovery power of this tool. Our results revealed previously unappreciated Lck PPI which may be associated with cytoskeletal rearrangement, ubiquitination of TCR signaling proteins, activation of the mitogen-activated protein kinase cascade, coalescence of the LAT signalosome, and formation of the immunological synapse. In this study, we demonstrated for the first time in immune cells and for the kinase Lck that TurboID can be utilized to unveil PPI dynamics in living cells at a time scale consistent with early TCR signaling. Data are available via ProteomeXchange with identifier PXD020759.

Background: There has been ongoing research impetus to uncover novel blood-based diagnostic and prognostic biomarkers for Alzheimer's disease (AD), vascular dementia (VaD), and related cerebrovascular disease (CEVD)-associated conditions within the spectrum of vascular cognitive impairment (VCI). Sphingosine-1-phosphates (S1Ps) are signaling lipids which act on the S1PR family of cognate G-protein-coupled receptors and have been shown to modulate neuroinflammation, a process known to be involved in both neurodegenerative and cerebrovascular diseases. However, the status of peripheral S1P in AD and VCI is at present unclear., Methods: We obtained baseline bloods from individuals recruited into an ongoing longitudinal cohort study who had normal cognition (N = 80); cognitive impairment, no dementia (N = 160); AD (N = 113); or VaD (N = 31), along with neuroimaging assessments of cerebrovascular diseases. Plasma samples were processed for the measurements of major S1P species: d16:1, d17:1, d18:0, and d18:1, along with pro-inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF). Furthermore, in vitro effects of S1Ps on cytokine expression were also studied in an astrocytoma cell line and in rodent primary astrocytes., Results: Of the S1Ps species measured, only d16:1 S1P was significantly reduced in the plasma of VaD, but not AD, patients, while the d18:1 to d16:1 ratios were increased in all cognitive subgroups (CIND, AD, and VaD). Furthermore, d18:1 to d16:1 ratios correlated with levels of IL-6, IL-8, and TNF. In both primary astrocytes and an astroglial cell line, treatment with d16:1 or d18:1 S1P resulted in the upregulation of mRNA transcripts of pro-inflammatory cytokines, with d18:1 showing a stronger effect than d16:1. Interestingly, co-treatment assays showed that the addition of d16:1 reduced the extent of d18:1-mediated gene expression, indicating that d16:1 may function to "fine-tune" the pro-inflammatory effects of d18:1., Conclusion: Taken together, our data suggest that plasma d16:1 S1P may be useful as a diagnostic marker for VCI, while the d18:1 to d16:1 S1P ratio is an index of dysregulated S1P-mediated immunomodulation leading to chronic inflammation-associated neurodegeneration and cerebrovascular damage.

Background: By 2050, 80% of the world's older population will reside in developing countries. There is a need for culturally appropriate training programs to increase awareness of eldercare issues, promote knowledge of how to better allocate resources to geriatric services, and promulgate elder-friendly policies. A monthly distance geriatric education programme between a public hospital in Singapore and health institute in Uganda was implemented. This study explored the enablers and barriers to the delivery of culturally appropriate geriatric education programmes via a videoconferencing platform., Methods: We conducted 12 in-depth interviews with six teachers from Singapore and six learners from Uganda. The interviews were audio-recorded, transcribed and analyzed using an inductive thematic approach to analysis with the aid of the NVivo software., Results: Enablers included inter-personal real-time interactions between teachers and learners whereas misaligned perceptions of cross-cultural differences between Singaporean teachers and Ugandan learners were a barrier. Rapport building, teacher motivation and institutional support were perceived to contribute to the programme's sustainability. Overall, Ugandan learners perceived that the training improved knowledge, skills, attitude and practice of geriatric care. Participants suggested that future initiatives consider aligning cross-cultural perceptions between partners, conducting a training needs analysis, exploring complementary modes of information dissemination, and allotting time for more interaction, thereby reinforcing mutual sharing. Adequate publicity and appropriate incentivisation may also better sustain the programme., Conclusions: Our findings suggest that cross-cultural training via a videoconferencing platform was feasible. Our results inform planners of future distance educational programmes of how to improve standards of cross-cultural competency and forge promising international partnerships.

Thanks to sequencing technology, modern molecular bioscience datasets are often compositions of counts, e.g. counts of amplicons, mRNAs, etc. While there is growing appreciation that compositional data need special analysis and interpretation, less well understood is the discrete nature of these count compositions (or, as we call them, lattice compositions) and the impact this has on statistical analysis, particularly log-ratio analysis (LRA) of pairwise association. While LRA methods are scale-invariant, count compositional data are not; consequently, the conclusions we draw from LRA of lattice compositions depend on the scale of counts involved. We know that additive variation affects the relative abundance of small counts more than large counts; here we show that additive (quantization) variation comes from the discrete nature of count data itself, as well as (biological) variation in the system under study and (technical) variation from measurement and analysis processes. Variation due to quantization is inevitable, but its impact on conclusions depends on the underlying scale and distribution of counts. We illustrate the different distributions of real molecular bioscience data from different experimental settings to show why it is vital to understand the distributional characteristics of count data before applying and drawing conclusions from compositional data analysis methods., (© The Author(s) 2019. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Background Drug-related problems are relatively common among hospitalised patients and may be detrimental to patients and even increase healthcare costs. Characterising drug-related problems allows remedial actions to be in place to minimise the frequency and ensure higher medication safety for the patients involved. Currently, the incidence of drug-related problems among hospitalised paediatric patients in Malaysia is unknown. Objective To determine the incidence, types and intervention acceptance, as well as the risk factors associated with drug-related problems in a Malaysian general paediatric ward. Setting General paediatric ward in Universiti Kebangsaan Malaysia Medical Centre. Method A cross-sectional observational study was conducted from March to May 2019. Data were collected from patients' medical charts, clinical notes and medical records for problem identification based on the modified Pharmaceutical Care Network Europe (PCNE) classification V 8.02. The incidence, types, causes and intervention acceptance as well as the risk factors were assessed. Main outcome measure Drug-related problems based on the modified PCNE classification V 8.02. Results In total, 225 paediatric patients with a total of 694 prescriptions were included in this study. The incidence of drug-related problems was 52.9% (198 prescriptions with problems in 119 patients). The main types of problems were treatment safety (68 prescriptions, 34.3%), incomplete prescription (56 prescriptions, 28.3%) and un-optimised drug treatment (35 prescriptions, 17.7%). The main causes for the problems were necessary information not provided (n = 63, 30.1%), inappropriate dose selection (n = 47, 22.5%) and medication served without a valid prescription (n = 40, 19.1%). Of the 143 interventions proposed to prescribers by the pharmacist, 117 (81.8%) were accepted. The number of prescriptions was found to be the only risk factor associated with DRPs (odds ratio, 1.91; 95% confidence interval, 1.54-2.38; p < 0.001). Conclusion The frequency of drug-related problems among general paediatric ward patients is high and with each increase in prescription a patient received, the risk of a DRP doubles.

Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, the BOOST ( B road-spectrum O ptimization O f S elective T riggering) method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of pervanadate (PV) boost channels (cells treated with the broad-spectrum tyrosine phosphatase inhibitor PV) selectively increased the relative abundance of pTyr-containing peptides. After PV boost channels facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered accurate quantitation of each peptide for the experimental samples while the quantitation from PV boost channels was ignored. This method yielded up to 6.3-fold boost in pTyr quantification depth of statistically significant data derived from contrived ratios, compared with TMT without PV boost channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The BOOST strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly elevate the levels of those modifications across the proteome are available., (© 2020 Chua et al.)

Background: While frailty status is an attractive risk stratification tool, the evaluation of frailty in acute care can be challenging as some inpatients are unable to complete performance-based tests as part of frailty assessment and some tools may lack discriminative ability and categorize majority of cohorts as "frail". In this study, we evaluated the feasibility of frailty screening with the simple clinical frailty scale (CFS) by different clinicians, and its association with mortality and rehospitalization in a geriatric acute care setting., Methods: This study took place in Geriatric Medicine Department of a General Hospital in Singapore. We analysed records of 314 inpatients aged 70 years and older. At baseline, premorbid frailty was assessed using the CFS of the Canadian Study on Health and Aging. Demographic characteristics and other variables were retrieved from their medical records. Primary outcomes were mortality and rehospitalization during the 6-month follow-up. Survival analysis was used to compare the time to death and rehospitalization among CFS categories (1-4: nonfrail, 5-6: mild-moderate frail, and 7-8: severe frail)., Results: CFS showed a high inter-rater reliability when used by different clinicians. In the Cox proportional hazard model controlling for age, gender, Charlson comorbidity index, modified severity of illness index, and discharge placements, severe frailty determined by CFS (HR = 2.09, 95% CI = 1.01-4.33, P = 0.047) and CFS scores (HR = 1.27, 95% CI = 1.05-1.53, P = 0.012) were significantly associated with higher mortality until 6-month postdischarge, but not rehospitalization., Conclusion: Frailty status determined by CFS adds to disease severity and comorbidity in predicting short-term mortality but not rehospitalization in older inpatients who received geriatric acute care in our setting. CFS is reliable and has the potential to be incorporated into routine screening to better identify, communicate, and address frailty in the acute settings., (© 2019 John Wiley & Sons, Ltd.)

We use micromanipulation techniques and real-time particle tracking to develop an approach to study specific attributes of neuron mechanics. We use a mechanical probe composed of a hollow micropipette with its tip fixed to a functionalized bead to induce the formation of a neurite in a sample of rat hippocampal neurons. We then move the sample relative to the pipette tip, elongating the neurite while simultaneously measuring its tension by optically tracking the deflection of the beaded tip. By calibrating the spring constant of the pipette, we can convert this deflection to a force. We use this technique to obtain uniaxial strain measurements of induced neurites and investigate the dependence of the force-extension relationship on mechanical pull speed. We show that in the range of pull speeds studied (0.05-1.8 μm/s), the variation in the work to extend a neurite 10 μm is consistent across pull speeds. We do not observe statistically significant rate-dependent effects in the force-extension profiles; instead we find the same quadratic behaviour (with parameters drawn from the same distributions) at each pull speed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Metagenomic sequencing is an increasingly common tool in environmental and biomedical sciences.  While software for detailing the composition of microbial communities using 16S rRNA marker genes is relatively mature, increasingly researchers are interested in identifying changes exhibited within microbial communities under differing environmental conditions. In order to gain maximum value from metagenomic sequence data we must improve the existing analysis environment by providing accessible and scalable computational workflows able to generate reproducible results. Here we describe a complete end-to-end open-source metagenomics workflow running within Galaxy for 16S differential abundance analysis. The workflow accepts 454 or Illumina sequence data (either overlapping or non-overlapping paired end reads) and outputs lists of the operational taxonomic unit (OTUs) exhibiting the greatest change under differing conditions. A range of analysis steps and graphing options are available giving users a high-level of control over their data and analyses. Additionally, users are able to input complex sample-specific metadata information which can be incorporated into differential analysis and used for grouping / colouring within graphs.  Detailed tutorials containing sample data and existing workflows are available for three different input types: overlapping and non-overlapping read pairs as well as for pre-generated Biological Observation Matrix (BIOM) files. Using the Galaxy platform we developed MetaDEGalaxy, a complete metagenomics differential abundance analysis workflow. MetaDEGalaxy is designed for bench scientists working with 16S data who are interested in comparative metagenomics.  MetaDEGalaxy builds on momentum within the wider Galaxy metagenomics community with the hope that more tools will be added as existing methods mature., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Thang MWC et al.)

We are amidst an ongoing flood of sequence data arising from the application of high-throughput technologies, and a concomitant fundamental revision in our understanding of how genomes evolve individually and within the biosphere. Workflows for phylogenomic inference must accommodate data that are not only much larger than before, but often more error prone and perhaps misassembled, or not assembled in the first place. Moreover, genomes of microbes, viruses and plasmids evolve not only by tree-like descent with modification but also by incorporating stretches of exogenous DNA. Thus, next-generation phylogenomics must address computational scalability while rethinking the nature of orthogroups, the alignment of multiple sequences and the inference and comparison of trees. New phylogenomic workflows have begun to take shape based on so-called alignment-free (AF) approaches. Here, we review the conceptual foundations of AF phylogenetics for the hierarchical (vertical) and reticulate (lateral) components of genome evolution, focusing on methods based on k-mers. We reflect on what seems to be successful, and on where further development is needed., (© The Author 2017. Published by Oxford University Press.)

Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal ( i ) is multipartite; ( ii ) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and ( iii ) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators., Competing Interests: JS, XC, TM, DN, TW, RM, RL No competing interests declared, (© 2019, Saba et al.)

ABSTRACTThe MMSE is used to screen for cognitive impairment and estimate dementia severity. In clinical settings, conventional cut-off scores have been used to distinguish between dementia stages. However, these scores have not been validated for different populations. This study maps scores from the modified version of the MMSE to dementia stages delineated by the Diagnostic and Statistical Manual of Mental Disorders-3rd revised edition (DSM-III-R), the Clinical Dementia Rating (CDR) and Functional Assessment Staging Test (FAST). We used cross-sectional data from a tertiary hospital memory clinic. Subjects were stratified into "primary education and below" (PE) or "secondary education and above" (SE). Receiving operating characteristic (ROC) analyses and Cohen's κ were performed to determine MMSE cut-off scores for dementia stages. Our derived cut-off scores were lower compared to the conventional scores. Scores also differed between subjects of different education levels. MMSE cut-off scores were 19, 15, and 9 for CDR stages 1, 2, and 3 respectively in PE subjects, and 23, 17, and 10 for SE subjects. Cut-off scores were comparable for staging by DSM-III-R Criteria and FAST. There is a need for locally derived stage-specific MMSE cut-off scores for the Asian population adjusted for education.

Objectives: Memory clinics play an important role in enabling early dementia diagnosis and intervention. Few studies have investigated the changing patient profiles at memory clinics over time. We studied the trend of patient characteristics in a geriatric medicine-led memory clinic over 12 years to improve services and care to meet their needs., Setting and Participants: Data from 2340 first-visit patients seen at a memory clinic from 2005-2017 were extracted from a registered database and analysed., Design: ANOVA, Pearson chi-square and non-parametric tests were used to describe and compare between patients with dementia (PWD) and patients with no dementia (PND)., Measurements: Data included diagnoses of dementia and mild cognitive impairment, age, education, MMSE scores and comorbidities., Results: Patients averaged 77.2 ± 8.3 years of age with mean MMSE score of 16.2 ± 6.7. Those diagnosed with dementia were older (78.3 ± 7.9 years) and almost half (48.4%) had moderate or moderately severe dementia (FAST 5-6). Over time, there was a growing proportion of patients with mild cognitive impairment (MCI) and mild Alzheimer's dementia. Many PWD had co-morbidities of hypertension (65.9%), hyperlipidemia (55.1%), diabetes (33.5%) and 28.4% were frail., Conclusions: The findings call for services to better diagnose and manage patients at the earlier stages of cognitive impairment and provide holistic interventions for those with frailty and other co-morbidities. The continued rise in number of patients presenting to memory clinics provides impetus to expedite integration of tertiary-based memory clinics with primary and community care providers to better support PWD and their families., Competing Interests: None.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h) 28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development 4 . First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

Culture independent techniques, such as shotgun metagenomics and 16S rRNA amplicon sequencing have dramatically changed the way we can examine microbial communities. Recently, changes in microbial community structure and dynamics have been associated with a growing list of human diseases. The identification and comparison of bacteria driving those changes requires the development of sound statistical tools, especially if microbial biomarkers are to be used in a clinical setting. We present mixMC, a novel multivariate data analysis framework for metagenomic biomarker discovery. mixMC accounts for the compositional nature of 16S data and enables detection of subtle differences when high inter-subject variability is present due to microbial sampling performed repeatedly on the same subjects, but in multiple habitats. Through data dimension reduction the multivariate methods provide insightful graphical visualisations to characterise each type of environment in a detailed manner. We applied mixMC to 16S microbiome studies focusing on multiple body sites in healthy individuals, compared our results with existing statistical tools and illustrated added value of using multivariate methodologies to fully characterise and compare microbial communities.

Biogenesis of chloroplasts involves a series of protein trafficking events. Nuclear-encoded proteins are imported into the organelle, and then trafficked to various chloroplast locations by systems that are directly homologous to bacterial systems. Although the thylakoid-based systems have been studied extensively, much less is known about the systems that reside and function in the inner envelope membrane. One such system, the Sec2 system, is homologous to both the thylakoid-based Sec1 system and bacterial Sec systems, and may mediate both integration and translocation across the inner envelope. At a minimum, this system is expected to include three components, but only two, SCY2 and SECA2, have been identified in Arabidopsis. Bioinformatics and protein modeling were used to identify the protein encoded by At4g38490 as a candidate for the missing component (SECE2). Cellular localization, biochemistry, protein interaction assays in yeast, and co-immunoprecipitation experiments were used to establish that this protein is an integral membrane protein of the inner envelope, and specifically interacts with the SCY2 component in vivo. Sequence analyses indicated that SECE2 proteins are found in a variety of plants, and differ from the thylakoid SECE1 proteins in a stroma-exposed helical domain, which may contribute to their specificity. Finally, a genetic analysis indicated that SECE2 plays an essential role in plant growth and development., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)