First study demonstrating speckle tracking echocardiography has prognostic value in patients with idiopathic inflammatory myopathies.

To measure left ventricular (LV) global longitudinal strain (GLS) using speckle tracking echocardiography in idiopathic inflammatory myopathy (IIM) patients and to determine whether the LV GLS predicts outcomes in those patients. Prospective study consisted of a cross-sectional phase with 61 IIM patients and 32 individuals without IIM and longitudinal phase, in which patients were divided into two subgroups: 26 with reduced LV GLS and 35 with normal LV GLS; patients were followed for a mean of 25 months, and the occurrence of cardiovascular events and criteria for IIM activity were compared. The mean LV GLS (18.5 ± 2.9% vs. 21.6 ± 2.5%; p < 0.001) and right ventricle free wall strain (21.9 ± 6.1% vs. 27.5 ± 4.7%; p < 0.001) were lower in patients than in controls. The mean N-terminal pro B-type natriuretic peptide level was higher in patients than in controls. There were no differences regarding other cardiac involvement. Anti-Jo1 antibody was associated with general electrocardiographic abnormality and LV diastolic dysfunction. The subgroup with reduced GLS progressed with higher mean creatine phosphokinase, myositis disease activity assessment visual analogue scales, the physician's and patient's visual analogue scales, the health assessment questionnaire, and a higher proportion of relapses than the subgroup with normal GLS. There was no difference between the subgroups regarding cardiovascular events. The LV GLS appears to be useful for evaluating patients with IIM. Abnormal values are associated with more frequent relapses and increased disease activity during follow-up.

Exercise intolerance in post-coronavirus disease 2019 survivors after hospitalisation.

Rationale: Post-coronavirus disease 2019 (COVID-19) survivors frequently have dyspnoea that can lead to exercise intolerance and lower quality of life. Despite recent advances, the pathophysiological mechanisms of exercise intolerance in the post-COVID-19 patients remain incompletely characterised. The objectives of the present study were to clarify the mechanisms of exercise intolerance in post-COVID-19 survivors after hospitalisation. Methods: This prospective study evaluated consecutive patients previously hospitalised due to moderate-to-severe/critical COVID-19. Within mean±sd 90±10 days of onset of acute COVID-19 symptoms, patients underwent a comprehensive cardiopulmonary assessment, including cardiopulmonary exercise testing with earlobe arterialised capillary blood gas analysis. Measurements and main results: 87 patients were evaluated; mean±sd peak oxygen consumption was 19.5±5.0 mL·kg-1·min-1, and the tertiles were ≤17.0, 17.1-22.2 and ≥22.3 mL·kg-1·min-1. Hospitalisation severity was similar among the three groups; however, at the follow-up visit, patients with peak oxygen consumption ≤17.0 mL·kg-1·min-1 reported a greater sensation of dyspnoea, along with indices of impaired pulmonary function, and abnormal ventilatory, gas-exchange and metabolic responses during exercise compared to patients with peak oxygen consumption >17 mL·kg-1·min-1. By multivariate logistic regression analysis (receiver operating characteristic curve analysis) adjusted for age, sex and prior pulmonary embolism, a peak dead space fraction of tidal volume ≥29 and a resting forced vital capacity ≤80% predicted were independent predictors of reduced peak oxygen consumption. Conclusions: Exercise intolerance in the post-COVID-19 survivors was related to a high dead space fraction of tidal volume at peak exercise and a decreased resting forced vital capacity, suggesting that both pulmonary microcirculation injury and ventilatory impairment could influence aerobic capacity in this patient population.

The COVID-19 pandemic in Brazilian pregnant and postpartum women: results from the REBRACO prospective cohort study.

Brazil presented a very high number of maternal deaths and evident delays in healthcare. We aimed at evaluating the characteristics of SARS-CoV-2 infection and associated outcomes in the obstetric population. We conducted a prospective cohort study in 15 Brazilian centers including symptomatic pregnant or postpartum women with suspected COVID-19 from Feb/2020 to Feb/2021. Women were followed from suspected infection until the end of pregnancy. We analyzed maternal characteristics and pregnancy outcomes associated with confirmed COVID-19 infection and SARS, determining unadjusted risk ratios. In total, 729 symptomatic women with suspected COVID-19 were initially included. Among those investigated for COVID-19, 51.3% (n = 289) were confirmed COVID-19 and 48% (n = 270) were negative. Initially (before May 15th), only 52.9% of the suspected cases were tested and it was the period with the highest proportion of ICU admission and maternal deaths. Non-white ethnicity (RR 1.78 [1.04-3.04]), primary schooling or less (RR 2.16 [1.21-3.87]), being overweight (RR 4.34 [1.04-19.01]) or obese (RR 6.55 [1.57-27.37]), having public prenatal care (RR 2.16 [1.01-4.68]), planned pregnancies (RR 2.09 [1.15-3.78]), onset of infection in postpartum period (RR 6.00 [1.37-26.26]), chronic hypertension (RR 2.15 [1.37-4.10]), pre-existing diabetes (RR 3.20 [1.37-7.46]), asthma (RR 2.22 [1.14-4.34]), and anaemia (RR 3.15 [1.14-8.71]) were associated with higher risk for SARS. The availability of tests and maternal outcomes varied throughout the pandemic period of the study; the beginning was the most challenging period, with worse outcomes. Socially vulnerable, postpartum and previously ill women were more likely to present SARS related to COVID-19.

Preeclampsia among women with COVID-19 during pregnancy and its impact on maternal and perinatal outcomes: Results from a national multicenter study on COVID in Brazil, the REBRACO initiative.

OBJECTIVE: To evaluate the prevalence of preeclampsia among cases of COVID-19 infection during pregnancy and the association between both conditions, in a multicenter cohort of Brazilian women with respiratory symptoms. STUDY DESIGN: Ancillary analysis of the Brazilian Network of COVID-19 in Obstetrics (REBRACO) study. We performed a nested case-control analysis selecting all women with COVID-19 and compared outcomes between women with and without PE. MAIN OUTCOMES: Maternal, gestational, and clinical characteristics and perinatal outcomes. MEASURES: Prevalence ratio (PR) and its 95%CI for each of the predictors and outcomes. RESULTS: A total of 203 women were included: 21 (10.3%) in PE group and 182 (89.7%) in non-PE group. Preeclampsia was not different among women with and without COVID-19 (10.3% vs 13.1%, p-value = 0.41), neither complication such as eclampsia and HELLP syndrome. Chronic hypertension (33.4%) (p < 0.01) and obesity (60.0%) (p = 0.03) were the most frequent comorbidities in PE group, and they were significantly more frequent in this group. Women with PE had more cesarean section (RR 5.54 [1.33 - 23.14]) and their neonates were more frequently admitted to neonatal intensive care unit (PR 2.46[1.06 - 5.69]), most likely due to preterm-birth-related complications. CONCLUSION: The prevalence of PE among women with COVID-19 infection during pregnancy was around 10%; women with COVID-19 and a history of chronic hypertension or obesity are more likely to have preeclampsia. Cesarean section is increased among women with PE and COVID-19, with increased rates of neonatal admission to intensive care units, mostly due to prematurity.

Invariant Natural Killer T-cells and their subtypes may play a role in the pathogenesis of endometriosis.

OBJECTIVE: To evaluate the frequencies of iNKT cells and their subsets in patients with deep endometriosis. METHODS: A case-control study was conducted between 2013 and 2015, with 73 patients distributed into two groups: 47 women with a histological diagnosis of endometriosis and 26 controls. Peripheral blood, endometriosis lesions, and healthy peritoneal samples were collected on the day of surgery to determine the frequencies of iNKT cells and subtypes via flow cytometry analysis. RESULTS: The authors observed a lower number of iNKT (p = 0.01) and Double-Negative (DN) iNKT cells (p = 0.02) in the blood of patients with endometriosis than in the control group. The number of DN iNKT IL-17+ cells in the secretory phase was lower in the endometriosis group (p = 0.049). There was an increase in the secretion of IL-17 by CD4+ iNKT cells in the blood of patients with endometriosis and severe dysmenorrhea (p = 0.038), and severe acyclic pelvic pain (p = 0.048). Patients with severe dysmenorrhea also had a decreased number of CD4+ CCR7+ cells (p = 0.022). CONCLUSION: The decreased number of total iNKT and DN iNKT cells in patients with endometriosis suggests that iNKT cells play a role in the pathogenesis of endometriosis and can be used to develop new diagnostic and therapeutic agents.

Invariant Natural Killer T-cells and their subtypes may play a role in the pathogenesis of endometriosis

Abstract Objective To evaluate the frequencies of iNKT cells and their subsets in patients with deep endometriosis. Methods A case-control study was conducted between 2013 and 2015, with 73 patients distributed into two groups: 47 women with a histological diagnosis of endometriosis and 26 controls. Peripheral blood, endometriosis lesions, and healthy peritoneal samples were collected on the day of surgery to determine the frequencies of iNKT cells and subtypes via flow cytometry analysis. Results The authors observed a lower number of iNKT (p= 0.01) and Double-Negative (DN) iNKT cells (p= 0.02) in the blood of patients with endometriosis than in the control group. The number of DN iNKT IL-17+ cells in the secretory phase was lower in the endometriosis group (p= 0.049). There was an increase in the secretion of IL-17 by CD4+ iNKT cells in the blood of patients with endometriosis and severe dysmenorrhea (p= 0.038), and severe acyclic pelvic pain (p= 0.048). Patients with severe dysmenorrhea also had a decreased number of CD4+ CCR7+ cells (p= 0.022). Conclusion The decreased number of total iNKT and DN iNKT cells in patients with endometriosis suggests that iNKT cells play a role in the pathogenesis of endometriosis and can be used to develop new diagnostic and therapeutic agents.

Study Protocol for "Psilocybin as a Treatment for Anorexia Nervosa: A Pilot Study".

Background: Anorexia nervosa (AN) is a serious and life-threatening psychiatric condition. With a paucity of approved treatments, there is a desperate need for novel treatment avenues to be explored. Here, we present (1) an overview of the ways through which Public Patient Involvement (PPI) has informed a trial of psilocybin-assisted therapy for AN and (2) a protocol for a pilot study of psilocybin-assisted therapy in AN currently underway at Imperial College London. The study aims to assess the feasibility, brain mechanisms and preliminary outcomes of treating anorexia nervosa with psilocybin. Methods: (1) PPI: Across two online focus groups, eleven individuals with lived experience of AN were presented with an overview of the protocol. Their feedback not only identified solutions to possible barriers for future participants, but also helped the research team to better understand the concept of "recovery" from the perspective of those with lived experience. (2) Protocol: Twenty female participants [21-65 years old, body mass index (BMI) 15 kg/m2 or above] will receive three oral doses of psilocybin (up to 25 mg) over a 6-week period delivered in a therapeutic environment and enveloped by psychological preparation and integration. We will work with participant support networks (care teams and an identified support person) throughout and there will be an extended remote follow-up period of 12 months. Our two-fold primary outcomes are (1) psychopathology (Eating Disorder Examination) across the 6-month follow-up and (2) readiness and motivation to engage in recovery (Readiness and Motivation Questionnaire) across the 6-week trial period. Neurophysiological outcome measures will be: (1) functional magnetic resonance imaging (fMRI) brain changes from baseline to 6-week endpoint and (2) post-acute changes in electroencephalography (EEG) activity, including an electrophysiological marker of neuronal plasticity. Discussion: The results of this pilot study will not only shed light on the acceptability, brain mechanisms, and impression of the potential efficacy of psilocybin as an adjunct treatment for AN but will be essential in shaping a subsequent Randomised Control Trial (RCT) that would test this treatment against a suitable control condition. Clinical Trial Registration: identifier: NCT04505189.

Facing the COVID-19 pandemic inside maternities in Brazil: A mixed-method study within the REBRACO initiative.

INTRODUCTION: COVID-19 pandemic posed major challenges in obstetric health care services. Preparedness, development, and implementation of new protocols were part of the needed response. This study aims to describe the strategies implemented and the perspectives of health managers on the challenges to face the pandemic in 16 different maternity hospitals that comprise a multicenter study in Brazil, called REBRACO (Brazilian network of COVID-19 during pregnancy). METHODS: Mixed-method study, with quantitative and qualitative approaches. Quantitative data on the infrastructure of the units, maternal and perinatal health indicators, modifications on staff and human resources, from January to July/2020. Also, information on total number of cases, and availability for COVID-19 testing. A qualitative study by purposeful and saturation sampling was undertaken with healthcare managers, to understand perspectives on local challenges in facing the pandemic. RESULTS: Most maternities early implemented their contingency plan. REBRACO centers reported 338 confirmed COVID-19 cases among pregnant and post-partum women up to July 2020. There were 29 maternal deaths and 15 (51.8%) attributed to COVID-19. All maternities performed relocation of beds designated to labor ward, most (75%) acquired mechanical ventilators, only the minority (25%) installed new negative air pressure rooms. Considering human resources, around 40% hired extra health professionals and increased weekly workload and the majority (68.7%) also suspended annual leaves. Only one center implemented universal screening for childbirth and 6 (37.5%) implemented COVID-19 testing for all suspected cases, while around 60% of the centers only tested moderate/severe cases with hospital admission. Qualitative results showed that main challenges experienced were related to the fear of the virus, concerns about reliability of evidence and lack of resources, with a clear need for mental health support among health professionals. CONCLUSION: Study findings suggest that maternities of the REBRACO initiative underwent major changes in facing the pandemic, with limitations on testing, difficulties in infrastructure and human resources. Leadership, continuous training, implementation of evidence-based protocols and collaborative initiatives are key to transpose the fear of the virus and ascertain adequate healthcare inside maternities, especially in low and middle-income settings. Policy makers need to address the specificities in considering reproductive health and childbirth during the COVID-19 pandemic and prioritize research and timely testing availability.

The tug of war between Al3+ and Na+ for order-disorder transitions in lipid-A membranes.

Cations play a critical role in the stability and morphology of lipid-A aggregates by neutralizing, hydrating and cross-linking these glycolipid molecules. Monophosphorylated lipid-A is the major immunostimulatory principle in commercially available adjuvants containing Al3+ such as adjuvant system 04 (AS04). The antagonist/agonist immunomodulatory properties of lipid-A are associated with chemical variations (e.g. the number of acyl chains and phosphate groups) and their aggregate arrangements (e.g. lamellar, nonlamellar or mixed). Therefore, the identification of the active form of lipid-A can provide valuable guidance in the development of vaccine adjuvants capable of boosting the immune system with decreased reactogenicity. Although the effect of mono and divalent cations on the structural polymorphism and endotoxicity of LPS has been previously investigated, much less is known about the effect of trivalent cations. We have investigated the effect of NaCl and AlCl3 salt solutions on the structural dynamics and stability of mono and diphosphorylated lipid-A membranes via atomistic MD simulations. The Al3+ ion exerts two major effects on the structural dynamics of lipid-A membranes. It acts as an efficient cross-linker of mono or diphosphorylated lipid-A molecules, thus stabilizing the lamellar arrangement of these glycolipids. It also alters the lipid-A packing and membrane fluidity, inducing disorder → order structural transitions of the membrane. This effect is promptly reversed upon the addition of NaCl solution, which promotes a nearly threefold increase in the amount of water in the carbohydrate moiety of the Al3+-containing lipid-A membranes. The exchange dynamics and residence times of cation-coordinated water molecules in these membranes provide insights into the molecular mechanism for the Na+-induced transition from a densely packed ordered phase to a disordered one. Al3+ counter-ions favor ordered lamellar aggregates, which has been previously associated with the lack of endotoxic activity and cytokine-inducing action. The resulting microscopic understanding of the structure and dynamics of lipid-A aggregates in the presence of Al3+ and Na+ salts can provide valuable guidance in the development of vaccine adjuvants capable of boosting the immune system with decreased reactogenicity.

Identification of clinical and radiographic predictors of central nervous system injury in genetic skeletal disorders.

Some studies report neurological lesions in patients with genetic skeletal disorders (GSDs). However, none of them describe the frequency of neurological lesions in a large sample of patients or investigate the associations between clinical and/or radiological central nervous system (CNS) injury and clinical, anthropometric and imaging parameters. The project was approved by the institution's ethics committee (CAAE 49433215.5.0000.0022). In this cross-sectional observational analysis study, 272 patients aged four or more years with clinically and radiologically confirmed GSDs were prospectively included. Genetic testing confirmed the diagnosis in the FGFR3 chondrodysplasias group. All patients underwent blinded and independent clinical, anthropometric and neuroaxis imaging evaluations. Information on the presence of headache, neuropsychomotor development (NPMD), low back pain, joint deformity, ligament laxity and lower limb discrepancy was collected. Imaging abnormalities of the axial skeleton and CNS were investigated by whole spine digital radiography, craniocervical junction CT and brain and spine MRI. The diagnostic criteria for CNS injury were abnormal clinical and/or radiographic examination of the CNS. Brain injury included malacia, encephalopathies and malformation. Spinal cord injury included malacia, hydrosyringomyelia and spinal cord injury without radiographic abnormalities. CNS injury was diagnosed in more than 25% of GSD patients. Spinal cord injury was found in 21.7% of patients, and brain injury was found in 5.9%. The presence of low back pain, os odontoideum and abnormal NPMD remained independently associated with CNS injury in the multivariable analysis. Early identification of these abnormalities may have some role in preventing compressive CNS injury, which is a priority in GSD patients.

The Division Defect of a Bacillus subtilis minD noc Double Mutant Can Be Suppressed by Spx-Dependent and Spx-Independent Mechanisms.

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, a ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a minD noc double mutant indicated that FtsZ formed proto-Z-rings at periodic interchromosome locations but that the rings failed to mature and become functional. Extragenic suppressor analysis indicated that a variety of mutations restored high temperature growth to the minD noc double mutant, and while many were likely pleiotropic, others implicated the proteolysis of the transcription factor Spx. Further analysis indicated that a Spx-dependent pathway activated the expression of ZapA, a protein that primarily compensates for the absence of Noc. In addition, an Spx-independent pathway reduced the length of the cytokinetic period, perhaps by increasing divisome activity. Finally, we provide evidence of an as-yet-unidentified protein that is activated by Spx and governs the frequency of polar division and minicell formation. IMPORTANCE Bacteria must properly position the location of the cell division machinery in order to grow, divide, and ensure each daughter cell receives one copy of the chromosome. In Bacillus subtilis, cell division site selection depends on the Min and Noc systems, and while neither is individually essential, cells fail to grow at high temperature when both are mutated. Here, we show that cell division fails in the absence of Min and Noc, due not to a defect in FtsZ localization but rather to a failure in the maturation of the cell division machinery. Suppressor mutations that restored growth were selected, and while some activated the expression of ZapA via the Spx stress response pathway, others appeared to directly enhance divisome activity.

Noc Corrals Migration of FtsZ Protofilaments during Cytokinesis in Bacillus subtilis.

Bacteria that divide by binary fission form FtsZ rings at the geometric midpoint of the cell between the bulk of the replicated nucleoids. In Bacillus subtilis, the DNA- and membrane-binding Noc protein is thought to participate in nucleoid occlusion by preventing FtsZ rings from forming over the chromosome. To explore the role of Noc, we used time-lapse fluorescence microscopy to monitor FtsZ and the nucleoid of cells growing in microfluidic channels. Our data show that Noc does not prevent de novo FtsZ ring formation over the chromosome nor does Noc control cell division site selection. Instead, Noc corrals FtsZ at the cytokinetic ring and reduces migration of protofilaments over the chromosome to the future site of cell division. Moreover, we show that FtsZ protofilaments travel due to a local reduction in ZapA association, and the diffuse FtsZ rings observed in the Noc mutant can be suppressed by ZapA overexpression. Thus, Noc sterically hinders FtsZ migration away from the Z-ring during cytokinesis and retains FtsZ at the postdivisional polar site for full disassembly by the Min system.IMPORTANCE In bacteria, a condensed structure of FtsZ (Z-ring) recruits cell division machinery at the midcell, and Z-ring formation is discouraged over the chromosome by a poorly understood phenomenon called nucleoid occlusion. In B. subtilis, nucleoid occlusion has been reported to be mediated, at least in part, by the DNA-membrane bridging protein, Noc. Using time-lapse fluorescence microscopy of cells growing in microchannels, we show that Noc neither protects the chromosome from proximal Z-ring formation nor determines the future site of cell division. Rather, Noc plays a corralling role by preventing protofilaments from leaving a Z-ring undergoing cytokinesis and traveling over the nucleoid.

Many birds with one stone: targeting the (p)ppGpp signaling pathway of bacteria to improve antimicrobial therapy.

Winning the war against resistant bacteria will require a change of paradigm in antibiotic discovery. A promising new direction is the targeting of non-essential pathways required for successful infection, such as quorum-sensing, virulence, and biofilm formation. Similarly important will be strategies to prevent or revert antibiotic resistance. Here, we argue that the (p)ppGpp signaling pathway should be a prime target of this effort, since its inactivation could potentially achieve all these goals simultaneously. The hyperphosphorylated guanine nucleotide (p)ppGpp is an ancient and universal second messenger of bacteria that has pleotropic effects on the physiology of these organisms. Initially described as a stress signal-an alarmone-it is now clear that (p)ppGpp plays a more general and fundamental role in bacterial adaptation, by integrating multiple internal and environmental signals to establish the optimal balance between growth and maintenance functions at any given time. Given such centrality, perturbation of the (p)ppGpp pathway will affect bacteria in multiple ways, from the ability to adjust metabolism to the available nutrients to the capacity to differentiate into developmental forms adapted to colonize different niches. Here, we provide an overview of the (p)ppGpp pathway, how it affects bacterial growth, survival and virulence, and its connection with antibiotic tolerance and persistence. We will emphasize the dysfunctions of cells living without (p)ppGpp and finalize by reviewing the efforts and prospects of developing inhibitors of this pathway, and how these could be employed to improve current antibiotic therapy.

Out of Sight, Out of Mind: The Effect of the Equilibration Protocol on the Structural Ensembles of Charged Glycolipid Bilayers.

Molecular dynamics (MD) simulations represent an essential tool in the toolbox of modern chemistry, enabling the prediction of experimental observables for a variety of chemical systems and processes and majorly impacting the study of biological membranes. However, the chemical diversity of complex lipids beyond phospholipids brings new challenges to well-established protocols used in MD simulations of soft matter and requires continuous assessment to ensure simulation reproducibility and minimize unphysical behavior. Lipopolysaccharides (LPS) are highly charged glycolipids whose aggregation in a lamellar arrangement requires the binding of numerous cations to oppositely charged groups deep inside the membrane. The delicate balance between the fully hydrated carbohydrate region and the smaller hydrophobic core makes LPS membranes very sensitive to the choice of equilibration protocol. In this work, we show that the protocol successfully used to equilibrate phospholipid bilayers when applied to complex lipopolysaccharide membranes occasionally leads to a small expansion of the simulation box very early in the equilibration phase. Although the use of a barostat algorithm controls the system dimension and particle distances according to the target pressure, fluctuation in the fleeting pressure occasionally enables a few water molecules to trickle into the hydrophobic region of the membrane, with spurious solvent buildup. We show that this effect stems from the initial steps of NPT equilibration, where initial pressure can be fairly high. This can be solved with the use of a stepwise-thermalization NVT/NPT protocol, as demonstrated for atomistic MD simulations of LPS/DPPE and lipid-A membranes in the presence of different salts using an extension of the GROMOS forcefield within the GROMACS software. This equilibration protocol should be standard procedure for the generation of consistent structural ensembles of charged glycolipids starting from atomic coordinates not previously pre-equilibrated. Although different ways to deal with this issue can be envisioned, we investigated one alternative that could be readily available in major MD engines with general users in mind.

Inoculation with the endophytic bacterium Herbaspirillum seropedicae promotes growth, nutrient uptake and photosynthetic efficiency in rice.

MAIN CONCLUSION: Higher vacuolar proton pump activity may increase plant energy and nutrient use efficiency and provide the nexus between plant inoculation with Herbaspirillum seropedicae and growth promotion. Global change and growing human population are exhausting arable land and resources, including water and fertilizers. We present inoculation with the endophytic plant-growth promoting bacterium (PGPB) Herbaspirillum seropedicae as a strategy for promoting growth, nutrient uptake and photosynthetic efficiency in rice (Oryza sativa L.). Because plant nutrient acquisition is coordinated with photosynthesis and the plant carbon status, we hypothesize that inoculation with H. seropedicae will stimulate proton (H+) pumps, increasing plant growth nutrient uptake and photosynthetic efficiency at low nutrient levels. Plants were inoculated and grown in pots with sterile soil for 90 days. Herbaspirillum seropedicae endophytic colonization was successful and, as hypothesized, inoculation (1) stimulated root vacuolar H+ pumps (vacuolar H+-ATPase and vacuolar H+-PPase), and (2) increased plant growth, nutrient contents and photosynthetic efficiency. The results showed that inoculation with the endophytic bacterium H. seropedicae can promote plant growth, nutrient uptake and photosynthetic efficiency, which will likely result in a more efficient use of resources (nutrients and water) and higher production of nutrient-rich food at reduced economic and environmental costs.

The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis.

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

SuAVE: A Tool for Analyzing Curvature-Dependent Properties in Chemical Interfaces.

Curvature is an intrinsic feature of biological membranes underlying vital cellular processes such as endocytosis, membrane fusion-fission, trafficking, and remodeling. The continuous expansion of the spatiotemporal scales accessible to computational simulations nowadays makes possible quasi-atomistic molecular dynamics simulations of these processes. In despite of that, computation of the shapes and curvatures associated with the dynamics of biological membranes remains challenging. For this reason, the effect of curvature is often neglected in the analysis of quantities essential for the accurate description of membrane properties (e.g., area and volume per lipid, density profiles, membrane thickness). We propose an algorithm for surface assessment via grid evaluation (SuAVE) that relies on the application of a radial base function to interpolate points scattered across an interface of any shape. This enables the representation of the chemical interface as fully differentiable so that related geometrical properties can be calculated through the straightforward employment of well-established differential geometry techniques. Hence, the effect of different types or degrees of curvature can be accurately taken into account in the calculations of structural properties of any interfaces regardless of chemical composition, asymmetry, and level of atom coarseness. The main functionalities implemented in SuAVE are featured for a number of tetraacylated and hexaacylated Lipid-A membranes of distinct curvatures and a surfactant micelle. We show that the properties calculated for moderately to highly curved membranes differ significantly between curvature-dependent and -independent algorithms. The SuAVE software is freely available from www.biomatsite.net/suave-software .

Violacein Targets the Cytoplasmic Membrane of Bacteria.

Violacein is a tryptophan-derived purple pigment produced by environmental bacteria, which displays multiple biological activities, including strong inhibition of Gram-positive pathogens. Here, we applied a combination of experimental approaches to identify the mechanism by which violacein kills Gram-positive bacteria. Fluorescence microscopy showed that violacein quickly and dramatically permeabilizes B. subtilis and S. aureus cells. Cell permeabilization was accompanied by the appearance of visible discontinuities or rips in the cytoplasmic membrane, but it did not affect the cell wall. Using in vitro experiments, we showed that violacein binds directly to liposomes made with commercial and bacterial phospholipids and perturbs their structure and permeability. Furthermore, molecular dynamics simulations were employed to reveal how violacein inserts itself into lipid bilayers. Thus, our combined results demonstrate that the cytoplasmic membrane is the primary target of violacein in bacteria. The implications of this finding for the development of violacein as a therapeutic agent are discussed.