Mycobacterium tuberculosis cords within lymphatic endothelial cells to evade host immunity

The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis remains obscure. Here, we show that several M. tuberculosis clinical isolates form intracellular cords in primary human lymphatic endothelial cells (hLECs) in vitro and in the lymph nodes of patients with tuberculosis. We identified via RNA-Seq a transcriptional program that activated, in infected-hLECs, cell survival and cytosolic surveillance of pathogens pathways. Consistent with this, cytosolic access was required for intracellular M. tuberculosis cording. Mycobacteria lacking ESX-1 type VII secretion system or phthiocerol dimycocerosates expression, which failed to access the cytosol, were indeed unable to form cords within hLECs. Finally, we show that M. tuberculosis cording is a size-dependent mechanism used by the pathogen to avoid its recognition by cytosolic sensors and evade either resting or IFN-γ–induced hLEC immunity. These results explain the long-standing association between M. tuberculosis cording and virulence and how virulent mycobacteria use intracellular cording as strategy to successfully adapt and persist in the lymphatic tracts

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocytederived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research

Motives and comprehension in a public goods game with induced emotions

This study analyses the sensitivity of public goods contributions through the lens of psychological motives. We report the results of a public goods experiment in which subjects were induced with the motives of care and anger through autobiographical recall. Subjects' preferences, beliefs, and perceptions under each motive are compared with those of subjects experiencing a neutral autobiographical recall control condition. We find, but only for those subjects with the highest comprehension of the game, that care elicits significantly higher contributions than anger, with the control treatment in between. This positive influence of the care motive on unconditional giving is accounted for partly by preferences for giving and partly by the beliefs concerning greater contributions by others. Anger also affects attention to own and other's payoffs (using mouse tracking) and perceptions of the game's incentive structure (cooperative or competitive)

Self-Reported Functional Status as Predictor of Observed Functional Capacity in Subjects with Early Osteoarthritis of the Hip and Knee: A Diagnostic Study in the CHECK Cohort

Objectives Patients with hip or knee osteoarthritis (OA) may experience functional limitations in work settings. In the Cohort Hip and Cohort Knee study (CHECK) physical function was both self-reported and measured performance-based, using Functional Capacity Evaluation (FCE). Relations between self-reported scores on SF-36 and WOMAC (Western Ontario and McMaster Arthritis Index, function scales) and FCE performance were studied, and their diagnostic value for clinicians in predicting observed physical work limitations was assessed. Methods Ninety-two subjects scored physical function on SF-36 (scale 0–100, 100 indicating the best health level) and WOMAC (scale 0–68, 68 indicates maximum restriction) and performed the FCE. Correlations were calculated between all scores. Cross-tables were constructed using both questionnaires as diagnostic tests to identify work limitations. Subjects lifting <22.5 kg on the FCE-test ‘lifting-low’ were labeled as having physical work limitations. Diagnostic aspects at different cut-off scores for both questionnaires were analysed. Results Statistically significant correlations (Spearman’s ρ 0.34–0.49) were found between questionnaire scores and lifting and carrying tests. Results of a diagnostic cross-table with cut-off point <60 on SF-36 ‘physical functioning’ were: sensitivity 0.34, specificity 0.97 and positive predictive value (PV+) 0.95. Cut-off point ≥21 on WOMAC ‘function’ resulted in sensitivity 0.51, specificity 0.88 and PV+ 0.88. Conclusion Low self-reported function scores on SF-36 and WOMAC diagnosed subjects with limitations on the FCE. However, high scores did not guarantee performance without physical work limitations. These results are specific to the tested persons with early OA, in populations with a different prevalence of limitations, different diagnostic values will be found. FCE may be indicated to help clinicians to assess actual work capacity

Ankyrin-mediated self-protection during cell invasion by the bacterial predator Bdellovibrio bacteriovorus

Predatory Bdellovibrio bacteriovorus are natural antimicrobial organisms, killing other bacteria by whole-cell invasion. Self-protection against prey-metabolizing enzymes is important for the evolution of predation. Initial prey entry involves the predator’s peptidoglycan DD-endopeptidases, which decrosslink cell walls and prevent wasteful entry by a second predator. Here we identify and characterize a self-protection protein from B. bacteriovorus, Bd3460, which displays an ankyrin-based fold common to intracellular pathogens of eukaryotes. Co-crystal structures reveal Bd3460 complexation of dual targets, binding a conserved epitope of each of the Bd3459 and Bd0816 endopeptidases. Complexation inhibits endopeptidase activity and cell wall decrosslinking in vitro. Self-protection is vital — DBd3460 Bdellovibrio deleteriously decrosslink self-peptidoglycan upon invasion, adopt a round morpholog, and lose predatory capacity and cellular integrity. Our analysis provides the first mechanistic examination of self-protection in Bdellovibrio, documents protection-multiplicity for products of two different genomic loci, and reveals an important evolutionary adaptation to an invasive predatory bacterial lifestyle

Measuring and modelling the response of Klebsiella pneumoniae KPC prey to Bdellovibrio bacteriovorus predation, in human serum and defined buffer

In worldwide conditions of increasingly antibiotic-resistant hospital infections, it is important to research alternative therapies. Bdellovibrio bacteriovorus bacteria naturally prey on Gram-negative pathogens, including antibiotic-resistant strains and so B. bacteriovorus have been proposed as "living antibiotics" to combat antimicrobially-resistant pathogens. Predator-prey interactions are complex and can be altered by environmental components. To be effective B. bacteriovorus predation needs to work in human body fluids such as serum where predation dynamics may differ to that studied in laboratory media. Here we combine mathematical modelling and lab experimentation to investigate the predation of an important carbapenem-resistant human pathogen, Klebsiella pneumoniae, by B. bacteriovorus in human serum versus buffer. We show experimentally that B. bacteriovorus is able to reduce prey numbers in each environment, on different timescales. Our mathematical model captures the underlying dynamics of the experimentation, including an initial predation-delay at the predator-prey-serum interface. Our research shows differences between predation in buffer and serum and highlights both the potential and limitations of B. bacteriovorus acting therapeutically against K. pneumoniae in serum, informing future research into the medicinal behaviours and dosing of this living antibacterial

A Bayesian Partition Method for Detecting Pleiotropic and Epistatic eQTL Modules

Studies of the relationship between DNA variation and gene expression variation, often referred to as “expression quantitative trait loci (eQTL) mapping”, have been conducted in many species and resulted in many significant findings. Because of the large number of genes and genetic markers in such analyses, it is extremely challenging to discover how a small number of eQTLs interact with each other to affect mRNA expression levels for a set of co-regulated genes. We present a Bayesian method to facilitate the task, in which co-expressed genes mapped to a common set of markers are treated as a module characterized by latent indicator variables. A Markov chain Monte Carlo algorithm is designed to search simultaneously for the module genes and their linked markers. We show by simulations that this method is more powerful for detecting true eQTLs and their target genes than traditional QTL mapping methods. We applied the procedure to a data set consisting of gene expression and genotypes for 112 segregants of S. cerevisiae. Our method identified modules containing genes mapped to previously reported eQTL hot spots, and dissected these large eQTL hot spots into several modules corresponding to possibly different biological functions or primary and secondary responses to regulatory perturbations. In addition, we identified nine modules associated with pairs of eQTLs, of which two have been previously reported. We demonstrated that one of the novel modules containing many daughter-cell expressed genes is regulated by AMN1 and BPH1. In conclusion, the Bayesian partition method which simultaneously considers all traits and all markers is more powerful for detecting both pleiotropic and epistatic effects based on both simulated and empirical data

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorous predation

Modification of essential bacterial peptidoglycan (PG) containing cell walls can lead to antibiotic resistance, for example β-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG labelling approach utilizing timed pulses of multiple fluorescent D-amino acids (FDAAs), we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall; L,D-transpeptidaseBd mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion and a zonal mode of predator-elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division

Interrupting peptidoglycan deacetylation during Bdellovibrio predator-prey interaction prevents ultimate destruction of prey wall, liberating bacterial-ghosts

The peptidoglycan wall, located in the periplasm between the inner and outer membranes of the cell envelope in Gram-negative bacteria, maintains cell shape and endows osmotic robustness. Predatory Bdellovibrio bacteria invade the periplasm of other bacterial prey cells, usually crossing the peptidoglycan layer, forming transient structures called bdelloplasts within which the predators replicate. Prey peptidoglycan remains intact for several hours, but is modified and then degraded by predators escaping. Here we show predation is altered by deleting two Bdellovibrio N-acetylglucosamine (GlcNAc) deacetylases, one of which we show to have a unique two domain structure with a novel regulatory-”plug”. Deleting the deacetylases limits peptidoglycan degradation and rounded prey cell “ghosts” persist after mutant-predator exit. Mutant predators can replicate unusually in the periplasmic region between the peptidoglycan wall and the outer membrane rather than between wall and inner-membrane, yet still obtain nutrients from the prey cytoplasm. Deleting two further genes encoding DacB/PBP4 family proteins, known to decrosslink and round prey peptidoglycan, results in a quadruple mutant Bdellovibrio which leaves prey-shaped ghosts upon predation. The resultant bacterial ghosts contain cytoplasmic membrane within bacteria-shaped peptidoglycan surrounded by outer membrane material which could have promise as “bacterial skeletons” for housing artificial chromosomes

Long-Stay Psychiatric Patients: A Prospective Study Revealing Persistent Antipsychotic-Induced Movement Disorder

OBJECTIVE: The purpose of this study was to assess the frequency of persistent drug-induced movement disorders namely, tardive dyskinesia (TD), parkinsonism, akathisia and tardive dystonia in a representative sample of long-stay patients with chronic severe mental illness. METHOD: Naturalistic study of 209, mainly white, antipsychotic-treated patients, mostly diagnosed with psychotic disorder. Of this group, the same rater examined 194 patients at least two times over a 4-year period, with a mean follow-up time of 1.1 years, with validated scales for TD, parkinsonism, akathisia, and tardive dystonia. RESULTS: The frequencies of persistent movement disorders in the sample were 28.4% for TD, 56.2% for parkinsonism, 4.6% for akathisia and 5.7% for tardive dystonia. Two-thirds of the participants displayed at least one type of persistent movement disorder. CONCLUSIONS: Persistent movement disorder continues to be the norm for long-stay patients with chronic mental illness and long-term antipsychotic treatment. Measures are required to remedy this situation