Parasite-induced TH1 cells and intestinal dysbiosis cooperate in IFN-γ-dependent elimination of Paneth cells.

Activation of Toll-like receptors (TLRs) by pathogens triggers cytokine production and T cell activation, immune defense mechanisms that are linked to immunopathology. Here we show that IFN-γ production by CD4(+) T(H)1 cells during mucosal responses to the protozoan parasite Toxoplasma gondii resulted in dysbiosis and the elimination of Paneth cells. Paneth cell death led to loss of antimicrobial peptides and occurred in conjunction with uncontrolled expansion of the Enterobacteriaceae family of Gram-negative bacteria. The expanded intestinal bacteria were required for the parasite-induced intestinal pathology. The investigation of cell type-specific factors regulating T(H)1 polarization during T. gondii infection identified the T cell-intrinsic TLR pathway as a major regulator of IFN-γ production in CD4(+) T cells responsible for Paneth cell death, dysbiosis and intestinal immunopathology.

Availability of Environmental Iron Influences the Performance of Biological Adhesives Produced by Blue Mussels.

Animals incorporate metals within the materials they manufacture, such as protective armor and teeth. Iron is an element used for adding strength and self-healing properties to load-bearing materials. Incorporation of iron is found beyond hard, brittle materials, even within the soft adhesive produced by marine mussels. Such findings suggest that the bioavailability of iron may have an influence on the properties of a biological material. Experiments were conducted using live mussels in which seawater iron levels were deficient, normal, or in excess of typical concentrations. The weakest adhesive strengths were produced in iron-deficient waters. Increasing seawater iron brought about more robust bonding. Changes in strengths correlated with varied adhesive morphology, color, and microstructural features, likely a result of variations in the degree of iron-induced protein cross-linking. This study provides the first whole animal scale data on how the manipulation of bioavailable iron influences the performance of a biological material. Changing ocean chemistries will alter the iron bioavailability when a decrease in pH shifts elemental speciation from particulate to dissolved, hindering the ability of filtering organisms to capture nutrients. These results show future implications of changing ocean chemistry as well as of the resulting abilities of marine organisms to construct essential materials.

Guidance for the Evaluation of Tuberculosis Diagnostics That Meet the World Health Organization (WHO) Target Product Profiles: An Introduction to WHO Process and Study Design Principles.

Existing high-priority target product profiles (TPPs) of the World Health Organization (WHO) establish important needs for tuberculosis (TB) diagnostic development. Building on this earlier work, this guidance series aims to provide study guidance for performing accuracy studies of novel diagnostic products that may meet the 4 high-priority WHO TPPs and thus enable adequate evidence generation to inform a WHO evidence review process. Diagnostic accuracy studies represent a fundamental step in the validation of all tests. Unfortunately, such studies often have limitations in design, execution, and reporting, leading to low certainty of the evidence about true test performance, which can delay or impede policy and scale-up decisions. This introductory paper outlines the following: (1) the purpose of this series of papers on study guidance; (2) WHO evidence needs and process for the development of policy guidelines for new TB diagnostic tests; and (3) study design considerations, ie, general diagnostic study considerations, intended use of test and role in the clinical pathway, choice of population and setting, index-test specific issues, suitable reference standard and comparators, study flow and specimen issues, and finally key issues beyond accuracy that should be considered. The other 4 papers in this series will provide more detailed guidance for each of the 4 WHO high-priority TPPs. By increasing the clarity around the clinical evaluation needs for tests that have the potential to meet the TPP specifications, we hope to support harmonized evidence generation and enable the WHO review process towards meeting the WHO End TB Strategy targets for reducing the incidence and mortality associated with TB.

Guidance for Studies Evaluating the Accuracy of Sputum-Based Tests to Diagnose Tuberculosis.

Tests that can replace sputum smear microscopy have been identified as a top priority diagnostic need for tuberculosis by the World Health Organization. High-quality evidence on diagnostic accuracy for tests that may meet this need is an essential requirement to inform decisions about policy and scale-up. However, test accuracy studies are often of low and inconsistent quality and poorly reported, leading to uncertainty about true test performance. Here we provide guidance for the design of diagnostic test accuracy studies of sputum smear-replacement tests. Such studies should have a cross-sectional or cohort design, enrolling either a consecutive series or a random sample of patients who require evaluation for tuberculosis. Adults with respiratory symptoms are the target population. The reference standard should at a minimum be a single, automated, liquid culture, but additional cultures, follow-up, clinical case definition, and specific measures to understand discordant results should also be included. Inclusion of smear microscopy and Xpert MTB/RIF (or MTB/RIF Ultra) as comparators is critical to allow broader comparability and generalizability of results, because disease spectrum can vary between studies and affects relative test performance. Given the complex nature of sputum (the primary specimen type used for pulmonary TB), careful design and reporting of the specimen flow is essential. Test characteristics other than accuracy (such as feasibility, implementation considerations, and data on impact on patient, population and health systems outcomes) are also important aspects.

Guidance for Studies Evaluating the Accuracy of Biomarker-Based Nonsputum Tests to Diagnose Tuberculosis.

The World Health Organization's (WHO) "End TB" strategy calls for development and implementation of novel tuberculosis (TB) diagnostics. Sputum-based diagnostics are challenging to implement and often less sensitive in high-priority populations. Nonsputum, biomarker-based tests may facilitate TB testing at lower levels of the healthcare system, accelerate treatment initiation, and improve outcomes. We provide guidance on the design of diagnostic accuracy studies evaluating nonsputum, biomarker-based tests within the context of WHO's target product profile for such tests. Study designs should account for the intended use when choosing the study population, setting, and reference standards. Although adults with respiratory symptoms may be an initial target population, other high-priority populations regardless of symptoms-including people living with human immunodeficiency virus, those unable to produce sputum samples or with extrapulmonary TB, household contacts, and children-should be considered. Studies beyond diagnostic accuracy that evaluate feasibility and population-level impacts are also needed. A biomarker-based diagnostic may be critical to ending the TB epidemic, but requires appropriate validation before implementation.

Guidance for Studies Evaluating the Accuracy of Rapid Tuberculosis Drug-Susceptibility Tests.

The development and implementation of rapid molecular diagnostics for tuberculosis (TB) drug-susceptibility testing is critical to inform treatment of patients and to prevent the emergence and spread of resistance. Optimal trial planning for existing tests and those in development will be critical to rapidly gather the evidence necessary to inform World Health Organization review and to support potential policy recommendations. The evidence necessary includes an assessment of the performance for TB and resistance detection as well as an assessment of the operational characteristics of these platforms. The performance assessment should include analytical studies to confirm the limit of detection and assay ability to detect mutations conferring resistance across globally representative strains. The analytical evaluation is typically followed by multisite clinical evaluation studies to confirm diagnostic performance in sites and populations of intended use. This paper summarizes the considerations for the design of these analytical and clinical studies.

Guidance for Studies Evaluating the Accuracy of Tuberculosis Triage Tests.

Approximately 3.6 million cases of active tuberculosis (TB) go potentially undiagnosed annually, partly due to limited access to confirmatory diagnostic tests, such as molecular assays or mycobacterial culture, in community and primary healthcare settings. This article provides guidance for TB triage test evaluations. A TB triage test is designed for use in people with TB symptoms and/or significant risk factors for TB. Triage tests are simple and low-cost tests aiming to improve ease of access and implementation (compared with confirmatory tests) and decrease the proportion of patients requiring more expensive confirmatory testing. Evaluation of triage tests should occur in settings of intended use, such as community and primary healthcare centers. Important considerations for triage test evaluation include study design, population, sample type, test throughput, use of thresholds, reference standard (ideally culture), and specimen flow. The impact of a triage test will depend heavily on issues beyond accuracy, primarily centered on implementation.

Skeletal muscle-derived exosomes regulate endothelial cell functions via reactive oxygen species-activated nuclear factor-κB signalling.

NEW FINDINGS: What is the central question of this study? Capillary rarefaction is found in diabetic and aged muscle, whereas exercise increases skeletal muscle angiogenesis. The association implies a crosstalk between muscle cells and endothelial cells. The underlying mechanisms mediating the crosstalk between these cells remains to be elucidated fully. What is the main finding and its importance? Endothelial cell functions are regulated by skeletal muscle cell-derived exosomes via a vascular endothelial growth factor-independent pathway. This study reveals a new mechanism mediating the crosstalk between skeletal muscle cells and endothelial cells. ABSTRACT: Loss of skeletal muscle capillarization, known as capillary rarefaction, is found in type 2 diabetes, chronic heart failure and healthy ageing and is associated with impaired delivery of substrates to the muscle. However, the interaction and communication of skeletal muscle with endothelial cells in the regulation of capillaries surrounding the muscle remains elusive. Exosomes are a type of secreted extracellular vesicle containing mRNAs, proteins and, especially, microRNAs that exert paracrine and endocrine effects. In this study, we investigated whether skeletal muscle-derived exosomes (SkM-Exo) regulate the endothelial cell functions of angiogenesis. We demonstrated that C2C12 myotube-derived exosomes improved endothelial cell functions, assessed by the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs), which were increased by 20, 23 and 40%, respectively, after SkM-Exo exposure. The SkM-Exo failed to activate HUVEC vascular endothelial growth factor (VEGF) signalling. The SkM-Exo increased HUVEC reactive oxygen species and activated the nuclear factor-κB pathway, suggesting that SkM-Exo-induced angiogenesis was mediated by a VEGF-independent pathway. In addition, several angiogenic microRNAs were packaged in SkM-Exo, with miR-130a being particularly enriched and successfully transferred from SkM-Exo to HUVECs. Delivery of miRNAs into endothelial cells might explain the enhancement of reactive oxygen species production and angiogenesis by SkM-Exo. The potential angiogenic effect of SkM-Exo could provide an effective therapy for promoting skeletal muscle angiogenesis in diseases characterized by capillary rarefaction or inadequate angiogenesis.

An evaluation framework for new tests that predict progression from tuberculosis infection to clinical disease.

Novel accurate tests are needed that identify individuals infected with Mycobacterium tuberculosis who have incipient disease and are likely to develop clinical tuberculosis (TB) in the near future to allow for targeted preventive treatment beyond the current risk groups. Recently, a target product profile was developed that outlines the minimal and optimal characteristics for such an incipient TB test. We describe an evaluation framework for generating evidence to inform the development of policy guidance for the use of such a new test by the World Health Organization. Two research objectives are addressed. 1) The predictive ability of an incipient TB test should be assessed in clinical evaluation studies that include the intended target population and follow-up of sufficient duration to observe whether individuals do or do not progress to clinical TB disease. 2) Studies are needed to evaluate the test under routine programmatic conditions and measure its impact on patient- or health-system-important outcomes. For both research objectives, study designs, methods and analysis are described, with the intent to inform the clinical development plans of test manufacturers, researchers and funders.

Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis.

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.

A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis.

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis.

Understanding the Impact of Water on the Miscibility and Microstructure of Amorphous Solid Dispersions: An AFM-LCR and TEM-EDX Study.

Miscibility is critical for amorphous solid dispersions (ASDs). Phase-separated ASDs are more prone to crystallization, and thus can lose their solubility advantage leading to product failure. Additionally, dissolution performance can be diminished as a result of phase separation in the ASD matrix. Water is known to induce phase separation during storage for some ASDs. However, the impact of water introduced during preparation has not been as thoroughly investigated to date. The purpose of this study was to develop a mechanistic understanding of the effect of water on the phase behavior and microstructure of ASDs. Evacetrapib and two polymers were selected as the model system. Atomic force microscopy coupled with Lorentz contact resonance, and transmission electron microscopy with energy dispersive X-ray spectroscopy were employed to evaluate the microstructure and composition of phase-separated ASDs. It was found that phase separation could be induced via two routes: solution-state phase separation during ASD formation caused by water absorption during film formation by a hydrophilic solvent, or solid-phase separation following exposure to high RH during storage. Water contents of as low as 2% in the organic solvent system used to dissolve the drug and polymer were found to result in phase separation in the resultant ASD film. These findings have profound implications on lab-scale ASD preparation and potentially also for industrial production. Additionally, these high-resolution imaging techniques combined with orthogonal analyses are powerful tools to visualize structural changes in ASDs, which in turn will enable better links to be made between ASD structure and performance.

Image-based genome-wide siRNA screen identifies selective autophagy factors.

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. Here, to identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide small interfering RNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to messenger RNA processing, interferon signalling, vesicle trafficking, cytoskeletal motor function and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, SMURF1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, SMURF1-deficient mice accumulate damaged mitochondria in the heart, brain and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines SMURF1 as a newly recognized mediator of both viral autophagy and mitophagy.

Eco-friendly (green) synthesis of magnetically active gold nanoclusters.

Au-FexOy composite nanoparticles (NPs) are of great technological interest due to their combined optical and magnetic properties. However, typical syntheses are neither simple nor ecologically friendly, creating a challenging situation for process scale-up. Here we describe conditions for preparing Au-FexOy NPs in aqueous solutions and at ambient temperatures, without resorting to solvents or amphiphilic surfactants with poor sustainability profiles. These magnetic gold nanoclusters (MGNCs) are prepared in practical yields with average sizes slightly below 100 nm, and surface plasmon resonances that extend to near-infrared wavelengths, and sufficient magnetic moment (up to 6 emu g-1) to permit collection within minutes by handheld magnets. The MGNCs also produce significant photoluminescence when excited at 488 nm. Energy dispersive X-ray (EDX) analysis indicates a relatively even distribution of Fe within the MGNCs, as opposed to a central magnetic core.

Implementation of HIV and Tuberculosis Diagnostics: The Importance of Context.

BACKGROUND: Novel diagnostics have been widely applied across human immunodeficiency virus (HIV) and tuberculosis prevention and treatment programs. To achieve the greatest impact, HIV and tuberculosis diagnostic programs must carefully plan and implement within the context of a specific healthcare system and the laboratory capacity. METHODS: A workshop was convened in Cape Town in September 2014. Participants included experts from laboratory and clinical practices, officials from ministries of health, and representatives from industry. RESULTS: The article summarizes best practices, challenges, and lessons learned from implementation experiences across sub-Saharan Africa for (1) building laboratory programs within the context of a healthcare system; (2) utilizing experience of clinicians and healthcare partners in planning and implementing the right diagnostic; and (3) evaluating the effects of new diagnostics on the healthcare system and on patient health outcomes. CONCLUSIONS: The successful implementation of HIV and tuberculosis diagnostics in resource-limited settings relies on careful consideration of each specific context.

Soft landing of polyatomic anions onto three-dimensional semiconductive and conductive substrates.

Soft landing of well-characterized polyoxometalate anions, PW12O40 3- (WPOM) and PMo12O40 3- (MoPOM), was carried out to explore the distribution of anions in the semiconducting 10 and 6 µm-long vertically aligned TiO2 nanotubes as well as 300 µm-long conductive vertically aligned carbon nanotubes (VACNTs). The distribution of soft-landed anions on the surfaces and their penetration into the nanotubes were studied using energy dispersive X-ray spectroscopy (EDX) and scanning electron microscopy (SEM). We observe that soft landed anions generate microaggregates on the TiO2 nanotubes and only reside in the top 1.5 µm of the nanotube height. Meanwhile, soft landed anions are uniformly distributed on top of VACNTs and penetrate into the top 40 µm of the sample. We propose that both the aggregation and limited penetration of POM anions into TiO2 nanotubes is attributed to the lower conductivity of this substrate as compared to VACNTs. This study provides first insights into the controlled modification of three dimensional (3D) semiconductive and conductive interfaces using soft landing of mass-selected polyatomic ions, which is of interest to the rational design of 3D interfaces for electronics and energy applications.

Diagnostic accuracy of three computer-aided detection systems for detecting pulmonary tuberculosis on chest radiography when used for screening: Analysis of an international, multicenter migrants screening study.

The aim of this study was to independently evaluate the diagnostic accuracy of three artificial intelligence (AI)-based computer aided detection (CAD) systems for detecting pulmonary tuberculosis (TB) on global migrants screening chest x-ray (CXR) cases when compared against both microbiological and radiological reference standards (MRS and RadRS, respectively). Retrospective clinical data and CXR images were collected from the International Organization for Migration (IOM) pre-migration health assessment TB screening global database for US-bound migrants. A total of 2,812 participants were included in the dataset used for analysis against RadRS, of which 1,769 (62.9%) had accompanying microbiological test results and were included against MRS. All CXRs were interpreted by three CAD systems (CAD4TB v6, Lunit INSIGHT v4.9.0, and qXR v2) in offline setting, and re-interpreted by two expert radiologists in a blinded fashion. The performance was evaluated using receiver operating characteristics curve (ROC), estimates of sensitivity and specificity at different CAD thresholds against both microbiological and radiological reference standards (MRS and RadRS, respectively), and was compared with that of the expert radiologists. The area under the curve against MRS was highest for Lunit (0.85; 95% CI 0.83-0.87), followed by qXR (0.75; 95% CI 0.72-0.77) and then CAD4TB (0.71; 95% CI 0.68-0.73). At a set specificity of 70%, Lunit had the highest sensitivity (81.4%; 95% CI 77.9-84.6); at a set sensitivity of 90%, specificity was also highest for Lunit (54.5%; 95% CI 51.7-57.3). The CAD systems performed comparable to the sensitivity (98.3%), and except CAD4TB, to specificity (13.7%) of the expert radiologists. Similar trends were observed when using RadRS. Area under the curve against RadRS was highest for CAD4TB (0.87; 95% CI 0.86-0.89) and Lunit (0.87; 95% CI 0.85-0.88) followed by qXR (0.81; 95% CI 0.80-0.83). At a set specificity of 70%, CAD4TB had highest sensitivity (84.1%; 95% CI 82.3-85.8) followed by Lunit (80.9%; 95% CI 78.9-82.7); and at a set sensitivity of 90%, specificity was also highest for CAD4TB (54.6%; 95% CI 51.3-57.8). In conclusion, the study demonstrated that the three CAD systems had broadly similar diagnostic accuracy with regard to TB screening and comparable accuracy to an expert radiologist against MRS. Compared with different reference standards, Lunit performed better than both qXR and CAD4TB against MRS, and CAD4TB and Lunit better than qXR against RadRS. Moreover, the performance of the CADs can be impacted by characteristics of subgroup of population. The main limitation was that our study relied on retrospective data and MRS was not routinely done in individuals with a low suspicion of TB and a normal CXR. Our findings suggest that CAD systems could be a useful tool for TB screening programs in remote, high TB prevalent places where access to expert radiologists may be limited. However, further large-scale prospective studies are needed to address outstanding questions around the operational performance and technical requirements of the CAD systems.