High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials

Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using forty plasma samples from convalescent individuals with mild-to-moderate COVID-19: four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate ELISA-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor, human angiotensin converting enzyme 2 (hACE2). Vero, Vero E6, HEK293T expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81–0.89) and ranged within 3.4-fold. The live-virus assay and LV-pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers: 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike and RBD (r = 0.63–0.89), but moderately correlated with nucleoprotein IgG (r = 0.46–0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV-pseudovirus assay and LV-pseudovirus assay with HEK293T/hACE2 cells in low and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. 24

Immune Cell Recruitment and Cell-Based System for Cancer Therapy

Immune cells, such as cytotoxic T lymphocytes, natural killer cells, B cells, and dendritic cells, have a central role in cancer immunotherapy. Conventional studies of cancer immunotherapy have focused mainly on the search for an efficient means to prime/activate tumor-associated antigen-specific immunity. A systematic understanding of the molecular basis of the trafficking and biodistribution of immune cells, however, is important for the development of more efficacious cancer immunotherapies. It is well established that the basis and premise of immunotherapy is the accumulation of effective immune cells in tumor tissues. Therefore, it is crucial to control the distribution of immune cells to optimize cancer immunotherapy. Recent characterization of various chemokines and chemokine receptors in the immune system has increased our knowledge of the regulatory mechanisms of the immune response and tolerance based on immune cell localization. Here, we review the immune cell recruitment and cell-based systems that can potentially control the systemic pharmacokinetics of immune cells and, in particular, focus on cell migrating molecules, i.e., chemokines, and their receptors, and their use in cancer immunotherapy

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Serum concentration of antigen-specific IgG can substantially bias interpretation of antibody-dependent phagocytosis assay readout

Summary: Because virus neutralization cannot solely explain vaccine-induced, antibody-mediated protection, antibody effector functions are being considered as a potential correlate of protection (CoP). However, measuring effector functions at a fixed serum dilution for high throughput purposes makes it difficult to distinguish between the effect of serum antibody concentration and antibody properties such as epitopes, subclass, and glycosylation. To address this issue, we evaluated antibody-dependent cellular phagocytosis (ADCP) assay against SARS-CoV-2 spike. Adjustment of serum samples to the same concentration of antigen-specific IgG prior to the ADCP assay revealed concentration-independent differences in ADCP after mRNA vaccination in subjects with and without prior SARS-CoV-2 infection not detectable in assay performed with fixed serum dilution. Phagocytosis measured at different concentrations of spike-specific IgG strongly correlated with the area under the curve (AUC) indicating that ADCP assay can be performed at a standardized antibody concentration for the high throughput necessary for vaccine trial analyses

Dataset associated with “Context dependency of disease-mediated competitive release in bat assemblages following white-nose syndrome”

Dataset of bat acoustic detections across four study areas in the US to study the impact of white-nose syndrome on bat community interactions. Data collected from 2003-2017 at four study locations in the United States, including Fenrnow Experimental Forest, WV; Fort Pickett, VA; Fort Drum, NY; two locations in Wisconsin.White-nose syndrome (WNS) has caused dramatic declines of several cave-hibernating bat species in North America since 2006, which has increased the activity of non-susceptible species in some geographic areas or during times of night formerly occupied by susceptible species - indicative of disease-mediated competitive release (DMCR). Yet, this pattern has not been evaluated across multiple bat assemblages simultaneously or across multiple years since WNS onset. We evaluated whether WNS altered spatial and temporal niche partitioning in bat assemblages at four locations in the eastern United States using long-term datasets of bat acoustic activity collected before and after WNS arrival. Activity of WNS-susceptible bat species decreased by 79-98% from pre-WNS levels across the four study locations, but only one of our four study sites provided strong evidence supporting the DMCR hypothesis in bats post-WNS. These results suggest that DMCR is likely dependent on the relative difference in activity by susceptible and non-susceptible species groups pre-WNS and the relative decline of susceptible species post-WNS allowing for competitive release, as well as the amount of time that had elapsed post-WNS. Our findings challenge the generality of WNS-mediated competitive release between susceptible and non-susceptible species and further highlight declining activity of some non-susceptible species, especially Lasiurus borealis, across three of four locations in the eastern US. These results underscore the broader need for conservation efforts to address the multiple potential interacting drivers of bat declines on both WNS susceptible and non-susceptible species.Funding for this study was provided by the U.S. Fish and Wildlife Service White-nose Syndrome Grant Program Agreement #4500900398 to the USGS South Carolina Cooperative Fish and Wildlife Research Unit and the Virginia Cooperative Fish and Wildlife Research Unit. Additional support for work at Fort Drum came from the U.S. Army Corps of Engineers’ Cooperative Agreement W9126G-15-2-0005 through the Southern Appalachian Cooperative Ecosystems Study Unit Program to the Virginia Tech Department of Fish and Wildlife Conservation. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government

Determination of Alternaria spp. Habitats Using 7-Day Volumetric Spore Trap, Hybrid Single Particle Lagrangian Integrated Trajectory Model and Geographic Information System

There are many species among the Alternaria genus, which hosts on economically important crops causing significant yield losses. Less attention has been paid to fungi hosting on plants constituting substantial components of pastures and meadows. Alternaria spp. spores are also recognised as important allergens. A 7-day volumetric spore trap was used to monitor the concentration of airborne fungal spores. Air samples were collected in Worcester, England (2006–2010). Days with a high spore count were then selected. The longest episode that occurred within a five year study was chosen for modelling. Two source maps presenting distribution of crops under rotation and pastures in the UK were produced. Back trajectories were calculated using the HYSPLIT model. In ArcGIS clusters of trajectories were studied in connection with source maps by including the height above ground level and the speed of the air masses. During the episode no evidence for a long distance transport from the continent of Alternaria spp. spores was detected. The overall direction of the air masses fell within the range from South-West to North. The back trajectories indicated that the most important sources of Alternaria spp. spores were located in the West Midlands of England

Critical Mass, Deliberation and the Substantive Representation of Women: Evidence from the UK's Devolution Programme

This article provides empirical evidence to support recent assertions that the substantive representation of women depends not only on the numbers of women elected representatives in national legislatures, but also who they are. In this case study of one of the UK's devolved legislatures, analysis was undertaken of the transcripts of 327 plenary debates held during the first term of the National Assembly for Wales, where women constituted 42 percent of elected members (1999–2003). The gender dynamics of political debate around key equality topics reveal that the link between descriptive and substantive representation of women is complex. When a ‘critical mass’ of women is achieved the substantive representation of women is affirmed as ‘probabilistic’ rather than ‘deterministic’ for it is shaped by the institutional context, the gender dynamics of debate and, importantly, the actions of individual ‘equality champions’. While women representatives exhibited a greater propensity to advance gender equality in debate than their male colleagues, the present findings also show the disproportionate influence of ‘equality champions’: women who are able to draw upon earlier feminist activism and act as ‘strategic insiders’ who make a difference to women's issues in a parliamentary context