Salmonella Typhimurium impairs glycolysismediated acidification of phagosomes to evade macrophage defense

Regulation of cellular metabolism is now recognized as a crucial mechanism for the activation of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. Despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggest that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolysis facilitates glycolytic enzyme aldolase A mediated v- ATPase assembly and the acidification of phagosomes which is critical for lysosomal degradation. Thus, impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in murine macrophages (BMDM). Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense. Copyright

3D-printed microplate inserts for long term high-resolution imaging of live brain organoids

Background: Organoids are a reliable model used in the study of human brain development and under pathological conditions. However, current methods for brain organoid culture generate tissues that range from 0.5 to 2mm of size, which need to be constantly agitated to allow proper oxygenation. The culture conditions are, therefore, not suitable for whole-brain organoid live imaging, required to study developmental processes and disease progression within physiologically relevant time frames (i.e. days, weeks, months). Results: Here we designed 3D-printed microplate inserts adaptable to standard 24 multi-well plates, which allow the growth of multiple organoids in pre-defined and fixed XYZ coordinates. This innovation facilitates highresolution imaging of whole-cerebral organoids, allowing precise assessment of organoid growth and morphology, as well as cell tracking within the organoids, over long periods. We applied this technology to track neocortex development through neuronal progenitors in brain organoids, as well as the movement of patient-derived glioblastoma stem cells within healthy brain organoids. Conclusions: This new bioengineering platform constitutes a significant advance that permits long term detailed analysis of whole-brain organoids using multimodal inverted fluorescence microscopy.Mariana Oksdath Mansilla, Camilo Salazar-Hernandez, Sally L. Perrin, Kaitlin G. Scheer, Gökhan Cildir, John Toubia, Kristyna Sedivakova, Melinda N. Tea, Sakthi Lenin, Elise Ponthier, Erica C. F. Yeo, Vinay Tergaonkar, Santosh Poonnoose, Rebecca J. Ormsby, Stuart M. Pitson, Michael P. Brown, Lisa M. Ebert, and Guillermo A. Gome

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Understanding mast cell heterogeneity at single cell resolution

Mast cells (MC)s are evolutionarily conserved, tissue-resident immune cells with diverse roles in allergy, cancer, and protection from infection by helminths and microorganisms. The significant diversity in MC development and tissue-specific functional characteristics has recently begun to be understood. Exciting developments in single-cell-based RNA, protein, and chromatin profiling technologies offer new opportunities to characterize MC heterogeneity and to uncover novel MC functions and subtypes; these developments might lead to new and clinically effective therapies for certain pathologies. In this review, we provide an overview of the current understanding of MC development and heterogeneity and discuss new insights gained from single-cell-based studies that may lead to future research directions and therapeutic opportunities.Gökhan Cildir, Kwok Ho Yip, Harshita Pant, Vinay Tergaonkar, Angel F. Lopez, and Damon J. Tume

Genome-wide analyses of chromatin state in human mast cells reveal molecular drivers and mediators of allergic and inflammatory diseases

Mast cells (MCs) are versatile immune cells capable of rapidly responding to a diverse range of extracellular cues. Here, we mapped the genomic and transcriptomic changes in human MCs upon diverse stimuli. Our analyses revealed broad H3K4me3 domains and enhancers associated with activation. Notably, the rise of intracellular calcium concentration upon immunoglobulin E (IgE)-mediated crosslinking of the high-affinity IgE receptor (FcεRI) resulted in genome-wide reorganization of the chromatin landscape and was associated with a specific chromatin signature, which we term Ca²⁺-dependent open chromatin (COC) domains. Examination of differentially expressed genes revealed potential effectors of MC function, and we provide evidence for fibrinogen-like protein 2 (FGL2) as an MC mediator with potential relevance in chronic spontaneous urticaria. Disease-associated single-nucleotide polymorphisms mapped onto cis-regulatory regions of human MCs suggest that MC function may impact a broad range of pathologies. The datasets presented here constitute a resource for the further study of MC function.Gökhan Cildir, John Toubia, Kwok Ho Yip, Mingyan Zhou, Harshita Pant, Pravin Hissaria, Jingxian Zhang, Wanjin Hong, Nirmal Robinson, Michele A. Grimbaldeston, Angel F. Lopez, and Vinay Tergaonka