QB3

Fibrosis involves sustained changes in fibroblast gene expression, leading to excessive extracellular matrix (ECM) deposition and progressive tissue stiffening. Although matrix stiffness is a potent regulator of cell fate and transcription, it is not clear how nuclear mechanosensing contributes to fibrosis. Here, we define a central role for SUN2, a component of linker of nucleoskeleton and cytoskeleton (LINC) complexes, as a mediator of stiffness-dependent nuclear and chromatin responses during skin fibrosis. SUN2 transcripts are upregulated in dermal fibroblasts of patients with systemic sclerosis and Sun2 protein is elevated in fibrotic mouse skin. Nuclear size, A-type lamins and Sun2 are elevated in dermal fibroblasts plated on stiff substrates. Loss of Sun2 protects against bleomycin-induced skin fibrosis in vivo and abolishes stiffness-induced changes in nuclear size and fibrotic gene expression in vitro. Mechanistically, we identify three Sun2-dependent mechanosensitive chromatin states and show that mechanical induction of the histone methyltransferase Ezh2 requires Sun2. These findings define SUN2 as a nuclear mechanosensor that couples matrix stiffness to chromatin regulation and transcriptional programs that drive fibrosis, identifying it as a potential therapeutic target pathway in fibrotic disease.

Accurate diagnostics are essential for disease control and elimination efforts. However, access to diagnostics for neglected tropical diseases (NTDs) is hindered by limited healthcare infrastructure in many NTD-endemic regions, as well as by reliance on time- and labor-intensive diagnostic methods, such as smear microscopy. New diagnostic tools that are portable, rapid, low-cost, and meet World Health Organization (WHO) sensitivity and specificity targets are urgently needed to accelerate NTD control and elimination programs. Here, we introduce the NTDscope, a portable microscopy platform that enables point-of-care imaging and automated detection of parasites and other pathogens in patient samples. The NTDscope builds on and extends the capabilities of the LoaScope, a device that turned the camera of a mobile phone into a microscope and used on-board image processing to automatically quantify Loa loa microfilariae burden in whole blood samples. The NTDscope replaces the mobile phone of the LoaScope with a system-on-module (SOM) that enables the integration of multiple imaging modalities in a single package designed to improve robustness and expand applications. In this work, we demonstrate the use of the NTDscope as a portable brightfield, darkfield, and fluorescence microscope for samples including microfilariae and helminth eggs. We also show that the device can be used to quantify molecular assays, such as a lateral flow test and a CRISPR-Cas13a-based assay. The ability to combine the diagnostic capabilities of conventional microscopy with molecular assays and machine learning in a single device could expand access to diagnostics for populations in NTD-endemic areas and beyond.

Many G-protein–coupled receptors (GPCRs) use endocytosis to promote gene transcription by prolonging signaling through the Gs-coupled cAMP/cAMP-dependent PKA cascade. However, not all GPCRs efficiently internalize after agonist-induced activation, and, among those that do, considerable differences have been observed in the ability of different GPCRs to stimulate endosomal cAMP production in different cell types. We asked if endocytosis distinguishes the signaling profiles of GPCRs that are naturally coexpressed in the same cells, focusing on three Gs-coupled GPCRs endogenous to human embryonic kidney–derived (293) cells: the vasoactive intestinal peptide receptor-1 (VIPR1/VPAC1) and β2-adrenergic receptor (β2AR/ADRB2), which both rapidly internalize after activation, and the adenosine-2B receptor (A2BR/ADORA2B) which we show here does not. For VIPR1, endocytosis significantly prolongs both the global cAMP elevation and cytoplasmic PKA activity increase. For β2AR, endocytosis has little effect on the global cAMP elevation but, nonetheless, it significantly prolongs the cytoplasmic PKA activity increase. A2BR differs still further, with endocytosis having little effect on signal duration measured at either intermediate step. We then show that further downstream steps in the cascade, nuclear PKA activation and transcriptional induction, are significantly endocytosis dependent when stimulated through VIPR1 and β2AR but endocytosis independent through A2BR. We conclude that endocytosis indeed distinguishes the signaling profiles of endogenously coexpressed GPCRs. We propose that quantitative differences in GPCR internalization and activation in endosomes program in cells a GPCR-selective, spatiotemporal “cAMP code” that is spatially “decoded” by proximity to local cytoplasmic PKA stores and then temporally interpreted by the nucleus.

The Mycobacteriales are an order of diverse bacteria that thrive in many environmental and host-associated niches. Because the most notorious member of this clade, Mycobacterium tuberculosis, is a major human pathogen, research on Mycobacteriales has focused on pathogenesis, and, as a consequence, many fundamental aspects of Mycobacterial biology remain understudied. Here, we address this gap by performing a genome-wide CRISPRi chemical genomics screen using a diverse set of >35 antibiotics, detergents, and other anti-microbials predominantly targeting the cell envelope of Mycobacterium smegmatis, a saprophytic model Mycobacterium. We highlight new information derived from this screen, including the identification of novel functions for previously uncharacterized conserved and essential genes (in mycolic acid and arabinogalactan synthesis), the discovery of a new drug scaffold/protein target pair, and insights into the mechanism of action of two commonly used antibiotics. These data are also a valuable resource for the mycobacterial research community, as they provide thousands of novel phenotypes for uncharacterized genes and meaningful phenotypic correlations between annotated and uncharacterized genes.

Enzyme catalysis has been shown to depend on distal pathways that channel thermal energy from solvent to the active site. In soybean lipoxygenase (SLO), experiments identified a cone-shaped network connecting loop residue Gln322 to Leu546 but not Leu754 in the active site. Here, microsecond molecular dynamics and a correlated motion-based protocol provide an atomistic analysis of such long-range communication. The developed approach enables systematic screening of communication between active site-specific residues that directly contact bound substrate and surface-exposed residues on the protein-solvent interface. In doing so, it provides a deeper molecular insight into experimentally mapped networks by resolving communication trends across diverse conformational ensembles. The simulations recover the experimentally demonstrated thermal initiation loop and the Leu546-directed cone in SLO, exclude the negative-control Ser596, and explain the preference for Leu546 over Leu754 through shorter, more correlated helical pathways. Mutational analysis further reveals the impact of single-site mutations on the network preference between Leu546 and Leu754. These results unify experiments and computation, corroborating an anisotropic channeling of thermal energy in SLO and establishing a general framework for computing distal intra-protein pathways that may enable the thermal activation of enzyme function.

Terpenoid natural products and their derivatives exhibit bioactivity that is utilized in FDA-approved drugs and candidates for future drugs; however, the widespread utilization of terpenoids has been limited by complex, low-yielding native biosyntheses and chemical syntheses. Microbial total/semi-biosynthesis of natural and new-to-nature terpenoids from sustainable feedstocks is scalable and can achieve economically viable cost targets. Herein, this review describes foundational advances in synthetic biology and metabolic engineering as exemplified by efforts to biosynthesize prominent terpenoids (i.e.artemisinin, taxol, vinblastine, QS-21, and cyclopamine) in engineered microorganisms (e.g.Escherichia coli and Saccharomyces cerevisiae). Emerging methods that accelerate microbial biosynthesis campaigns (i.e.automation, machine learning, artificial intelligence, and combinatorial screening) are then discussed.

The unfolded protein response (UPR) was discovered in budding yeast as a mechanism that allows cells to adapt to endoplasmic reticulum (ER) stressors. Although the UPR is not thought to be necessary for cellular fitness of wild-type cells in the absence of stress, we found that UPR deficiency led to poor growth in cycling mitotic yeast cells. This led to pervasive adaptive aneuploidy of specific chromosomes that was seen in divergent strain backgrounds, indicating an important basal role for this pathway that was missed by studies of the most common laboratory-derived strains. Aneuploid UPR-deficient cells grew better than euploid UPR-deficient cells but exhibited heightened general proteostatic stress, a hallmark of aneuploidy in wild-type cells. Modulation of key genes involved in ER proteostasis that were encoded on aneuploid chromosomes could phenocopy the effects of aneuploidy, indicating that the reason UPR-deficient cells become aneuploid is to counteract protein folding stress in the ER. Proteomic analyses indicate that expression of a small subset of stress-induced UPR targets is supported by basal UPR activity, including the chaperone Kar2/BiP. Together, our results reveal an unexpected role for the UPR in baseline ER folding that is important enough to safeguard cellular fitness that cells tolerate the substantial proteostatic costs that result from aneuploidy to counteract its loss.

ObjectivesPediatric asthma is multifactorial: social, environmental, and infectious exposures trigger exacerbations. School COVID-19 policies, including closures, mask mandates, and reopenings, provide a natural experiment to discover associations between the severity of pediatric asthma exacerbations, viral transmission, and exposure to wildfire smoke. We investigated the epidemiology of acute unscheduled care for asthma in children aged 5 to 17 years, including emergency department (ED) visits, hospital and intensive care unit (ICU) admissions, and viral testing.MethodsThis observational study used a retrospective review of all electronic health records from New York City’s largest health system over 5 school years, from 2018 to 2023. Comparison groups included citywide surveillance data and all-cause visits. We investigated population-level exposures, including COVID-19 mitigation policies and wildfire smoke events.ResultsIn the postpandemic period, fewer children presented to the ED for asthma (P < .0001); however, more children required hospital or ICU admission (P < .001). Viral testing was employed more frequently and was more often positive in the postpandemic period (P < .0001). Rhinovirus was positive in a greater proportion of pediatric asthma visits than in all-cause adult or pediatric visits (P = .02). After mask mandates ended in early 2022, pediatric asthma visits peaked during the return-to-school fall period, slightly preceding the winter 2022 viral illness peak. Wildfire smoke events were not associated with pediatric asthma visits.ConclusionViral transmission and school policies were associated with pediatric asthma exacerbations during and after the COVID-19 pandemic. Increased acuity, despite lower volumes, may help guide health systems as they strive to increase readiness to care for pediatric populations in response to future pandemics or environmental exposures.