Serotonin reduction in post-acute sequelae of viral infection.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

ß-Hydroxybutyrate suppresses colorectal cancer.

Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed1. Here we identify a metabolite signalling pathway that provides actionable insights towards this goal. We perform a dietary screen in autochthonous animal models of CRC and find that ketogenic diets exhibit a strong tumour-inhibitory effect. These properties of ketogenic diets are recapitulated by the ketone body ß-hydroxybutyrate (BHB), which reduces the proliferation of colonic crypt cells and potently suppresses intestinal tumour growth. We find that BHB acts through the surface receptor Hcar2 and induces the transcriptional regulator Hopx, thereby altering gene expression and inhibiting cell proliferation. Cancer organoid assays and single-cell RNA sequencing of biopsies from patients with CRC provide evidence that elevated BHB levels and active HOPX are associated with reduced intestinal epithelial proliferation in humans. This study thus identifies a BHB-triggered pathway regulating intestinal tumorigenesis and indicates that oral or systemic interventions with a single metabolite may complement current prevention and treatment strategies for CRC.

Caenorhabditis elegans saposin-like spp-9 is involved in specific innate immune responses.

Animals counter specific environmental challenges with a combination of broad and tailored host responses. One protein family enlisted in the innate immune response includes the saposin-like antimicrobial proteins. We investigated the expression of a Caenorhabditis elegans saposin-like gene, spp-9, in response to different stresses. spp-9 expression was detected in the intestine and six amphid neurons, including AWB and AWC. spp-9 expression is increased in response to starvation stress. In addition, we discovered pathogen-specific regulation of spp-9 that was not clearly demarcated by Gram nature of the bacterial challenge. Multiple molecular innate immune response pathways, including DBL-1/TGF-ß-like, insulin-like, and p38/MAPK, regulate expression of spp-9. Our results suggest spp-9 is involved in targeted responses to a variety of abiotic and bacterial challenges that are coordinated by multiple signaling pathways.

The DBL-1/TGF-ß signaling pathway tailors behavioral and molecular host responses to a variety of bacteria in Caenorhabditis elegans.

Generating specific, robust protective responses to different bacteria is vital for animal survival. Here, we address the role of transforming growth factor ß (TGF-ß) member DBL-1 in regulating signature host defense responses in Caenorhabditis elegans to human opportunistic Gram-negative and Gram-positive pathogens. Canonical DBL-1 signaling is required to suppress avoidance behavior in response to Gram-negative, but not Gram-positive bacteria. We propose that in the absence of DBL-1, animals perceive some bacteria as more harmful. Animals activate DBL-1 pathway activity in response to Gram-negative bacteria and strongly repress it in response to select Gram-positive bacteria, demonstrating bacteria-responsive regulation of DBL-1 signaling. DBL-1 signaling differentially regulates expression of target innate immunity genes depending on the bacterial exposure. These findings highlight a central role for TGF-ß in tailoring a suite of bacteria-specific host defenses.

Single-cell analysis and spatial resolution of the gut microbiome.

Over the past decade it has become clear that various aspects of host physiology, metabolism, and immunity are intimately associated with the microbiome and its interactions with the host. Specifically, the gut microbiome composition and function has been shown to play a critical role in the etiology of different intestinal and extra-intestinal diseases. While attempts to identify a common pattern of microbial dysbiosis linked with these diseases have failed, multiple studies show that bacterial communities in the gut are spatially organized and that disrupted spatial organization of the gut microbiome is often a common underlying feature of disease pathogenesis. As a result, focus over the last few years has shifted from analyzing the diversity of gut microbiome by sequencing of the entire microbial community, towards understanding the gut microbiome in spatial context. Defining the composition and spatial heterogeneity of the microbiome is critical to facilitate further understanding of the gut microbiome ecology. Development in single cell genomics approach has advanced our understanding of microbial community structure, however, limitations in approaches exist. Single cell genomics is a very powerful and rapidly growing field, primarily used to identify the genetic composition of microbes. A major challenge is to isolate single cells for genomic analyses. This review summarizes the different approaches to study microbial genomes at single-cell resolution. We will review new techniques for microbial single cell sequencing and summarize how these techniques can be applied broadly to answer many questions related to the microbiome composition and spatial heterogeneity. These methods can be used to fill the gaps in our understanding of microbial communities.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA.

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Reduced bone morphogenic protein signaling along the gut-neuron axis by heat shock factor promotes longevity.

Aging is a complex and highly regulated process of interwoven signaling mechanisms. As an ancient transcriptional regulator of thermal adaptation and protein homeostasis, the Heat Shock Factor, HSF-1, has evolved functions within the nervous system to control age progression; however, the molecular details and signaling dynamics by which HSF-1 modulates age across tissues remain unclear. Herein, we report a nonautonomous mode of age regulation by HSF-1 in the Caenorhabditis elegans nervous system that works through the bone morphogenic protein, BMP, signaling pathway to modulate membrane trafficking in peripheral tissues. In particular, HSF-1 represses the expression of the neuron-specific BMP ligand, DBL-1, and initiates a complementary negative feedback loop within the intestine. By reducing receipt of DBL-1 in the periphery, the SMAD transcriptional coactivator, SMA-3, represses the expression of critical membrane trafficking regulators including Rab GTPases involved in early (RAB-5), late (RAB-7), and recycling (RAB-11.1) endosomal dynamics and the BMP receptor binding protein, SMA-10. This reduces cell surface residency and steady-state levels of the type I BMP receptor, SMA-6, in the intestine and further dampens signal transmission to the periphery. Thus, the ability of HSF-1 to coordinate BMP signaling along the gut-brain axis is an important determinate in age progression.

Genetic interactions between the DBL-1/BMP-like pathway and dpy body size-associated genes in Caenorhabditis elegans.

Bone morphogenetic protein (BMP) signaling pathways control many developmental and homeostatic processes, including cell size and extracellular matrix remodeling. An understanding of how this pathway itself is controlled remains incomplete. To identify novel regulators of BMP signaling, we performed a forward genetic screen in Caenorhabditis elegans for genes involved in body size regulation, a trait under the control of BMP member DBL-1. We isolated mutations that suppress the long phenotype of lon-2, a gene that encodes a negative regulator that sequesters DBL-1. This screen was effective because we isolated alleles of several core components of the DBL-1 pathway, demonstrating the efficacy of the screen. We found additional alleles of previously identified but uncloned body size genes. Our screen also identified widespread involvement of extracellular matrix proteins in DBL-1 regulation of body size. We characterized interactions between the DBL-1 pathway and extracellular matrix and other genes that affect body morphology. We discovered that loss of some of these genes affects the DBL-1 pathway, and we provide evidence that DBL-1 signaling affects many molecular and cellular processes associated with body size. We propose a model in which multiple body size factors are controlled by signaling through the DBL-1 pathway and by DBL-1-independent processes.