Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms.

BACKGROUND & AIMS: The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism. METHODS: We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites. RESULTS: Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells. CONCLUSIONS: Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Ontogeny and function of the circadian clock in intestinal organoids.

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses to TcdB. RNA-Seq analysis shows that ~3-10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti-phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock-dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock-controlled genes in both mouse and human enteroids governing organism-specific, circadian phase-dependent necrotic cell death responses, and lay a foundation for human organ- and disease-specific investigation of clock functions using human organoids for translational applications.

An integrated microfluidic bubble pocket for long-term perfused three-dimensional intestine-on-a-chip model.

Perfused three-dimensional (3D) cultures enable long-term in situ growth and monitoring of 3D organoids making them well-suited for investigating organoid development, growth, and function. One of the limitations of this long-term on-chip perfused 3D culture is unintended and disruptive air bubbles. To overcome this obstacle, we invented an imaging platform that integrates an innovative microfluidic bubble pocket for long-term perfused 3D culture of gastrointestinal (GI) organoids. We successfully applied 3D printing technology to create polymer molds that cast polydimethylsiloxane (PDMS) culture chambers in addition to bubble pockets. Our developed platform traps unintended, or induced, air bubbles in an integrated PDMS pocket chamber, where the bubbles diffuse out across the gas permeable PDMS or an outlet tube. We demonstrated that our robust platform integrated with the novel bubble pocket effectively circumvents the development of bubbles into human and mouse GI organoid cultures during long-term perfused time-course imaging. Our platform with the innovative integrated bubble pocket is ideally suited for studies requiring long-term perfusion monitoring of organ growth and morphogenesis as well as function.

Dehydration prompts increased activity and blood feeding by mosquitoes.

Current insights into the mosquito dehydration response rely on studies that examine specific responses but ultimately fail to provide an encompassing view of mosquito biology. Here, we examined underlying changes in the biology of mosquitoes associated with dehydration. Specifically, we show that dehydration increases blood feeding in the northern house mosquito, Culex pipiens, which was the result of both higher activity and a greater tendency to land on a host. Similar observations were noted for Aedes aegypti and Anopheles quadrimaculatus. RNA-seq and metabolome analyses in C. pipiens following dehydration revealed that factors associated with carbohydrate metabolism are altered, specifically the breakdown of trehalose. Suppression of trehalose breakdown in C. pipiens by RNA interference reduced phenotypes associated with lower hydration levels. Lastly, mesocosm studies for C. pipiens confirmed that dehydrated mosquitoes were more likely to host feed under ecologically relevant conditions. Disease modeling indicates dehydration bouts will likely enhance viral transmission. This dehydration-induced increase in blood feeding is therefore likely to occur regularly and intensify during periods when availability of water is low.

Intercellular Coupling of the Cell Cycle and Circadian Clock in Adult Stem Cell Culture.

Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted WNT as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species.

BACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control. RESULTS: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A high-quality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT. CONCLUSIONS: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution.

Rhythm and bugs: circadian clocks, gut microbiota, and enteric infections.

PURPOSE OF REVIEW: To highlight recent developments in understanding the dynamic relationship between circadian rhythms, the gut microbiome, and gastrointestinal infections. RECENT FINDINGS: In humans and mice, the composition and functions of the intestinal microbiome display diurnal rhythms orchestrated by feeding behaviors and host circadian gene expression. Jet lag, or circadian disruption, perturbs these rhythms to produce gut dysbiosis. When mice are orally infected with Salmonella typhimurium in the morning (the beginning of their rest period) they show higher levels of colonization and gut inflammation vs. infection at other times of day. At the cellular level, recent studies highlight circadian regulation of innate and adaptive gut immunity in coordination with the microbiome, as well as intestinal stem cell growth and regeneration. SUMMARY: Taken together, these reports support a key role for circadian rhythms in regulating the gut microbiome and host responses to gastrointestinal pathogens. Further research is needed to translate these findings to improving outcomes for patients with gastrointestinal infections by guiding the right interventions for the right patients at the right time.

Fungicide contamination reduces beneficial fungi in bee bread based on an area-wide field study in honey bee, Apis mellifera, colonies.

Fermentation by fungi converts stored pollen into bee bread that is fed to honey bee larvae, Apis mellifera, so the diversity of fungi in bee bread may be related to its food value. To explore the relationship between fungicide exposure and bee bread fungi, samples of bee bread collected from bee colonies pollinating orchards from 7 locations over 2 years were analyzed for fungicide residues and fungus composition. There were detectable levels of fungicides from regions that were sprayed before bloom. An organic orchard had the highest quantity and variety of fungicides, likely due to the presence of treated orchards within bees' flight range. Aspergillus, Penicillium, Rhizopus, and Cladosporium (beneficial fungi) were the primary fungal isolates found, regardless of habitat differences. There was some variation in fungal components amongst colonies, even within the same apiary. The variable components were Absidia, Alternaria, Aureobasidium, Bipolaris, Fusarium, Geotrichum, Mucor, Nigrospora, Paecilomyces, Scopulariopsis, and Trichoderma. The number of fungal isolates was reduced as an effect of fungicide contamination. Aspergillus abundance was particularly affected by increased fungicide levels, as indicated by Simpson's diversity index. Bee bread showing fungicide contamination originated from colonies, many of which showed chalkbrood symptoms.

Water balance characteristics of pupae developing in different size maggot masses from six species of forensically important flies.

The impact of maggot mass size on body water content, net transpiration rate, and dehydration tolerance of fly pupae was examined in six species of necrophagous flies. Species that spent more time on food as larvae, produced pupae with high body water contents. Dehydration tolerance limits of pupae were modest, matching the moisture-rich conditions of decaying carrion for larvae. Protophormia terraenovae pupariates on food as it dries, and this was reflected by pupae having the highest body water content and lowest net transpiration rate. Megaselia scalaris featured the lowest body water content and highest dehydration tolerance, implying that this species is arid-suited, which matches its ability to feed and colonize on post-decay carrion. Lucilia illustris was the most sensitive to larval overcrowding, resulting in a dramatic decrease in pupal size, early dispersal from food, fed less and had fast net transpiration rates. By contrast, Lucilia sericata was the most resistant, by showing no pupal size decrease and no change in net transpiration rate. Other species were between these extremes, requiring larger maggot mass sizes to produce the effect of decreasing pupal size and increasing net transpiration rate. We conclude: (1) the pupa's response to overcrowding and water balance profile are species-specific, varying according to pupal size and net transpiration rate as independent characteristics; (2) water balance profile of the pupae reflects the behavior and microhabitat of the larva; and (3) danger of lethal desiccation to smaller-sized pupae is circumvented by a faster developmental rate rather than enhanced water conservation.