Alterations in the fecal microbiota in patients with advanced cystic fibrosis liver disease after 6 months of elexacaftor/tezacaftor/ivacaftor.

BACKGROUND: Cystic fibrosis associated liver disease (CFLD) carries a significant disease burden with no effective preventive therapies. According to the gut-liver axis hypothesis for CFLD pathogenesis, dysbiosis and increased intestinal inflammation and permeability permit pathogenic bacterial translocation into the portal circulation, leading to hepatic inflammation and fibrosis. Evaluating the effect of CFTR (cystic fibrosis transmembrane conductance regulator) modulation with elexacaftor/tezacaftor/ivacaftor (ETI) may help determine the role of CFTR in CFLD and increase understanding of CFLD pathogenesis, which is critical for developing therapies. We aimed to characterize the fecal microbiota in participants with CF with and without advanced CFLD (aCFLD) before and after ETI. METHODS: This is an ancillary analysis of stool samples from participants ages ≥12 y/o enrolled in PROMISE (NCT04038047). Included participants had aCFLD (cirrhosis with or without portal hypertension, or non-cirrhotic portal hypertension) or CF without liver disease (CFnoLD). Fecal microbiota were defined by shotgun metagenomic sequencing at baseline and 1 and 6 months post-ETI. RESULTS: We analyzed 93 samples from 34 participants (11 aCFLD and 23 CFnoLD). Compared to CFnoLD, aCFLD had significantly higher baseline relative abundances of potential pathogens Streptococcus salivarius and Veillonella parvula. Four of 11 aCFLD participants had an initially abnormal fecal calprotectin that normalized 6 months post-ETI, correlating with a significant decrease in S. salivarius and a trend towards decreasing V. parvula. CONCLUSIONS: These results support an association between dysbiosis and intestinal inflammation in CFLD with improvements in both post-ETI, lending further support to the gut-liver axis in aCFLD.

Genome sequence of Microbacterium foliorum phage CandC.

We report the discovery and genome sequence of CandC, a lytic bacteriophage with siphovirus morphology. CandC was isolated from a soil sample from Plattsburgh, NY, USA (Fall 2021). It has a genome size of 62,344 bp with 106 predicted protein-encoding genes, 30 of which are assigned putative functions.

The global anaerobic metabolism regulator fnr is necessary for the degradation of food dyes and drugs by Escherichia coli.

IMPORTANCE: This work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context.

Anthropogenic impacts on tidal creek sedimentation since 1900.

Land cover and use around the margins of estuaries has shifted since 1950 at many sites in North America due to development pressures from higher population densities. Small coastal watersheds are ubiquitous along estuarine margins and most of this coastal land-cover change occurred in these tidal creek watersheds. A change in land cover could modify the contribution of sediments from tidal creek watersheds to downstream areas and affect estuarine habitats that rely on sediments to persist or are adversely impacted by sediment loading. The resilience of wetlands to accelerating relative sea-level rise depends, in part, on the supply of lithogenic sediment to support accretion and maintain elevation; however, subtidal habitats such as oyster reefs and seagrass beds are stressed under conditions of high turbidity and sedimentation. Here we compare sediment accumulation rates before and after 1950 using 210Pb in 12 tidal creeks across two distinct regions in North Carolina, one region of low relief tidal-creek watersheds where land cover change since 1959 was dominated by fluctuations in forest, silviculture, and agriculture, and another region of relatively high relief tidal-creek watersheds where land-use change was dominated by increasing suburban development. At eight of the creeks, mass accumulation rates (g cm-2 y-1) measured at the outlet of the creeks increased contemporaneously with the largest shift in land cover, within the resolution of the land-cover data set (~5-years). All but two creek sites experienced a doubling or more in sediment accumulation rates (cm yr-1) after 1950 and most sites experienced sediment accumulation rates that exceeded the rate of local relative sea-level rise, suggesting that there is an excess of sediment being delivered to these tidal creeks and that they may slowly be infilling. After 1950, land cover within one creek watershed changed little, as did mass accumulation rates at the coring location, and another creek coring site did not record an increase in mass accumulation rates at the creek outlet despite a massive increase in development in the watershed that included the construction of retention ponds. These abundant tidal-creek watersheds have little relief, area, and flow, but they are impacted by changes in land cover more, in terms of percent area, than their larger riverine counterparts, and down-stream areas are highly connected to their associated watersheds. This work expands the scientific understanding of connectivity between lower coastal plain watersheds and estuaries and provides important information for coastal zone managers seeking to balance development pressures and environmental protections.

Mismatch repair is a double-edged sword in the battle against microsatellite instability.

Roughly 3% of the human genome consists of microsatellites or tracts of short tandem repeats (STRs). These STRs are often unstable, undergoing high-frequency expansions (increases) or contractions (decreases) in the number of repeat units. Some microsatellite instability (MSI) is seen at multiple STRs within a single cell and is associated with certain types of cancer. A second form of MSI is characterised by expansion of a single gene-specific STR and such expansions are responsible for a group of 40+ human genetic disorders known as the repeat expansion diseases (REDs). While the mismatch repair (MMR) pathway prevents genome-wide MSI, emerging evidence suggests that some MMR factors are directly involved in generating expansions in the REDs. Thus, MMR suppresses some forms of expansion while some MMR factors promote expansion in other contexts. This review will cover what is known about the paradoxical effect of MMR on microsatellite expansion in mammalian cells.

Bile salt hydrolase profiling by fluorogenic probes in the human gut microbiome.

Bile is a digestive fluid produced in the liver and stored in the gallbladder. It participates in absorption of fatty nutrients and vitamins, and aids in elimination of metabolic waste and toxins. The major chemical components of bile are bile salts that, apart from their function in digestion, are also known to participate in cell signaling by binding host farnesoid X (FXR), vitamin D (VDR), and G-protein coupled bile acid (TGR5) receptors. Microbial bile salt hydrolases (BSHs) catalyze bile salt deconjugation, a gatekeeper reaction that is a prerequisite for all subsequent microbial transformations of bile acids. As a result, BSH determines the composition of the bile salt and acid pools, which in turn affects its nutrient absorption and signaling capabilities. BSH profiling remains a challenge due to a paucity of tools that enable scientists to study its function. In this chapter, we discuss current BSH profiling approaches and demonstrate a novel fluorogenic probe-based assay that circumvents laborious and resource intensive BSH quantification methods. Alongside our assay protocol, we provide the reader with a detailed method for microbial cell extraction from fecal matter. We also cover probe validation protocols that can be adapted for Michaelis-Menten analysis with any BSH expressing strain.

Profiling How the Gut Microbiome Modulates Host Xenobiotic Metabolism in Response to Benzo[a]pyrene and 1-Nitropyrene Exposure.

The gut microbiome is a key contributor to xenobiotic metabolism. Polycyclic aromatic hydrocarbons (PAHs) are an abundant class of environmental contaminants that have varying levels of carcinogenicity depending on their individual structures. Little is known about how the gut microbiome affects the rates of PAH metabolism. This study sought to determine the role that the gut microbiome has in determining the various aspects of metabolism in the liver, before and after exposure to two structurally different PAHs, benzo[a]pyrene and 1-nitropyrene. Following exposures, the metabolic rates of PAH metabolism were measured, and activity-based protein profiling was performed. We observed differences in PAH metabolism rates between germ-free and conventional mice under both unexposed and exposed conditions. Our activity-based protein profiling (ABPP) analysis showed that, under unexposed conditions, there were only minor differences in total P450 activity in germ-free mice relative to conventional mice. However, we observed distinct activity profiles in response to corn oil vehicle and PAH treatment, primarily in the case of 1-NP treatment. This study revealed that the repertoire of active P450s in the liver is impacted by the presence of the gut microbiome, which modifies PAH metabolism in a substrate-specific fashion.

Modifiers of Somatic Repeat Instability in Mouse Models of Friedreich Ataxia and the Fragile X-Related Disorders: Implications for the Mechanism of Somatic Expansion in Huntington's Disease.

Huntington's disease (HD) is one of a large group of human disorders that are caused by expanded DNA repeats. These repeat expansion disorders can have repeat units of different size and sequence that can be located in any part of the gene and, while the pathological consequences of the expansion can differ widely, there is evidence to suggest that the underlying mutational mechanism may be similar. In the case of HD, the expanded repeat unit is a CAG trinucleotide located in exon 1 of the huntingtin (HTT) gene, resulting in an expanded polyglutamine tract in the huntingtin protein. Expansion results in neuronal cell death, particularly in the striatum. Emerging evidence suggests that somatic CAG expansion, specifically expansion occurring in the brain during the lifetime of an individual, contributes to an earlier disease onset and increased severity. In this review we will discuss mouse models of two non-CAG repeat expansion diseases, specifically the Fragile X-related disorders (FXDs) and Friedreich ataxia (FRDA). We will compare and contrast these models with mouse and patient-derived cell models of various other repeat expansion disorders and the relevance of these findings for somatic expansion in HD. We will also describe additional genetic factors and pathways that modify somatic expansion in the FXD mouse model for which no comparable data yet exists in HD mice or humans. These additional factors expand the potential druggable space for diseases like HD where somatic expansion is a significant contributor to disease impact.

Activity-Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta-Lactam or Acrylamide-Based Probes.

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine- and glycine-linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much-improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity-based probes with two different reactive groups to target bile salt hydrolases. These probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.

Simple Analysis of Primary and Secondary Bile Salt Hydrolysis in Mouse and Human Gut Microbiome Samples by Using Fluorogenic Substrates.

Animals produce bile to act as an antibacterial agent and to maximize the absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also participate in signaling pathways by modulating physiological processes upon binding host receptors. Upon excretion of bile salts from the gall bladder into the intestine, the gut microbiota can create metabolites with modified signaling capabilities. The category and magnitude of bile salt metabolism can have positive or negative effects on the host. A key modification is bile salt hydrolysis, which is a prerequisite for all additional microbial transformations. We have synthesized five different fluorogenic bile salts for simple and continuous reporting of hydrolysis in both murine and human fecal samples. Our data demonstrate that most gut microbiomes have the highest capacity for hydrolysis of host-produced primary bile salts, but some microbially modified secondary bile salts also display significant turnover.

CGG Repeat Expansion, and Elevated Fmr1 Transcription and Mitochondrial Copy Number in a New Fragile X PM Mouse Embryonic Stem Cell Model.

The Fragile-X related disorders (FXDs) are Repeat Expansion Diseases (REDs) that result from expansion of a CGG-repeat tract located at the 5' end of the FMR1 gene. While expansion affects transmission risk and can also affect disease risk and severity, the underlying molecular mechanism responsible is unknown. Despite the fact that expanded alleles can be seen both in humans and mouse models in vivo, existing patient-derived cells do not show significant repeat expansions even after extended periods in culture. In order to develop a good tissue culture model for studying expansions we tested whether mouse embryonic stem cells (mESCs) carrying an expanded CGG repeat tract in the endogenous Fmr1 gene are permissive for expansion. We show here that these mESCs have a very high frequency of expansion that allows changes in the repeat number to be seen within a matter of days. CRISPR-Cas9 gene editing of these cells suggests that this may be due in part to the fact that non-homologous end-joining (NHEJ), which is able to protect against expansions in some cell types, is not effective in mESCs. CRISPR-Cas9 gene editing also shows that these expansions are MSH2-dependent, consistent with those seen in vivo. While comparable human Genome Wide Association (GWA) studies are not available for the FXDs, such studies have implicated MSH2 in expansion in other REDs. The shared unusual requirement for MSH2 for this type of microsatellite instability suggests that this new cell-based system is relevant for understanding the mechanism responsible for this peculiar type of mutation in humans. The high frequency of expansions and the ease of gene editing these cells should expedite the identification of factors that affect expansion risk. Additionally, we found that, as with cells from human premutation (PM) carriers, these cell lines have elevated mitochondrial copy numbers and Fmr1 hyperexpression, that we show here is O2-sensitive. Thus, this new stem cell model should facilitate studies of both repeat expansion and the consequences of expansion during early embryonic development.

All three mammalian MutL complexes are required for repeat expansion in a mouse cell model of the Fragile X-related disorders.

Expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene causes the fragile X-related disorders (FXDs; aka the FMR1 disorders). The expansion mechanism is likely shared by the 35+ other diseases resulting from expansion of a disease-specific microsatellite, but many steps in this process are unknown. We have shown previously that expansion is dependent upon functional mismatch repair proteins, including an absolute requirement for MutLγ, one of the three MutL heterodimeric complexes found in mammalian cells. We demonstrate here that both MutLα and MutLß, the two other MutL complexes present in mammalian cells, are also required for most, if not all, expansions in a mouse embryonic stem cell model of the FXDs. A role for MutLα and MutLß is consistent with human GWA studies implicating these complexes as modifiers of expansion risk in other Repeat Expansion Diseases. The requirement for all three complexes suggests a novel model in which these complexes co-operate to generate expansions. It also suggests that the PMS1 subunit of MutLß may be a reasonable therapeutic target in those diseases in which somatic expansion is an important disease modifier.

Probe-enabled approaches for function-dependent cell sorting and characterization of microbiome subpopulations.

Understanding the roles that individual species or communities play within a microbiome is a significant challenge. The complexity and heterogeneity of microbiomes presents a challenge to researchers looking to unravel the function that microbiomes serve within larger environments. While identification of the species and proteins present in a microbiome can be accomplished through genomics approaches, strategies that report on enzyme activity are limited. In this chapter, we describe the application of small molecule chemical probes in the isolation and subsequent characterization of microbiome subpopulations based on enzymatic function. We will cover protocols for labeling microbes with appropriate probes, microbiome sample preparation, and using fluorescence-activated cell sorting to isolate subpopulations based on function. We hope that the strategies outlined here will serve as a resource for researchers studying the functional role that microbiomes play in the gut and soil.

Programmable Topology in New Families of Heterobimetallic Metal-Organic Frameworks.

Using diverse building blocks, such as different heterometallic clusters, in metal-organic framework (MOF) syntheses greatly increases MOF complexity and leads to emergent synergistic properties. However, applying reticular chemistry to syntheses involving more than two molecular building blocks is challenging and there is limited progress in this area. We are therefore motivated to develop a strategy for achieving systematic and differential control over the coordination of multiple metals in MOFs. Herein, we report the design and synthesis of a diverse series of heterobimetallic MOFs with different metal ions and clusters severally distributed throughout two or three inorganic secondary building units (SBUs). By taking advantage of the bifunctional isonicotinate linker and its derivatives, which can coordinatively distinguish between early and late transition metals, we control the assembly and topology of up to three different inorganic SBUs in one-pot solvothermal reactions. Specifically, M6(µ3-O) n(µ3-OH)8- n(CO2)12 (M = Zr4+, Hf4+, Dy3+) SBUs are formed along with metal-pyridyl complexes. By controlling the geometry of the metal-pyridyl complexes, we direct the overall topology to produce eight new MOFs with fcu, ftw, and previously unreported trinodal pfm crystallographic nets.

Regulation of odor receptor genes in trichoid sensilla of the Drosophila antenna.

This study concerns the problem of odor receptor gene choice in the fruit fly Drosophila melanogaster. From a family of 60 Odor receptor genes, only one or a small number are selected for expression by each olfactory receptor neuron. Little is known about how an olfactory receptor neuron selects a receptor, or how the nucleotide sequences flanking a receptor gene dictate its expression in a particular neuron. Previous investigation has primarily concerned the maxillary palp, the simpler of the fly's two olfactory organs. Here we focus on genes encoding four antennal receptors that respond to fly odors in an in vivo expression system. To investigate the logic of odor receptor expression, we carry out a genetic analysis of their upstream regulatory sequences. Deletion analysis reveals that relatively short regulatory regions are sufficient to confer expression in the appropriate neurons, with limited if any misexpression. We find evidence for both positive and negative regulation. Multiple repressive functions restrict expression to the antenna, to a region of the antenna, and to neurons. Through deletion and base substitution mutagenesis we identify GCAATTA elements and find evidence that they act in both positive and negative regulation.

Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate.

Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male's strategy to suppress reproduction by competing males.

Differential experimental micrometastasis to lung, liver, and bone with lacZ-tagged CWR22R prostate carcinoma cells.

LacZ-tagged human prostate carcinoma CWR22Rv1 cells metastasize spontaneously to lung, liver, and bone from subcutaneous primary tumors in athymic nude mice; these organs are 'natural' targets of metastasis for the human disease. To evaluate the mechanism(s) of metastasis to these organs, an experimental metastasis model was used by taking advantage of the ultrasensitive detection of lacZ. Within I h after tail vein injection, micrometastases were forming in lung, liver, bone, kidney, and brain with very different quantitative levels. The kinetics of loss of unstable micrometastases and retention of stable ones were also very different in these organs. After injecting suspensions of single cells, both whole-organ and serial-section staining for lacZ revealed considerable heterogeneity in cell number of individual lung micrometastases while micrometastases in liver contained only I or 2 cells. The size of individual bone micrometastases also suggested only 1 or 2 cells. Tumor cells could also be detected in the small blood vessels of the lung within minutes after injection. These studies indicate that lacZ-tagged CWR22Rv1 cells after tissue culturing contain subsets of cells capable of establishing transient micrometastases in lung, liver, and bone after direct injection into the animal's circulation. Moreover, the quantitative and qualitative properties of the micrometastases in the three organs differ significantly, suggesting different mechanisms for stabilization and fates of micrometastases in these organs.