Variant calling enhances the identification of cancer cells in single-cell RNA sequencing data.

Single-cell RNA-sequencing is an invaluable research tool that allows for the investigation of gene expression in heterogeneous cancer cell populations in ways that bulk RNA-seq cannot. However, normal (i.e., non tumor) cells in cancer samples have the potential to confound the downstream analysis of single-cell RNA-seq data. Existing methods for identifying cancer and normal cells include copy number variation inference, marker-gene expression analysis, and expression-based clustering. This work aims to extend the existing approaches for identifying cancer cells in single-cell RNA-seq samples by incorporating variant calling and the identification of putative driver alterations. We found that putative driver alterations can be detected in single-cell RNA-seq data obtained with full-length transcript technologies and noticed that a subset of cells in tumor samples are enriched for putative driver alterations as compared to normal cells. Furthermore, we show that the number of putative driver alterations and inferred copy number variation are not correlated in all samples. Taken together, our findings suggest that augmenting existing cancer-cell filtering methods with variant calling and analysis can increase the number of tumor cells that can be confidently included in downstream analyses of single-cell full-length transcript RNA-seq datasets.

Gut Site and Gut Morphology Predict Microbiome Structure and Function in Ecologically Diverse Lemurs.

Most studies of wildlife gut microbiotas understandably rely on feces to approximate consortia along the gastrointestinal tract. We therefore compared microbiome structure and predicted metagenomic function in stomach, small intestinal, cecal, and colonic samples from 52 lemurs harvested during routine necropsies. The lemurs represent seven genera (Cheirogaleus, Daubentonia, Varecia, Hapalemur, Eulemur, Lemur, Propithecus) characterized by diverse feeding ecologies and gut morphologies. In particular, the hosts variably depend on fibrous foodstuffs and show correlative morphological complexity in their large intestines. Across host lineages, microbiome diversity, variability, membership, and function differed between the upper and lower gut, reflecting regional tradeoffs in available nutrients. These patterns related minimally to total gut length but were modulated by fermentation capacity (i.e., the ratio of small to large intestinal length). Irrespective of feeding strategy, host genera with limited fermentation capacity harbored more homogenized microbiome diversity along the gut, whereas those with expanded fermentation capacity harbored cecal and colonic microbiomes with greater diversity and abundant fermentative Ruminococcaceae taxa. While highlighting the value of curated sample repositories for retrospective comparisons, our results confirm that the need to survive on fibrous foods, either routinely or in hypervariable environments, can shape the morphological and microbial features of the lower gut.

Michaelis-like complex of mouse ketohexokinase isoform C.

Over the past forty years there has been a drastic increase in fructose-related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP-dependent phosphorylation of fructose to fructose 1-phosphate. Understanding the role of KHK in disease-related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure-function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK-A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK-C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK-A and KHK-C are known, as well as structures of unliganded and inhibitor-bound mouse KHK-C (mKHK-C), which shares 90% sequence identity with human KHK-C. Here, a high-resolution X-ray crystal structure of mKHK-C refined to 1.79 Šresolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis-like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the ß-sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose-related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground-state, Michaelis-like complex suggests that a conformational change plays a role in the catalytic function of KHK-C.

Antibiotic-induced gut dysbiosis elicits gut-brain axis relevant multi-omic signatures and behavioral and neuroendocrine changes in a nonhuman primate model.

Emerging evidence indicates that antibiotic-induced dysbiosis can play an etiological role in the pathogenesis of neuropsychiatric disorders. However, most of this evidence comes from rodent models. The objective of this study was to evaluate if antibiotic-induced gut dysbiosis can elicit changes in gut metabolites and behavior indicative of gut-brain axis disruption in common marmosets (Callithrix jacchus) - a nonhuman primate model often used to study sociability and stress. We were able to successfully induce dysbiosis in marmosets using a custom antibiotic cocktail (vancomycin, enrofloxacin and neomycin) administered orally for 28 days. This gut dysbiosis altered gut metabolite profiles, behavior, and stress reactivity. Increase in gut Fusobacterium spp. post-antibiotic administration was a novel dysbiotic response and has not been observed in any rodent or human studies to date. There were significant changes in concentrations of several gut metabolites which are either neurotransmitters (e.g., GABA and serotonin) or have been found to be moderators of gut-brain axis communication in rodent models (e.g., short-chain fatty acids and bile acids). There was an increase in affiliative behavior and sociability in antibiotic-administered marmosets, which might be a coping mechanism in response to gut dysbiosis-induced stress. Increase in urinary cortisol levels after multiple stressors provides more definitive proof that this model of dysbiosis may cause disrupted communication between gut and brain in common marmosets. This study is a first attempt to establish common marmosets as a novel model to study the impact of severe gut dysbiosis on gut-brain axis cross-talk and behavior.

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

Exploring the Readability of Ingredients Lists of Food Labels with Existing Metrics.

Healthy diet and dietary behaviors are key components in prevention of chronic disease and management of chronic illness. Nutritional literacy has been associated with dietary behaviors and consumer choice of healthy foods. Nutritional literacy can be measured, for example, by examining consumer food label use, but current research focuses largely on the Nutrition Facts panel of a food product. Ingredients lists are critical for communicating food composition but are relatively unstudied in existing literature. The goal of this work is to measure the readability of ingredients lists on branded food products in the United States using existing metrics. We examined ingredients lists for all 495,646 products listed in the USDA Food Data Central database using four existing readability measures for text written in natural language. Each of these indices approximates the grade level that would be expected to comprehend a text; comparatively, patient consent forms are considered acceptable at an 8th grade reading level or lower. We report a broad variability for in readability using different metrics: ingredients lists recorded at a 9th grade reading level or higher to comprehend are found at rates of 16.5% (Automated Reading Index) to 74.9% (Gunning-Fog Index). Ingredients lists recorded at a 10th grade reading level or higher to comprehend are found at rates of 84.2% (using FRE Index). These results demonstrate the need to further explore how ingredients lists can be measured for readability, both for the purposes of consumer understanding as well as for supporting future nutrition research involving text mining.

Host Identity and Geographic Location Significantly Affect Gastrointestinal Microbial Richness and Diversity in Western Lowland Gorillas (Gorilla gorilla gorilla) under Human Care.

The last few decades have seen an outpouring of gastrointestinal (GI) microbiome studies across diverse host species. Studies have ranged from assessments of GI microbial richness and diversity to classification of novel microbial lineages. Assessments of the "normal" state of the GI microbiome composition across multiple host species has gained increasing importance for distinguishing healthy versus diseased states. This study aimed to determine baselines and trends over time to establish "typical" patterns of GI microbial richness and diversity, as well as inter-individual variation, in three populations of western lowland gorillas (Gorilla gorilla gorilla) under human care at three zoological institutions in North America. Fecal samples were collected from 19 western lowland gorillas every two weeks for seven months (n = 248). Host identity and host institution significantly affected GI microbiome community composition (p < 0.05), although host identity had the most consistent and significant effect on richness (p = 0.03) and Shannon diversity (p = 0.004) across institutions. Significant changes in microbial abundance over time were observed only at Denver Zoo (p < 0.05). Our results suggest that individuality contributes to most of the observed GI microbiome variation in the study populations. Our results also showed no significant changes in any individual's microbial richness or Shannon diversity during the 7-month study period. While some microbial taxa (Prevotella, Prevotellaceae and Ruminococcaceae) were detected in all gorillas at varying levels, determining individual baselines for microbial composition comparisons may be the most useful diagnostic tool for optimizing non-human primate health under human care.

Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: identifying ChIP-quality p300 monoclonal antibodies.

Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physiological states call for higher throughput and consistency afforded by automation. Such automation advances, when provided by multiuser facilities, could also improve the quality and efficiency of individual small-scale projects. The immunoprecipitation process has become rate-limiting, and is a source of substantial variability when performed manually. Here we report a fully automated robotic ChIP (R-ChIP) pipeline that allows up to 96 reactions. A second bottleneck is the dearth of renewable ChIP-validated immune reagents, which do not yet exist for most mammalian transcription factors. We used R-ChIP to screen new mouse monoclonal antibodies raised against p300, a histone acetylase, well-known as a marker of active enhancers, for which ChIP-competent monoclonal reagents have been lacking. We identified, validated for ChIP-seq, and made publicly available a monoclonal reagent called ENCITp300-1.

X-ray crystal structure of the UCS domain-containing UNC-45 myosin chaperone from Drosophila melanogaster.

UCS proteins, such as UNC-45, influence muscle contraction and other myosin-dependent motile processes. We report the first X-ray crystal structure of a UCS domain-containing protein, the UNC-45 myosin chaperone from Drosophila melanogaster (DmUNC-45). The structure reveals that the central and UCS domains form a contiguous arrangement of 17 consecutive helical layers that arrange themselves into five discrete armadillo repeat subdomains. Small-angle X-ray scattering data suggest that free DmUNC-45 adopts an elongated conformation and exhibits flexibility in solution. Protease sensitivity maps to a conserved loop that contacts the most carboxy-terminal UNC-45 armadillo repeat subdomain. Amino acid conservation across diverse UCS proteins maps to one face of this carboxy-terminal subdomain, and the majority of mutations that affect myosin-dependent cellular activities lie within or around this region. Our crystallographic, biophysical, and biochemical analyses suggest that DmUNC-45 function is afforded by its flexibility and by structural integrity of its UCS domain.