Research on the human microbiome, the microbiota that live in, on, and around the human person, has revolutionized our understanding of the complex interactions between microbial life and human ...health and disease. The microbiome may also provide a valuable tool in forensic death investigations by helping to reveal the postmortem interval (PMI) of a decedent that is discovered after an unknown amount of time since death. Current methods of estimating PMI for cadavers discovered in uncontrolled, unstudied environments have substantial limitations, some of which may be overcome through the use of microbial indicators. In this project, we sampled the microbiomes of decomposing human cadavers, focusing on the skin microbiota found in the nasal and ear canals. We then developed several models of statistical regression to establish an algorithm for predicting the PMI of microbial samples. We found that the complete data set, rather than a curated list of indicator species, was preferred for training the regressor. We further found that genus and family, rather than species, are the most informative taxonomic levels. Finally, we developed a k-nearest- neighbor regressor, tuned with the entire data set from all nasal and ear samples, that predicts the PMI of unknown samples with an average error of ±55 accumulated degree days (ADD). This study outlines a machine learning approach for the use of necrobiome data in the prediction of the PMI and thereby provides a successful proof-of- concept that skin microbiota is a promising tool in forensic death investigations.
The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and ...metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism
. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB
, EccC
, and EccE
and two copies of the EccD
protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.
Direct evidence in humans for the impact of the microbiome on nutrient absorption is lacking. We conducted an extended inpatient study using two interventions that we hypothesized would alter the gut ...microbiome and nutrient absorption. In each, stool calorie loss, a direct proxy of nutrient absorption, was measured. The first phase was a randomized cross-over dietary intervention in which all participants underwent in random order 3 d of over- and underfeeding. The second was a randomized, double-blind, placebo-controlled pharmacologic intervention using oral vancomycin or matching placebo (NCT02037295). Twenty-seven volunteers (17 men and 10 women, age 35.1 ± 7.3, BMI 32.3 ± 8.0), who were healthy other than having impaired glucose tolerance and obesity, were enrolled and 25 completed the entire trial. The primary endpoints were the effects of dietary and pharmacological intervention on stool calorie loss. We hypothesized that stool calories expressed as percentage of caloric intake would increase with underfeeding compared with overfeeding and increase during oral vancomycin treatment. Both primary endpoints were met. Greater stool calorie loss was observed during underfeeding relative to overfeeding and during vancomycin treatment compared with placebo. Key secondary endpoints were to evaluate the changes in gut microbial community structure as evidenced by amplicon sequencing and metagenomics. We observed only a modest perturbation of gut microbial community structure with under- versus overfeeding but a more widespread change in community structure with reduced diversity with oral vancomycin. Increase in Akkermansia muciniphila was common to both interventions that resulted in greater stool calorie loss. These results indicate that nutrient absorption is sensitive to environmental perturbations and support the translational relevance of preclinical models demonstrating a possible causal role for the gut microbiome in dietary energy harvest.
Mutations in transporters can impact an individual’s response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine ...saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.
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•Deep mutational scan of OCT1 reveals the determinants of biogenesis and substrate uptake•Discovery of a conserved motif, the stability helix, essential to SLC22 biogenesis•AI structure prediction and mutational data for accurate structure-function modeling•Integrating genomic-health records reveals variants effect on human physiology
Yee et al. create a complete functional map of how genetic variants in the OCT1 transporter alter biogenesis and substrate uptake. This work provides a framework for biophysically informed precision medicine by integrating functional genomics, mechanistic modeling, and human genetics to predict variant effects on disease and drug efficacy.
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