Machine Learning and Augmented Intelligence Enables Prognosis of Type 2 Diabetes Prior to Clinical Manifestation.

BACKGROUND: The global incidence of type 2 diabetes (T2D) persists at epidemic proportions. Early diagnosis and/or preventive efforts are critical to attenuate the multi-systemic clinical manifestation and consequent healthcare burden. Despite enormous strides in the understanding of pathophysiology and on-going therapeutic development, effectiveness and access are persistent limitations. Among the greatest challenges, the extensive research efforts have not promulgated reliable predictive biomarkers for early detection and risk assessment. The emerging fields of multi-omics combined with machine learning (ML) and augmented intelligence (AI) have profoundly impacted the capacity for predictive, preventive, and personalized medicine. OBJECTIVE: This paper explores the current challenges associated with the identification of predictive biomarkers for T2D and discusses potential actionable solutions for biomarker identification and validation. METHODS: The articles included were collected from PubMed queries. The selected topics of inquiry represented a wide range of themes in diabetes biomarker prediction and prognosis. RESULTS: The current criteria and cutoffs for T2D diagnosis are not optimal nor consider a myriad of contributing factors in terms of early detection. There is an opportunity to leverage AI and ML to significantly enhance the understanding of the underlying mechanisms of the disease and identify prognostic biomarkers. The innovative technologies being developed by GATC are expected to play a crucial role in this pursuit via algorithm training and validation, enabling comprehensive and in-depth analysis of complex biological systems. CONCLUSION: GATC is an emerging leader guiding the establishment of a systems approach towards research and predictive, personalized medicine. The integration of these technologies with clinical data can contribute to a more comprehensive understanding of T2D, paving the way for precision medicine approaches and improved patient outcomes.

A Randomized, Triple-Blind, Comparator-Controlled Parallel Study Investigating the Pharmacokinetics of Cannabidiol and Tetrahydrocannabinol in a Novel Delivery System, Solutech, in Association with Cannabis Use History.

Background: An oral route of administration for tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) eliminates the harmful effects of smoking and has potential for efficacious cannabis delivery for therapeutic and recreational applications. We investigated the pharmacokinetics of CBD, Δ9-THC, 11-OH-THC, and 11-nor-9-carboxy-Δ9-THC (THC-COOH) in a novel oral delivery system, Solutech™, compared to medium-chain triglyceride-diluted cannabis oil (MCT-oil) in a healthy population. Materials and Methods: Thirty-two participants were randomized and divided into two study arms employing a comparator-controlled, parallel-study design. To evaluate the pharmacokinetics of Δ9-THC, CBD, 11-OH-THC, and THC-COOH, blood was collected at pre-dose (t=0) and 10, 20, 30, and 45, min and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, and 48 h post-dose after a single dose of Solutech (10.0 mg Δ9-THC, 9.76 mg CBD) or MCT (10.0 mg Δ9-THC, 9.92 mg CBD). Heart rate and blood pressure were measured at 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h. Relationships between cannabis use history, body mass index, sex, and pharmacokinetic parameters were investigated. Safety was assessed before and at 48 h post-acute dose. Results: Acute consumption of Solutech provided a significantly greater maximum concentration (Cmax), larger elimination and absorption rate constants, faster time to Cmax and lag time, and half-life for all analytes compared to MCT-oil (p<0.001). In addition, cannabis use history had a significant influence on the pharmacokinetic parameters of CBD, Δ9-THC, 11-OH-THC, and THC-COOH. On average, participants with later age of first use had higher Δ9-THC, CBD, and THC-COOH Cmax and later time-to-Cmax and half-life for Δ9-THC, CBD, THC-COOH, and 11-OH-THC than those with earlier age of first use (p≤0.032). Those with more years of recreational cannabis use had higher area under the curve for Δ9-THC and CBD, Cmax for CBD, and longer 11-OH-THC half-life than those with less (p≤0.048). Conclusion: This study demonstrated that consumption of Solutech enhanced most pharmacokinetics parameters measured compared to MCT-oil. Participant's cannabis use history, including their age of first use and number of years using cannabis significantly impacted pharmacokinetic parameters investigated. Acute consumption of both products was found to be safe and well tolerated. The results suggest that Solutech may optimize bioavailability from cannabis formulations.

Yellowstone Lake: high-energy geochemistry and rich bacterial diversity.

Yellowstone Lake is central to the balanced functioning of the Yellowstone ecosystem, yet little is known about the microbial component of its food chain. A remotely operated vehicle provided video documentation (http://www.tbi.montana.edu/media/videos/) and allowed sampling of dilute surface zone waters and enriched lake floor hydrothermal vent fluids. Vent emissions contained substantial H(2)S, CH(4), CO(2) and H(2), although CH(4) and H(2) levels were also significant throughout the lake. Pyrosequencing and near full-length sequencing of Bacteria 16S rRNA gene diversity associated with two vents and two surface water environments demonstrated that this lake contains significant bacterial diversity. Biomass was size-fractionated by sequentially filtering through 20-µm-, 3.0-µm-, 0.8-µm- and 0.1-µm-pore-size filters, with the >0.1 to <0.8 µm size class being the focus of this study. Major phyla included Acidobacteria, Actinobacteria, Bacteroidetes, α- and ß-Proteobacteria and Cyanobacteria, with 21 other phyla represented at varying levels. Surface waters were dominated by two phylotypes: the Actinobacteria freshwater acI group and an α-Proteobacteria clade tightly linked with freshwater SAR11-like organisms. We also obtained evidence of novel thermophiles and recovered Prochlorococcus phylotypes (97-100% identity) in one near surface photic zone region of the lake. The combined geochemical and microbial analyses suggest that the foundation of this lake's food chain is not simple. Phototrophy presumably is an important driver of primary productivity in photic zone waters; however, chemosynthetic hydrogenotrophy and methanotrophy are likely important components of the lake's food chain.

Functional characterization and high-throughput proteomic analysis of interrupted genes in the archaeon Sulfolobus solfataricus.

Sequenced genomes often reveal interrupted coding sequences that complicate the annotation process and the subsequent functional characterization of the genes. In the past, interrupted genes were generally considered to be the result of sequencing errors or pseudogenes, that is, gene remnants with little or no biological importance. However, recent lines of evidence support the hypothesis that these coding sequences can be functional; thus, it is crucial to understand whether interrupted genes are expressed in vivo. We addressed this issue by experimentally demonstrating the existence of functional disrupted genes in archaeal genomes. We discovered previously unknown disrupted genes that have interrupted homologues in distantly related species of archaea. The combination of a RT-PCR strategy with shotgun proteomics demonstrates that interrupted genes in the archaeon Sulfolobus solfataricus are expressed in vivo. In addition, the sequence of the peptides determined by LCMSMS and experiments of in vitro translation allows us to identify a gene expressed by programmed -1 frameshifting. Our findings will enable an accurate reinterpretation of archaeal interrupted genes shedding light on their function and on archaeal genome evolution.

Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function.

The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14-15,000 Sanger reads per site) was obtained for five high-temperature (>65 degrees C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O2) control microbial community structure and function in YNP geothermal springs.

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.

Proteorhodopsin in the ubiquitous marine bacterium SAR11.

Proteorhodopsins are light-dependent proton pumps that are predicted to have an important role in the ecology of the oceans by supplying energy for microbial metabolism. Proteorhodopsin genes were first discovered through the cloning and sequencing of large genomic DNA fragments from seawater. They were later shown to be widely distributed, phylogenetically diverse, and active in the oceans. Proteorhodopsin genes have not been found in cultured bacteria, and on the basis of environmental sequence data, it has not yet been possible to reconstruct the genomes of uncultured bacterial strains that have proteorhodopsin genes. Although the metabolic effect of proteorhodopsins is uncertain, they are thought to function in cells for which the primary mode of metabolism is the heterotrophic assimilation of dissolved organic carbon. Here we report that SAR11 strain HTCC1062 ('Pelagibacter ubique'), the first cultivated member of the extraordinarily abundant SAR11 clade, expresses a proteorhodopsin gene when cultured in autoclaved seawater and in its natural environment, the ocean. The Pelagibacter proteorhodopsin functions as a light-dependent proton pump. The gene is expressed by cells grown in either diurnal light or in darkness, and there is no difference between the growth rates or cell yields of cultures grown in light or darkness.

High-throughput cultivation of microorganisms using microcapsules.

This chapter describes a universal and novel method that provides access to the immense reservoir of untapped microbial diversity by cultivation. This technique uses microcapsules to encapsulate single cells combined with parallel microbial cultivation under low nutrient flux conditions. Under these conditions, single encapsulated cells grow and form microcolonies within the microcapsules. Flow cytometry is used as a sensitive tool to detect growth within the microcapsules. Microcapsules that contain microcolonies (originated from a single encapsulated cell) are sorted individually into microtiter dishes containing organic-rich medium. This high-throughput cultivation can provide more than 10,000 bacterial and fungal isolates per environmental sample.

Genome streamlining in a cosmopolitan oceanic bacterium.

The SAR11 clade consists of very small, heterotrophic marine alpha-proteobacteria that are found throughout the oceans, where they account for about 25% of all microbial cells. Pelagibacter ubique, the first cultured member of this clade, has the smallest genome and encodes the smallest number of predicted open reading frames known for a free-living microorganism. In contrast to parasitic bacteria and archaea with small genomes, P. ubique has complete biosynthetic pathways for all 20 amino acids and all but a few cofactors. P. ubique has no pseudogenes, introns, transposons, extrachromosomal elements, or inteins; few paralogs; and the shortest intergenic spacers yet observed for any cell.

Comparative metagenomics of microbial communities.

The species complexity of microbial communities and challenges in culturing representative isolates make it difficult to obtain assembled genomes. Here we characterize and compare the metabolic capabilities of terrestrial and marine microbial communities using largely unassembled sequence data obtained by shotgun sequencing DNA isolated from the various environments. Quantitative gene content analysis reveals habitat-specific fingerprints that reflect known characteristics of the sampled environments. The identification of environment-specific genes through a gene-centric comparative analysis presents new opportunities for interpreting and diagnosing environments.

Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric.

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.

Unusual microbial xylanases from insect guts.

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Exploring nitrilase sequence space for enantioselective catalysis.

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10(6) to 10(10) members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.

An evolutionary route to xylanase process fitness.

Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90 degrees C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61 degrees C to as high as 96 degrees C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).

The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism.

The hyperthermophile Nanoarchaeum equitans is an obligate symbiont growing in coculture with the crenarchaeon Ignicoccus. Ribosomal protein and rRNA-based phylogenies place its branching point early in the archaeal lineage, representing the new archaeal kingdom Nanoarchaeota. The N. equitans genome (490,885 base pairs) encodes the machinery for information processing and repair, but lacks genes for lipid, cofactor, amino acid, or nucleotide biosyntheses. It is the smallest microbial genome sequenced to date, and also one of the most compact, with 95% of the DNA predicted to encode proteins or stable RNAs. Its limited biosynthetic and catabolic capacity indicates that N. equitans' symbiotic relationship to Ignicoccus is parasitic, making it the only known archaeal parasite. Unlike the small genomes of bacterial parasites that are undergoing reductive evolution, N. equitans has few pseudogenes or extensive regions of noncoding DNA. This organism represents a basal archaeal lineage and has a highly reduced genome.

Cultivating the uncultured.

The recent application of molecular phylogeny to environmental samples has resulted in the discovery of an abundance of unique and previously unrecognized microorganisms. The vast majority of this microbial diversity has proved refractory to cultivation. Here, we describe a universal method that provides access to this immense reservoir of untapped microbial diversity. This technique combines encapsulation of cells in gel microdroplets for massively parallel microbial cultivation under low nutrient flux conditions, followed by flow cytometry to detect microdroplets containing microcolonies. The ability to grow and study previously uncultured organisms in pure culture will enhance our understanding of microbial physiology and metabolic adaptation and will provide new sources of microbial metabolites. We show that this technology can be applied to samples from several different environments, including seawater and soil.