Mapping microhabitats of lignocellulose decomposition by a microbial consortium.

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

Proteome Mapping of the Human Pancreatic Islet Microenvironment Reveals Endocrine-Exocrine Signaling Sphere of Influence.

The need for a clinically accessible method with the ability to match protein activity within heterogeneous tissues is currently unmet by existing technologies. Our proteomics sample preparation platform, named microPOTS (Microdroplet Processing in One pot for Trace Samples), can be used to measure relative protein abundance in micron-scale samples alongside the spatial location of each measurement, thereby tying biologically interesting proteins and pathways to distinct regions. However, given the smaller pixel/voxel number and amount of tissue measured, standard mass spectrometric analysis pipelines have proven inadequate. Here we describe how existing computational approaches can be adapted to focus on the specific biological questions asked in spatial proteomics experiments. We apply this approach to present an unbiased characterization of the human islet microenvironment comprising the entire complex array of cell types involved while maintaining spatial information and the degree of the islet's sphere of influence. We identify specific functional activity unique to the pancreatic islet cells and demonstrate how far their signature can be detected in the adjacent tissue. Our results show that we can distinguish pancreatic islet cells from the neighboring exocrine tissue environment, recapitulate known biological functions of islet cells, and identify a spatial gradient in the expression of RNA processing proteins within the islet microenvironment.

Computational tools and algorithms for ion mobility spectrometry-mass spectrometry.

Ion mobility spectrometry-mass spectrometry (IMS-MS or IM-MS) is a powerful analytical technique that combines the gas-phase separation capabilities of IM with the identification and quantification capabilities of MS. IM-MS can differentiate molecules with indistinguishable masses but different structures (e.g., isomers, isobars, molecular classes, and contaminant ions). The importance of this analytical technique is reflected by a staged increase in the number of applications for molecular characterization across a variety of fields, from different MS-based omics (proteomics, metabolomics, lipidomics, etc.) to the structural characterization of glycans, organic matter, proteins, and macromolecular complexes. With the increasing application of IM-MS there is a pressing need for effective and accessible computational tools. This article presents an overview of the most recent free and open-source software tools specifically tailored for the analysis and interpretation of data derived from IM-MS instrumentation. This review enumerates these tools and outlines their main algorithmic approaches, while highlighting representative applications across different fields. Finally, a discussion of current limitations and expectable improvements is presented.

A fast and sensitive size-exclusion chromatography method for plasma extracellular vesicle proteomic analysis.

Extracellular vesicles (EVs) carry diverse biomolecules derived from their parental cells, making their components excellent biomarker candidates. However, purifying EVs is a major hurdle in biomarker discovery since current methods require large amounts of samples, are time-consuming and typically have poor reproducibility. Here we describe a simple, fast, and sensitive EV fractionation method using size exclusion chromatography (SEC) on a fast protein liquid chromatography (FPLC) system. Our method uses a Superose 6 Increase 5/150, which has a bed volume of 2.9 mL. The FPLC system and small column size enable reproducible separation of only 50 µL of human plasma in 15 min. To demonstrate the utility of our method, we used longitudinal samples from a group of individuals who underwent intense exercise. A total of 838 proteins were identified, of which, 261 were previously characterized as EV proteins, including classical markers, such as cluster of differentiation (CD)9 and CD81. Quantitative analysis showed low technical variability with correlation coefficients greater than 0.9 between replicates. The analysis captured differences in relevant EV proteins involved in response to physical activity. Our method enables fast and sensitive fractionation of plasma EVs with low variability, which will facilitate biomarker studies in large clinical cohorts.

Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer's Disease Across Distinct Brain Regions.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex etiology influenced by confounding factors such as genetic polymorphisms, age, sex, and race. Traditionally, AD research has not prioritized these influences, resulting in dramatically skewed cohorts such as three times the number of Apolipoprotein E (APOE) ε4-allele carriers in AD relative to healthy cohorts. Thus, the resulting molecular changes in AD have previously been complicated by the influence of apolipoprotein E disparities. To explore how apolipoprotein E polymorphism influences AD progression, 62 post-mortem patients consisting of 33 AD and 29 controls (Ctrl) were studied to balance the number of ε4-allele carriers and facilitate a molecular comparison of the apolipoprotein E genotype. Lipid and protein perturbations were assessed across AD diagnosed brains compared to Ctrl brains, ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s and APOE4- for non-ε4 carriers), and differences in ε3ε3 and ε3ε4 Ctrl brains across two brain regions (frontal cortex (FCX) and cerebellum (CBM)). The region-specific influences of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction and cell proteostasis at the core of AD pathophysiology in the post-mortem brains, indicating these two processes may be influenced by genotypic differences and brain morphology.

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment.

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 µm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

Assessing Digital Breast Tomosynthesis Impact on Early Cancer Detection: Insights from Consecutive Screening.

Background Analysis of how digital breast tomosynthesis (DBT) screening affects consecutive screening performance is important to estimate its future value in screening. Purpose To evaluate whether DBT contributes to early detection of breast cancer by assessing cancer detection rates (CDRs), including the fraction of invasive cancers and cancer subtypes in consecutive routine digital mammography (DM). Materials and Methods The paired prospective Malmö Breast Tomosynthesis Screening Trial (MBTST) was performed between January 2010 and February 2015. Participating women underwent one-view DBT and two-view DM at one screening occasion. In this secondary analysis, women were followed up through their first (DM1) and second (DM2) consecutive two-view DM screening rounds after MBTST participation. Cancer diagnoses were identified by referencing records. A logistic regression model, adjusted for age, was used to calculate the odds of luminal A-like cancers with use of the MBTST as reference. Results There were 14 848 final participants in the MBTST (median age, 57 years [IQR, 49-65 years]). Of those, 12 876 women were screened in DM1 (median age, 58 years [IQR, 50-66 years]) and 10 883 were screened in DM2 (median age, 59 years [IQR, 51-67 years]). Compared with CDRs in the trial of 6.5 of 1000 women (95% CI: 5.2, 7.9) for DM and 8.7 of 1000 women (95% CI: 7.3, 10.3) for DBT, the CDR was lower in DM1 (4.6 of 1000 women [95% CI: 3.6, 5.9]) and DM2 (5.3 of 1000 women [95% CI: 4.1, 6.9]). The proportion of invasive cancers was 84.9% (118 of 139 cancers) in the MBTST; the corresponding numbers were 66% (39 of 59 cancers) for DM1 and 83% (50 of 60 cancers) for DM2. The odds of luminal A-like cancers were lower in DM1 at 0.28 (95% CI: 0.12, 0.66 [P = .004]) but not in DM2 at 0.80 (95% CI: 0.40, 1.58 [P = .52]) versus screening in the MBTST. Conclusion CDR and the fraction of invasive cancers were lower in DM1 and then increased in DM2 following the MBTST, indicating earlier cancer detection mainly due to increased detection of luminal A-like cancers in DBT screening. Clinical trials registration no. NCT01091545 © RSNA, 2024 See also the editorial by Hooley and Philpotts in this issue.

Regulation of ß-cell death by ADP-ribosylhydrolase ARH3 via lipid signaling in insulitis.

BACKGROUND: Lipids are regulators of insulitis and ß-cell death in type 1 diabetes development, but the underlying mechanisms are poorly understood. Here, we investigated how the islet lipid composition and downstream signaling regulate ß-cell death. METHODS: We performed lipidomics using three models of insulitis: human islets and EndoC-ßH1 ß cells treated with the pro-inflammatory cytokines interlukine-1ß and interferon-γ, and islets from pre-diabetic non-obese mice. We also performed mass spectrometry and fluorescence imaging to determine the localization of lipids and enzyme in islets. RNAi, apoptotic assay, and qPCR were performed to determine the role of a specific factor in lipid-mediated cytokine signaling. RESULTS: Across all three models, lipidomic analyses showed a consistent increase of lysophosphatidylcholine species and phosphatidylcholines with polyunsaturated fatty acids and a reduction of triacylglycerol species. Imaging assays showed that phosphatidylcholines with polyunsaturated fatty acids and their hydrolyzing enzyme phospholipase PLA2G6 are enriched in islets. In downstream signaling, omega-3 fatty acids reduce cytokine-induced ß-cell death by improving the expression of ADP-ribosylhydrolase ARH3. The mechanism involves omega-3 fatty acid-mediated reduction of the histone methylation polycomb complex PRC2 component Suz12, upregulating the expression of Arh3, which in turn decreases cell apoptosis. CONCLUSIONS: Our data provide insights into the change of lipidomics landscape in ß cells during insulitis and identify a protective mechanism by omega-3 fatty acids. Video Abstract.

New Views of Old Proteins: Clarifying the Enigmatic Proteome.

All human diseases involve proteins, yet our current tools to characterize and quantify them are limited. To better elucidate proteins across space, time, and molecular composition, we provide a >10 years of projection for technologies to meet the challenges that protein biology presents. With a broad perspective, we discuss grand opportunities to transition the science of proteomics into a more propulsive enterprise. Extrapolating recent trends, we describe a next generation of approaches to define, quantify, and visualize the multiple dimensions of the proteome, thereby transforming our understanding and interactions with human disease in the coming decade.

Impact of breast density on diagnostic accuracy in digital breast tomosynthesis versus digital mammography: results from a European screening trial.

BACKGROUND: The diagnostic accuracy of digital breast tomosynthesis (DBT) and digital mammography (DM) in breast cancer screening may vary per breast density subgroup. The purpose of this study was to evaluate which women, based on automatically assessed breast density subgroups, have the greatest benefit of DBT compared with DM in the prospective Malmö Breast Tomosynthesis Screening Trial. MATERIALS AND METHODS: The prospective European, Malmö Breast Tomosynthesis Screening Trial (n = 14,848, Jan. 27, 2010-Feb. 13, 2015) compared one-view DBT and two-view DM, with consensus meeting before recall. Breast density was assessed in this secondary analysis with the automatic software Laboratory for Individualized Breast Radiodensity Assessment. DBT and DM's diagnostic accuracies were compared by breast density quintiles of breast percent density (PD) and absolute dense area (DA) with confidence intervals (CI) and McNemar's test. The association between breast density and cancer detection was analyzed with logistic regression, adjusted for ages < 55 and ≥ 55 years and previous screening participation. RESULTS: In total, 14,730 women (median age: 58 years; inter-quartile range = 16) were included in the analysis. Sensitivity was higher and specificity lower for DBT compared with DM for all density subgroups. The highest breast PD quintile showed the largest difference in sensitivity and specificity at 81.1% (95% CI 65.8-90.5) versus 43.2% (95% CI 28.7-59.1), p < .001 and 95.5% (95% CI 94.7-96.2) versus 97.2% (95% CI 96.6-97.8), p < 0.001, respectively. Breast PD quintile was also positively associated with cancer detected via DBT at odds ratio 1.24 (95% CI 1.09-1.42, p = 0.001). CONCLUSION: Women with the highest breast density had the greatest benefit from digital breast tomosynthesis compared with digital mammography with increased sensitivity at the cost of slightly lower specificity. These results may influence digital breast tomosynthesis's use in an individualized screening program stratified by, for instance, breast density. TRIAL REGISTRATION: Trial registration at https://www. CLINICALTRIALS: gov : NCT01091545, registered March 24, 2010.

Biomarkers for rapid H-reflex operant conditioning among females.

Operant conditioning of a spinal monosynaptic pathway using the Hoffman reflex (H-reflex) is well established in animal and human studies. There is a subset within the human population (∼20% nonresponders) who are unable to up train this pathway suggesting some distinct or unique identifying characteristics. Importantly, females, who have a nine times higher rate of injury during human performance activities than men, have been understudied in areas of CNS neuroplasticity. Our long-term goal is to understand if innate ability to rapidly up train the H-reflex is predictive of future performance-based injury among females. In this study, we primarily determined whether healthy, young females could rapidly increase the H-reflex within a single session of operant conditioning and secondarily determined if electro-physiological, humoral, cognitive, anthropometric, or anxiety biomarkers distinguished the responders from nonresponders. Eighteen females (mean age: 24) participated in the study. Overall, females showed a group main effect for up training the H-reflex (P < 0.05). Of the cohort, 10 of 18 females met the criteria for up training the H-reflex (responders). The responders showed lower levels of estradiol (P < 0.05). A multivariate stepwise regression model supported that extracellular to intracellular water ratio (ECW/ICW) and H-max/M-max ratio explained 60% of the variation in up training among females. These findings support that females can acutely upregulate the H-reflex with training and that electro-physiological and hormonal factors may be associated with the up training.NEW & NOTEWORTHY Young females who acutely increase their H-reflexes with operant conditioning had lower levels of estradiol. However, the best predictors of those who could up-train the H-reflex were baseline H-reflex excitability (H-max/M-max) and extracellular to intracellular water ratio (ECW/ICW). Future studies are warranted to understand the complex relationship between operant conditioning, human performance, and injury among active young females.

Trial of Sodium Phenylbutyrate-Taurursodiol for Amyotrophic Lateral Sclerosis.

BACKGROUND: Sodium phenylbutyrate and taurursodiol have been found to reduce neuronal death in experimental models. The efficacy and safety of a combination of the two compounds in persons with amyotrophic lateral sclerosis (ALS) are not known. METHODS: In this multicenter, randomized, double-blind trial, we enrolled participants with definite ALS who had had an onset of symptoms within the previous 18 months. Participants were randomly assigned in a 2:1 ratio to receive sodium phenylbutyrate-taurursodiol (3 g of sodium phenylbutyrate and 1 g of taurursodiol, administered once a day for 3 weeks and then twice a day) or placebo. The primary outcome was the rate of decline in the total score on the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R; range, 0 to 48, with higher scores indicating better function) through 24 weeks. Secondary outcomes were the rates of decline in isometric muscle strength, plasma phosphorylated axonal neurofilament H subunit levels, and the slow vital capacity; the time to death, tracheostomy, or permanent ventilation; and the time to death, tracheostomy, permanent ventilation, or hospitalization. RESULTS: A total of 177 persons with ALS were screened for eligibility, and 137 were randomly assigned to receive sodium phenylbutyrate-taurursodiol (89 participants) or placebo (48 participants). In a modified intention-to-treat analysis, the mean rate of change in the ALSFRS-R score was -1.24 points per month with the active drug and -1.66 points per month with placebo (difference, 0.42 points per month; 95% confidence interval, 0.03 to 0.81; P = 0.03). Secondary outcomes did not differ significantly between the two groups. Adverse events with the active drug were mainly gastrointestinal. CONCLUSIONS: Sodium phenylbutyrate-taurursodiol resulted in slower functional decline than placebo as measured by the ALSFRS-R score over a period of 24 weeks. Secondary outcomes were not significantly different between the two groups. Longer and larger trials are necessary to evaluate the efficacy and safety of sodium phenylbutyrate-taurursodiol in persons with ALS. (Funded by Amylyx Pharmaceuticals and others; CENTAUR ClinicalTrials.gov number, NCT03127514.).

Identification of a specific exporter that enables high production of aconitic acid in Aspergillus pseudoterreus.

Aconitic acid is an unsaturated tricarboxylic acid that is attractive for its potential use in manufacturing biodegradable and biocompatible polymers, plasticizers, and surfactants. Previously Aspergillus pseudoterreus was engineered as a platform to produce aconitic acid by deleting the cadA (cis-aconitic acid decarboxylase) gene in the itaconic acid biosynthetic pathway. In this study, the aconitic acid transporter gene (aexA) was identified using comparative global discovery proteomics analysis between the wild-type and cadA deletion strains. The protein AexA belongs to the Major Facilitator Superfamily (MFS). Deletion of aexA almost abolished aconitic acid secretion, while its overexpression led to a significant increase in aconitic acid production. Transportation of aconitic acid across the plasma membrane is a key limiting step in its production. In vitro, proteoliposome transport assay further validated AexA's function and substrate specificity. This research provides new approaches to efficiently pinpoint and characterize exporters of fungal organic acids and accelerate metabolic engineering to improve secretion capability and lower the cost of bioproduction.

Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate.

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomics and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4 g/L 3HP production. This represents one of the highest 3HP titers reported in yeast from lignocellulosic feedstocks. This work establishes R. toruloides as a host for 3HP production from lignocellulosic hydrolysate at high titers, and paves the way for further strain and process optimization towards enabling industrial production of 3HP in the future.

Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440.

Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional ß-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.

False-positive recalls in the prospective Malmö Breast Tomosynthesis Screening Trial.

OBJECTIVES: To evaluate the total number of false-positive recalls, including radiographic appearances and false-positive biopsies, in the Malmö Breast Tomosynthesis Screening Trial (MBTST). METHODS: The prospective, population-based MBTST, with 14,848 participating women, was designed to compare one-view digital breast tomosynthesis (DBT) to two-view digital mammography (DM) in breast cancer screening. False-positive recall rates, radiographic appearances, and biopsy rates were analyzed. Comparisons were made between DBT, DM, and DBT + DM, both in total and in trial year 1 compared to trial years 2 to 5, with numbers, percentages, and 95% confidence intervals (CI). RESULTS: The false-positive recall rate was higher with DBT, 1.6% (95% CI 1.4; 1.8), compared to screening with DM, 0.8% (95% CI 0.7; 1.0). The proportion of the radiographic appearance of stellate distortion was 37.3% (91/244) with DBT, compared to 24.0% (29/121) with DM. The false-positive recall rate with DBT during trial year 1 was 2.6% (95% CI 1.8; 3.5), then stabilized at 1.5% (95% CI 1.3; 1.8) during trial years 2 to 5. The percentage of stellate distortion with DBT was 50% (19/38) trial year 1 compared to 35.0% (72/206) trial years 2 to 5. CONCLUSIONS: The higher false-positive recall rate with DBT compared to DM was mainly due to an increased detection of stellate findings. The proportion of these findings, as well as the DBT false-positive recall rate, was reduced after the first trial year. CLINICAL RELEVANCE STATEMENT: Assessment of false-positive recalls gives information on potential benefits and side effects in DBT screening. KEY POINTS: • The false-positive recall rate in a prospective digital breast tomosynthesis screening trial was higher compared to digital mammography, but still low compared to other trials. • The higher false-positive recall rate with digital breast tomosynthesis was mainly due to an increased detection of stellate findings; the proportion of these findings was reduced after the first trial year.

Engineering transcriptional regulation of pentose metabolism in Rhodosporidium toruloides for improved conversion of xylose to bioproducts.

Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R. toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R. toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R. toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.

Effect of 5 years of exercise training on the cardiovascular risk profile of older adults: the Generation 100 randomized trial.

AIMS: The aim of this study was to compare the effects of 5 years of supervised exercise training (ExComb), and the differential effects of subgroups of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT), with control on the cardiovascular risk profile in older adults. METHODS AND RESULTS: Older adults aged 70-77 years from Trondheim, Norway (n = 1567, 50% women), able to safely perform exercise training were randomized to 5 years of two weekly sessions of HIIT [∼90% of peak heart rate (HR), n = 400] or MICT (∼70% of peak HR, n = 387), together forming ExComb (n = 787), or control (instructed to follow physical activity recommendations, n = 780). The main outcome was a continuous cardiovascular risk score (CCR), individual cardiovascular risk factors, and peak oxygen uptake (VO2peak). CCR was not significantly lower [-0.19, 99% confidence interval (CI) -0.46 to 0.07] and VO2peak was not significantly higher (0.39 mL/kg/min, 99% CI -0.22 to 1.00) for ExComb vs. control. HIIT showed higher VO2peak (0.76 mL/kg/min, 99% CI 0.02-1.51), but not lower CCR (-0.32, 99% CI -0.64 to 0.01) vs. control. MICT did not show significant differences compared to control or HIIT. Individual risk factors mostly did not show significant between-group differences, with some exceptions for HIIT being better than control. There was no significant effect modification by sex. The number of cardiovascular events was similar across groups. The healthy and fit study sample, and contamination and cross-over between intervention groups, challenged the possibility of detecting between-group differences. CONCLUSIONS: Five years of supervised exercise training in older adults had little effect on cardiovascular risk profile and did not reduce cardiovascular events. REGISTRATION: ClinicalTrials.gov: NCT01666340.

Uncovering Hidden Members and Functions of the Soil Microbiome Using De Novo Metaproteomics.

Metaproteomics has been increasingly utilized for high-throughput characterization of proteins in complex environments and has been demonstrated to provide insights into microbial composition and functional roles. However, significant challenges remain in metaproteomic data analysis, including creation of a sample-specific protein sequence database. A well-matched database is a requirement for successful metaproteomics analysis, and the accuracy and sensitivity of PSM identification algorithms suffer when the database is incomplete or contains extraneous sequences. When matched DNA sequencing data of the sample is unavailable or incomplete, creating the proteome database that accurately represents the organisms in the sample is a challenge. Here, we leverage a de novo peptide sequencing approach to identify the sample composition directly from metaproteomic data. First, we created a deep learning model, Kaiko, to predict the peptide sequences from mass spectrometry data and trained it on 5 million peptide-spectrum matches from 55 phylogenetically diverse bacteria. After training, Kaiko successfully identified organisms from soil isolates and synthetic communities directly from proteomics data. Finally, we created a pipeline for metaproteome database generation using Kaiko. We tested the pipeline on native soils collected in Kansas, showing that the de novo sequencing model can be employed as an alternative and complementary method to construct the sample-specific protein database instead of relying on (un)matched metagenomes. Our pipeline identified all highly abundant taxa from 16S rRNA sequencing of the soil samples and uncovered several additional species which were strongly represented only in proteomic data.

Quantitative analysis of transcriptome dynamics provides novel insights into developmental state transitions.

BACKGROUND: During embryogenesis, the developmental potential of initially pluripotent cells becomes progressively restricted as they transit to lineage restricted states. The pluripotent cells of Xenopus blastula-stage embryos are an ideal system in which to study cell state transitions during developmental decision-making, as gene expression dynamics can be followed at high temporal resolution. RESULTS: Here we use transcriptomics to interrogate the process by which pluripotent cells transit to four different lineage-restricted states: neural progenitors, epidermis, endoderm and ventral mesoderm, providing quantitative insights into the dynamics of Waddington's landscape. Our findings provide novel insights into why the neural progenitor state is the default lineage state for pluripotent cells and uncover novel components of lineage-specific gene regulation. These data reveal an unexpected overlap in the transcriptional responses to BMP4/7 and Activin signaling and provide mechanistic insight into how the timing of signaling inputs such as BMP are temporally controlled to ensure correct lineage decisions. CONCLUSIONS: Together these analyses provide quantitative insights into the logic and dynamics of developmental decision making in early embryos. They also provide valuable lineage-specific time series data following the acquisition of specific lineage states during development.