Philip D. Charles

Missing values (MVs) remain a significant barrier to reliable proteomics analysis, particularly in single-cell proteomics, where small amounts of starting material and limits in detection drive Missing-Not-At-Random (MNAR) sparsity. Commonly used bulk proteomic imputation approaches typically address Missing-At-Random (MAR) MVs and improve replicate consistency at the expense of sensitivity for biological variation, whereas MNAR-specific strategies preserve group differences but compromise cross-replicate reproducibility. Existing imputation methods are commonly applied to bulk data and do not offer a generalised ‘off-the-shelf’ implementation that robustly addresses the significant sparsity observed in single-cell studies. Here, we introduce SoftHybrid, a continuous weighting framework that automatically balances MAR- and MNAR-oriented imputation across a dataset-derived model between missing rate and protein abundance model. Benchmarking across known ground truth samples (three-species mix) and real single-cell proteomics data showed that SoftHybrid outperforms existing methods at low inputs while meeting or exceeding previous state-of-the-art performance at the mini-bulk level. Preservation of proteomic patterns and enhanced replicate consistency improve testing significance and boost recovery of biological signals. SoftHybrid is implemented as an R package and freely available on GitHub.

Background: White adipose tissue dysfunction has emerged as a critical factor in cardiometabolic disease development, yet the cellular microstructure and genetic architecture of adipocyte morphology remain poorly explored. Methods: We introduce Adipocyte U-Net 2.0, an advanced deep learning method for the semantic segmentation of adipose tissue histology, enabling analysis of over 27 million adipocytes from 2,667 individuals. Findings: Our approach revealed that adipocyte hypertrophy associates with metabolic dysfunction, including increased fasting glucose, glycated hemoglobin, leptin, and triglycerides, with decreased adiponectin and HDL cholesterol levels. Through the largest genome-wide association study of adipocyte size to date (N _Subcutaneous = 2,066, N _Visceral = 1,878), we identified four genome-wide significant loci: two in sex-combined analysis (rs73184721 in NAALADL2 and rs200047724 in NRXN3 ) and two female-specific variants (rs140503338 and rs11656704 in ULK2 ). Notably, these genetic associations showed congruent relationships with cardiometabolic traits, suggesting shared biological mechanisms. Interpretation: Our findings demonstrate the utility of deep learning for adipocyte phenotyping at scale and provide novel insights into the genetic basis of adipocyte morphology and its relationship to metabolic disease.

Early life conditions can have long-lasting fitness effects on organisms. In insects, crowding during larval stages impose a significant constraint on adult phenotypes due to increased intraspecific competition for resources, which can modulate males’ success in pre- and post-mating competition in adulthood. Evidence for larval crowding effects on male seminal fluid allocation exists but is limited to a small subset of well-known seminal fluid proteins (Sfps), and often overlooks the interactions between male and female phenotypes. We currently lack a comprehensive understanding of how male and female larval crowding interact to affect production, composition, and transfer of the wider seminal proteome. Here, we manipulated Drosophila melanogaster larval crowding (low versus high) of males and females to generate individuals with large and small body size, respectively. We mated individuals in a fully factorial design, and measured the abundance, composition, and transfer of Sfps. Large males produced Sfps in significantly higher abundances, yet this difference was marginal and not detected when Sfps were analysed individually. Conversely, small males transferred greater quantities of much of their seminal proteome to females than did large males. When analysing proteins individually, 10 Sfps were transferred in significantly higher abundances by small males than large males. Our results suggest that small males invest more on each mating opportunity, potentially as a response of overall fewer mating opportunities due to their reduced size, or due to the larval cues of high population density. This work provides an insight into early life effects on ejaculate allocation in D. melanogaster and sheds light on the physiological and behavioural responses to developmental conditions in insects.

Overall adiposity and body fat distribution are heritable traits associated with altered risk of cardiometabolic disease and mortality. Performing rare variant (minor allele frequencyP -7 ) and 50 additional genes at FDR≤1% ( P ≤4.37×10 ^-5 ). These 69 genes exhibited significantly higher (one-sided t -test P =3.58×10 ^-18 ) common variant prioritisation scores than genes not significantly enriched for rare putatively damaging variation, with evidence of monotonic allelic series (dose-response relationships) among ultra-rare variants (minor allele count≤10) in 22 genes. Combining rare and common variation evidence, allelic series and longitudinal analysis, we selected 14 genes for CRISPR knockdown in human white adipose tissue cell lines. In three previously uncharacterised target genes, knockdown increased (two-sided t -test P COL5A3 (fold change [FC]=1.72, P =0.0028), EXOC7 (FC=1.35, P =0.0096), and TRIP10 (FC=1.39, P =0.0157); furthermore, knockdown of PPARG (FC=0.25, P =5.52×10 ^-7 ) and SLTM (FC=0.51, P =1.91×10 ^-4 ) resulted in reduced lipid accumulation. Integrating across population-based genetic and in vitro functional evidence, we highlight therapeutic avenues for altering obesity and body fat distribution by modulating lipid accumulation.

SUMMARY: The capacity of host cells to sustain or restrict virus infection is influenced by their proteome. Understanding the compendium of proteins defining cellular permissiveness is key to many questions in fundamental virology. Here, we apply a multiomic approach to determine the proteins that are associated with highly permissive, intermediate, and hostile cellular states. We observed two groups of differentially regulated genes: i) with robust changes in mRNA and protein levels, and ii) with protein/RNA discordances. Many of the latter are classified as interferon stimulated genes (ISGs) but have no reported antiviral activity. This suggests that IFN-dependent changes in mRNA levels do not imply antiviral function. Phosphoproteomics revealed an additional regulatory layer involving non-signalling proteins with altered phosphorylation. Indeed, we confirmed that several permissiveness-associated proteins with changes in abundance or phosphorylation regulate infection fitness. Altogether, our study provides a comprehensive and systematic map of the cellular alterations driving virus susceptibility.

Sepsis, the dysregulated host response to infection causing life-threatening organ dysfunction, is a global health challenge requiring better understanding of pathophysiology and new therapeutic approaches. Here, we applied high-throughput tandem mass spectrometry to delineate the plasma proteome for sepsis and comparator groups (noninfected critical illness, postoperative inflammation, and healthy volunteers) involving 2612 samples (from 1611 patients) and 4553 liquid chromatography–mass spectrometry analyses acquired through a single batch of continuous measurements, with a throughput of 100 samples per day. We show how this scale of data can delineate proteins, pathways, and coexpression modules in sepsis and be integrated with paired leukocyte transcriptomic data (837 samples from n = 649 patients). We mapped the plasma proteomic landscape of the host response in sepsis, including changes over time, and identified features relating to etiology, clinical phenotypes (including organ failures), and severity. This work reveals subphenotypes informative for sepsis response state, disease processes, and outcome; identifies potential biomarkers; and advances opportunities for a precision medicine approach to sepsis.

The spatial organisation of cellular protein expression profiles within tissue determines cellular function and is key to understanding disease pathology. To define molecular phenotypes in the spatial context of tissue, there is a need for unbiased, quantitative technology capable of mapping proteomes within tissue structures. Here, we present a workflow for spatially-resolved, quantitative proteomics of tissue that generates maps of protein abundance across tissue slices derived from a human atypical teratoid-rhabdoid tumour at three spatial resolutions, the highest being 40 µm, to reveal distinct abundance patterns of thousands of proteins. We employ spatially-aware algorithms that do not require prior knowledge of the fine tissue structure to detect proteins and pathways with spatial abundance patterns and correlate proteins in the context of tissue heterogeneity and cellular features such as extracellular matrix or proximity to blood vessels. We identify PYGL, ASPH and CD45 as spatial markers for tumour boundary and reveal immune response-driven, spatially-organised protein networks of the extracellular tumour matrix. Overall, we demonstrate spatially-aware deep proteo-phenotyping of tissue heterogeneity, to re-define understanding tissue biology and pathology at the molecular level.

Summary: Marine phytoplankton can interchange trace metals in various biochemical functions, particularly under metal‐limiting conditions. Here, we investigate the stimulating and toxicity effect of chromium (Cr) on a marine Chlorophyceae Osetreococcus tauri under Fe‐replete and Fe‐deficient conditions. We determined the growth, photosynthesis, and proteome expressions of Osetreococcus tauri cultured under different Cr and Fe concentrations. In Fe‐replete conditions, the presence of Cr(VI) stimulated significantly the growth rate and the maximum yield of photochemistry of photosystem II (F_v/F_m) of the phytoplankton, while the functional absorption cross‐section of photosystem II (σ_PSII) did not change. Minor additions of Cr(VI) partially rescued phytoplankton growth under Fe‐limited conditions. Proteomic analysis of this alga grown in Fe‐replete normal and Fe‐replete with Cr addition media (10 μM Cr) showed that the presence of Cr significantly decreased the expression of phosphate‐transporting proteins and photosynthetic proteins, while increasing the expression of proteins related to carbon assimilation. Cr can stimulate the growth and photosynthesis of O. tauri, but the effects are dependent on both the Cr(VI) concentration and the availability of Fe. The proteomic results further suggest that Cr(VI) addition might significantly increase starch production and carbon fixation.