Overall adiposity and body fat distribution are heritable traits associated with altered risk of cardiometabolic disease and mortality. Performing rare-variant (minor allele frequency <1%) association testing using exome-sequencing data from 402,375 participants of European ancestry in the UK Biobank for nine overall and tissue-specific fat distribution traits, we identified 19 genes where putatively damaging rare variation associated with at least one trait (Bonferroni-adjusted p < 1.58 × 10-7) and 50 additional genes at false discovery rate (FDR) ≤1% (p ≤ 4.37 × 10-5). These 69 genes exhibited significantly higher (one-sided t test p = 3.58 × 10-18) common-variant prioritization scores for association with body mass index (BMI), waist-to-hip ratio adjusted for BMI, and body fat percentage 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 two target genes, knockdown significantly (two-sided t test p < 0.05/14) decreased lipid accumulation: PPARG (fold change [FC] = 0.25, p = 5.52 × 10-7) and SLTM (FC = 0.51, p = 1.91 × 10-4); knockdown of COL5A3 (FC = 1.72, p = 0.0028) resulted in significantly increased 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.

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 (NSubcutaneous = 2,066, NVisceral = 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 effects on fitness. In insects, larval crowding increases intraspecific competition, shaping adult phenotypes and influencing male reproductive success in pre- and post-mating competition. Although some larval crowding effects on seminal fluid protein (Sfp) allocation are known, studies often focus on a small subset of Sfps and overlook male-female interactions. A comprehensive understanding of how male and female larval environments interact to influence seminal proteome composition and transfer is still lacking. Here, we manipulated Drosophila melanogaster larval crowding (low vs. high) to generate large and small adults and mated individuals in a fully factorial design. We then measured Sfp production, composition and transfer. Large males produced relatively higher quantities of Sfps. However, small males transferred greater quantities of a subset of Sfps. When examining proteins individually, 10 Sfps were transferred at significantly higher abundances by small males than large males. Our findings suggest that small males invest more per mating, potentially due to fewer mating opportunities or cues of high larval density influencing reproductive strategies. This study provides new insights into early life effects on ejaculate allocation in D. melanogaster, highlighting physiological and behavioural responses to developmental conditions. Understanding these mechanisms offers valuable perspectives on reproductive strategies and fitness trade-offs in insects.

Abstract Background and Aims Cystatin-C is an established marker of glomerular filtration rate in kidney disease. High levels of cystatin-C have been associated with unfavorable outcomes (acute kidney injury, cardiovascular morbidity) in critically ill patients. The aim of this study was to evaluate the levels of Cystatin-C in donors after brain (DBD) and circulatory death (DCD) and investigate its association with posttransplant graft function. Method Plasma samples from 303 donors (283 deceased, 20 living donors; LD) were obtained from the Quality in Organ Donation (QUOD) and the Oxford Transplant (OTB) biobanks. QUOD is a national multi-center UK wide bioresource of deceased donor clinical samples collected during donor management and organ procurement. OTB is a living donor biobank established to obtain clinical samples as control cohorts. Only deceased donors with both grafts transplanted were included in the study. Plasma Cystatin-C from LD samples as assessed as an optimal control group. The samples were linked to donor and recipient metadata obtained from the National Transplant Registry (NHSBT). Using Luminex assays, plasma Cystatin-C was quantified in samples collected during donor management (prior to cross clamp in DBDs and LDs and at withdrawal of support in DCDs). We performed descriptive statistics and multivariate linear and logistic analysis to determine the association between Cystatin-C, primary non-function (PNF), delayed graft function (DGF), acute rejection (AR) and 12-month posttransplant graft function. Results Among 303 donors (median age: 50 years), 126 were DCDs, 157 DBDs (20.1% of them were expanded criteria donors, ECD) and 20 LDs. ECDs and DCDs had significantly higher levels of Cystatin-C, compared to DBDs. In a total of 606 recipients, 11 (1.9%) developed PNF, and 128 (21%) developed DGF (24 as pairs, 80 non-pairs). Forty-six recipients (7.6%) experienced at least one AR episode and 60 (10%) donors had lost the graft during follow-up. Donor levels of Cystatin-C were associated with DGF and were further elevated in donors of kidneys which developed PNF (P-value < .0001, Fig. 1). Grafts with acute rejections were retrieved from donors with higher levels of Cystatin-C, but the difference did not meet statistical significance. Cystatin-C levels were significantly lower (P-value < .0001) in donors who offered concordant grafts with optimal 12-month post-transplant function (eGFR >60 ml/min), compared with grafts with intermediate function (GFR 30-60 ml/min) or eGFR <30ml/min (Fig. 2). LDs had significantly lower levels of Cystatin-C compared to deceased donors in all studied outcomes (Figs 1 and 2). Cystatin-C during donor management was an independent risk factor for inferior 12-month paired graft function, after the implementation of three different multivariate models, including donor (β-coef = −11.3, P-value = .001), recipient (β-coef = −16, P-value < .001) and post-transplant characteristics (β-coef = −13.1, P-value = .002). Conclusion In a large cohort of deceased and living donors, we were able to evaluate the circulating levels of Cystatin-C during donor management with the use of high-end performance technology. The concentration of Cystatin-C was notably different among donor types and was significantly higher in donors with kidneys that developed PNF or had suboptimal graft function 12-month posttransplant. Our findings suggest that Cystatin-C may have a role in identifying higher-risk donors during donor management and offers the opportunity for granular assessment of donor kidney quality prior to transplantation.

KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute to ∼30% of human solid tumours, including lung adenocarcinoma, pancreatic, and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. However, the precise signalling cascades these mutations affect are poorly understood. Here, APEX2 proximity labelling was used to profile the molecular environment of WT, G12D, G13D, and Q61H-activating KRAS mutants under starvation and stimulation conditions. Through quantitative proteomics, we demonstrate the presence of known KRAS interactors, including ARAF and LZTR1, which are differentially captured by WT and KRAS mutants. Notably, the KRAS mutations G12D, G13D, and Q61H abrogate their association with LZTR1, thereby affecting turnover. Elucidating the implications of LZTR1-mediated regulation of KRAS protein levels in cancer may offer insights into therapeutic strategies targeting KRAS-driven malignancies.

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. ### Competing Interest Statement The authors have declared no competing interest.

Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL facilitate the elimination of damaged membranes, protein aggregates, membranous organelles and play an important role in calcium signaling. The specific role of EL in cardiac atrial fibrillation (AF) is not well understood. We isolated atrial EL organelles from AF goat biopsies and conducted a comprehensive integrated omics analysis to study the EL-specific proteins and pathways. We also performed electron tomography, protein and enzyme assays on these biopsies. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates, including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophagic-vacuole formation, irregularly shaped mitochondria, and glycogen deposition. Our results provide molecular information suggesting EL play a role in AF disease process over extended time frames.

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.

Some accreting binary systems containing a white dwarf (such as classical novae or persistent supersoft sources) are seen to emit low-energy X-rays with temperatures of similar to 106 K and luminosities exceeding 1035 erg s-1. These X-rays are thought to originate from nuclear burning on the white dwarf surface, either caused by a thermonuclear runaway (classical novae) or a high mass-accretion rate that sustains steady nuclear burning (persistent sources). The discovery of transient supersoft X-rays from ASASSN-16oh challenged these ideas, as no clear signatures of mass ejection indicative of a classical nova eruption were detected, and the origin of these X-rays remains controversial. It was unclear whether this star was one of a kind or representative of a larger, as yet undiscovered, group. Here, we present the discovery of 29 stars located in the direction of the Magellanic Clouds exhibiting long-duration, symmetrical optical outbursts similar to that seen in ASASSN-16oh. We observed one of these objects during an optical outburst and found it to be emitting transient supersoft X-rays, while no signatures of mass ejection (indicative of a classical nova eruption) were detected. We therefore propose that these objects form a homogeneous group of transient supersoft X-ray sources, which we dub "millinovae" because their optical luminosities are approximately a 1000 times fainter than those of ordinary classical novae.

BACKGROUND:The widespread use of the antifibrinolytic agent, tranexamic acid (TXA), interferes with the quantification of fibrinolysis by dynamic laboratory assays such as clot lysis, making it difficult to measure fibrinolysis in many trauma patients. At the final stage of coagulation, factor (F)XIIIa catalyzes the formation of fibrin-fibrin and fibrin-α2-antiplasmin (α2AP) cross-links, which increases clot mechanical strength and resistance to fibrinolysis. OBJECTIVES:Here, we developed a method to quantify fibrin-fibrin and fibrin-α2AP cross-links that avoids the challenges posed by TXA in determining fibrinolytic resistance in conventional assays. METHODS:Fibrinogen alpha (FGA) chain (FGA-FGA), fibrinogen gamma (FGG) chain (FGG-FGG), and FGA-α2AP cross-links were quantified using liquid chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring in paired plasma samples from trauma patients prefibrinogen and postfibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analyzed. RESULTS:The abundance of cross-links was significantly increased in trauma patients postcryo, but not Fg-C transfusion (P < .0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibers, the peak thrombin concentration, and FXIII antigen (P < .05). CONCLUSION:We have developed a novel method that allows us to quantify fibrin cross-links in trauma patients who have received TXA, providing an indirect measure of fibrinolytic resistance. Using this novel approach, we have avoided the effect of TXA and shown that cryo increases fibrin-fibrin and fibrin-α2AP cross-linking when compared with Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients.

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/RT). 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. We identified 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, this work informs on methods for spatially resolved deep proteo-phenotyping of tissue heterogeneity, to push the boundaries of understanding tissue biology and pathology at the molecular level.

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 (Fv /Fm ) of the phytoplankton, while the functional absorption cross-section of photosystem II (σPSII ) 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.

Introduction and Problem Statement:The aging US population has led to the increased prevalence of neurodegenerative diseases and the critical need for specialists with advanced training in the management of these conditions. Focus on Common Movement Disorders (FOCMD), a 2-day educational course hosted by Vanderbilt University, was started 16 years ago to provide neurology residents with exposure to the diagnosis and treatment of movement disorders. Objectives:The aim of the course was to provide early-career neurology residents with relevant exposure to the field of movement disorders, through which we hope to increase medical knowledge of movement disorders and common Food and Drug Administration (FDA)-approved therapies and inspire residents to pursue fellowship training in the field. Methods and Curriculum Description:FOCMD consists of lectures and small-group workshops that provide an overview of common movement disorders and approved therapies. All North American neurology residency program directors are invited to nominate a first-year or second-year resident. Attendees are administered standardized multiple-choice precourse and postcourse examinations to assess foundational knowledge of common movement disorders and FDA-approved therapies and to measure acquisition of the course material. Past participants are regularly surveyed to gauge their impression of the course's effect on fellowship selection and their utilization of therapies common in the treatment of movement disorders. Results and Assessment Data:Since 2008, FOCMD has trained 854 neurology residents from 113 programs. Between 2010 and 2020, 507 residents completed the precourse and postcourse examinations. There was an increase of 22.4 (95% CI 20.67-24.49; p < 0.001) percentage points between the precourse and postcourse examinations or an additional 3.8 questions were answered correctly. Follow-up surveys were sent to 414 past participants, and 116 were completed. Survey responses revealed that 84% of past attendees completed a fellowship, 44% of which were in the field of movement disorders. In addition, 82% of past participants reported that the course affected subspecialty selection and 63% reported treating patients with movement disorders in their current practice. Discussion and Lessons Learned:Experience with FOCMD has shown it to be successful in introducing neurology residents to the subspecialty early in their career and increasing medical knowledge on movement disorders. An educational program of this format may also positively influence fellowship selection.

OBJECTIVE:To provide mechanistic insight into key biological alterations in donation after circulatory death kidneys during continuous pefusion we performed mass spectrometry profiling of perfusate samples collected during a phase 3 randomized double-blind paired clinical trial of hypothermic machine perfusion with and without oxygen (COMPARE).BACKGROUND:Despite the clinical benefits of novel perfusion technologies aiming to better preserve donor organs, biological processes that may be altered during perfusion have remained largely unexplored. The collection of serial perfusate samples during the COMPARE clinical trial provided a unique resource to study perfusate proteomic profiles, with the hypothesis that in-depth profiling may reveal biologically meaningful information on how donor kidneys benefit from this intervention.METHODS:Multiplexed liquid chromatography-tandem mass spectrometry was used to obtain a proteome profile of 210 perfusate samples. Partial least squares discriminant analysis and multivariate analysis involving clinical and perfusion parameters were used to identify associations between profiles and clinical outcomes.RESULTS:Identification and quantitation of 1716 proteins indicated that proteins released during perfusion originate from the kidney tissue and blood, with blood-based proteins being the majority. Data show that the overall hypothermic machine perfusion duration is associated with increasing levels of a subgroup of proteins. Notably, high-density lipoprotein and complement cascade proteins are associated with 12-month outcomes, and blood-derived proteins are enriched in the perfusate of kidneys that developed acute rejection.CONCLUSIONS:Perfusate profiling by mass spectrometry was informative and revealed proteomic changes that are biologically meaningful and, in part, explain the clinical observations of the COMPARE trial.

Background Organ availability limits kidney transplantation, the best treatment for end-stage kidney disease. Deceased donor acceptance criteria have been relaxed to include older donors with higher risk of inferior posttransplant outcomes. Donor age, although significantly correlates with transplant outcomes, lacks granularity in predicting graft dysfunction. Better characterization of the biological mechanisms associated with deceased donor organ damage and specifically predictive of transplant outcome in recipients is key to developing new assessment criteria for donor kidneys and developing function-preserving interventions. Methods 185 deceased donor pretransplant biopsies with clinical and demographic donor and recipient metadata were obtained from the Quality in Organ Donation biobank (QUOD), selected on the basis of 12-month paired posttransplant function and deep proteomic profiles acquired by mass spectrometry. Using a 2/3rd:1/3rd training:test data split, sampling equally across posttransplant function, we applied machine learning feature selection followed by protein-wise relaxed LASSO regression modeling, assessing the performance of the final set of protein models on the test data. Western blotting validated protein changes, and the biological relevance of the final set of protein models was externally validated by contextualization against a published dataset of human healthy and disease kidney transcriptomes. Results Our analysis revealed 144 proteins carrying outcome-predictive information, all of which showed donor-age modulated associations with posttransplant function, as opposed to age and protein/gene effects being independent terms. Observed associations with inflammatory, metabolic, protein processing and cell cycle pathways suggest biological targets for possible interventions pretransplant. Contextualization of our results against external spatial transcriptomic data suggest a sub-nephrotic spatial localization of the predictive signal. Conclusions Integrating kidney proteome information with clinical metadata enhances the resolution of donor kidney quality stratification, and the highlighted biological mechanisms open new research directions in developing predictive models and novel interventions during donor management or preservation to improve kidney transplant outcome. SIGNIFICANCE STATEMENT Currently, organ quality assessment pretransplant relies on key factors such as donor age or clinical information, these lack granularity in depicting graft susceptibility and capacity to function posttransplant. A high-resolution proteomic profiling of 185 pretransplant biopsies of kidneys with known posttransplant function and complete metadata was performed. Integration of donor kidney proteomes with 56 clinical metadata variables using regularized regression modelling resulted in enhancing the resolution of donor kidney quality stratification. Immunometabolic and catabolic processes contributed to donor kidney susceptibility and worse transplant outcomes in an age modulated pattern, validated by western blotting. Comparison of kidney proteomes with a recent transcriptomics dataset of healthy and diseased kidneys provide an additional special single cell resolution to the findings of this study. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by NHS Blood and Transplant WP15-07 awarded to MK & RJP. SF was supported by Kidney Research UK, grant reference KS\_RP\_002_20210111 awarded to MK. PDC was supported by a Chinese Academy of Medical Sciences 2018-I2M-2-002 awarded to BMK. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: NHS Health Authority, National Research Ethics Service, gave ethical approval for IRAS project ID: 87824, Quality in Organ Donation (QUOD) (NW/18/0187), sponsored by University of Oxford I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 67 partner repository with the dataset identifier PXD033428. All other data produced are contained in the manuscript or supplemental materials.

Background: Abdominal aortic aneurysms (AAA) are pathological dilatations of the aorta which can result in rupture and mortality. Novel methods of predicting AAA growth is a recognised priority in AAA research. Patient with AAAs have increased risk of cardiovascular morbidity. We have previously observed accelerated systemic endothelial dysfunction (measured by brachial artery FMD) in AAA patients and FMD correlates with future AAA growth. Further, systemic endothelial dysfunction is reversed by AAA repair. AAAs contain intra-luminal thrombus (ILT). Since ILT is either removed or excluded from circulation after successful repair of AAAs, we hypothesise that ILT to be the source of mediators that contribute to AAA growth. Methods: Patients were prospectively recruited to the Study (Ethics Ref SC/13/0250). Plasma samples were collected at baseline and at 1 year from each patient. Plasma samples were also collected before and at 10-12 weeks after surgery from each patient (n=29). Paired aneurysm wall, ILT, omental biopsies were collected intra-operatively during open surgical repair (n=3). In addition to analyses of the tissue, supernatant was obtained from ex vivo culture of these paired tissue samples. Samples were subjected to non-targeted LC-MSMS workflow after trypsin digest, using the Universal method to discover novel proteins. LC-MSMS data was analysed using the Progenesis QI pipeline. Results: The median AAA size at baseline was 48 mm. 59 patients were prospectively followed for 12 months. The median growth rate of AAA was 3.8%/year (IQR 1.9% to 6.8%). Comparison between patients with the fastest vs the slowest (n=10 each) showed 116 proteins to be differentially expressed in their plasma. Among these proteins, 35 also changed significantly before and after AAA repair, suggesting their origin to from the AAA complex. Comparison of the proteomics profile of aneurysm tissue, ILT, and omental artery show 128 proteins to be uniquely present in ILT. Analyses of the tissue culture supernatant further revealed 3 proteins that are: (i) uniquely present in ILT; (ii) released by ILT; (iii) systemic levels reduced after AAA surgery; (iv) differs between fast and slow growth AAAs. One of these proteins is attractin. To validate the LC-MSMS data, attractin level in individual patient was measured by ELISA. Consistent with the LC-MSMS data, plasma attractin level is higher in patients with fast AAA growth. Plasma attractin level correlates significantly with future AAA growth rate (Spearman r=0.35, P<;0.005). Using attractin and AAA diameter as input variables, the AUROC for predicting no growth of AAA at 12 months is 85% (P<0.001). Conclusion: We show that ILT of AAAs releases mediators (such as attractin) during the natural history of AAA growth. These are novel biomarkers for AAA growth prediction in humans.

Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behavior. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterized protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term postmating responses, including increased ovulation, elevated feeding, and reduced receptivity to remating, primarily signaling through the SP receptor (SPR). Here, we demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Its absence leads to major changes in the seminal proteome transferred to females upon mating. Males expressing nonfunctional SP mutant proteins that affect SP's binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread abnormalities in ejaculate function. Our data therefore reveal a role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species that lack the signaling functions seen in D. melanogaster.

Background Amyotrophic lateral sclerosis is a clinical syndrome with complex biological determinants, but which in most cases is characterized by TDP-43 pathology. The identification in CSF of a protein signature of TDP-43 network dysfunction would have the potential to inform the identification of new biomarkers and therapeutic targets. Methods We compared CSF proteomic data from patients with ALS ( n = 41), Parkinson’s disease ( n = 19) and healthy control participants ( n = 20). Weighted correlation network analysis was used to identify modules within the CSF protein network and combined with gene ontology enrichment analysis to functionally annotate module proteins. Analysis of module eigenproteins and differential correlation analysis of the CSF protein network was used to compare ALS and Parkinson’s disease protein co-correlation with healthy controls. In order to monitor temporal changes in the CSF proteome, we performed longitudinal analysis of the CSF proteome in a subset of ALS patients. Results Weighted correlation network analysis identified 10 modules, including those enriched for terms involved in gene expression including nucleic acid binding, RNA metabolism and translation; humoral immune system function, including complement pathways; membrane proteins, axonal outgrowth and adherence; and glutamatergic synapses. Immune system module eigenproteins were increased in ALS, whilst axonal module eigenproteins were decreased in ALS. The 19 altered protein correlations in ALS were enriched for gene expression (OR 3.05, p = 0.017) and membrane protein modules (OR 17.48, p = 0.011), including intramodular hub proteins previously identified as TDP-43 interactors. Proteins decreasing over longitudinal analysis ALS were enriched in glutamatergic synapse and axonal outgrowth modules. Protein correlation network disruptions in Parkinson’s disease showed no module enrichment. Conclusions Alterations in the co-correlation network in CSF samples identified a set of pathways known to be associated with TDP-43 dysfunction in the pathogenesis of ALS, with important implications for therapeutic targeting and biomarker development.

The transcription factor FOXN1 is a master regulator of thymic epithelial cell development and function. Here we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multi-molecular nuclear condensates essential for the factor’s transcriptional activity. FOXN1’s C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are significantly altered in a patient with a mutant FOXN1 which is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog, selectively impairs mouse thymic epithelial cell (TEC) differentiation revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.

The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p-TDP-43, p-tau, alpha-synuclein, and beta-amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed the synthesis of heavy isotope-labeled peptides for absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson's, 8 Alzheimer's disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson's and Alzheimer's disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent limbic TDP-43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.