Agnostic identification of plasma biomarkers for postpartum hemorrhage risk.

BACKGROUND: Postpartum hemorrhage (PPH) is difficult to predict, is associated with significant maternal morbidity, and is the leading cause of maternal mortality worldwide. The identification of maternal biomarkers that can predict increased PPH risk would enhance clinical care and may uncover mechanisms that lead to PPH. OBJECTIVE: This retrospective case-control study employed agnostic proteomic profiling of maternal plasma samples to identify differentially abundant proteins in controls and PPH cases. STUDY DESIGN: Maternal plasma samples were procured from a cohort of >60,000 participants in a single institution's perinatal repository. PPH was defined as a decrease in hematocrit of ≥10% or receipt of transfusion within 24 hours of delivery. PPH cases (N=30) were matched by maternal age and delivery mode (vaginal or cesarean) with controls (N=56). Mass spectrometry was used to identify differentially abundant proteins using integrated peptide peak areas. Statistically significant differences between groups were defined by a p-value of <0.05 after controlling for multiple comparisons. RESULTS: By study design, cases and controls did not differ in race, ethnicity, gestational age at delivery, blood type, or pre-delivery platelet count. Cases had slightly, but significantly lower pre- and post-delivery hematocrit and hemoglobin. Mass spectrometry detected 1140 proteins, including 77 proteins for which relative abundance differed significantly between cases and controls (fold change >1.15, P<0.05). Of these differentially abundant plasma proteins, most had likely liver or placental origins. Gene ontology term analysis mapped to protein clusters involved in responses to wound healing, stress response, and host immune defense. Significantly differentially abundant proteins with the highest fold change (prostaglandin D2 synthase, periostin, and several serine protease inhibitors) did not correlate with pre-delivery hematocrit or hemoglobin, but identified PPH cases with logistic regression modeling revealing good-to-excellent area under the operator receiver characteristic curves (AUROC 0.802-0.874). Incorporating pre-delivery hemoglobin with these candidate proteins further improved identification of PPH cases. CONCLUSION: Agnostic analysis of maternal plasma samples identified differentially abundant proteins in controls and PPH cases. Several of these proteins are known to participate in biologically plausible pathways for PPH risk and have potential value for predicting PPH. These findings identify candidate protein biomarkers for future validation and mechanistic studies.

Serine protease inhibitor, SerpinA3n regulates cardiac remodeling after myocardial infarction.

Following myocardial infarction, the heart repairs itself via a fibrotic repair response. The degree of fibrosis is determined by the balance between deposition of extracellular matrix by activated fibroblasts and breakdown of nascent scar tissue by proteases that are secreted predominantly by inflammatory cells. Excessive proteolytic activity and matrix turnover has been observed in human heart failure and protease inhibitors in the injured heart regulate matrix breakdown. Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of evolutionary conserved protease inhibitors and levels of the specific Serpin, SerpinA3, have been strongly associated with clinical outcomes in human myocardial infarction as well as non-ischemic cardiomyopathies. Yet, the role of Serpins in regulating cardiac remodeling is poorly understood. We observed the robust expression of Serpins in the infarcted murine heart and demonstrate that genetic deletion of SerpinA3n (mouse homolog of SerpinA3) leads to increased activity of substrate proteases, poorly compacted matrix and significantly worse post infarct cardiac function. Single cell transcriptomics complemented with histology in SerpinA3n deficient animals, demonstrated increased inflammation, adverse myocyte hypertrophy and expression of pro-hypertrophic genes. Proteomic analysis of scar tissue demonstrated decreased cross linking of extracellular matrix peptides consistent with increased proteolysis in SerpinA3n deficient animals. Taken together these observations demonstrate a hitherto unappreciated causal role of Serpins in regulating matrix function and post infarct cardiac remodeling.

ASSESSING TARGET SPECIFICITY OF THE SMALL MOLECULE INHIBITOR MARIMASTAT TO SNAKE VENOM TOXINS: A NOVEL APPLICATION OF THERMAL PROTEOME PROFILING.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation, and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including Thermal Proteome Profiling (TPP) and Proteome Integral Solubility Alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply TPP and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Intra-individual changes in sperm parameters and total motile count with time among infertile men.

BACKGROUND: Paternal age association with sperm parameters has been previously studied, demonstrating a decrease in semen volume, sperm motility, and sperm morphology, but not in sperm concentration. However, scarce data exists on the individual intra-personal changes in semen parameters with time. STUDY DESIGN: Retrospective cohort study. OBJECTIVE: To evaluate the changes in semen parameters and total motile count of infertile men over time. MATERIALS AND METHODS: In this retrospective cohort study, infertile men without known risk factors for sperm quality deterioration and at least two semen analyses done > 3 months apart, between 2005 and 2021, were evaluated. Allocation to groups was according to time between first and last semen analyses - 3-12 months, 1-3 years, 3-5 years, and > 5 years. Basic characteristics and first and last semen analyses were compared. The primary outcome was the change in sperm parameters and the secondary outcome was the occurrence of a total motile count < 5 million in men with an initial total motile count > 10 million. RESULTS: A total of 2018 men were included in the study. The median age at first semen analyses was 36.2 (interquartile range: 32.8-40.1) years and the median time between semen analyses was 323 days (range 90-5810 days). The overall trend demonstrated an increase in concentration in the 3-12 months and the 1-3 years groups, whereas volume, motility, and morphology remained similar in these time groups. Semen analyses done more than 5 years apart showed decreased volume (p < 0.05), motility (p < 0.05) morphology (p < 0.05), and steady sperm concentration. Significant declines in TMCs were found over time (p < 0.001), with 18% and 22% of infertile men with an initial total motile count > 10 million dropping to < 5 million after 3 and 5 years, respectively. The factors independently predictive of total motile count < 5 M in the last semen analyses in men with an initial total motile count of > 10 M in a multivariate logistic regression model were baseline volume (odds ratio 0.80, p = 0.03), baseline total motile count (odds ratio 0.98, p = 0.01) and time between semen analyses - 3-5 years (odds ratio 3.79, p < 0.001) and > 5 years (odds ratio 3.49, p = 0.04) DISCUSSION: Our study demonstrates, at the individual level, that while improvement in sperm concentration is observed in the first year and between 1 and 3 years, possibly due to fertility treatments, fertility-related counseling, and lifestyle changes, semen parameters decline with time over 3 years in individuals. Of significance, close to 22% of men with an initial total motile count > 10 million (a range where spontaneous pregnancy is attainable) declined to < 5 million (a range usually indicating a need for in-vitro fertilization/intracytoplasmic sperm injection) over 5 years. This data could contribute to individualized family planning for infertile men regarding the mode and timing of conception and the need for sperm banking, in order to minimize the need for future fertility treatments.

Assessing Heterogeneity in the N-Telopeptides of Type I Collagen by Mass Spectrometry.

Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.

Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

Recent large-scale multi-omics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on two separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured L-carnitine and acyl-carnitines in 13,091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation (REDS) study. Genome wide association studies against 879,000 polymorphisms identified critical genetic factors contributing to inter-donor heterogeneity in end-of-storage carnitine levels, including common non-synonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, SLC16A9); carnitine synthesis (FLVCR1, MTDH) and metabolism (CPT1A, CPT2, CRAT, ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying two alleles of the rs12210538 SLC22A16 Single Nucleotide Polymorphism exhibited the lowest L-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice and Percoll density gradients of human RBCs, showed age-dependent depletions of L-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process following chemically induced membrane lipid damage. Supplementation of stored murine RBCs with L-carnitine boosted post-transfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

A Multiomics Assessment of Preoperative Exercise in Pancreatic Cancer Survivors Receiving Neoadjuvant Therapy: A Case Series.

To molecularly characterize the impact of exercise on mitigating neoadjuvant treatment (NAT)-induced physical decline in pancreatic ductal adenocarcinoma (PDAC) patients, a multi-omics approach was employed for the analysis of plasma samples before and after a personalized exercise intervention. Consisting of personalized aerobic and resistance exercises, this intervention was associated with significant molecular changes that correlated with improvements in lean mass, appendicular skeletal muscle index (ASMI), and performance in the 400-m walk test (MWT) and sit-to-stand test. These alterations indicated exercise-induced modulation of inflammation and mitochondrial function markers. This case study provides proof-of-principal application for multiomics-based assessments of supervised exercise, thereby supporting this intervention as a feasible and beneficial intervention for PDAC patients to potentially enhance treatment response and patient quality of life. The molecular changes observed here underscore the importance of physical activity in cancer treatment protocols, advocating for the development of accessible multiomics-guided exercise programs for cancer patients.

Multiomics Signatures of Coagulopathy in a Polytrauma Swine Model Contrasted with Severe Multisystem Injured Patients.

Trauma-induced coagulopathy (TIC) is a leading contributor to preventable mortality in severely injured patients. Understanding the molecular drivers of TIC is an essential step in identifying novel therapeutics to reduce morbidity and mortality. This study investigated multiomics and viscoelastic responses to polytrauma using our novel swine model and compared these findings with severely injured patients. Molecular signatures of TIC were significantly associated with perturbed coagulation and inflammation systems as well as extensive hemolysis. These results were consistent with patterns observed in trauma patients who had multisystem injuries. Here, intervention using resuscitative endovascular balloon occlusion of the aorta following polytrauma in our swine model revealed distinct multiomics alterations as a function of placement location. Aortic balloon placement in zone-1 worsened ischemic damage and mitochondrial dysfunction, patterns that continued throughout the monitored time course. While placement in zone-III showed a beneficial effect on TIC, it showed an improvement in effective coagulation. Taken together, this study highlights the translational relevance of our polytrauma swine model for investigating therapeutic interventions to correct TIC in patients.

More Similar than Different: Memory, Executive Functions, Cortical Thickness, and Glucose Metabolism in Biomarker-Positive Alzheimer's Disease and Behavioral Variant Frontotemporal Dementia.

Background: Alzheimer's disease (AD) and behavioral variant frontotemporal dementia (bvFTD) are typically associated with very different clinical and neuroanatomical presentations; however, there is increasing recognition of similarities. Objective: To examine memory and executive functions, as well as cortical thickness, and glucose metabolism in AD and bvFTD signature brain regions. Methods: We compared differences in a group of biomarker-defined participants with Alzheimer's disease and a group of clinically diagnosed participants with bvFTD. These groups were also contrasted with healthy controls (HC). Results: As expected, memory functions were generally more impaired in AD, followed by bvFTD, and both clinical groups performed more poorly than the HC group. Executive function measures were similar in AD compared to bvFTD for motor sequencing and go/no-go, but bvFTD had more difficulty with a set shifting task. Participants with AD showed thinner cortex and lower glucose metabolism in the angular gyrus compared to bvFTD. Participants with bvFTD had thinner cortex in the insula and temporal pole relative to AD and healthy controls, but otherwise the two clinical groups were similar for other frontal and temporal signature regions. Conclusions: Overall, the results of this study highlight more similarities than differences between AD and bvFTD in terms of cognitive functions, cortical thickness, and glucose metabolism. Further research is needed to better understand the mechanisms mediating this overlap and how these relationships evolve longitudinally.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo and during storage in vitro in the blood bank. Here we identify an association between blood donor age, sex, ethnicity and end-of-storage levels of glycolytic metabolites in 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study. Associations were also observed to ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (which we detected in mature RBCs), hexokinase 1, and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP levels, breakdown, and deamination into hypoxanthine were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;RBC PFKP boosts glycolytic fluxes when ATP is low, such as in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Temporal Trends in Semen Quality, Hormone Levels, and Substance Use Among Infertile Men in Pre- and Post-Cannabis Legalization Eras in Canada.

Background: The Cannabis Act (Bill C-45) was enacted in 2018, to legalize and regulate the use, production, and sale of nonmedical cannabis in Canada. While public health and safety implications of cannabis legalization have yet to be elucidated, the wide availability of cannabis necessitates health care providers to be knowledgeable about therapeutic potential and side effects of use. This study aimed to examine the temporal trends over two decades and the impact of the Cannabis Act in Canada, implemented in October 2018, on substance use, semen parameters, and testosterone levels of infertile men. Methods: We conducted a retrospective cohort study from a prospectively maintained database of a single infertility clinic. Demographic, fertility, and substance use history were correlated with semen and hormone assessments. Temporal trends in cannabis use and semen quality between 2001 and 2021 were investigated and compared between pre-cannabis legalization eras (PRCL) and post-cannabis legalization eras (POCL). Results: Our cohort included 11,630 patients (9411 PRCL and 2230 POCL). Cannabis use increased by 8.4% per year (p<0.001), while alcohol and tobacco consumption declined (0.8% and 1.5% per year, p<0.05 and p=0.004, respectively). Similar trends were noticed in the POCL, with higher rates of cannabis use (22.4% vs. 12.9%, p<0.001) and decreased tobacco and alcohol intake (15.2% vs. 17.7%, p=0.005 and 50.5% vs. 55.2%, p<0.001, respectively) compared to the PRCL group. Semen concentration was lower in the POCL group (24.8±44.8 vs. 28.7±48.3 million/mL, p=0.03). Testosterone did not differ between the cohorts. Comparison between cannabis users (n=1715) and nonusers (n=9924) demonstrated a slight increase in sperm motility (25.9%±15.3% vs. 23.9%±15.0%, p=0.002) and decreased sperm concentration among users (27.6±53.5 vs. 23.9±15.0 million/mL, p=0.03). Conclusion: A nearly 10% rise in cannabis use in the POCL era was observed among men being investigated for infertility. Our data suggest cannabis use may be associated with an increase in testosterone, slightly improved sperm motility, and decreased sperm concentration.

Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Genetic polymorphisms and expression of Rhesus blood group RHCE are associated with 2,3-bisphosphoglycerate in humans at high altitude.

Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity.

Endothelial Cell Calcium Influx Mediates Trauma-induced Endothelial Permeability.

OBJECTIVE: We aimed to investigate if ex vivo plasma from injured patients causes endothelial calcium (Ca2+) influx as a mechanism of trauma-induced endothelial permeability. SUMMARY BACKGROUND DATA: Endothelial permeability after trauma contributes to post-injury organ dysfunction. While the mechanisms remain unclear, emerging evidence suggests intracellular Ca2+ signaling may play a role. METHODS: Ex vivo plasma from injured patients with "Low Injury/Low Shock" (injury severity score [ISS]<15, base excess [BE])≥-6mEq/L) and "High Injury/High Shock" (ISS≥15, BE<-6mEq/L) were used to treat endothelial cells. Experimental conditions included Ca2+ removal from the extracellular buffer, cyclopiazonic acid pre-treatment to deplete intracellular Ca2+ stores, and GSK2193874 pre-treatment to block the TRPV4 Ca2+ channel. Live cell fluorescence microscopy and ECIS were used to assess cytosolic Ca2+ increases and permeability, respectively. Western blot and live cell actin staining were used to assess myosin light chain (MLC) phosphorylation and actomyosin contraction. RESULTS: Compared to Low Injury/Low Shock plasma, High Injury/High Shock induced greater cytosolic Ca2+ increase. Cytosolic Ca2+ increase, MLC phosphorylation, and actin cytoskeletal contraction were lower without extracellular Ca2+ present. High Injury/High Shock plasma did not induce endothelial permeability without extracellular Ca2+ present. TRPV4 inhibition lowered trauma plasma-induced endothelial Ca2+ influx and permeability. CONCLUSIONS: This study illuminates a novel mechanism of post-injury endotheliopathy involving Ca2+ influx via the TRPV4 channel. TRPV4 inhibition mitigates trauma-induced endothelial permeability. Moreover, widespread endothelial Ca2+ influx may contribute to trauma-induced hypocalcemia. This study provides the mechanistic basis for the development of Ca2+-targeted therapies and interventions in the care of severely injured patients.

Carabidae and Tenebrionidae diversity in the Great Basin Province of California.

The high desert regions of eastern California within the Great Basin are vast areas of shrub-dominated habitat heavily impacted by invasive exotic grasses and forbs. Trapping efforts within these areas provided distributional information about various surface-active arthropod taxa. Two groups with high species diversity and abundance encountered at our sites were the coleopteran families Carabidae and Tenebrionidae. Here, we report trapping of 45 species of carabids and 46 species of tenebrionids, along with mitochondrial cytochrome c oxidase subunit 1 (COI) sequence data for 65 of these 92 species. These results build upon existing distributional information regarding these families in California and further refine our knowledge of the biodiversity of the understudied Great Basin provinces.

Improved sperm DNA fragmentation levels in infertile men following very short abstinence of 3-4 hours.

Background: Limited data exists on possible approaches to improve sperm DNA fragmentation index (DFI) when no identifiable cause is found. The effect of short abstinence on sperm parameters has been extensively studied, but rarely reported on the effect on DFI in infertile men. In this study, we aimed to determine whether a second ejaculate provided after very short abstinence demonstrates lower DFI rates in infertile men. Methods: This prospective cohort study was conducted at Mount Sinai Hospital, Toronto, Canada, a tertiary university affiliated hospital. All men having DFI testing in addition to the standard semen analysis were identified via a prospectively collected database. Infertile men were instructed to provide two semen samples 3-4 hours apart (the first sample was given after 2-5 days of abstinence) to test the effect on DFI levels. Data analysis was performed for the comparison of the change in sperm parameters and DFI between samples and between men with DFI above and under 30%. Results: A total of 52 men provided double ejaculates 3-4 hours apart. In the entire group, DFI decreased from 38.9%±21.4% to 35.1%±21.6% in the second sample (P<0.001). Semen volume was lower on the second sample (2.3±1.4 vs. 1.5±0.9 mL, P<0.001), while the remaining parameters did not change. Forty out of 52 patients (76.9%) had improved DFI (average of 6.0±4.0 percentage points). Change in DFI varied with 22/52 (42.3%) and 7/52 (13.5%) of patients found to have decreases in DFI >5% and >10% in the second ejaculate, respectively. For men with DFI of 30-40%, 64% (7/11) of DFIs reduced to the under 30% range. First DFI value was the only parameter associated with DFI decrease to under 30% in multivariate models [odds ratio (OR), 0.62; 95% confidence interval (CI): 0.39-0.98; P=0.04]. Conclusions: This study identified significant improvements in DFI in infertile men providing a second sample after 3-4 hours. Controlled trials are needed to determine if reproductive outcomes are improved using a second ejaculate for infertile men with high initial sperm DFI values.

Employing direct electromagnetic coupling to assess acute fracture healing: An ovine model assessment.

OBJECTIVES: This study explored the efficacy of collecting temporal fracture site compliance data via an advanced direct electromagnetic coupling (DEC) system equipped with a Vivaldi-type antenna, novel calibration technique, and multi-antenna setup (termed maDEC) as an approach to monitor acute fracture healing progress in a translational large animal model. The overarching goal of this approach was to provide insights into the acute healing dynamics, offering a promising avenue for optimizing fracture management strategies. METHODS: A sample of twelve sheep, subjected to ostectomies and intramedullary nail fixations, was divided into two groups, simulating normal and impaired healing scenarios. Sequential maDEC compliance or stiffness measurements and radiographs were taken from the surgery until euthanasia at four or eight weeks and were subsequently compared with post-sacrifice biomechanical, micro-CT, and histological findings. RESULTS: The results showed that the maDEC system offered straightforward quantification of fracture site compliance via a multiantenna array. Notably, the rate of change in the maDEC-measured bending stiffness significantly varied between normal and impaired healing groups during both the 4-week (p = 0.04) and 8-week (p = 0.02) periods. In contrast, radiographically derived mRUST healing measurements displayed no significant differences between the groups (p = 0.46). Moreover, the cumulative normalized stiffness maDEC data significantly correlated with post-sacrifice mechanical strength (r2 = 0.80, p < 0.001), micro-CT measurements of bone volume fraction (r2 = 0.60, p = 0.003), and density (r2 = 0.60, p = 0.003), and histomorphometric measurements of new bone area fraction (r2 = 0.61, p = 0.003) and new bone area (r2 = 0.60, p < 0.001). CONCLUSIONS: These data indicate that the enhanced maDEC system provides a non-invasive, accurate method to monitor fracture healing during the acute healing phase, showing distinct stiffness profiles between normal and impaired healing groups and offering critical insights into the healing process's progress and efficiency.

ECM-Focused Proteomic Analysis of Ear Punch Regeneration in Acomys Cahirinus.

In mammals, significant injury is generally followed by the formation of a fibrotic scar which provides structural integrity but fails to functionally restore damaged tissue. Spiny mice of the genus Acomys represent the first example of full skin autotomy in mammals. Acomys cahirinus has evolved extremely weak skin as a strategy to avoid predation and is able to repeatedly regenerate healthy tissue without scar after severe skin injury or full-thickness ear punches. Extracellular matrix (ECM) composition is a critical regulator of wound repair and scar formation and previous studies have suggested that alterations in its expression may be responsible for the differences in regenerative capacity observed between Mus musculus and A. cahirinus , yet analysis of this critical tissue component has been limited in previous studies by its insolubility and resistance to extraction. Here, we utilize a 2-step ECM-optimized extraction to perform proteomic analysis of tissue composition during wound repair after full-thickness ear punches in A. cahirinus and M. musculus from weeks 1 to 4 post-injury. We observe changes in a wide range of ECM proteins which have been previously implicated in wound regeneration and scar formation, including collagens, coagulation and provisional matrix proteins, and matricryptic signaling peptides. We additionally report differences in crosslinking enzyme activity and ECM protein solubility between Mus and Acomys. Furthermore, we observed rapid and sustained increases in CD206, a marker of pro-regenerative M2 macrophages, in Acomys, whereas little or no increase in CD206 was detected in Mus. Together, these findings contribute to a comprehensive understanding of tissue cues which drive the regenerative capacity of Acomys and identify a number of potential targets for future pro-regenerative therapies.

Col1a2-Deleted Mice Have Defective Type I Collagen and Secondary Reactive Cardiac Fibrosis with Altered Hypertrophic Dynamics.

RATIONALE: The adult cardiac extracellular matrix (ECM) is largely comprised of type I collagen. In addition to serving as the primary structural support component of the cardiac ECM, type I collagen also provides an organizational platform for other ECM proteins, matricellular proteins, and signaling components that impact cellular stress sensing in vivo. OBJECTIVE: Here we investigated how the content and integrity of type I collagen affect cardiac structure function and response to injury. METHODS AND RESULTS: We generated and characterized Col1a2-/- mice using standard gene targeting. Col1a2-/- mice were viable, although by young adulthood their hearts showed alterations in ECM mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included augmented TGFß activity, increases in fibroblast number, and progressive cardiac hypertrophy, with reduced functional performance by 9 months of age. Col1a2-loxP-targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer mice to delete the Col1a2 gene in myofibroblasts with pressure overload injury. Interestingly, while germline Col1a2-/- mice showed gradual pathologic hypertrophy and fibrosis with aging, the acute deletion of Col1a2 from activated adult myofibroblasts showed a loss of total collagen deposition with acute cardiac injury and an acute reduction in pressure overload-induce cardiac hypertrophy. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was lost after 2 and 6 weeks of pressure overload, as fibrotic deposition accumulated. CONCLUSIONS: Defective type I collagen in the heart alters the structural integrity of the ECM and leads to cardiomyopathy in adulthood, with fibroblast expansion, activation, and alternate fibrotic ECM deposition. However, acute inhibition of type I collagen production can have an anti-fibrotic and anti-hypertrophic effect.

METABOLOMIC AND PROTEOMIC CHANGES IN TRAUMA-INDUCED HYPOCALCEMIA.

ABSTRACT: Background: Trauma-induced hypocalcemia is common and associated with adverse outcomes, but the mechanisms remain unclear. Thus, we aimed to characterize the metabolomic and proteomic differences between normocalcemic and hypocalcemic trauma patients to illuminate biochemical pathways that may underlie a distinct pathology linked with this clinical phenomenon. Methods: Plasma was obtained on arrival from injured patients at a Level 1 Trauma Center. Samples obtained after transfusion were excluded. Multiple regression was used to adjust the omics data for injury severity and arrival base excess before metabolome- and proteome-wide comparisons between normocalcemic (ionized Ca 2+ > 1.0 mmol/L) and hypocalcemic (ionized Ca 2+ ≤ 1.0 mmol/L) patients using partial least squares-discriminant analysis. OmicsNet and Gene Ontology were used for network and pathway analyses, respectively. Results: Excluding isolated traumatic brain injury and penetrating injury, the main analysis included 36 patients (n = 14 hypocalcemic, n = 22 normocalcemic). Adjusted analyses demonstrated distinct metabolomic and proteomic signatures for normocalcemic and hypocalcemic patients. Hypocalcemic patients had evidence of mitochondrial dysfunction (tricarboxylic acid cycle disruption, dysfunctional fatty acid oxidation), inflammatory dysregulation (elevated damage-associated molecular patterns, activated endothelial cells), aberrant coagulation pathways, and proteolytic imbalance with increased tissue destruction. Conclusions: Independent of injury severity, hemorrhagic shock, and transfusion, trauma-induced hypocalcemia is associated with early metabolomic and proteomic changes that may reflect unique pathology in hypocalcemic trauma patients. This study paves the way for future experiments to investigate mechanisms, identify intervenable pathways, and refine our management of hypocalcemia in severely injured patients.