Exposure to systemic heat stress in male mammals adversely affects sperm production, fertility, and DNA integrity. To date, few studies have investigated this phenomenon in horses, particularly in industry-relevant environments. Therefore, this study examined the relationship between ambient climatic conditions and fertility within a population of commercially fertile stallions. Post-coital semen samples (n = 804) were collected weekly from 46 Thoroughbred stallions during two successive breeding seasons (NSW, Australia; 22 weeks total). Semen samples were processed via single-layer colloidal centrifugation on-site to remove contaminants, seminal plasma and poor-quality sperm cells. The remaining sperm fraction was resuspended in Biggers, Whitten and Whittingham (BWW) medium for analyses, including sperm concentration, motility, and DNA damage. Fertility data (first cycle and per-cycle pregnancy rates) were collected from farms. Loggers were placed in the stables and paddocks of stallions to record ambient temperature and humidity, on 3-min cycles. Our results indicate that current management regimens, involving the stabling of stallions at night, with paddock access during the day, expose stallions to highest ambient climatic conditions. Polynomial distributed lag modelling identified 18 stallions whose fertility was adversely affected by heat stress, based on correlations between fertility and ambient climatic conditions (p ≤ 0.05). Of these 18 candidate stallions, six (13

Antibiotic resistance is surging, demanding approaches that detect resistance before genetic fixation, since MIC, sequencing, and culture assays detect resistance late. We present Metabolomics-Driven Intervention Antibiotic Design (MDAD), which stages resistance evolution from metabolic compensation to sub-lethal adaptation to genetic fixation. A brief pulse challenge with a targeted metabolomics panel yields a pre-genetic risk index, interpreted with time-kill assays to situate early survival behaviours and guide mechanism-aware treatment and surveillance.

The escalating spread of multidrug-resistant (MDR) Gram-negative pathogens, particularly Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, has severely undermined the efficacy of polymyxin B, one of the few remaining last-line antibiotics. Here, we identify hyperforin, a natural product derived from Hypericum perforatum, as a potent polymyxin B adjuvant capable of restoring its antibacterial activity against clinical Gram-negative isolates with high-level polymyxin resistance. Using a suite of in vitro assays, including fractional inhibitory concentration index, time-kill kinetics, biofilm eradication, membrane integrity assays, and ultrastructural imaging of the bacterial outer membrane, we show that hyperforin significantly enhances polymyxin B-mediated bacterial killing. Microscopy and N-phenyl-1-naphthylamine uptake are consistent with membrane perturbation, and increased intracellular reactive oxygen species is consistent with oxidative stress under combination treatment. Taken together, these orthogonal readouts support a working model of complementary membrane perturbation and oxidative stress. Remarkably, the observed synergistic effects occur at concentrations potentially achievable in the epithelial lining fluid of the lungs, providing a testable rationale for localized lung delivery (e.g., via a dry-powder inhaler). These findings unveil a promising therapeutic strategy that repurposes a bioactive phytochemical to potentiate polymyxins against otherwise untreatable polymyxin-resistant MDR Gram-negative infections.

The pharmacokinetics, drug–drug interaction potential, and safety of a new HIV-1 capsid inhibitor, VH4004280 (VH-280), are described in this first-time-in-human study. This randomized, double-blind, placebo-controlled, phase 1 study assessed oral VH-280 in adults without HIV. In parts 1 and 3, VH-280 was administered as powder-in-bottle (PiB) and tablet formulations, respectively, in single ascending doses. In part 2, VH-280 was administered as a PiB formulation once daily for 14 days in multiple ascending doses. In addition, in part 2, the effect of VH-280 on cytochrome P450 3A (CYP3A) activity was evaluated using midazolam as a probe substrate. In total, 73 participants were included (VH-280, n = 57; placebo, n = 16). Plasma exposures for VH-280 were broadly dose-proportional, and median time to maximum observed concentration was 9.0–17.0 h for the PiB and tablet formulations. Geometric mean terminal half-life was 145.8–207.8 h (> 6 days). Compared with PiB, exposures for the tablet formulation were 45–56

Data-independent acquisition mass spectrometry (DIA-MS) is a powerful tool for quantitative proteomics, but a well-constructed reference spectral library is crucial to optimize DIA analysis, particularly for low-abundance proteins. In this study, we evaluate the efficacy of a recombinant protein spectral library (rPSL), generated from tryptic digestion of 42 human recombinant proteins, in enhancing the detection and quantification of lower-abundance cancer-associated proteins. Additionally, we generated a combined sample-specific biological-rPSL by integrating the rPSL with a spectral library derived from pooled biological samples. We compared the performance of these libraries for DIA data extraction with standard methods, including sample-specific biological spectral library and library-free DIA methods. Our specific focus was on quantifying cancer-associated proteins, including key enzymes involved in kynurenine pathway, across patient-derived tissues and cell lines. Both rPSL and biological-rPSL DIA approaches provided significantly improved coverage of lower-abundance proteins, enhancing sensitivity and more consistent protein quantification across matched tumour and adjacent noncancerous tissues from breast and colorectal cancer patients and in cancer cell lines. Overall, our study demonstrates that rPSL and biological-rPSL coupled with DIA-MS workflows, can address the limitations of both biological library-based and library-free DIA methods, offering a robust approach for quantifying low-abundance cancer-associated proteins in complex biological samples.

This untargeted metabolomics study investigated the synergistic antibacterial activity of polymyxin B and Leu10-teixobactin, a depsipeptide inhibitor of cell wall biosynthesis. Checkerboard microdilution assays revealed a significant synergy against polymyxin-susceptible and -resistant A. baumannii, excluding lipopolysaccharide-deficient variants. Time-kill assays confirmed bactericidal synergy, reducing bacterial burden by approximately 4-6-log10CFU/mL. The combination (2xMIC polymyxin B and 0.5xMIC Leu10-teixobactin) prevented bacterial regrowth after 24 h, indicating sustained efficacy against the emergence of resistant mutants. The analysis of A. baumannii ATCC (TM) 19606 metabolome demonstrated that the polymyxin B-Leu10-teixobactin combination produced more pronounced perturbation compared to the individual antibiotics across all time points (1, 3 and 6 h). Pathway analysis revealed that lipid metabolism, cell envelope biogenesis, and cellular respiration were predominantly impacted by the combination, and to a lesser extent by polymyxin B monotherapy. Leu10-teixobactin treatment alone had only a minor impact on the metabolome, primarily at the 6 h time point. Peptidoglycan assays confirmed the combination's concerted deleterious effects on bacterial cell envelope integrity. Electron microscopy further substantiated these findings, revealing pronounced cell envelope damage, membrane blebbing, and vacuole formation. These findings highlight the potential of the polymyxin B-Leu10-teixobactin combination as an effective treatment in preventing resistance in A. baumannii.

The transition zone is a specialised gate at the base of cilia/flagella, which separates the ciliary compartment from the cytoplasm and strictly regulates protein entry. We identified a potential new regulator of the male germ cell transition zone, CEP76. We demonstrated that CEP76 was involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In the mutant, sperm tails were shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulated at the sperm neck in the mutant. Severe annulus, fibrous sheath, and outer dense fibre abnormalities were also detected in sperm lacking CEP76. Finally, we identified that CEP76 dictates annulus positioning and structure. This study suggests CEP76 as a male germ cell transition zone protein and adds further evidence to the hypothesis that the spermatid transition zone and annulus are part of the same functional structure.

Non-invasive vagus nerve stimulation (nVNS) is an established neurostimulation therapy used in the treatment of epilepsy, migraine and cluster headache. In this randomized, double-blind, sham-controlled trial we explored the role of nVNS in the treatment of gait and other motor symptoms in Parkinson’s disease (PD) patients. In a subgroup of patients, we measured selected neurotrophins, inflammatory markers and markers of oxidative stress in serum. Thirty-three PD patients with freezing of gait (FOG) were randomized to either active nVNS or sham nVNS. After baseline assessments, patients were instructed to deliver six 2 min stimulations (12 min/day) of the active nVNS/sham nVNS device for 1 month at home. Patients were then re-assessed. After a one-month washout period, they were allocated to the alternate treatment arm and the same process was followed. Significant improvements in key gait parameters (speed, stance time and step length) were observed with active nVNS. While serum tumor necrosis factor- α decreased, glutathione and brain-derived neurotrophic factor levels increased significantly (p < 0.05) after active nVNS treatment. Here we present the first evidence of the efficacy and safety of nVNS in the treatment of gait in PD patients, and propose that nVNS can be used as an adjunctive therapy in the management of PD patients, especially those suffering from FOG.Clinical trial registration: identifier ISRCTN14797144.

One of the major consequences of the COVID-19 pandemic has been the significant incidence of persistent fatigue following resolution of an acute infection (i.e. post-COVID fatigue). We have shown previously that, in comparison to healthy controls, those suffering from post-COVID fatigue exhibit changes in muscle physiology, cortical circuitry, and autonomic function. Whether these changes preceded infection, potentially predisposing people to developing post-COVID fatigue, or whether the changes were a consequence of infection was unclear. Here we present results of a 12-month longitudinal study of 18 participants from the same cohort of post-COVID fatigue sufferers to investigate these correlates of fatigue over time. We report improvements in self-perception of the impact of fatigue via questionnaires, as well as significant improvements in objective measures of peripheral muscle fatigue and autonomic function, bringing them closer to healthy controls. Additionally, we found reductions in muscle twitch tension rise times, becoming faster than controls, suggesting that the improvement in muscle fatigability might be due to a process of adaptation rather than simply a return to baseline function.

Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.

Colorectal cancer (CRC) poses a significant threat to many human lives worldwide and survival following resection is predominantly stage dependent. For early-stage cancer, patients are not routinely advised to undergo additional post-operative adjuvant chemotherapy. Acceptable clinical management guidelines are well established for patients in pTNM stages I, III and IV. However, recommendations for managing CRC stage II patients remain controversial and many studies have been conducted to segregate stage II patients into low- and high-risk of recurrence using genomic, transcriptomic and proteomic molecular markers. As proteins provide valuable insights into cellular functions and disease state and have a relatively easy translation to the clinic, this review aims to discuss potential prognostic protein biomarkers proposed for predicting tumour relapse in early-stage II CRC. It is suggested that a panel of markers may be more effective than a single marker and further evaluation is required to translate these into clinical practice.

Thirteen pairs of bull semen samples were assessed. One of each pair of samples was inadvertently stored in phosphate buffered saline (PBS) that was used for analysis of motility immediately after collection. Other sample of the pair that was correctly stored in buffered formol saline (BFS) was assessed later. Proportion of morphologically normal sperm was different (p = 0.0021) between the 2 storage methods. Most of the abnormalities in the PBS samples were loose and detached heads. Closer examination of these sperm illustrated changes in the composition of the tail, with an apparent loss of the plasma membrane. This serendipitous error allowed documentation of the importance of correct storage of semen samples for morphological assessment of sperm, and the tertiary defects detected with the incorrect storage of semen samples in semen extender. Tertiary defects should always be considered a possibility during morphological assessment of sperm via spermiogram.

BackgroundSporadic parathyroid adenoma (PA) is the most common cause of hyperparathyroidism, yet the mechanisms involved in its pathogenesis remain incompletely understood.MethodsSurgically removed PA samples, along with normal parathyroid gland (PG) tissues that were incidentally dissected during total thyroidectomy, were analysed using single-cell RNA-sequencing with the 10x Genomics Chromium Droplet platform and Cell Ranger software. Gene set variation analysis was conducted to characterise hallmark pathway gene signatures, and single-cell regulatory network inference and clustering were utilised to analyse transcription factor regulons. Immunohistochemistry and immunofluorescence were performed to validate cellular components of PA tissues. siRNA knockdown and gene overexpression, alongside quantitative polymerase chain reaction, Western blotting and cell proliferation assays, were conducted for functional investigations.ResultsThere was a pervasive increase in gene transcription in PA cells (PACs) compared with PG cells. This is associated with high expression of histone-lysine N-methyltransferase 2A (KMT2A). High KMT2A levels potentially contribute to promoting PAC proliferation through upregulation of the proto-oncogene CCND2, which is mediated by the transcription factors signal transducer and activator of transcription 3 (STAT3) and GATA binding protein 3 (GATA3). PA tissues are heavily infiltrated with myeloid cells, while fibroblasts, endothelial cells and macrophages in PA tissues are commonly enriched with proinflammatory gene signatures relative to their counterparts in PG tissues.ConclusionsWe revealed the previously underappreciated involvement of the KMT2A-STAT3/GATA3-CCND2 axis and chronic inflammation in the pathogenesis of PA. These findings underscore the therapeutic promise of KMT2A inhibition and anti-inflammatory strategies, highlighting the need for future investigations to translate these molecular insights into practical applications.Highlights Single-cell RNA-sequencing reveals a transcriptome catalogue comparing sporadic parathyroid adenomas (PAs) with normal parathyroid glands. PA cells show a pervasive increase in gene expression linked to KMT2A upregulation. KMT2A-mediated STAT3 and GATA3 upregulation is key to promoting PA cell proliferation via cyclin D2. PAs exhibit a proinflammatory microenvironment, suggesting a potential role of chronic inflammation in PA pathogenesis. Single-cell RNA-sequencing reveals a transcriptome catalogue comparing sporadic parathyroid adenomas (PAs) with normal parathyroid glands. PA cells show a pervasive increase in gene expression linked to KMT2A upregulation. KMT2A-mediated STAT3 and GATA3 upregulation is key to promoting PA cell proliferation via cyclin D2. PAs exhibit a proinflammatory microenvironment, suggesting a potential role of chronic inflammation in PA pathogenesis. image

Male subfertility or infertility is a common condition often characterized by men producing a low number of sperm with poor quality. To gain insight into this condition, we performed a quantitative proteomic analysis of semen samples obtained from infertile and fertile men. At least 6 proteins showed significant differences in regulation of alternatively spliced isoforms. To investigate this link between aberrant alternative splicing and production of poor-quality spermatozoa, we overexpressed the hnrnpH/F-orthologue Glorund (Glo) in Drosophila, which was also found to be abundant in poor quality human sperm. Transgenic animals produced low numbers of morphologically defective spermatozoa and aberrant formation of the "dense body," an organelle akin to the mammalian manchette. Furthermore, fertility trials demonstrated that transgenic flies were either completely infertile or highly subfertile. These findings suggest that dysregulation of hnrnpH/F is likely to result in the production of low-quality semen, leading to subfertility or infertility in men.

Background: Recent advancements in understanding plasma extracellular vesicles (EVs) and their role in disease biology have provided additional unique insights into the study of Colorectal Cancer (CRC). Methods: This study aimed to gain biological insights into disease progression from plasma-derived extracellular vesicle proteomic profiles of 80 patients (20 from each CRC stage I-IV) against 20 healthy age- and sex-matched controls using a high-resolution SWATH-MS proteomics with a reproducible centrifugation method to isolate plasma EVs. Results: We applied the High-Stringency Human Proteome Project (HPP) guidelines for SWATH-MS analysis, which refined our initial EV protein identification from 1362 proteins (10,993 peptides) to a more reliable and confident subset of 853 proteins (6231 peptides). In early-stage CRC, we identified 11 plasma EV proteins with differential expression between patients and healthy controls (three up-regulated and eight down-regulated), many of which are involved in key cancer hallmarks. Additionally, within the same cohort, we analysed EV proteins associated with tumour recurrence to identify potential prognostic indicators for CRC. A subset of up-regulated proteins associated with extracellular vesicle formation (GDI1, NSF, and TMED9) and the down-regulation of TSG101 suggest that micro-metastasis may have occurred earlier than previously anticipated. Discussion: By employing stringent proteomic analysis and a robust SWATH-MS approach, we identified dysregulated EV proteins that potentially indicate early-stage CRC and predict recurrence risk, including proteins involved in metabolism, cytoskeletal remodelling, and immune response. While our findings underline discrepancies with other studies due to differing isolation and stringency parameters, they provide valuable insights into the complexity of the EV proteome, emphasising the need for standardised protocols and larger, well-controlled studies to validate potential biomarkers.