Protein Abundance Research Articles

6PPD, Not 6PPD-Quinone, Induced Serious Zebrafish Eye Damage by Disrupting the Thyroid Signaling Pathway.

N-(1,3-Dimethylbutyl)-N'-phenyl-1,4-phenylenediamine (6PPD) and its oxidation product 6PPD-quinone (6PPDQ) showed different acute toxicities and bioaccumulation potencies in fish. In this study, we compared the thyroid disrupting effects of 6PPD and 6PPDQ through in vitro, in silico, and in vivo assays. Interestingly, although 6PPD and 6PPDQ showed similar docking affinities with thyroid hormone receptor (TR) isoforms and GH3 cell inhibition effects, the thyroid signaling pathway, eye development, phototactic behaviors, and cell density in the retinal layer in the larval zebrafish were significantly affected only following 6PPD exposure. Further investigation demonstrates that 6PPD can act as a TR antagonist to reduce the opsin protein abundance and inhibit the cone photoreceptor cell proliferation, which finally alters the retinal layer structure and causes microphthalmus in zebrafish. Especially, under environmental relevant concentration exposure, 6PPD induced alterations of trβ, opn1lw1, opn1mw1, rpe65a, nr2e3 gene expressions although no significant eye histopathological change was observed. This study illustrates for the first time the more serious visual system impairment of 6PPD compared to 6PPDQ, with thyroid signaling disruption being a contributing factor, while other important toxic targets still require further research.

Proteomics in Acute Heart Transplant Rejection, On Behalf of the GRAfT Investigators.

Proteomic phenotyping can provide insights into rejection pathophysiology, novel biomarkers, and therapeutic targets. Within the prospective, multicenter Genomic Research Alliance for Transplantation study, 181 proteins were evaluated from blood drawn at the time of endomyocardial biopsy; protein fold change, logistic regression, and pathway analyses were conducted, with protein discovery adjusted for a 5% false discovery rate. Among 104 adult heart transplant patients (31% female sex, 53% Black race, median age 52 y), 74 had no rejection, 18 developed acute cellular rejection (ACR), and 12 developed antibody-mediated rejection (AMR). Differential expression was found in 2 proteins during ACR (inflammatory proteins CXCL10 and CD5) and 73 proteins during AMR. The most abundant AMR proteins were the heart failure biomarkers N-terminal pro-B-type natriuretic peptide and suppression of tumorigenicity 2. In univariate logistic regression, odds of identifying ACR on endomyocardial biopsy increased with doubling of CXCL10 (odds ratio [OR] 2.2 [95% confidence interval (CI), 1.3-3.6]) and CD5 (OR 4.7 [95% CI, 1.7-12.9]) concentrations, and odds of AMR increased with doubling of N-terminal pro-B-type natriuretic peptide (OR 13.0 [95% CI, 2.7-62.7) and suppression of tumorigenicity 2 (OR 4.8 [95% CI, 2.1-10.7]) concentrations. After multivariable analysis with clinical covariates, these proteins showed similar odds of ACR or AMR on biopsy. Pathway analysis identified T cell-receptor signaling and cell differentiation as key pathways in ACR and cardiovascular disease and cell turnover in AMR. Proteomic analysis reveals unique biomarkers and biological pathway expression in ACR and AMR. Cardiac injury-associated biomarkers were more pronounced in AMR, whereas inflammatory biomarkers were more pronounced in ACR. Proteomic analysis may provide insights into rejection pathophysiology, detection, and therapy.

Necroptosis contributes to deoxynivalenol-induced liver injury and inflammation in weaned piglets

BackgroundThe aim of this study was to investigate the role of necroptosis in deoxynivalenol (DON)-induced liver injury and inflammation in weaned piglets.MethodsIn Exp. 1, 12 weaned piglets were divided into 2 groups including pigs fed basal diet and pigs fed diet contaminated with 4 mg/kg DON for 21 d. In Exp. 2, 12 weaned piglets were divided into 2 groups including control piglets and piglets given a gavage of 2 mg/kg body weight (BW) DON. In Exp. 3, 24 weaned piglets were used in a 2 × 2 factorial design and the main factors including necrostatin-1 (Nec-1) (DMSO or 0.5 mg/kg BW Nec-1) and DON challenge (saline or 2 mg/kg BW DON gavage). On 21 d in Exp. 1, or at 6 h post DON gavage in Exp. 2 and 3, pigs were killed for blood samples and liver tissues. Liver histology, blood biochemical indicators, and liver inflammation and necroptosis signals were tested.ResultsDietary or oral gavage with DON caused liver morphological damage in piglets. Dietary DON led to hepatocyte damage indicated by increased aspartate transaminase (AST) activity and AST/alanine aminotransferase (ALT) ratio, and DON gavage also caused hepatocyte damage and cholestasis indicated by increased AST and alkaline phosphatase (AKP) activities. Dietary DON caused liver necroptosis indicated by increased protein abundance of total receptor interacting protein kinase 3 (t-RIP3) and total mixed lineage kinase domain-like protein (t-MLKL). Moreover, DON gavage increased mRNA expression of interleukin (IL)-6 and IL-1β in liver. DON gavage also induced liver necroptosis demonstrated by increased protein abundance of t-RIP3, phosphorylated-RIP3 (p-RIP3), t-MLKL and p-MLKL. However, pretreatment with Nec-1, a specific inhibitor of necroptosis, inhibited liver necroptosis indicated by decreased protein expression of t-RIP3, p-RIP3, t-MLKL and p-MLKL. Nec-1 pretreatment reduced liver morphological damage after DON gavage. Pretreatment with Nec-1 also attenuated liver damage induced by DON indicated by decreased activities of AST and AKP. Furthermore, Nec-1 pretreatment inhibited liver mRNA expression of IL-6 and IL-1β after DON challenge.ConclusionsOur data demonstrate for the first time that necroptosis contributes to DON-induced liver injury and inflammation in piglets.

Design of fluorescent sensors for the detection of human serum albumin structure employing nitrogen-by modulation of molecular containing heterocycles

Serum albumin is the most abundant protein in mammalian plasma. It occupies an extremely important position in organisms as an early indicator of the development of many diseases. The development of fluorescence sensing technology has revolutionized the drawbacks of traditional methods. In this strategy, we designed and synthesized three dual fluorophore organic sensors TPA-IPA-T1, TPA-QL-T2, and TPA-Py-T3 for the detection of serum albumin. The rotation of the C–C bond of sensors is restricted and the molecular structure forms a rigid plane that contributes to the fluorescent emission intensity of the sensor. The fluorescent skeletons were successfully constructed by triphenylamine composed the electron-withdrawing nitrogen-containing compounds benzopyrrole (indole), benzopyridine (quinoline), and pyridine, respectively, with piperazine as a rigid connecting bridge. The addition of human serum albumin (HSA) turned on the fluorescent emission of the sensor near 580–600 nm. The color of the sensor solution changed from colorless to pink and bright orange, respectively. The fluorescent intensity increased by 24, 67, and 31 fold, respectively. The sensors have the advantages of sensitivity and fast response, strong anti-interference ability, low detection limit and good water solubility. Drug experiments in artificial urine demonstrate the characteristics of sensors, which has a very high potential for biological feasibility and medical applications. The sensors facilitaty the binding of the HSA through electrostatic interactions. The sensors were explored to be able to efficiently bind the IB structural domain of HSA, which had a good meaningful of avoiding the interference of commonly drugs in clinical medicine and in artificial urine on the detection of HSA.

Glutinous rice gel as all-natural ink supply for extrusion-based food 3D printing – the chemical basis of gel printability

In this study, four varieties of glutinous rice were screened out of 18 varieties as representative model inks. Gel prepared using variety WN9612 of high amylose content displayed high viscosity and large overall print deviation (OPD = 17.4 ± 0.4 %) due to difficulties in extrusion. Printed item by variety 19,415 of medium amylose content showed good printability (OPD = 4.4 ± 0.4 %) and highest hardness (197.5 ± 7.3 N). Similarly good printability was demonstrated by variety 19,416 (OPD = 4.1 ± 0.2 %) containing low amylose content but abundant protein and bound-water. Variety WN9446 lacking protein and moisture created item with fluid structure, lowest gel strength (738.5 ± 20.9 Pa) and standing ability. Therefore, low to medium amylose content rendered suitable viscosity, extrudability and printability of rice gel while non-starchy components predominated water mobility and texture of 3D printed items. This study for the first time establishes theoretical groundwork of selecting glutinous rice varieties with appropriate chemical composition as all-natural materials in 3D printing and food customization.

The crucial quality marker of Panax ginseng: Glycosylated modified ribonuclease-like storage protein

Panax ginseng C.A.Mey is a famous natural herbal medicine worldwide. Mountain-cultivated ginseng (MCG) and garden-cultivated ginseng (GCG) are two types of Panax ginseng. There is a significant difference in economic benefits between MCG and GCG, which can always lead to problems such as adulteration and substitution of MCG with lower-priced alternatives. We explored the quality marker of ginseng at the intact protein level and established a foundation for the quality control of ginseng. Cellulose nanocrystal assisted sample preparation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) equipped with a high mass detector was performed to analyze intact proteins in ginseng. The results revealed that the ribonuclease-like storage protein is the most abundant protein in MCG and GCG. Meanwhile, the molecular weight of the ribonuclease-like storage protein showed great difference between different ginseng species, which is 26.2 kDa in MCG and 24.2 kDa in GCG. The ribonuclease-like storage protein glycosylation modification difference provides data support for the differentiation between MCG and GCG. This study showed that glycosylated modified ribonuclease-like storage protein can be a crucial quality marker of ginseng, facilitating the rapid distinction between MCG and GCG.

Homozygous variant in translocase of outer mitochondrial membrane 7 leads to metabolic reprogramming and microcephalic osteodysplastic dwarfism with moyamoya disease

Impaired mitochondrial protein import machinery leads to phenotypically heterogeneous diseases. Here, we report a recurrent homozygous missense variant in the gene that encodes the translocase of outer mitochondrial membrane 7 (TOMM7) in nine patients with microcephaly, short stature, facial dysmorphia, atrophic macular scarring, and moyamoya disease from seven unrelated families. To prove the causality of the TOMM7 variant, mitochondrial morphology, proteomics, and respiration were investigated in CRISPR/Cas9-edited iPSCs-derived endothelial cells. Cerebrovascular defects and mitochondrial respiration were also examined in CRISPR/Cas9-edited zebrafish. iPSC-derived endothelial cells with homozygous TOMM7 p.P29L showed increased TOM7 stability, enlarged mitochondria, increased senescence, and defective tube formation. In addition, proteomic analysis revealed a reduced abundance of mitochondrial proteins involved in ATP synthesis or coordinating TCA cycle and gluconeogenesis. Moreover, mitochondrial respiration was slightly decreased while ATP production from glycolysis was significantly increased. Furthermore, deletion of tomm7 in zebrafish caused craniofacial and cerebrovascular defects that recapitulated human phenotypes. Notably, homozygous iPSCs differentially expressed genes involved in glycolysis and response to hypoxia. Finally, the metabolic imbalance was evidenced by decreased oxygen consumption, increased level of hexokinase 2, and enhanced glycolysis in endothelial cells derived from the patient's iPSCs. These results revealed the essential role of TOMM7 in balancing cellular sources of energy production at both proteomic and transcriptomic levels and provided the molecular mechanisms through which TOMM7 p.P29L variant leads to an autosomal recessive microcephalic osteodysplastic dwarfism with moyamoya disease. This work is supported by National Science and Technology Council grants and National Cheng Kung University Hospital.

Exploration of age-related changes in reproductive parameters of female Japanese quail (Coturnix japonica)

The decline in reproductive efficiency during post-peak period of production in poultry species holds significant economic implications. This study aimed to investigate the productive and reproductive performance of Japanese quails across distinct production stages and the association between these parameters and some genes expression and histometric alterations within the reproductive system. A total of 180 quails from a commercial flock were selected at varying egg production stages, including young, mature, and old, with 45 female and 15 male quails allocated to each group. The quails were maintained for six weeks. During recording period, daily records of egg production and egg weight were recorded. Additionally, oviduct histometric and Follicle biometric measurements, along with mRNA transcript abundance assessments related to follicular selection and yolk accumulation, were conducted on the oviduct, ovary, and small yellow follicles at the end of the experimental period. The results revealed a decrease in egg production in the old group compared to the young and mature groups (P < 0.05); meanwhile, the old group had the highest egg weight, and F1 follicle weight (P < 0.05). Additionally, the number of prehierarchical follicles was lower in the mature and old groups compared to the young group (P < 0.05). The lowest oviduct length, primary and secondary fold height, and thickness of the isthmus and magnum were noted in the old group (P < 0.05). Fertility and hatchability were lower in the old group compared to the other groups (P < 0.05). The mRNA transcript abundance of anti-Mullerian hormone (AMH), was highest in the old group and lowest in the young group (P < 0.05), while the mRNA transcript abundance of bone morphogenetic protein 15 (BMP15) was higher in the mature group compared to the other groups (P < 0.05). Additionally, the young quails had the highest occludin (OCLN) mRNA transcript abundance compared to other groups (P < 0.05). Overall, the study findings indicate decreased production and reproductive performance, as well as reduced hatchling quality over the production period, attributed to a declining number of follicles, noncooperative gene expression related to follicle selection and yolk accumulation, and diminishing oviduct fold size.

Genome-wide identification and expression analysis of genes encoding late embryogenesis proteins in Cicer arietinum

Late embryogenesis abundant (LEA) proteins play defensive roles during seed maturation and seed germination processes. However, there is no such investigation was carried out in chickpea. In present study, genome wide identification and characterization of LEA encoding genes has been investigated, and identified 65 and 74 LEA encoding genes in desi and kabuli cultivar of chickpea, respectively. All these genes have been classified into eight subfamilies on the bases of their phylogenetic analysis and conserved domain. Maximum members of LEA encoding genes were found to be a part of the LEA_2 gene family. The analysis of physicochemical properties of LEAs was also conducted. LEA encoding genes have been found to be located in all chromosomes (8 chr) of chickpea and identified as involved in response to stimulus, biological processes, molecular functions and cellular components based upon gene ontology analysis. Gene expression analysis of randomly selected 8 LEA encoding genes has been carried out during different seed developmental stages which revealed the higher expression of LEA encoding genes during later stage of seed development in chickpea and proved their potential role in desiccation process during seed maturation. During seed germination, expression analysis of LEA encoding genes was found to be higher during the initial stages of seed germination. In conclusion, this work highlights the genome wide identification and characterization of LEA encoding genes in chickpea and proposed potential roles during seed developmental processes. This information could also be useful as a reference investigation for molecular breeding of chickpea for recalcitrant behaviour of seed.

Identification of biological pathways and putative candidate genes for residual feed intake in a tropically adapted beef cattle breed by plasma proteome analysis

This study identified potential biomarkers for feed efficiency by blood plasma proteome analysis of a tropically adapted beef cattle breed. Two experimental groups were selected based on residual feed intake (RFI). The proteome was investigated by LC-MS/MS in a data-dependent acquisition mode. After quality control, 123 differentially abundant proteins (DAPs) were identified between the two experimental groups. Among DAPs with the highest absolute log-fold change values, the PRDM2, KRT5, UGGT1, DENND5B, B2M, SLC44A2, SLC7A2, PTPRC, and FETUB were highlighted as potential biomarkers because of their functions that may contribute to RFI. Furthermore, functional enrichment analysis revealed several biological processes, molecular functions and pathways that contributes to RFI, such as cell signaling, cellular responses to stimuli, immune system, calcium, hormones, metabolism and functions of proteins, lipids and carbohydrates. Protein-protein interaction analysis identified 32 and 11 DAPs as important nodes based on their interactions in the high- and low-RFI groups, respectively. This study represents the first comprehensive profiling of the blood plasma proteome of a tropically adapted beef cattle breed and provides valuable insights into the potential roles of these DAPs in key biological processes and pathways, contributing to our understanding of the mechanisms underlying feed efficiency in tropically adapted beef cattle. SignificanceLC-MS/MS analysis was performed to investigate changes in the blood plasma proteome associated with residual feed intake (RFI) in a tropically adapted beef cattle breed (Bos taurus taurus). Some putative biomarkers were identified to distinguish the high-RFI to low-RFI animals, based on their log-fold change value or on their protein-protein interaction network, which provide helpful sources in developing novel selection strategies for breeding programs. Our findings also revealed valuable insights into the metabolic pathways and biological processes that contribute to RFI in beef cattle, such as those closely linked to cell signaling, cellular responses to stimuli, immune system, calcium, hormones, metabolism and functions of proteins, lipids and carbohydrates.

Bone marrow mesenchymal stem cell-derived exosomal miR-181a-5p promotes M2 macrophage polarization to alleviate acute pancreatitis through ZEB2-mediated RACK1 ubiquitination.

As a common digestive disease, acute pancreatitis (AP) often threatens the life of patients. Bone marrow mesenchymal stem cells (BMSCs) derived exosomes have exhibited some benefits for AP. However, the mechanism remains unclear and deserves to be further investigated. The characteristics of BMSCs-exosomes (BMSCs-Exos) were identified. The abundance of genes and proteins was evaluated using quantitative real-time PCR (RT-qPCR), western blot, enzyme-linked immunosorbent assay (ELISA) and IF assay. Cell apoptosis and CD206-positive cells were measured by flow cytometry. The interactions among miR-181a-5p, Zinc finger E-box binding homeobox2 (ZEB2) and Receptor for Activated C Kinase 1 (RACK1) were verified using dual luciferase reporter assay, RNA immunoprecipitation (RIP), coimmunoprecipitation (Co-IP). BMSCs-Exos effectively improved AP injury through restraining AR42J cell apoptosis and promoting M2 macrophage polarization, which was realized due to BMSCs-Exos harboring an abundance of miR-181a-5p. Further experiments validated miR-181a-5p silenced ZEB2 and ZEB2 reduced RACK1 expression through mediating RACK1 ubiquitination. ZEB2 knockdown decreased AR42J cell apoptosis and induced M2 macrophage polarization to alleviate AP injury, whereas RACK1 downregulation abolished these phenomena. BMSCs-Exos harboring miR-181a-5p suppressed AR42J cell apoptosis and promoted M2 macrophage polarization to delay AP progression through ZEB2-mediated RACK1 ubiquitination.

Elucidating loss-of-function mechanisms of monoallelic EPAS1 mutations underlying congenital hypoplastic anaemia in a paediatric anaemia cohort.

HIF-2α, encoded by EPAS1, plays a dominant role in regulating erythropoietin (EPO) production, maintaining the dynamic balance of erythropoiesis. Gain-of-function mutations in EPAS1 cause erythrocytosis. However, anaemia caused by EPAS1 loss-of-function mutations has been confined to only one case report, and the underlying mechanism remains unclear. Herein, the reanalysis of high-throughput sequencing data from 311 patients with anaemia identified three monoallelic EPAS1 variants from three unrelated families in a paediatric anaemia cohort. The probands showed highly consistent clinical phenotypes with normocytic and normochromic anaemia, reticulocytopenia and relative deficiency of serum EPO, characterised as congenital hypoplastic anaemia. Invitro studies suggested that defects in steady-state protein abundance, nuclear localisation and binding with co-activator in EPAS1 variants lead to impaired EPO transcriptional activation. Therefore, loss-of-function mutations in EPAS1 can cause erythroid hypoplasia in an EPO-dependent manner. This study identified a new causative gene for congenital hypoplastic anaemia and clarified the molecular aetiology of loss-of-function EPAS1 mutations.

Unraveling the uterine fluid proteome of mares diagnosed with post-breeding and infectious endometritis

Endometritis is the leading cause of subfertility in mares and a significant challenge to equine reproduction. Unraveling uterine fluid proteome may promote advancements in the knowledge of endometritis etiopathogeneses and its diagnosis and therapeutic practices. Therefore, we aimed to characterize and compare the protein profile of the uterine fluid from healthy mares and animals diagnosed with endometritis. Mangalarga Marchador breed mares from Muriaé - Brazil were divided into control, infectious endometritis, and post-breeding endometritis groups (n = 8/ group). Uterine fluid was collected via low-volume lavage and subjected to protein identification and relative abundance counting. From the 549 proteins detected, 279 were in the uterine fluid of mares from the three experimental groups. Thirteen proteins expressed mostly in healthy mares were associated with endometrial remodeling and early embryonic development. Albumin and uteroglobin presented higher relative abundance in healthy mares and animals with infectious endometritis. Infectious endometritis exhibited proteins related to innate immune and inflammatory responses, including annexin and glutathione S-transferase, and the highest abundance of lipocalins. Fifty-five proteins detected in mares with post-breeding endometritis showed signaling pathways and biological processes related to the innate immune response. These animals also presented the highest abundance of PIGR proteins, which promote IgA transport from plasma into the endometrial mucosa. In conclusion, our results revealed distinct protein profiles from the uterine fluid of mares with infections and post-breeding endometritis. These findings provided valuable insights into the molecular alterations during the establishment and progression of endometritis, contributing to further identification of potential biomarkers.

Tandem upregulation of ion transporters in thick ascending limb of Henle's loop of young Milan hypertensive strain of rats.

Milan hypertensive strain (MHS) of rat represents as one of the ideal rat models to study the genetic form of hypertension associated with aberrant renal salt reabsorption. In contrast to Milan normotensive strain (MNS), MHS rats possess missense mutations in three adducin genes and develop hypertension at 3 months old due to upregulation of sodium-chloride cotransporter (NCC). At pre-hypertensive stage (23-25 days old), MHS rats show enhanced protein abundance of Na+-K+-2Cl- cotransporter (NKCC2) but retain blood pressure comparable to MNS probably through enhanced GFR and reduced NCC and α-subunit of epithelial sodium channel (α-ENaC) expressed in distal convoluted tubule (DCT) and collecting duct (CD). In the present study, mRNA and protein expressions of ion transporters in thick ascending limb of Henle's loop (TAL) of young MHS rats were investigated. Protein abundance of core-glycosylated form of renal outer medullary potassium channel (ROMK) in inner stripe of outer medulla (ISOM) is remarkably increased in MHS rats at prehypertensive stage. Furthermore, basolaterally expressed Na+-K+-ATPase and Barttin were upregulated. These results may indicate that in TAL of MHS rats at this age, both total NKCC2 and core-glycosylated ROMK are upregulated in tandem potentially to balance the luminal potassium concentration. On the basolateral side, upregulation of Na+-K+-ATPase and CLC-Ka/b may energize the excretion of sodium and chloride out from the cells. These data may suggest the interplay of apical and basolateral ion transporters in TAL for the modulation of TAL function in favor of enhancing the transepithelial sodium reabsorption, although this seems compensated by NCC and ENaC expressed at the downstream nephron segments in young MHS rats.

The transcription factor FgSfp1 orchestrates mycotoxin deoxynivalenol biosynthesis in Fusarium graminearum

Fusarium graminearum (F. graminearum) and its derivative mycotoxin deoxynivalenol (DON) are highly concerned with food safety and sustainability worldwide. Although several transcription factors (TFs) had been elucidated, molecular mechanism participates in DON biosynthesis regulation remains largely unrevealed. Here, we first characterized a zinc finger-contained TF in F. graminearum, FgSfp1, which is indispensable for DON production since its depletion resulting in a 95.4% DON yielding reduction. Interestingly, contrast to previous knowledge, all TRI-cluster genes were abnormally upregulated in ΔFgSfp1 while Tri proteins abundance rationally decreased simultaneously. Further evidence show FgSfp1 could coordinate genetic translation pace by manipulating ribosomal biogenesis process. Specifically, FgSfp1-depletion leads to ribosome biogenesis assembly factor (RiBi) expression attenuation along with DON precursor acetyl-CoA synthase reduction since FgSfp1 actively interacts with RNA 2’-O-methylation enzyme FgNop1 revealed by Bi-FC. It subsequently influences mRNA translation pace. In conclusion, we elucidated that the FgSfp1 orchestrates DON biosynthesis via participating RNA posttranscriptional modification for ribosomal RNA maturation, offering insights into the DON biosynthesis regulation. Ultimately, this TF might be a key regulator for DON contamination control in the whole food chain.

Integrating proteomics and metabolomics to evaluate impact of semen collection techniques on the quality and cryotolerance of goat semen

sResults of artificial insemination (AI) are affected by changes in sperm quality and the function throughout collection and preservation procedures. Proteome and metabolome alterations of sperm treated with the different procedures in goat, however, aren’t fully understood. To this end, we sought to investigate the impacts of rectal probe electrostimulation (EE) and artificial vagina (AV) semen collection methods on the quality and the cryotolerance of goat sperm, with additional focus on proteomic and metabolomic analyses. Semen samples were collected from Yunshang black goats and categorized into four groups: fresh sperm collected via AV (XAZ), fresh sperm collected via EE (XEZ), frozen sperm post-AV collection (DAZ) and frozen sperm post-EE collection (DEZ). Four comparisons (XAZ vs. XEZ, DAZ vs. XAZ, DEZ vs. XEZ, DAZ vs. DEZ) were performed, respectively. This study first evaluated sperm motility, acrosome integrity, plasma membrane integrity, mitochondrial activity, and reactive oxygen species (ROS) levels. The results indicated that there were no significant differences in fresh sperm quality parameters between the EE and AV methods. However, notable differences emerged post-cryopreservation. Specifically, the AV method proved more advantageous in preserving the motility, integrities of acrosome and plasma membrane, mitochondrial activity of frozen sperm compared to the EE method. Through the multi-omics approaches, a total of 210 differentially abundant proteins (DAPs) related to sperm characteristics and function were identified across the four comparations. Moreover, 32 differentially abundant metabolites (DAMs) were detected. Comprehensive bioinformatics analysis underscored significant molecular pathways in the co-enrichment of DAPs and DAMs, particularly focusing on the citrate cycle, ROS, oxidative phosphorylation, and glycine, serine, and threonine metabolism etc. We elucidated the differential impacts of AV and EE collection methods on the quality and cryotolerance of goat semen from omics perspectives, which offer a critical foundation for further exploration into optimizing semen collection and cryopreservation techniques in goat breeding program.

Coordinated protein modules define DNA damage responses to carboplatin at single cell resolution in human ovarian carcinoma models.

Tubo-ovarian high-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy and frequently responds to platinum-based chemotherapy because of common genetic and somatic impairment of DNA damage repair (DDR) pathways. The mechanisms of clinical platinum resistance are diverse and poorly molecularly defined. Consequently, there are no biomarkers or medicines that improve patient outcomes. Herein we use single cell mass cytometry (CyTOF) to systematically evaluate the phosphorylation and abundance of proteins known to participate in the DNA damage response (DDR). Single cell analyses of highly characterized HGSC cell lines that phenocopy human patients show that cells with comparable levels of intranuclear platinum, a proxy for carboplatin uptake, undergo different cell fates. Unsupervised analyses revealed a continuum of DDR responses. Decompositional methods were used to identify eight distinct protein modules of carboplatin resistance and sensitivity at single cell resolution. CyTOF profiling of primary and secondary platinum-resistance patient models shows that a complex DDR sensitivity module is strongly associated with response, suggesting it as a potential tool to clinically characterize complex drug resistance phenotypes.

Tamm-Horsfall protein augments neutrophil NETosis during urinary tract infection.

Urinary neutrophils are a hallmark of urinary tract infection (UTI), yet the mechanisms governing their activation, function, and efficacy in controlling infection remain incompletely understood. Tamm-Horsfall glycoprotein (THP), the most abundant protein in urine, uses terminal sialic acids to bind an inhibitory receptor and dampen neutrophil inflammatory responses. We hypothesized that neutrophil modulation is an integral part of THP-mediated host protection. In a UTI model, THP-deficient mice showed elevated urinary tract bacterial burdens, increased neutrophil recruitment, and more severe tissue histopathological changes compared to WT mice. Furthermore, THP-deficient mice displayed impaired urinary NETosis during UTI. To investigate the impact of THP on NETosis, we coupled in vitro fluorescence-based NET assays, proteomic analyses, and standard and imaging flow cytometry with peripheral human neutrophils. We found that THP increases proteins involved in respiratory chain, neutrophil granules, and chromatin remodeling pathways, enhances NETosis in an ROS-dependent manner, and drives NET-associated morphologic features including nuclear decondensation. These effects were observed only in the presence of a NETosis stimulus and could not be solely replicated with equivalent levels of sialic acid alone. We conclude that THP is a critical regulator of NETosis in the urinary tract, playing a key role in host defense against UTI.

Comprehensive Study of Sperm Proteins and Metabolites Potentially Associated with Higher Fertility of Zebu Cattle (Bos indicus) in Tropical Areas.

Crossbreeding of zebu cattle (Bos indicus) with European breeds (Bos taurus) producing crossbred cattle was performed to overcome the low growth rates and milk production of indigenous tropical cattle breeds. However, zebu cattle fertility is higher than those of crossbred cattle and European breeds under warm conditions. Combination study of proteomics and metabolomics toward Malaysian indigenous breed Kedah × Kelantan-KK (B. indicus) and crossbreed Mafriwal-M (B. taurus × B. indicus) to understand physiological reasons for higher thermotolerance and fertility in Zebu cattle sperm. 161 regulated metabolites and 96 regulated proteins in KK and M (p < 0.05) showed more efficient carbohydrate and energy metabolism, higher integrity of the DNA and plasma membrane, a lower level of reactive oxygen species, and higher levels of phospholipids, which confirmed higher sperm plasma membrane integrity in KK. A stronger antioxidant system and lower polyunsaturated fatty acids help KK sperm cope with oxidative stress under warm conditions. The higher abundance of flagella structural proteins in KK provides a stronger structure that supports sperm motility. Abnormality of flagella, plasma membrane disruption, and DNA fragmentation were higher in M. These findings provide selective molecular markers for developing high-producing and more thermotolerant cattle breeds in tropical areas (197 words).

H3K27 trimethylation-mediated downregulation of miR-216a-3p in sensory neurons regulates neuropathic pain behaviors via targeting STIM1.

Although the therapeutic potential of miRNA-mediated gene regulation has been investigated, its precise functional regulatory mechanism in neuropathic pain remains incompletely understood. In this study, we elucidate that miR-216a-3p serves as a critical non-coding RNA involved in the modulation of trigeminal-mediated neuropathic pain. By conducting RNA-seq and qPCR analysis, we observed a notable decrease of miR-216a-3p in the injured trigeminal ganglia (TG) of male rats. Intra-TG administration of miR-216a-3p agomir or lentiviral-mediated overexpression of miR-216a-3p specifically in sensory neurons of injured TGs alleviated established neuropathic pain behaviors, while downregulation of miR-216a-3p (pharmacologically or genetically) in naïve rats induced pain behaviors. Moreover, nerve injury significantly elevated the H3K27 trimethylation (H3K27me3) levels in the ipsilateral TG, thereby suppressing the SRY-box transcription factor 10 (SOX10) binding to the miR-216a-3p promoter and resulting in the reduction of miR-216a-3p. Inhibiting the enzymes that responsible for catalyzing H3K27me3 restored the nerve injury-induced reduction in miR-216a-3p expression and markedly ameliorated neuropathic pain behaviors. Furthermore, miR-216a-3p targeted stromal interaction molecule 1 (STIM1), and the decreased miR-216a-3p associated with neuropathic pain caused a significant upregulation in the protein abundance of STIM1. Conversely, overexpression of miR-216a-3p in the injured TG suppressed the upregulation of STIM1 expression and reversed the mechanical allodynia. Together, the mechanistic understanding of H3K27me3-dependent SOX10/miR-216a-3p/STIM1 signaling axial in sensory neurons may facilitate the discovery of innovative therapeutic strategies for neuropathic pain management.Significance Statement miRNAs are posttranscriptional regulators of gene expression that play critical roles in the pathogenesis of neuropathic pain. However, the detailed mechanisms by which most pain-associated miRNAs operate and their therapeutic potential are incompletely understood. Our present study revealed that nerve injury-induced trimethylation of lysine 27 on histone H3 (H3K27me3) reduces the binding of SOX10, a transcription factor, to the promoter region of the miR-216a-3p gene, leading to decreased expression of the microRNA, miR-216a-3p. This reduction subsequently promotes neuropathic pain by regulating STIM1. Given that miRNA-mediated gene regulation is a proposed therapeutic approach for treating neuropathic pain, our findings suggest that replenishing miR-216a-3p could serve as a novel strategy for treating chronic neuropathic pain.