Cysteine-rich Secretory Protein Research Articles

DNA Microarray and Bioinformatic Analysis Reveals the Potential of Whale Oil in Enhancing Hair Growth in a C57BL/6 Mice Dorsal Skin Model.

Much research has been conducted to determine how hair regeneration is regulated, as this could provide therapeutic, cosmetic, and even psychological interventions for hair loss. The current study focused on the hair growth effect and effective utilization of fatty oil obtained from Bryde's whales through a high-throughput DNA microarray approach in conjunction with immunohistochemical observations. The research also examined the mechanisms and factors involved in hair growth. In an experiment using female C57BL/6J mice, the vehicle control group (VC: propylene glycol: ethanol: water), the positive control group (MXD: 3% minoxidil), and the experimental group (WO: 20% whale oil) were topically applied to the dorsal skin of the mouse. The results showed that 3% MXD and 20% WO were more effective than VC in promoting hair growth, especially 20% WO. Furthermore, in hematoxylin and eosin-stained dorsal skin tissue, an increase in the number of hair follicles and subcutaneous tissue thickness was observed with 20% WO. Whole-genome transcriptome analysis also confirmed increases for 20% WO in filaggrin (Flg), a gene related to skin barrier function; fibroblast growth factor 21 (Fgf21), which is involved in hair follicle development; and cysteine-rich secretory protein 1 (Crisp1), a candidate gene for alopecia areata. Furthermore, the results of KEGG pathway analysis indicated that 20% WO may have lower stress and inflammatory responses than 3% MXD. Therefore, WO is expected to be a safe hair growth agent.

PI15, a novel secreted WNT-signaling antagonist, regulates chondrocyte differentiation

ABSTRACT Purpose/Aim of Study During the development of the vertebrate skeleton, the progressive differentiation and maturation of chondrocytes from mesenchymal progenitors is precisely coordinated by multiple secreted factors and signaling pathways. The WNT signaling pathway has been demonstrated to play a major role in chondrogenesis. However, the identification of secreted factors that fine-tune WNT activity has remained elusive. Here, in this study, we have identified PI15 (peptidase inhibitor 15, protease Inhibitor 15, SugarCrisp), a member of the CAP (cysteine rich secretory proteins, antigen 5, and pathogenesis related 1 proteins) protein superfamily, as a novel secreted WNT antagonist dynamically upregulated during chondrocyte differentiation. Materials and Methods ATDC5 cells, C3H10T1/2 micromass cultures and primary chondrocyte cells were used as in vitro models of chondrogenesis. PI15 levels were stably depleted or overexpressed by viral shRNA or expression vectors. Chondrogenesis was evaluated by qPCR gene expression analysis and Alcian blue staining. Protein interactions were determined by coimmunoprecipitation assays. Results and Conclusions shRNA-mediated knockdown of PI15 in ATDC5 cells, C3H10T1/2 cells or primary chondrocytes inhibits chondrogenesis, whereas the overexpression of PI15 strongly enhances chondrogenic potential. Mechanistically, PI15 binds to the LRP6 WNT co-receptor and blocks WNT-induced LRP6 phosphorylation, thus repressing WNT-induced transcriptional activity and alleviating the inhibitory effect of WNT signaling on chondrogenesis. Altogether, our findings suggest that PI15 acts as a key regulator of chondrogenesis and unveils a mechanism through which chondrocyte-derived molecules can modulate WNT activity as differentiation proceeds, thereby creating a positive feedback loop that further drives differentiation.

Dexamethasone-Integrated Mesenchymal Stem Cells for Systemic Lupus Erythematosus Treatment via Multiple Immunomodulatory Mechanisms.

The therapeutic application of mesenchymal stem cells (MSCs) has good potential as a treatment strategy for systemic lupus erythematosus (SLE), but traditional MSC therapy still has limitations in effectively modulating immune cells. Herein, we present a promising strategy based on dexamethasone liposome-integrated MSCs (Dexlip-MSCs) for treating SLE via multiple immunomodulatory pathways. This therapeutic strategy prolonged the circulation time of dexamethasone liposomes in vivo, restrained CD4+T-cell proliferation, and inhibited the release of proinflammatory mediators (IFN-γ and TNF-α) by CD4+T cells. In addition, Dexlip-MSCs initiated cellular reprogramming by activating the glucocorticoid receptor (GR) signaling pathway to upregulate the expression of anti-inflammatory factors such as cysteine-rich secretory protein LCCL-containing domain 2 (CRISPLD2) and downregulate the expression of proinflammatory factors. In addition, Dexlip-MSCs synergistically increased the anti-inflammatory inhibitory effect of CD4+T cells through the release of dexamethasone liposomes or Dex-integrated MSC-derived exosomes (Dex-MSC-EXOs). Based on these synergistic biological effects, we demonstrated that Dexlip-MSCs alleviated disease progression in MRL/lpr mice more effectively than Dexlip or MSCs alone. These features indicate that our stem cell delivery strategy is a promising therapeutic approach for clinical SLE treatment.

CRISPLD1 promotes gastric cancer progression by regulating the Ca2+/PI3K-AKT signaling pathway

Gastric cancer (GC) is a malignant tumor with poor prognosis. Studies have shown that cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is associated with tumor progression. However, its role in GC is unclear. The present study aimed to determine the pathogenic mechanism of CRISPLD1 in GC. Analysis of public databases revealed high mRNA expression of CRISPLD1 in GC, which was associated with poor prognosis. Additionally, CRISPLD1 expression levels showed significant correlations with T stage, overall survival events, and stage. Knockdown of CRISPLD1 reduced cell proliferation, invasion, and migration. Furthermore, CRISPLD1 knockdown decreased intracellular calcium levels in GC cells and inhibited the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Treatment with an AKT activator reversed the inhibitory effect of CRISPLD1 knockdown on GC cell migration and invasion. Our findings suggest that CRISPLD1 promotes tumor cell progression in GC by mediating intracellular calcium levels and activating the PI3K-AKT pathway, highlighting CRISPLD1 as a potential therapeutic target for GC.

The Eastern Bandy Bandy Vermicella annulata, expresses high abundance of SVMP, CRiSP and Kunitz protein families in its venom proteome

The Australian elapid snake radiation (Hydrophiinae) has evolved in the absence of competition from other advanced snakes. This has resulted in ecological specialisation in Australian elapids and the potential for venom proteomes divergent to other elapids. We characterised the venom of the Australian elapid Vermicella annulata (eastern bandy bandy). The venom was analysed using a two-dimensional fractionation process consisting of reverse-phase high-performance liquid chromatography then sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by bottom-up proteomics. Resulting peptides were matched to a species-specific transcriptome and 87% of the venom was characterised. We identified 11 toxins in the venom from six families: snake venom metalloproteinases (SVMP; 24.2%; two toxins) that are class P-III SVMPs containing a disintegrin-like domain, three-finger toxins (3FTx; 21.6%; five toxins), kunitz peptides (KUN; 19.5%; one toxin), cysteine-rich secretory proteins (CRiSP; 18%; one toxin), and phospholipase A2 (PLA2; 4%; two toxins). The venom had low toxin diversity with five protein families having one or two toxins, except for 3FTx with five different toxins. V. annulata expresses an unusual venom proteome, with high abundances of CRiSP, KUN and SVMP, which are not normally highly expressed in elapid venoms. This unusual venom composition could be an adaptation to its specialised diet. Biological significanceAlthough the Australian elapid radiation represents the most extensive speciation event of elapids on any continent, with 100 terrestrial species, the venom composition of these snakes has rarely been investigated, with only five species currently characterised. Here we provide the venom proteome of a sixth species, Vermicella annulata. The venom of this species could be particularly informative from an evolutionary perspective, as it is an extreme dietary specialist, only preying on blind snakes (Typhlopidae). We show that V. annulata expresses a highly unusual venom for an elapid, due to the high abundance of the protein families SVMP, CRiSP, and KUN, which together make up 61% of the venom. When averaged across all species, a typical elapid venom is 82% PLA2 and 3FTx. This is the second recorded instance of an Australian elapid having evolved highly divergent venom expression.

Proteomic Investigation of Cape Cobra (Naja nivea) Venom Reveals First Evidence of Quaternary Protein Structures.

Naja nivea (N. nivea) is classed as a category one snake by the World Health Organization since its envenomation causes high levels of mortality and disability annually. Despite this, there has been little research into the venom composition of N. nivea, with only one full venom proteome published to date. Our current study separated N. nivea venom using size exclusion chromatography before utilizing a traditional bottom-up proteomics approach to unravel the composition of the venom proteome. As expected by its clinical presentation, N. nivea venom was found to consist mainly of neurotoxins, with three-finger toxins (3FTx), making up 76.01% of the total venom proteome. Additionally, cysteine-rich secretory proteins (CRISPs), vespryns (VESPs), cobra venom factors (CVFs), 5'-nucleotidases (5'NUCs), nerve growth factors (NGFs), phospholipase A2s (PLA2), acetylcholinesterases (AChEs), Kunitz-type serine protease inhibitor (KUN), phosphodiesterases (PDEs), L-amino acid oxidases (LAAOs), hydrolases (HYDs), snake venom metalloproteinases (SVMPs), and snake venom serine protease (SVSP) toxins were also identified in decreasing order of abundance. Interestingly, contrary to previous reports, we find PLA2 toxins in N. nivea venom. This highlights the importance of repeatedly profiling the venom of the same species to account for intra-species variation. Additionally, we report the first evidence of covalent protein complexes in N. nivea venom, which likely contribute to the potency of this venom.

Valsa mali PR1-like protein modulates an apple valine-glutamine protein to suppress JA signaling-mediated immunity.

Apple Valsa canker (AVC) is a devastating disease of apple (Malus × domestica), caused by Valsa mali (Vm). The Cysteine-rich secretory protein, Antigen 5, and Pathogenesis-related protein 1 (CAP) superfamily protein PATHOGENESIS-RELATED PROTEIN 1-LIKE PROTEIN c (VmPR1c) plays an important role in the pathogenicity of Vm. However, the mechanisms through which it exerts its virulence function in Vm-apple interactions remain unclear. In this study, we identified an apple valine-glutamine (VQ)-motif-containing protein, MdVQ29, as a VmPR1c target protein. MdVQ29-overexpressing transgenic apple plants showed substantially enhanced AVC resistance as compared with the wild type. MdVQ29 interacted with the transcription factor MdWRKY23, which was further shown to bind to the promoter of the jasmonic acid (JA) signaling-related gene CORONATINE INSENSITIVE 1 (MdCOI1) and activate its expression to activate the JA signaling pathway. Disease evaluation in lesion areas on infected leaves showed that MdVQ29 positively modulated apple resistance in a MdWRKY23-dependent manner. Furthermore, MdVQ29 promoted the transcriptional activity of MdWRKY23 toward MdCOI1. In addition, VmPR1c suppressed the MdVQ29-enhanced transcriptional activation activity of MdWRKY23 by promoting the degradation of MdVQ29 and inhibiting MdVQ29 expression and the MdVQ29-MdWRKY23 interaction, thereby interfering with the JA signaling pathway and facilitating Vm infection. Overall, our results demonstrate that VmPR1c targets MdVQ29 to manipulate the JA signaling pathway to regulate immunity. Thus, this study provides an important theoretical basis and guidance for mining and utilizing disease-resistance genetic resources for genetically improving apples.

Snake venom cysteine-rich secretory protein from Mojave rattlesnake venom (Css-CRiSP) induces acute inflammatory responses on different experimental models

Snake venoms contain various molecules known for activating innate immunity and causing local effects associated with increased vascular permeability, such as vascular leakage and edema, common symptoms seen in snakebite envenomings. We have demonstrated that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability. This study aimed to explore the functional role of CRiSP isolated from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom (Css-CRiSP) on the activation of inflammatory responses in different models. We measured the release of inflammatory mediators in cultured human dermal blood endothelial cells (HDBEC), lymphatic endothelial cells (HDLEC) and monocyte-derived macrophages (MDM) at 0.5, 1, 3, 6, and 24 h after treatment with Css-CRiSP (1 μM). We also determined the acute inflammatory response in BALB/c mice 30 min after intraperitoneal injection of the toxin (2 μg/mouse). Css-CRiSP induced the production of IL-8 and IL-6, but not TNF-α, in HDBEC and HDLEC in a time-dependent manner. In addition, Css-CRiSP significantly enhanced the production of IL-6, TNF-α, IL-8, and IL-1β in MDM. Moreover, it caused a remarkable increase of chemotactic mediators in the exudates of experimental mice. Our results reveal that Css-CRiSPs can promote a sustained release of inflammatory mediators on cell lines and an acute activation of innate immunity in a murine model. These findings contribute to the growing body of evidence supporting the involvement of svCRiSPs in the augmentation of envenomation effects, specifically, the role of svCRiSPs in inducing vascular dysfunction, initiating early inflammatory responses, and facilitating the activation of leukocytes and releasing mediators. These findings will lead to a better understanding of the pathophysiology of envenoming by Mojave rattlesnakes, allowing the development of more efficient therapeutic strategies.

Unravelling the Proteomic Profiles of Bovine Colostrum and Mature Milk Derived from the First and Second Lactations.

Bovine colostrum (BC) and mature bovine milk are highly nutritious. In addition to being consumed by adults, these dairy products are also used as protein ingredients for infant formula. However, the differences in the nutritional composition of BC and mature milk, especially regarding proteins present in trace amounts, have not been comprehensively studied. Furthermore, the distinct proteomic profiles of mature milk derived from the first lactation (Milk-L1) and the second lactation (Milk-L2) are not fully understood. To address these gaps, this study aims to uncover the subtle differences in protein compositions of BC, Milk-L1, and Milk-L2 by proteomics. Compared with BC, anti-microbial proteins β-defensins and bovine hemoglobin subunit were up-regulated in Milk-L1, while Milk-L2 exhibited higher levels of enteric β-defensin, sterol regulatory element binding transcription factor 1, sydecan-2, and cysteine-rich secretory protein 2. Additionally, immune proteins such as vacuolar protein sorting-associated protein 4B, polymeric immunoglobulin receptor (PIGR), and Ig-like domain-containing protein were found at higher levels in Milk-L1 compared with Milk-L2. The study provides a comprehensive understanding of the distinct proteomic profiles of BC, Milk-L1, and Milk-L2, which contributes to the development of protein ingredients for infant formula.

Mammalian cysteine-rich secretory proteins interact with plasma membrane Ca2+ exporter PMCA4b.

Mammalian cysteine-rich secretory proteins (CRISPs) are predominantly expressed in the male reproductive tract. Knockout mice lacking two or more CRISPs show defects in sperm transport, sperm-egg interaction and Ca2+ homeostasis. CRISPs play redundant and specific roles via their binding partners. To understand this, a comprehensive analysis of CRISP interactome needs to be undertaken. This study aimed to analyse CRISP4 binding partners on the plasma membrane of rat caudal spermatozoa. Total proteins from rat caudal spermatozoa were subjected to immunoprecipitation using anti-CRISP4 antibody followed by liquid chromatography-mass spectrophotometry analysis. Plasma membrane localised proteins were shortlisted, and a key target was validated by co-immunoprecipitation and co-localisation. Co-transfection followed by co-immunoprecipitation was carried out for studying the interaction of full-length as well as deletion mutants of CRISPs with human plasma membrane calcium ATPase, isoform b (hPMCA4b). Calcium assays were performed using Fura-2-AM. The cholesterol binding ability of different CRISPs was evaluated in silico. The membrane-specific interactome of rat CRISP4 (rCRISP4) from caudal spermatozoa revealed PMCA4b as a novel binding partner, and their interaction was validated in rat spermatozoa. Human CRISP1 (hCRISP1) and hCRISP3 also interacted with PMCA4b via the N-terminal domain. Interestingly, hCRISP1 and rCRISP4 delayed PMCA4b-mediated calcium extrusion but hCRISP3 did not. In silico analysis demonstrated that hCRISP1 and rCRISP4 have higher binding affinity towards cholesterol than hCRISP3. The secretion profile of different CRISPs also showed that the ratio of secreted to cell-associated proteins was highest for hCRISP3. Our study identifies PMCA4b as a target of multiple mammalian CRISPs and unravels a new role of CRISPs in regulating calcium homeostasis. Differences in the interaction of different CRISPs with cholesterol may regulate their enrichment in the lipid rafts and redistribution in the membrane post-capacitation, thereby affecting their interaction with PMCA4b.

Corrigendum to "MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients".

Corrigendum to "MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients".

The CAP superfamily protein PsCAP1 secreted by Phytophthora triggers immune responses in Nicotiana benthamiana through a leucine-rich repeat receptor-like protein.

The role of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 (CAP) superfamily proteins in the innate immune responses of mammals is well characterized. However, the biological function of CAP superfamily proteins in plant-microbe interactions is poorly understood. We used proteomics and transcriptome analyses to dissect the apoplastic effectors secreted by the oomycete Phytophthora sojae during early infection of soybean leaves. By transiently expressing these effectors in Nicotiana benthamiana, we identified PsCAP1, a novel type of secreted CAP protein that triggers immune responses in multiple solanaceous plants including N. benthamiana. This secreted CAP protein is conserved among oomycetes, and multiple PsCAP1 homologs can be recognized by N. benthamiana. PsCAP1-triggered immune responses depend on the N-terminal immunogenic fragment (aa 27-151). Pretreatment of N. benthamiana with PsCAP1 or the immunogenic fragment increases plant resistance against Phytophthora. The recognition of PsCAP1 and different homologs requires the leucine-rich repeat receptor-like protein RCAP1, which associates with two central receptor-like kinases BRI1-associated receptor kinase 1 (BAK1) and suppressor of BIR1-1 (SOBIR1) in planta. These findings suggest that the CAP-type apoplastic effectors act as an important player in plant-microbe interactions that can be perceived by plant membrane-localized receptor to activate plant resistance.

Identification and Functional Analysis of CAP Genes from the Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici.

Cysteine-rich secretory proteins (C), antigen 5 (A), and pathogenesis-related 1 proteins (P) comprise widespread CAP superfamily proteins, which have been proven to be novel virulence factors of mammalian pathogenic fungi and some plant pathogens. Despite this, the identification and function of CAP proteins in more species of plant pathogens still need to be studied. This work presents the identification and functional analysis of CAP superfamily proteins from Puccinia striiformis f. sp. tritici (Pst), an important fungal pathogen that causes wheat stripe rust on wheat worldwide. A total of six CAP genes were identified in the Pst genome, designated as PsCAP1-PsCAP6. Five PsCAP proteins, including PsCAP1, PsCAP2, PsCAP3, PsCAP4, and PsCAP5, have N-terminal signal peptides secreted with the yeast signal sequence trap assay. Single-nucleotide polymorphism (SNP) analysis indicated that they showed a low level of intraspecies polymorphism. The expression abundance of PsCAP genes at different Pst infection stages was detected by RT-qPCR, and most of them were highly expressed during Pst infection on wheat and also Pst sexual reproduction on barberry (Berberis shensiana). Noticeably, the silencing of these six PsCAP genes by BSMV-mediated HIGS indicated that PsCAP1, PsCAP4, and PsCAP5 contribute significantly to Pst infection in wheat. These results indicate that PsCAP proteins may act as virulence factors during Pst infection, which also provides insights into Pst pathogenicity.

Proteomic Profiling of Extracellular Vesicles Isolated from Plasma and Peritoneal Exudate in Mice Induced by Crotalus scutulatus scutulatus Crude Venom and Its Purified Cysteine-Rich Secretory Protein (Css-CRiSP).

Increased vascular permeability is a frequent outcome of viperid snakebite envenomation, leading to local and systemic complications. We reported that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability both in vitro and in vivo. They also induce acute activation of several adhesion and signaling molecules that may play a critical role in the pathophysiology of snakebites. Extracellular vesicles (EVs) have gained interest for their diverse functions in intercellular communication, regulating cellular processes, blood-endothelium interactions, vascular permeability, and immune modulation. They also hold potential as valuable biomarkers for diagnosing, predicting, and monitoring therapeutic responses in different diseases. This study aimed to identify proteins in peritoneal exudate and plasma EVs isolated from BALB/c mice following a 30 min post-injection of Crotalus scutulatus scutulatus venom and its purified CRiSP (Css-CRiSP). EVs were isolated from these biofluids using the EVtrap method. Proteomic analysis of exudate- and plasma-derived EVs was performed using LC-MS/MS. We observed significant upregulation or downregulation of proteins involved in cell adhesion, cytoskeleton rearrangement, signal transduction, immune responses, and vesicle-mediated transports. These findings suggest that svCRiSPs play a crucial role in the acute effects of venom and contribute to the local and systemic toxicity of snakebites.

The Venom Composition of the Snake Tribe Philodryadini: 'Omic' Techniques Reveal Intergeneric Variability among South American Racers.

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Expression of genes associated with sperm-oocyte interaction in the stallion reproductive tract

Sperm-oocyte fusion is an essential function during the fertilization process. Membrane fusion occurs as a result of a complex series of intermolecular interactions between the sperm and the oocyte. Proteins involved in this interaction include izumo sperm-egg fusion 1 (IZUMO1), sperm acrosome associated 6 (SPACA6), transmembrane protein 95 (TMEM95), fertilization influencing membrane protein (FIMP) and sperm-oocyte fusion required 1 (SOF1), ADAM metallopeptidase domain 2 (ADAM2), cysteine rich secretory protein 1-3 (CRISP1-3), cation channel sperm associated 1-4 (CATSPER1-4) and polycystin family receptor for egg jelly (PKDREJ) (Rivera and Swanson, 2022. Front. Cell Dev. Biol., 10:827-827454) on the male gamete, and izumo 1 receptor (JUNO) and cluster of differentiation 9 (CD9) on the female gamete (Inoue et al., 2021. eLife; 10:e66313). The IZUMO1-JUNO interaction is essential for gamete fusion and the block of polyspermy (Bianchi et al., 2014: Nature, 508:483-487). Other proteins called dendrocyte expressed seven transmembrane protein (DC-STAMP) and homologue domain-containing 1 and 2 (DCST1/2) are also required for sperm-egg fusion in mice (Inoue et al., 2021. eLife; 10:e66313). The objective of this study was to identify and quantify the expression of genes associated with sperm-oocyte interaction during the fertilization process in tissue samples from the testis, head, body and tail of the epididymis. Tissues were collected after castration from three light-breed reproductively normal stallions(5-13 years). mRNA was extracted and libraries were generated using Next Generation Sequencing. FPKM of the genes of interest was compared between epididymal head, body and tail, and testis (control tissue) using in silico analysis (online databases). Expression of CRISP3, SPACA6, ADAM2, CATSPER1, CATSPER2, CATSPER3, CATSPER4, GLPR1L1, IZUMO1, PKDREJ, TMEM95, DCST1 and DCST2 genes were identified in the testis and all segments of the epididymis of the horse. All these genes, except for CRISP1, were up-regulated in the testis compared with the three segments of the epididymis, while CRISP1 was down-regulated in the testis compared with the epididymis. In this study, JUNO gene was expressed in the testis (FPKM 48.15), and was downregulated in the head, body and tail of the epididymis (FPKM 2.3, 3.2 and 2.9, respectively). In summary, genes encoding for proteins associated with sperm-oocyte interaction were more strongly expressed within the testis, suggesting that these proteins are acquired during spermatogenesis or spermiogenesis. Meanwhile, CRISP1 may be acquired during sperm transport along the epididymis. The expression of JUNO gene within the stallion reproductive tract is striking since the expression has not been reported in mice (RPKM 0) and human testis (RPKM 0.003). JUNO is a receptor only found on the oocyte membrane. The specific cells that express these genes, and their function within the stallion reproductive tract needs further studies.

Proteomic profile of stallion seminal plasma

Proteins mediate all physiological changes. Seminal plasma (SP), the product of testes, epididymis, and accessory sex glands, is a fluid released during ejaculation and represents up to 98% of the voluminous stallion ejaculate. The SP plays an essential role in reproduction as a transit medium and a source of energy, antioxidants, enzymes, and minerals. This study aimed to investigate proteomic profile of the seminal plasma of stallions. Twenty-four Criollo stallions with a known reproductive history and per-cycle concept rate data calculated from at least 30 inseminations were used. One ejaculate was collected from each stallion during the breeding season. Pregnancy rates ranged at day 16 after artificial insemination from 20.2% to 95.6%. The animals were divided into two groups: High Pregnancy (HP), with a pregnancy rate per cycle ≥51%, and Low Pregnancy (LP), with a pregnancy rate of <50%. Sperm concentration, kinetics, morphology, and plasma membrane integrity and functionality were evaluated. The sample was centrifuged at 400xg for 10min. SP was then transferred to a 2 mL microcentrifuge tube and centrifuged again (10.000xg, 60min, 4°C). Proteins were separated using 2D SDS PAGE gel electrophoresis in duplicate. Spots in at least 80% of the gels in one of the groups, with significant abundance (P<0.05) and at least a 1.5-fold magnitude difference between groups, were selected. Thespectra were acquired in a nano LC-MS/MS spectrometer, and proteins were identified by the MASCOT application and validated by SCAFFOLD. From a total of 716 analyzed spots, four were selected. For non-parametric data the Kruskal Wallis and for parametric data Anova and t-test were used. The cysteine-rich secretory protein 3 (CRISP3) and SP protein HSP-1-like (HSP1) showed higher abundance in the SP in group LP than HP (P< 0.05). However, kallikrein-1E2 (KLK) and phospholipase A2 (PLA2) showed higher abundance in HP than LP (P<0.05). In the horse, CRISP-3 is synthesized in great amounts in the accessory sexual glands, ampulla and seminal vesicle. HSP1 has an essential role as a molecular chaperone for sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume, and possibly in the interaction between gametes. KLK is one of the major proteins found in seminal equine plasma. The kallikreins are serine proteases, and the glandular kallikrein is regulated by androgens and secreted by the prostate. The PLA2 plays an essential role in the late maturational events of spermatozoa, the acrosomal reaction, and sperm egg fusion. In conclusion, the proteins are essential to the biological processes related to stallion fertility, providing insight into SP proteins that could serve as biomarkers for fertility in stallions.

Sparcl1 and Atherosclerosis.

Atherosclerosis and its complications constitute some of the major diseases affecting humans worldwide. A core component of atherogenesis is endothelial cell damage and dysfunction, which also includes factors such as adhesion and proliferation of various cells. Multiple studies have shown that atherosclerosis and cancer share a common pathophysiological process and exhibit a degree of similarity. Sparcl-1 is a cysteine-rich secretory stromal cell protein present in the extracellular matrix and belongs to the Sparc family of proteins. Its role in tumor development has been widely investigated; however, its role in cardiovascular diseases has rarely been studied. Sparcl-1 is considered an oncogene correlated with the regulation of cell adhesion, migration, and proliferation and is also related to blood vessel integrity. In this review, the potential link between Sparcl-1 and atherosclerosis development is investigated, and recommendations on future research on the role of Sparcl-1 in atherogenesis are provided.

BIOINFORMATICS EVALUATION OF CRISP2 GENE SNPs AND THEIR IMPACTS ON PROTEIN

This study evaluated the CRISP2 gene's functional single nucleotide polymorphisms, and its results may be advantageous for future population-based studies and early diagnostic discoveries, particularly in developing effective treatments. The CRISP2 gene encodes a secretory protein with a high cysteine content, which belongs to the family of cysteine-rich secretory proteins (CRISP). SNPs are genetic variations that may affect a protein's structure or functionality. Prior to carrying out a broader population investigation, it is possible to evaluate suspected functional SNPs since it is challenging to uncover functional SNPs in disease-linked genes. As a result, using various bioinformatic prediction models, the potentially harmful three SNPs of the CRISP2 gene were predicted in this in-silico study from the neutral ones. Out of a pool of 260 nsSNPs, three SNPs (L56V, M176I, and C196R) been selected to anticipate their impacts on functions and structures along with their capability to impair protein stability. Actually, two of the three SNPs in the CRISP2 gene L56V and C196R were identified as possibly detrimental, although M176I was not. But all of these SNPs dropped significantly protein stability, per the I-Mutant suite.

Exploring Toxin Genes of Myanmar Russell's Viper, Daboia siamensis, through De Novo Venom Gland Transcriptomics.

The Russell's viper (Daboia siamensis) is a medically important venomous snake in Myanmar. Next-generation sequencing (NGS) shows potential to investigate the venom complexity, giving deeper insights into snakebite pathogenesis and possible drug discoveries. mRNA from venom gland tissue was extracted and sequenced on the Illumina HiSeq platform and de novo assembled by Trinity. The candidate toxin genes were identified via the Venomix pipeline. Protein sequences of identified toxin candidates were compared with the previously described venom proteins using Clustal Omega to assess the positional homology among candidates. Candidate venom transcripts were classified into 23 toxin gene families including 53 unique full-length transcripts. C-type lectins (CTLs) were the most highly expressed, followed by Kunitz-type serine protease inhibitors, disintegrins and Bradykinin potentiating peptide/C-type natriuretic peptide (BPP-CNP) precursors. Phospholipase A2, snake venom serine proteases, metalloproteinases, vascular endothelial growth factors, L-amino acid oxidases and cysteine-rich secretory proteins were under-represented within the transcriptomes. Several isoforms of transcripts which had not been previously reported in this species were discovered and described. Myanmar Russell's viper venom glands displayed unique sex-specific transcriptome profiles which were correlated with clinical manifestation of envenoming. Our results show that NGS is a useful tool to comprehensively examine understudied venomous snakes.