Cysteine-rich Secretory Protein 3 Research Articles

EP300 promotes tumor stemness via epigenetic activation of CRISP3 leading to lobaplatin resistance in triple-negative breast cancer.

Lobaplatin shows antitumor activity against a wide range of tumors, including triple-negative breast cancer (TNBC), and has been linked to cancer stem cell pool. Here, we investigated the molecular mechanisms behind lobaplatin resistance and stemness in vitro and in vivo. Two chemoresistance-related GEO data sets (GSE70690 and GSE103115) were included to screen out relevant genes. Cysteine-rich secretory protein 3 (CRISP3) was found to be overexpressed in lobaplatin-resistant TNBC and related to poor diagnosis. CRISP3 expression was significantly correlated with tumor stemness markers in lobaplatin-resistant cells. E1A-associated protein p300 (EP300) regulated CRISP3 expression by affecting the H3K27ac modification of the CRISP3 promoter. In addition, knocking down EP300 curbed the malignant biological behavior of lobaplatin-resistant cells, which was antagonized by CRISP3 overexpression. Collectively, our results highlight the EP300/CRISP3 axis as a key driver of lobaplatin resistance in TNBC and suggest that therapeutic targeting of this axis may be an effective strategy for enhancing platinum sensitivity in TNBC.

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.

Bioinformatics identification of characteristic genes of cervical cancer via an artificial neural network.

Artificial neural networks (ANNs) have been extensively used in the field of medicine. The present hypothesis-free study sought to use an ANN to identify the characteristic genes of cervical cancer (CC). RNA sequencing profiles were obtained from the GSE7410, GSE9750, GSE63514, and GSE52903 datasets. The differentially expressed genes (DEGs) were identified and compared between the normal and CC tissues. An ANN analysis was conducted to obtain the random-forest tree and to examine differences in gene filtering. A neural network model was established using the characteristic genes of CC, while the verification accuracy of the model was examined by Cox regression. The differences in the immune infiltrating cells between the normal cervical and CC tissues were compared by CIBERSORT (an analytical tool can provide an estimation of the abundances of member cell types in a mixed cell population). Nine genes' characteristics for CC were identified: cyclin-dependent kinase inhibitor 2A (CDKN2A), chromosome 1 open reading frame 112 (C1orf112), helicase, lymphoid-specific (HELLS), mini-chromosome maintenance protein 5 (MCM5), mini-chromosome maintenance protein 2 (MCM2), kinetochore associated 1 (KNTC1), cysteine-rich secretory protein 3 (CRISP3), phytanoyl-CoA 2-hydroxylase interacting protein (PHYHIP), and cornulin (CRNN). ANN is a robust neural network model that can be used to potentially predict CC based on the gene score. It can provide novel insights into the pathogenesis and molecular mechanisms of CC.

S405 Gene Expression and Pathway Analyses Reveal Distinctions Between Eosinophilic Esophagitis Pre and Post Treatment With Glucocorticoids

Introduction: Eosinophilic esophagitis (EoE) is a distinct entity causing significant morbidity and that which responded to anti-allergic treatment. We performed a secondary analysis of gene expression microarray GSE36725 dataset published in the Gene Expression Omnibus using samples of patients with EoE before and after successful treatment with glucocorticoids. Methods: Total RNA was extracted from the formalin fixed tissue and hybridized to Affymetrix Gene ST 1.0 Arrays and microarray data analyzed for 10 samples (5 pairs). The Affymetrix probe IDs of all the rows were converted to their respective gene IDs by DAVID (Database for Annotation, Visualization and Integrated Discovery) (https://david.ncifcrf.gov/) tool. The converted dataset was analyzed using the Networkanalyst.ca software. Results: A total of 32 significant pathways were found after Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis by Welch’s t test ranking. P < 0.05 was regarded as indicating statistical significance. The top two enriched pathways were Mineral absorption (P=0.0067) and chemokine signaling (P=0.0084) and the top two enrichment categories were protein processing in endoplasmic reticulum (P< .05) and cell adhesion molecules (P< .05) (Figure). The top genes upregulated with glucocorticoid treatment included CRISP3 (5.49-fold change), SPINK7 (5.49-fold change), TGM3 (5.49-fold change), EPGN (5.49-fold change) and UPK1A (5.49-fold change). Among genes downregulated with glucocorticoid treatment included TNFAIP6 (-5.29-fold change), POSTN (-4.57-fold change), JCHAIN (-4.55-fold change), ALOX15 (-3.36-fold change) and CCL26(-3.07-fold change). Based on our results, CRISP3, SPINK7 and TGM3 genes seem to be upregulated with glucocorticoid treatment whereas, TNFAIP6, POSTN and JCHAIN are the top genes that are downregulated with glucocorticoid treatment that have the highest sensitivity and specificity as diagnostic markers for successful treatment of EoE. Conclusion: CRISP3(Cysteine-rich secretory protein 3) and SPINK7(serine peptidase inhibitor, kazal type 7) are part of the differentiation program of human esophageal epithelium and that SPINK7 depletion occurs in a human allergic, esophageal condition termed eosinophilic esophagitis and are called as key barrier genes. This study shows that Glucocorticoid treatment improves the esophageal epithelial barrier integrity in cases of EoE.Figure 1.: Network analysis of enriched pathways.

Conceptus interferon gamma is essential for establishment of pregnancy in the pig†.

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG-/- conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG-/- conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG-/- embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs.

LINC01342 silencing upregulates microRNA-508-5p to inhibit progression of lung cancer by reducing cysteine-rich secretory protein 3

Long noncoding RNAs (lncRNAs) are critical players during cancer progression. Nevertheless, the effect of most lncRNAs in lung cancer (LC) remains unclear. We aimed to explore the role of LINC01342 in LC development through the microRNA-508-5p (miR-508-5p)/cysteine-rich secretory protein 3 (CRISP3) axis. LINC01342, miR-508-5p, and CRISP3 expression in clinical samples and cell lines were determined, and their correlations in LC were analyzed. The prognostic role of LINC01342 in LC patients was evaluated. LC cells were screened and, respectively, transfected to alter the expression of LINC01342, miR-508-5p, and CRISP3. Then, proliferation, migration, invasion, and apoptosis of transfected LC cells were determined, and the in vivo tumor growth was observed as well. Binding relationships between LINC01342 and miR-508-5p, and between miR-508-5p and CRISP3 were identified. LINC01342 and CRISP3 were upregulated and miR-508-5p was downregulated in LC tissues and cells. High LINC01342 expression indicated a poor prognosis of LC patients. The LINC01342/CRISP3 silencing or miR-508-5p elevation inhibited proliferation, migration, and invasion of LC cells and promoted LC cell apoptosis, and also suppressed the in vivo tumor growth. LINC01342 bound to miR-508-5p and miR-508-5p targeted CRISP3. LINC01342 plays a prognostic role in LC and LINC01342 silencing upregulates miR-508-5p to inhibit the progression of LC by reducing CRISP3.

CRISP3 glycoprotein: a good biomarker for prostate cancer?

ABSTRACT Introduction: Cysteine-rich secretory protein 3 (CRISP3) is expressed at low levels in normal human prostate but often overexpressed in prostate cancer (PCa). The relevance of this overexpression for the malignancy of PCa is still unclear. The prognostic value of the currently used prostate specific antigen (PSA) test can be misleading under certain circumstances, resulting in overtreatment of indolent tumors. New biomarkers are needed to reduce overtreatment and improve quality of life of men. Objective: Evaluate if CRISP3 expression could be a good biomarker for PCa. Methods: CRISP3 expression was determined by immunohistochemistry in tissue sections of prostate cancer from twenty-five patients subjected to radical prostatectomy. Gleason grading system was used as prognostic indicator and the staging of PCa was defined using the TNM system. Clinical parameters and PSA levels before and after surgery were determined. Results: CRISP3 expression was strong in 14 (56%), moderate in four (16%) and weak in seven (28%) specimens. There was no correlation between the intensity of CRISP3 expression and pre- and post-treatment PSA levels. Fifteen (60%) of PCa biopsies showed extension of the primary tumor pT2. Seven patients (28%) showed Gleason score higher than 7; thirteen (52%) equal to 7, and five (20%) lower than 7. There were no significant statistical differences between Gleason score and CRISP3 expression. Conclusion: CRISP3 is expressed in prostate cancer at different levels. Additional studies are required to better evaluate if CRISP3 could be used as a biomarker.

CRISP3 expression drives prostate cancer invasion and progression.

Identifying the factors stimulating prostate cancer cells migration and invasion has the potential to bring new therapeutic targets to the clinic. Cysteine-rich secretory protein 3 (CRISP3) is one of the most highly upregulated proteins during the transition of a healthy human prostatic epithelium to prostate cancer. Here we show using a genetically engineered mouse model of prostate cancer that CRISP3 production greatly facilitates disease progression from carcinoma in situ to invasive prostate cancer in vivo. This interpretation was confirmed using both human and mouse prostate cancer cell lines, which showed that exposure to CRISP3 enhanced cell motility and invasion. Further, using mass spectrometry, we show that CRISP3 induces changes in abundance of a subset of cell-cell adhesion proteins, including LASP1 and TJP1 both in vivo and in vitro. Collectively, these data identify CRISP3 as being pro-tumorigenic in the prostate and validate it as a potential target for therapeutic intervention.

Low expression of CRISP3 predicts a favorable prognosis in patients with mammary carcinoma.

The discovery of cysteine-rich secretory protein 3 (CRISP3) has been made in human neutrophils for the first time. Cloning of the complementary DNA (cDNA) for CRISP3 was performed from a cDNA library of human bone marrow. In patients with mammary carcinoma, we found that lower expression of CRISP3 was connected to a significantly improved DFS (disease-free survival) and OS (overall survival). Furthermore, the CRISP3 protein level was significantly associated with negative ANXA1 protein level. In addition, the heterogeneous expression of CRISP3 had been exhibited in diverse mammary carcinoma cells. A significant higher mRNA and the protein level of CRISP3 were seen in T-47D as well as SK-BR-3 cells compared with those in other types of mammary carcinoma cells. Knockdown of CRISP3 in T-47D or SK-BR-3 cells resulted in the weakened migration or invasion abilities. Furthermore, CRISP3 knockdown significantly inhibited the ERK1/2 MAPK signaling pathway in T-47D or SK-BR-3 cells. Research results indicated that the lowering in the expression of CRISP3 is likely to serve as an unprecedented approach for the treatment of mammary carcinoma.

Relationship of cysteine-rich secretory protein-3 gene and protein with semen quality in stallions.

Cysteine-rich secretory protein-3 (CRISP-3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP-3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP-3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP-3 was identified and haplotypes were derived. Semen quality was assessed through the total motility (MOT), sperm vitality (SV), normal morphology (NM), functional integrity of membrane (MI) and a seminal quality index (SQi). CRISP-3 protein content of seminal plasma (SP) was determined by ELISA. The effect of the genotype, the haplotype and the concentration of the CRISP-3 protein on the seminal quality were evaluated through generalized linear models and linear regression analyses. Homozygous genotypes for SNP1, SNP2 and SNP3 and the heterozygous genotype for SNP4 showed a positive effect on seminal quality. Different haplotypes with positive effect on MOT, SV, NM, MI and SQi were identified. The allelic substitution analysis resulted in positive regression coefficients for MOT (SNP2) and MI (SNP2 and SNP3). A high level of CRISP-3 resulted in a higher MOT and SQi. It is concluded that the quality of stallion semen is influenced by the genotype of CRISP-3 and the concentration of CRISP-3 protein in SP.

PO-230 Cysteine-rich secretory protein 3 regulates progression from in situ to invasive prostate cancer

IntroductionOne of the most challenging aspects of prostate cancer diagnosis is predicting whether cancer will remain indolent or progress to invasive, aggressive and potentially lethal disease. Current markers such as PSA are unreliable and tumours requiring treatment may remain undetected while others are over-treated. Cysteine-rich secretory protein 3 (CRISP3) is a member of a poorly defined family of proteins that is highly up-regulated in human prostate cancer.Material and methodsWe sought to define the role of CRISP3 in the molecular pathology of prostate cancer through the generation of a Crisp3 knockout mouse line, which was crossed onto the Hi-MYC mouse model of prostatic adenocarcinoma. The pro-invasive actions of CRISP3 were also studied using human and mouse derived cell lines and purified recombinant CRISP3.Results and discussionsHere we show that CRISP3 induces migration and invasion of prostate cancer cells in vitro. Furthermore, and consistent with human expression data, CRISP3 was dramatically up-regulated with advanced disease in the Hi-MYC mouse model of prostatic adenocarcinoma and specifically associated with transformed and migratory cells both in vivo and in vitro. Importantly, Crisp3 deletion delayed the transition from prostatic intraepithelial neoplasia to carcinoma in situ and blocked the transition to the invasive disease. These effects are attributed to changes in the expression of EMT markers in response to CRISP3. We are currently validating potential CRISP3 binding partners identified by mass spectrometry.ConclusionCollectively, these data define CRISP3 as pro-tumourigenic in the prostate through a role in promoting cancer invasion.

Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells

Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR.

Association of the cysteine-rich secretory protein-3 (CRISP-3) and some of its polymorphisms with the quality of cryopreserved stallion semen.

Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A>G (SNP1), CRISP3c.+566C>A (SNP2), CRISP3c.+622G>A (SNP3) and CRISP3c.+716A>G (SNP4). CRISP-3 protein content in seminal plasma was determined by enzyme-linked immunosorbent assay. Semen from 30 stallions was cryopreserved and post-thaw motility, kinetics, abnormal morphology (AM), sperm vitality (SV) and membrane integrity (MI) were evaluated. Generalized linear models were fitted and means were compared using Tukey's test. Correlation and regression analyses were performed. For SNP1 and SNP3, the AA genotype had the highest results for motility and MI; for SNP2, the best results for motility and AM were obtained with the CC genotype. For SNP4, the GG genotype had the lowest results, except for MI. A high level of CRISP-3 protein in seminal plasma had the best results for motility, kinetics, SV and AM. In conclusion, there was a relationship between CRISP-3 genotype and seminal plasma protein and post-thawing semen quality in stallions.

Structural and functional analysis of CRISP3 protein in relation to diseases of exocrine pancreas

Introduction: Human cysteine rich secretory protein 3 (CRISP3), also known as specific granule protein (SGP28) is a small 28kDa protein. CRISP-3 is widely expressed in salivary glands, pancreas (particularly in pancreatic disease), lachrymal glands, prostate, myeloid precursors, human epididymis, ovary, thymus and colon. Human CRISP3 share similarity with snake venoms as well as Glioma Pathogenesis Related protein GLIPR1. Here we are reporting the extent of alignment of human CRISP3 protein with pancreatic enzymes and snake venom proteins and presence of GLIPR1 and CRISP3 genes containing conserved GHYTQVVW motifs in clinical samples of pancreatitis and pancreatic cancer. Material and Methods: Protein sequences with accession numbers for Human CRISP3, Snake Venom and enzymes present in pancreatic juices i.e. Trypsinogen, Cathepsin, Lipase, Amylase and Elastase were retrieved from swissprot, protein database. These sequences were aligned in silico for demonstrating the presence of 16 conserved cysteine residues and other motifs i.e. GENL and GHYTQVVW. The multiple sequence alignment (MSA) was performed. Presence of CRISP-3 conserved motifs in clinical samples of pancreatitis and pancreatic cancer was evaluated by PCR. DNA extracted from a small piece taken from inflamed pancreatic tail biopsy with confirmed diagnosis for pancreatitis and an excised pancreatic cancer tissue was subjected to PCR to conform presence of CRISP3 and GLIPR1 genes. Results: Our results show conservation of 9 Cysteine residues out of 16 Cysteine residues in majority of pancreatic enzymes and notable conservation of GENL and GHYTQVVW motifs across snake venom proteins. PCR yielded desired amplicon of expected size with the primers spanning conserved residues and core consensus motifs specifically designed to amplify CRISP3 gene in clinical samples of pancreatitis and pancreatic cancer. We also detected presence of GLIPR1 gene sequence in the DNA extracted from clinical samples of pancreatitis and pancreatic cancer thus confirming similarity between the pathogenesis related protein and CRISP3. Conclusion: Notable conservation of cysteine residues across pancreatic enzymes, snake venom and Human CRISP3 opens avenues of further research to evaluate the likely clinical potential of the conserved residues as putative biomarkers in exocrine pancreatic diseases. To the best of our knowledge this is the first report where primers designed around conserved GHYTQVVW motifs to amplify CRISP3 gene and its structural similarity with GLIPR1 gene sequence in clinical samples of pancreatitis and pancreatic cancer has been validated.

The effect of select seminal plasma proteins on endometrial mRNA cytokine expression in mares susceptible to persistent mating-induced endometritis.

In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating-induced endometritis PMIE Select seminal plasma proteins cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP-3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six mares classified as susceptible to PMIE were inseminated during four consecutive oestrous cycles with treatments in randomized order of: 1mg/ml CRISP-3, 150μg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution (LRS; negative control) to a total volume of 10ml combined with 1 × 109 spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis of selected genes associated with inflammation (pro-inflammatory cytokines interleukin (IL)-1β, IL-8, tumour necrosis factor (TNF)-α, interferon (INF)-γ, anti-inflammatory cytokines IL-1RN and IL-10, and inflammatory-modulating cytokine IL-6). Seminal plasma treatment increased the mRNA expression of IL-1β (p=.019) and IL-8 (p=.0068), while suppressing the mRNA expression of TNF (p=.0013). Lactoferrin also suppressed the mRNA expression of TNF (p=.0013). In conclusion, exogenous lactoferrin may be considered as one modulator of the complex series of events resulting in the poorly regulated pro-inflammatory response seen in susceptible mares.

Expression and localization of cysteine-rich secretory protein-3 (CRISP-3) in the prepubertal and postpubertal male horse

The seminal plasma protein, cysteine-rich secretory protein-3 (CRISP-3), has been correlated with increased fertility and first-cycle conception rates, and has been suggested to be involved in the modulation of polymorphonuclear neutrophil and phagocytosis of spermatozoa during the inflammatory response to breeding in the horse. Previous research demonstrated that equine CRISP-3 is located in both the ampulla of the vas deferens and the seminal vesicles. However, this was done with nonquantitative laboratory techniques. In humans and rodents, CRISP-3 has been described as an androgen-dependent protein, but the effect of androgens on the expression of CRISP-3 has not been investigated in the horse. The objectives of this study were to (a) confirm and quantify the expression of CRISP-3 in the male equine reproductive tract, (b) describe the localization of CRISP-3 within the specific tissues which express it, and (c) determine if expression of CRISP-3 increases after puberty. We hypothesized that expression of CRISP-3 would be expressed in both the ampulla of the vas deferens and the seminal vesicles, and expression would increase after puberty. Tissues were collected postmortem from three prepubertal colts (<6 months) and six postpubertal stallions (>3 years). Tissue samples were collected from the ampulla of vas deferens, seminal vesicles, bulbourethral gland, prostate gland, testis, as well as the cauda, corpus, and caput aspects of the epididymis. Quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) were performed using an equine-specific CRISP-3 designed primer and monocolonal antibody. A mixed linear additive model was used to compare mRNA expression between age groups, and significance was set to P < 0.05. There was a significant interaction between maturity and tissue type (P < 0.0001). Expression of CRISP-3 mRNA was found primarily in the ampulla of vas deferens with lesser expression in the seminal vesicles. Expression of CRISP-3 was higher in the postpubertal stallion when compared with the prepubertal colt for the ampulla (P < 0.0001) and seminal vesicles (P = 0.0013). IHC showed that equine CRISP-3 is primarily located in the glandular aspects of both the ampulla of vas deferens and the seminal vesicles, with staining concentrated in the cytoplasm of the epithelial cells that surrounded the glands of the mucosa. CRISP-3 was only observed in the postpubertal male horse suggesting that puberty plays a role in the activation of equine CRISP-3 expression.

Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers.

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers.

Prognostic value of ERG, PTEN, CRISP3 and SPINK1 in predicting biochemical recurrence in prostate cancer.

The established prognostic factors associated with prostatic adenocarcinoma are the Gleason score, pathological T staging and serum prostatic-specific antigen (PSA) level. However, these prognostic factors alone are not sufficient for predicting prognostic characteristics, including early stage or advanced prostate cancer, presence of metastasis or disease-related mortality. The purpose of the present study was to simultaneously evaluate the prognostic value and associations of four biomarkers, namely, transcriptional regulator ERG (ERG), phosphatase and tensin homolog (PTEN), cysteine-rich secretory protein 3 (CRISP3) and serine protease inhibitor Kazal type I (SPINK1), and to conduct risk stratification of prostate cancer for use in patient management. A total of 68 formalin-fixed, paraffin-embedded, prostate cancer samples from radical prostatectomies were obtained in the Kyung Hee University Hospital (Seoul, Korea) and were studied immunohistochemically for ERG, PTEN, CRISP3 and SPINK1 to determine the proportion and intensity of staining. SPINK1 expression was mutually exclusive of ERG expression (P=0.001). The loss of PTEN and high CRISP3 expression are unfavorable indicators for prostate cancer, as PTEN loss was associated with shorter biochemical recurrence (BCR) (P=0.039), and high CRISP3 expression was associated with increased BCR (P<0.001) and cancer-related mortalities (P=0.011). Using the combination of low PTEN and high CRISP3 expression enables attention to be focused on patients who exhibit a poor prognosis. Subgrouping of patients, into high-risk and low-risk categories, was correlated with BCR-free survival in prostate cancer upon multivariate analysis (P=0.030). Overall, low PTEN and high CRISP3 expression significantly characterize the subgroups of prostate cancer that have a poor prognosis for BCR.

The Effect of Cysteine-Rich Secretory Protein-3 and Lactoferrin on Endometrial Cytokine mRNA Expression After Breeding in the Horse

The equine uterus undergoes a transient innate immune response after breeding, also known as mating-induced endometritis. The deposition of spermatozoa triggers the expression of pro-inflammatory cytokines, which results in the migration of polymorphonuclear neutrophils (PMNs) into the endometrium and the uterine lumen. Select seminal plasma proteins, specifically cysteine-rich secretory protein 3 (CRISP-3) and lactoferrin, have been shown to affect the activity of the PMNs, either by suppressing (CRISP-3) or promoting (lactoferrin) the phagocytosis of spermatozoa based on their viability in vitro. Conjointly, many components of inseminate, including seminal plasma, bacteria, and spermatozoa itself, have shown to have an effect on the expression of endometrial cytokines after breeding. The objective of this study was to determine if select proteins affect the mRNA expression of endometrial cytokines after insemination. Six mares were bred during four consecutive estrous cycles with treatments in randomized order of: 1mg/mL CRISP-3, 150 ug/mL lactoferrin, seminal plasma, or Lactated Ringer’s Solution (LRS) to a total volume of 10 mL combined with 1×109 progressively motile spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis. No treatment effects were found for the mRNA expression of IL-1β, IL-6, IL-8, IL-10, TNFα, and IFNγ, while lactoferrin significantly suppressed the mRNA expression of IL-1RN when compared to LRS. In conclusion, the seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on the expression of select endometrial cytokines at 6 hours post breeding.

Cysteine-rich secretory protein 3 plays a role in prostate cancer cell invasion and affects expression of PSA and ANXA1.

Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.