In vivo modification of the enamel pellicle and saliva resveratrol levels after use of resveratrol-containing orodispersible capsules.

OBJECTIVES: To evaluate in vivo 1) the bioavailability of trans-resveratrol when administered through sublingual capsules; 2) the effect of resveratrol on the protein composition of the acquired enamel pellicle (AEP). DESIGN: Ten volunteers received a sublingual capsule containing 50 mg of trans-resveratrol. Unstimulated saliva was then collected after 0, 30, 60, and 120 min and AEP was collected after 120 min following administration of the capsule. In the next week, the volunteers received a placebo sublingual capsule, and saliva and AEP were collected again. Saliva samples were analyzed for free trans-resveratrol using high-performance liquid chromatopgraphy (HPLC), and AEP samples were subjected to proteomic analysis (nLC-ESI-MS/MS). RESULTS: Trans-resveratrol was detected in saliva at all the time points evaluated, with the peak at 30 min. A total of 242 proteins were identified in both groups. Ninety-six proteins were increased and 23 proteins were decreased in the Resveratrol group. Among the up-regulated proteins, isoforms of cystatins, PRPs, Mucin-7, Histatin-1, Lactotrasnferrin and Lysozyme-C were increased and the isoforms of Protein S100, Neutrophil defensins, Albumin, PRPs, and, Statherin were decreased in Resveratrol group. CONCLUSION: The sublingual capsule is effective at increasing the bioavailability of trans-resveratrol in saliva. Several proteins involved in important processes to maintain systemic and oral health homeostasis were identified. These proteins differently expressed due to the presence of trans-resveratrol deserve attention for future studies, since they have important functions, mainly related to antimicrobial action.

Mapping Conformational Changes in the Saliva Proteome Potentially Associated with Oral Cancer Aggressiveness.

Diverse proteomics-based strategies have been applied to saliva to quantitatively identify diagnostic and prognostic targets for oral cancer. Considering that these targets may be regulated by events that do not imply variation in protein abundance levels, we hypothesized that changes in protein conformation can be associated with diagnosis and prognosis, revealing biological processes and novel targets of clinical relevance. For this, we employed limited proteolysis-mass spectrometry in saliva samples to explore structural alterations, comparing the proteome of healthy control and oral squamous cell carcinoma (OSCC) patients with and without lymph node metastasis. Thirty-six proteins with potential structural rearrangements were associated with clinical patient features including transketolase and its interacting partners. Moreover, N-glycosylated peptides contribute to structural rearrangements of potential diagnostic and prognostic markers. Altogether, this approach utilizes saliva proteins to search for targets for diagnosing and prognosing oral cancer and can guide the discovery of potential regulated sites beyond protein-level abundance.

Comparative proteomic analysis of the hemolymph and salivary glands of Rhodnius prolixus and R. colombiensis reveals candidates associated with differential lytic activity against Trypanosoma cruzi Dm28c and T. cruzi Y.

BACKGROUND: Immune response of triatomines plays an important role in the success or failure of transmission of T. cruzi. Studies on parasite-vector interaction have shown the presence of trypanolytic factors and have been observed to be differentially expressed among triatomines, which affects the transmission of some T. cruzi strains or DTUs (Discrete Typing Units). METHODOLOGY/PRINCIPAL FINDINGS: Trypanolytic factors were detected in the hemolymph and saliva of R. prolixus against epimastigotes and trypomastigotes of the Y strain (T. cruzi II). To identify the components of the immune response that could be involved in this lytic activity, a comparative proteomic analysis was carried out, detecting 120 proteins in the hemolymph of R. prolixus and 107 in R. colombiensis. In salivary glands, 1103 proteins were detected in R. prolixus and 853 in R. colombiensis. A higher relative abundance of lysozyme, prolixin, nitrophorins, and serpin as immune response proteins was detected in the hemolymph of R. prolixus. Among the R. prolixus salivary proteins, a higher relative abundance of nitrophorins, lipocalins, and triabins was detected. The higher relative abundance of these immune factors in R. prolixus supports their participation in the lytic activity on Y strain (T. cruzi II), but not on Dm28c (T. cruzi I), which is resistant to lysis by hemolymph and salivary proteins of R. prolixus due to mechanisms of evading oxidative stress caused by immune factors. CONCLUSIONS/SIGNIFICANCE: The lysis resistance observed in the Dm28c strain would be occurring at the DTU I level. T. cruzi I is the DTU with the greatest geographic distribution, from the south of the United States to central Chile and Argentina, a distribution that could be related to resistance to oxidative stress from vectors. Likewise, we can say that lysis against strain Y could occur at the level of DTU II and could be a determinant of the vector inability of these species to transmit T. cruzi II. Future proteomic and transcriptomic studies on vectors and the interactions of the intestinal microbiota with parasites will help to confirm the determinants of successful or failed vector transmission of T. cruzi DTUs in different parts of the Western Hemisphere.

Changes in saliva protein profile throughout Rhipicephalus microplus blood feeding.

BACKGROUND: When feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding. METHODS: Ticks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species. RESULTS: Changes in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as "sialome switching." This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva. CONCLUSIONS: Overall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.

Proteomic profile of the acquired enamel pellicle of children with early childhood caries and caries-free children.

Acquired enamel pellicle plays an important role in the pathogenesis of early childhood caries (ECC), working as a protective interface between the tooth and the oral cavity. The aim of this cross-sectional in vivo proteomic study was to compare the acquired enamel pellicle protein profile of 3-5-year-old children with ECC (n = 10) and caries-free children (n = 10). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). In total, 241 proteins were identified. Basic salivary proline-rich protein 1 and 2, Cystatin-B, and SA were found only in the caries free group. When comparing caries free and ECC groups, lower protein levels were found in the caries free group for hemoglobin subunit beta, delta, epsilon, gamma-2, globin domain-containing protein and gamma-1, neutrophil defensin 3, serum albumin, protein S100-A8, and S100-A9. The proteins histatin-1, statherin, salivary acidic proline-rich phosphoprotein ½, proline-rich protein 4, submaxillary gland androgen-regulated protein 3B, alpha-amylase 1 and 2B were found at higher levels in the caries free group. The exclusive and the proteins found at higher levels in the caries free group might have protective functions that play a role in the prevention of caries, besides providing important insights to be evaluated in future studies for the possible development of new therapeutic strategies for ECC.

Gingivitis in cattle and supplemental protein diet: Insights from proteomic analysis.

Salivary proteins are essencial in the maintenance of oral homeostasis and can reflect systemic and localized processes, like gingivitis. However, little is known about the relationship between diet and the occurrence of gingivitis in cattle. The present study aimed to characterize the salivary proteomic profile of cattle (n = 12) fed hay (112.19 g/kg of crude protein) cultivated in reformed pastures, and, one group received protein supplement (PS, n = 6); the effect of the protein supplement on the gingival health of the cattle was determined by weekly intraoral examination and periodontal evaluation of the eight (deciduous) incisors. The whole saliva proteome of the two groups was evaluated after 20 and 60 days of confinement. In the periodontal clinical evaluation both groups had episodes of gingivitis; however, the average number of affected sites in the PS group was higher on day 60. The cattle fed exclusively hay, presented a lower average of affected gingival sites on day 60. After 60 days of experimentation, nine biological and 11 immunological processes were altered in bovine saliva. Proteins with multiple functions were detected in the saliva of the cattle; however, differences were observed in their regulation between the two groups. SIGNIFICANCE: In bovine populations, the relationship between diet and increased incidence of gingivitis is theorized. The results of the present pilot study, both diets caused episodes of gingivitis in the primary dentition of cattle and, apparently, diets with protein supplementation stimulate the expression of salivary proteins with a protective role in cattle that can act against infectious-inflammatory processes, such as gingivitis. However, it is plausible that over time, cattle will adapt to these diets and become more vulnerable to gingivitis.

A multiepitope chimeric antigen from Rhipicephalus microplus-secreted salivary proteins elicits anti-tick protective antibodies in rabbit.

Rhipicephalus (Boophilus) microplus, the Cattle Fever Tick, causes significant economic losses in livestock in tropical and subtropical regions of the world. As the usual control strategy based on chemical acaricides presents different drawbacks, alternative control strategies have been considered for tick control. In recent decades, several tick proteins have been evaluated as targets for the development of anti-tick vaccines. Thus, in the present work, coding sequences from three different proteins present in tick saliva were employed together to construct a recombinant chimeric protein that was evaluated as an antigen in rabbit immunization. Then, the elicited antibodies were tested in a tick artificial feeding experiment to verify the protective effect against the parasites. In addition to Rhipicephalus microplus subtilisin inhibitor 7 (RmSI-7), a serine protease inhibitor member of the TIL (Trypsin Inhibitory Like) family, an interdomain region from the Kunitz inhibitor BmTI-A, and a new cysteine-rich AMP-like microplusin, called RmSEI (previously identified as an elastase inhibitor), were selected to compose the chimeric protein. Anti-chimeric IgG antibodies were able to affect R. microplus female egg production after artificial feeding. Moreover, antibodies elicited in infested tick-resistant and tick-susceptible cattle recognized the recombinant chimera. Additionally, the functional characterization of recombinant RmSEI was performed and revealed antimicrobial activity against gram-positive bacteria. Moreover, the antimicrobial protein was also recognized by antibodies elicited in sera from cattle previously exposed to R. microplus bites. Together, these data suggest that the chimeric protein composed of three salivary antigens is suitable for anti-tick vaccine development.

Identification of early biomarkers in saliva in genetically engineered mouse model C(3)1-TAg of breast cancer.

Breast cancer is one of leading causes of death worldwide in the female population. Deaths from breast cancer could be reduced significantly through earlier and more efficient detection of the disease. Saliva, an oral fluid that contains an abundance of protein biomarkers, has been recognized as a promising diagnostic biofluid that is easy to isolate through non-invasive techniques. Assays on saliva can be performed rapidly and are cost-effective. Therefore, our work aimed to identify salivary biomarkers present in the initial stages of breast cancer, where cell alterations are not yet detectable by histopathological analysis. Using state-of-the-art techniques, we employed a transgenic mouse model of mammary cancer to identify molecular changes in precancerous stage breast cancer through protein analysis in saliva. Through corroborative molecular approaches, we established that proteins related to metabolic changes, inflammatory process and cell matrix degradation are detected in saliva at the onset of tumor development. Our work demonstrated that salivary protein profiles can be used to identify cellular changes associated with precancerous stage breast cancer through non-invasive means even prior to biopsy-evident disease.

Fructose and methylglyoxal-induced glycation alters structural and functional properties of salivary proteins, albumin and lysozyme.

Glycation process refers to reactions between reduction sugars and amino acids that can lead to formation of advanced glycation end products (AGEs) which are related to changes in chemical and functional properties of biological structures that accumulate during aging and diseases. The aim of this study was to perform and analyze in vitro glycation by fructose and methylglyoxal (MGO) using salivary fluid, albumin, lysozyme, and salivary α-amylase (sAA). Glycation effect was analyzed by biochemical and spectroscopic methods. The results were obtained by fluorescence analysis, infrared spectroscopy (total attenuated reflection-Fourier transform, ATR-FTIR) followed by multivariate analysis of principal components (PCA), protein profile, immunodetection, enzymatic activity and oxidative damage to proteins. Fluorescence increased in all glycated samples, except in saliva with fructose. The ATR-FTIR spectra and PCA analysis showed structural changes related to the vibrational mode of glycation of albumin, lysozyme, and salivary proteins. Glycation increased the relative molecular mass (Mr) in protein profile of albumin and lysozyme. Saliva showed a decrease in band intensity when glycated. The analysis of sAA immunoblotting indicated a relative reduction in intensity of its correspondent Mr after sAA glycation; and a decrease in its enzymatic activity was observed. Carbonylation levels increased in all glycated samples, except for saliva with fructose. Thiol content decreased only for glycated lysozyme and saliva with MGO. Therefore, glycation of salivary fluid and sAA may have the potential to identify products derived by glycation process. This opens perspectives for further studies on the use of saliva, an easy and non-invasive collection fluid, to monitor glycated proteins in the aging process and evolution of diseases.

Salivary antioxidant enzymes associated with oral toxicity in haematopoietic cell transplantation: An observational study.

BACKGROUND: In haematopoietic cell transplantation (HCT), oral mucositis and xerostomia are related to conditioning-related oxidative stress. The role of salivary antioxidant enzymes in oral toxicity is poorly described. The aim of this study was to verify the association between salivary antioxidant enzymes and oral mucositis and xerostomia in HCT. DESIGN: Saliva from autologous and allogeneic HCT patients (n = 77) was selected before conditioning (T0), during the neutropenia period (T1) and after marrow engraftment (T2). Salivary flow, total salivary proteins, and superoxide dismutase, catalase and glutathione reductase activities were measured. RESULTS: There were no significant differences in salivary flow, total salivary proteins and catalase at the three HCT time points. Glutathione reductase levels were reduced at T1 compared to T0 (P = .013) and T2 (P = .001). Superoxide dismutase levels were increased from T0 to T2 (P = .013). Neither of these enzymes was associated with oral mucositis. Increased superoxide dismutase levels were associated with xerostomia frequency. Levels of this enzyme also showed significant correlation with days of xerostomia in T2 (ρ = .40, P = .002). CONCLUSIONS: Salivary antioxidant enzymes changed before and during early periods after HCT. The increase in salivary superoxide dismutase suggested partial activation of the salivary antioxidant system and was associated with xerostomia.

Salivary flow, amylase, and total protein in hospitalized patients with HIV infection / AIDS complications.

BACKGROUND: Saliva is a complex secretion produced daily by the salivary glands. Saliva consists mainly of water, enzymes, ions and amino acids and performs several important functions in oral health. OBJECTIVE: The aim of this study was to investigate the flow rate and concentrations of amylase and total proteins in the saliva of hospitalized patients due to AIDS complications. METHODS: Ninety-three men and women (20-64 years of age) were divided into two groups (46 HIV-infected patients and 47 controls) and had salivary flow rate and levels of amylase enzyme and total proteins evaluated. RESULT: The mean salivary flow rate was lower in individuals with HIV when compared to controls (P < 0.05). No significant difference between amylase enzyme levels and total proteins were observed in the saliva of patients with HIV infection when compared to controls. CONCLUSION: Individuals with HIV / AIDS infection (in hospital treatment) suffer no interference in levels of amylase and total salivary proteins, but they have significantly reduced salivary flow.

Digestive activity and organic compounds of Nezara viridula watery saliva induce defensive soybean seed responses.

The stink bug Nezara viridula is one of the most threatening pests for agriculture in North and South America, and its oral secretion may be responsible for the damage it causes in soybean (Glycine max) crop. The high level of injury to seeds caused by pentatomids is related to their feeding behavior, morphology of mouth parts, and saliva, though information on the specific composition of the oral secretion is scarce. Field studies were conducted to evaluate the biochemical damage produced by herbivory to developing soybean seeds. We measured metabolites and proteins to profile the insect saliva in order to understand the dynamics of soybean-herbivore interactions. We describe the mouth parts of N. viridula and the presence of metabolites, proteins and active enzymes in the watery saliva that could be involved in seed cell wall modification, thus triggering plant defenses against herbivory. We did not detect proteins from bacteria, yeasts, or soybean in the oral secretion after feeding. These results suggest that the digestive activity and organic compounds of watery saliva may elicit a plant self-protection response. This study adds to our understanding of stink bug saliva plasticity and its role in the struggle against soybean defenses.

Development of 131I-ixolaris as a theranostic agent: metastatic melanoma preclinical studies.

Tissue factor (TF), a blood coagulation protein, plays an important role in tumor growth, invasion, and metastasis. Ixolaris, a tick-derived non-immunogenic molecule that binds to TF, has demonstrated in vivo inhibitory effect on murine models of melanoma, including primary growth and metastasis. This work aimed to: I) develop an efficient and stable labeling technique of ixolaris with Iodine-131(131I); II) compare the biodistribution of 131I and 131I-ixolaris in tumor-free and melanoma-bearing mice; III) evaluate whether 131I-ixolaris could serve as an antimetastatic agent. Ixolaris radioiodination was performed using iodogen, followed by liquid paper chromatography. Labeling stability and anticoagulant activity were measured. Imaging studies were performed after intravenous administration of free 131I or 131I-ixolaris in a murine melanoma model employing the B16-F10 cell line. Animals were divided in three experimental groups: the first experimental group, D0, received a single-dose of 9.25 MBq of 131I-ixolaris at the same day the animals were inoculated with melanoma cells. In the second group, D15, a single-dose of 9.25 MBq of 131I-ixolaris or free 131I was applied into mice on the fifteenth day after the tumor induction. The third group, D1-D15, received two therapeutic doses of 9.25 MBq of 131I-ixolaris or 131I. In vitro studies demonstrated that 131I-ixolaris is stable for up to 24 h and retains its inhibitory activity on blood coagulation. Biodistribution analysis and metastasis assays showed that all treatment regimens with 131I-ixolaris were effective, being the double-treatment (D1/D15) the most effective one. Remarkably, treatment with free 131I showed no anti-metastatic effect. 131I-ixolaris is a promising theranostic agent for metastatic melanoma.

Salivary protein biomarkers for head and neck cancer.

Introduction: Saliva has gained attention as an important diagnostic fluid because it contains biomolecules that have the potential to detect early-stage cancer or to monitor the response to treatment in patients. Several saliva-based proteins have been proposed as potential biomarkers for head and neck cancers (HNC).Areas covered: This review aims to provide an update on saliva-based protein biomarkers for HNC, often studied in observational research and clinical trials.Expert opinion: Despite the increasing number of studies relating to salivary proteins as biomarkers for HNC, there is no consensus regarding which proteins have the best clinical utility. Most studies have analyzed individual proteins and not a protein panel approach. It must be considered that combining different proteins as a panel can increase the accuracy and will have the potential to change the current clinical practice for HNC patients.

Aberrant MUC1 accumulation in salivary glands of Sjögren's syndrome patients is reversed by TUDCA in vitro.

OBJECTIVES: Xerostomia in SS patients has been associated with low quality and quantity of salivary mucins, which are fundamental for the hydration and protection of the oral mucosa. The aim of this study was to evaluate if cytokines induce aberrant mucin expression and whether tauroursodeoxycholic acid (TUDCA) is able to counteract such an anomaly. METHODS: Labial salivary glands from 16 SS patients and 15 control subjects, as well as 3D acini or human submandibular gland cells stimulated with TNF-α or IFN-γ and co-incubated with TUDCA, were analysed. mRNA and protein levels of Mucin 1 (MUC1) and MUC7 were determined by RT-qPCR and western blot, respectively. Co-immunoprecipitation and immunofluorescence assays for mucins and GRP78 [an endoplasmic reticulum (ER)-resident protein] were also performed. mRNA levels of RelA/p65 (nuclear factor-κB subunit), TNF-α, IL-1ß, IL-6, SEL1L and EDEM1 were determined by RT-qPCR, and RelA/p65 localization was evaluated by immunofluorescence. RESULTS: MUC1 is overexpressed and accumulated in the ER of labial salivary gland from SS patients, while MUC7 accumulates throughout the cytoplasm of acinar cells; however, MUC1, but not MUC7, co-precipitated with GRP78. TUDCA diminished the overexpression and aberrant accumulation of MUC1 induced by TNF-α and IFN-γ, as well as the nuclear translocation of RelA/p65, together with the expression of inflammatory and ER stress markers in 3D acini. CONCLUSION: Chronic inflammation alters the secretory process of MUC1, inducing ER stress and affecting the quality of saliva in SS patients. TUDCA showed anti-inflammatory properties decreasing aberrant MUC1 accumulation. Further studies are necessary to evaluate the potential therapeutic effect of TUDCA in restoring glandular homeostasis in SS patients.

Salivary extracellular vesicles can modulate purinergic signalling in oral tissues by combined ectonucleoside triphosphate diphosphohydrolases and ecto-5'-nucleotidase activities.

We reported previously that the rat submandibular gland is able to release nanovesicles capable to hydrolyse millimolar concentrations of ATP, ADP and AMP in vitro. Here, we show that rat saliva also contains nanovesicles with the ability to hydrolyse ATP. Our aim was to identify and characterize vesicular nucleotidases by using kinetic, immunological and in silico approaches. Nucleotidase activity in the absence or presence of specific inhibitors allowed us to assume the participation of NTPDase1, -2 and -3, together with ecto-5'-nucleotidase, confirmed using specific antibodies. At neutral pH, initial ATPase activity would be mostly due to NTPDase2, which was thereafter inactivated, leaving NTPDase1 and NTPDase3 to hydrolyse ATP and ADP with an efficacy ATPase/ADPase around 2. Ecto-5'nucleotidase would be mainly responsible for AMP hydrolysis and adenosine accumulation. We proposed a kinetic model for NTPDase2 as a tool to isolate and analyse the turnover of this enzyme in the presence of different ATP concentrations, including those expected in extracellular media. Our study characterizes the ectonucleotidases carried by extracellular vesicles which contribute to modulate ATP and adenosine concentrations in the oral cavity, essential players in purinergic signalling.

Relevance of CRISP proteins for epididymal physiology, fertilization, and fertility.

BACKGROUND: The molecular mechanisms involved in the acquisition of mammalian sperm fertilizing ability are still poorly understood, reflecting the complexity of this process. OBJECTIVES: In this review, we describe the role of Cysteine RIch Secretory Proteins (CRISP1-4) in different steps of the sperm journey to the egg as well as their relevance for fertilization and fertility. MATERIALS AND METHODS: We analyze bibliography reporting the phenotypes of CRISP KO mice models and combine this search with recent findings from our team. RESULTS: Generation of individual KO for CRISP proteins reveals they are key mediators in different stages of the fertilization process. However, in spite of their important functional roles, KO males for each of these proteins remain fertile, supporting the existence of compensatory mechanisms between homologous CRISP family members. The development of mice lacking epididymal CRISP1 and CRISP4 simultaneously (DKO) revealed that mutant males exhibit an impaired fertility due to deficiencies in the sperm ability to fertilize the eggs in vivo, consistent with the proposed roles of the two proteins in fertilization. Interestingly, DKO males show clear defects in both epididymal epithelium differentiation and luminal acidification known to be critical for sperm maturation and storage. Whereas in most of the cases, these epithelium defects seem to specifically affect the sperm fertilizing ability, some animals exhibit a disruption of the characteristic immune tolerance of the organ with clear signs of inflammation and sperm viability defects. DISCUSSION AND CONCLUSION: Altogether, these observations confirm the relevance of CRISP proteins for male fertility and contribute to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immune tolerance within the epididymis. Moreover, considering the existence of a human epididymal protein functionally equivalent to rodent CRISP1 and CRISP4, DKO mice may represent an excellent model for studying human epididymal physiology and pathology.

RNA-seq analysis of the salivary glands and midgut of the Argasid tick Ornithodoros rostratus.

Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.

Cysteine-rich secretory protein 3: inflammation role in adult varicocoele.

BACKGROUND: Cysteine-rich secretory protein (CRISP-3), a protein involved in inflammatory response, is highly increased in seminal plasma of adolescents with varicocoele and altered semen analysis, but not in adolescents with varicocoele and normal semen. It is not known, however, whether this increased seminal concentration occurs as an acute marker during the initial stages of varicocoele or whether this persists as an altered protein pathway. OBJECTIVE: The purpose of this study, thus, was to test the hypothesis that this inflammatory state persists through adulthood and the correction of varicocoele could correct this state, by identifying the levels of CRISP-3 in seminal plasma. MATERIALS AND METHODS: This study was carried out in two substudies: (i) to verify the effect of varicocoele and (ii) to verify the effect of varicocelectomy on seminal plasma CRISP-3 levels. Seminal plasma CRISP-3 levels (29 and 31 kDa isoforms) were assessed for each provided sample using standard Western blotting. RESULTS: The varicocoele group presented higher seminal levels of CRISP-3 when compared to controls, with a 67.5-fold increase in the unglycosylated isoform (29 kDa) and a 5.2-fold increase in the glycosylated isoform (31 kDa). In contrast, CRISP-3 levels decreased following varicocelectomy, both in the unglycosylated (5.6-fold decrease) and in the glycosylated (4.3-fold decrease) isoforms. DISCUSSION: CRISP-3, a protein involved in inflammation, is increased in seminal plasma of men with varicocoele and this is partially reversed by varicocelectomy. Monitoring its seminal levels may be useful for assessing inflammation-related alterations to fertility in men with varicocoele. CONCLUSION: We conclude that, in the presence of varicocoele, there is a marked increase in seminal CRISP-3 levels. Surgical intervention (varicocelectomy) decreases CRISP-3 levels and improves semen quality.

Positive Selection in the Evolution of Mammalian CRISPs.

Cysteine-RIch Secretory Proteins (CRISPs) constitute a versatile family, with functions in reptilian venom and mammalian reproduction. Mammals generally express three CRISPs, four in mice, and all are highly expressed in male reproductive tissues, either testis or accessory organs. Because reproductive proteins often evolve adaptively in response to post-copulatory sexual selection, we hypothesized that mammalian CRISPs, with important roles in male reproduction, could have undergone positive selection promoting their divergence. We explored the molecular adaptation of mammalian CRISPs applying phylogenetic methods. Our analyses revealed the evidence of positive selection in all mammalian CRISPs. The intensity of positive selection was heterogeneous among CRISP members, being stronger in CRISP3 than in CRISP1 and CRISP2, and also across functional domains, having stronger impact on Pathogenesis-Related 1 (PR-1) in CRISP2 and on Ion Channel Regulator (ICR) in CRISP1 and CRISP3. In addition, we discovered a new CRISP in some rodent species, suggesting that the acquisition of new CRISP components could contribute to male reproductive success or to acquire new physiological roles. Signatures of positive selection were not focused on any particular mammalian group, suggesting that adaptive evolution is a recurrent pattern in mammalian CRISPs. Our findings support a model of CRISP family diversification driven by episodes of duplication and posterior neofunctionalization, and provide potential adaptive changes responsible for interspecific differences in CRISPs activity.