Comparative Proteome Analysis of Cryopreserved Flagella and Head Plasma Membrane Proteins from Sea Bream Spermatozoa: Effect of Antifreeze Proteins

Palabras clave extraídas del título[EN] Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to the procedures of fish sperm cryopreservationSIThis work was supported by Apulia Region (Italy) under the program between the Apulia Region and the University of Salento. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Does Diabetes Induce the Vascular Endothelial Growth Factor (VEGF) Expression in Periodontal Tissues? A Systematic Review

Abstract: Aim: Diabetes and periodontal disease are both chronic pathological conditions linked by several underlying biological mechanisms, in which the inflammatory response plays a critical role, and their association has been largely recognized. Recently, attention has been given to diabetes as an important mediator of vascular endothelial growth factor (VEGF) overexpression in periodontal tissues, by virtue of its ability to affect microvasculature. This review aims to summarize the findings from studies that explored VEGF expression in diabetic patients with periodontitis, compared to periodontally healthy subjects. Materials and Methods: A systematic literature review was performed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A PubMed search of select medical subject heading (MeSH) terms was carried out to identify all studies reporting findings about VEGF expression in periodontal tissues of diabetic patients up to May 2018. The inclusion criteria were studies on VEGF expression in periodontally diseased tissues of diabetic patients compared with nondiabetic subjects, with any method of analysis, and published in the English language. Results: Eight articles met the inclusion criteria. Immunohistochemistry was used in six of the studies, reverse transcriptase polymerase chain reaction (real-time RT-PCR) aiming to quantify mRNA VEGF expression was used in one study, and ELISA analysis was used for one study. Compared with nondiabetic patients, a higher VEGF expression in gingival tissue and gingival crevicular fluid (GCF) samples in diabetic patients with periodontitis was reported. Conclusions: Overall, novel evidence for the VEGF expression within the periodontal tissue of diabetic patients paves the way for further studies on the role of this protein in neovascularization physiology and pathophysiology in microvasculature of the periodontium

Representative 2-D gels of proteins extracted from the plasma membrane head isolated from fresh (A), cryopreserved with DMSO (B), with DMSO plus AFPI (C) and with DMSO plus AFPIII (D) gilthead sea bream spermatozoa.

<p>Extracted flagellar proteins (60 µg) were loaded onto non-linear IPG strips pH 3–10, and following isoelectric focusing were separated by 12.5% SDS–PAGE. Gels were silver stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099992#s2" target="_blank">Materials and Methods</a>. Spots that changed their expression after cryopreservation procedure are highlighted with a circle.</p

Effects of different cryoprotectants on different motility and viability parameters in sea bream spermatozoa before freezing (fresh sperm) and after different thawing conditions.

<p>Values are expressed as means ± SD for % motility, motility duration and % vitality and as means ± SEM for VCL and VSL. Within each column, values with different superscript letters indicate significant statistical differences (<i>P</i><0.05).</p><p>VSL: Velocity Straight Line.</p><p>VCL: curvilinear velocity.</p

Differences in relative abundance of 10 sperm head membrane spots in fresh and cryopreserved sea bream spermatozoa.

<p>Spots are highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099992#pone-0099992-g002" target="_blank">Figure 2</a> and the <i>x</i> axis identified the experimental conditions (fresh semen or cryopreserved with DMSO, with DMSO plus AFPI and with DMSO plus AFPIII). The <i>y</i>-axis corresponds to the intensity of spots expressed as normalized spot volume. Spot intensity is expressed as mean (dark bars) ± SD (light grey bars) of normalized spot volume (N = 18, that correspond to 6 different fish and 3 replicates for each). Different letters in the same graph indicate that mean values are significantly different by Mann–Whitney test (p<0.05). The spots showing a decrease of their expression after cryopreservation procedure are reported in the group 1, and those showing an increase are reported in the group 2.</p

Effect of AFPI and AFPIII at three different concentrations (0.1, 1 and 10 µg/ml) on motility rate and viability before freezing (fresh sperm) and after thawing.

<p>Values are expressed as means ± SD.</p><p>Within each row, values with different superscript letters indicate significant statistical differences (<i>P</i><0.05).</p

Periodontal Microbiological Status Influences the Occurrence of Cyclosporine-A and Tacrolimus-Induced Gingival Overgrowth

Immune suppressed renal transplant patients are more prone to developing oral tissue alterations due to medications associated with a pleiotropic set of side effects involving the oral cavity. Drug-induced gingival overgrowth (DIGO) is the most commonly encountered side effect resulting from administration of calcineurin inhibitors such as cyclosporine-A (CsA), the standard first-line treatment for graft rejection prevention in transplant patients. Pathogenesis of gingival overgrowth (GO) is determined by the interrelation between medications and a pre-existing inflammatory periodontal condition, the main modifiable risk factor. Severity of gingival hyperplasia clinical manifestation is also related to calcium channel blocker association, frequently provided in addition to pharmacological therapy of transplant recipients. Specifically, nifedipine-induced enlargements have a higher prevalence rate compared to amlodipine-induced enlargements; 47.8% and 3.3% respectively. Available epidemiological data show a gender difference in prevalence, whereby males are generally more frequently affected than females. The impact of GO on the well-being of an individual is significant, often leading to complications related to masticatory function and phonation, a side effect that may necessitate switching to the tacrolimus drug that, under a similar regimen, is associated with a low incidence of gingival lesion. Early detection and management of GO is imperative to allow patients to continue life-prolonging therapy with minimal morbidity. The purpose of this study was threefold: firstly, to determine the prevalence and incidence of GO under the administration of CsA and Tacrolimus; secondly, to assess the correlation between periodontal status before and after periodontal therapy and medications on progression or recurrence of DIGO; and finally, to analyse the effect of immunosuppressant in association to the channel blocker agents on the onset and progression of gingival enlargement. We compared seventy-two renal transplant patients, including 33 patients who were receiving CsA, of which 25% were also receiving nifedipine and 9.72% also receiving amlodipine, and 39 patients who were receiving tacrolimus, of which 37.5% were also receiving nifedipine and 5.55% also receiving amlodipine, aged between 35 and 60 years. Medical and pharmacological data were recorded for all patients. Clinical periodontal examination, in order to establish the inflammatory status and degree of gingival enlargement, was performed at baseline (T0), 3 months (T1), 6 months (T2), and 9 months (T3). All patients were subjected to periodontal treatment. Statistically significant correlation between the reduction of the mean value of periodontal indices and degree of gingival hyperplasia at the three times was revealed. The prevalence of GO in patients taking cyclosporine was higher (33.3%) in comparison with those taking tacrolimus (14.7%). In accordance with previous studies, this trial highlighted the clinical significance of the pathological substrate on stimulating drug-induced gingival lesion, confirming the key role of periodontal inflammation in pathogenesis of gingival enlargement, but did not confirm the additional effect of calcium-channel blocker drugs in inducing gingival enlargement

Impact of Periodontal Inflammation on Nutrition and Inflammation Markers in Hemodialysis Patients

Background: Malnutrition-inflammation complex syndrome (MICS) is a common and usually concurrent condition occurring in patients undergoing hemodialysis (HD), with a pathogenesis linked to biological and in situ environmental traditional risk factors. Periodontitis, one of the major types of infection-driven inflammation, often co-occurs in the in the hemodialysis population and correlates with markers of malnutrition and inflammation, such as albumin, creatinine, and C-reactive protein. Aim: The present study aimed to determine whether the periodontal inflammatory status parameters correlate with the albumin, creatinine, and C-reactive protein serum concentrations in HD patients, and investigate whether periodontal treatment improves these markers of nutritional and systemic inflammation. Materials and Methods: The serum creatinine, albumin, and C-reactive Protein (CRP) levels were measured at baseline and after non-surgical periodontal treatment, at 3 months and 6 months. Results: At 3 months, a significant correlation between plaque index and C-reactive protein (p = 0.012), bleeding on probing and C- reactive protein (p &lt; 0.0019), and clinical attachment level and C-reactive protein (p = 0.022) was found. No significant correlation was found between clinical periodontal parameters and nutrition markers at each time. Conclusions: Our results confirmed the association between C-reactive protein serum concentration and periodontal inflammatory status, but further research is necessary to identify the contributing role of periodontitis on the onset and progression of MICS