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Objective To design protein phosphatase 1 (PP1)–disrupting peptides covalently coupled to inert cell-penetrating peptides (CPPs) as sychnologically organized bioportide constructs as a strategy to modulate sperm motility. Design Experimental study. Setting Academic research laboratory. Patient(s)/Animal(s) Normozoospermic men providing samples for routine analysis and Holstein Frisian bulls. Intervention(s) None. Main Outcome Measure(s) Effect of the bioportides on the activity and interactions of PP1γ2—a PP1 isoform expressed exclusively in testicular germ cells and sperm—and on sperm vitality and motility. Result(s) PP1-disrupting peptides were designed based on the sequences from: 1) a sperm-specific PP1 interactor (A kinase anchor protein 4); and 2) a PP1 inhibitor (protein phosphatase inhibitor 2). Those sequences were covalently coupled to inert CPPs as bioportide constructs, which were successfully delivered to the flagellum of sperm cells to induce a marked impact on PP1γ2 activity and sperm motility. Molecular modeling studies further facilitated the identification of an optimized PP1-binding sequence and enabled the development of a modified stop-sperm bioportide with reduced size and increased potency of action. In addition, a bioportide mimetic of the unique 22-amino acid C-terminus of PP1γ2 accumulated within spermatozoa to significantly reduce sperm motility and further define the PP1γ2-specific interactome. Conclusion(s) These investigations demonstrate the utility of CPPs to deliver peptide sequences that target unique protein-protein interactions in spermatozoa to achieve a significant impact upon spermatozoa motility, a key prognostic indicator of male fertility.

The mature spermatozoon, a highly differentiated cell equipped for the sole purpose of fertilization, lacks the protein machinery required for conventional endocytotic mechanisms. Perhaps contrary to expectation, cell-penetrating peptides (CPPs) rapidly translocate across the unique sperm plasma membrane to accrete within distinct intracellular compartments. Confocal microscopy, employing red-fluorescent CPPs and bioportides, is a convenient platform to study this membrane translocation process. In the virtual absence of genetic expression, rapid physiological responses of human sperm are dependent upon protein-protein interactions that may be regulated by posttranslational modifications including phosphorylation. This chapter provides an outline of the design of bioactive CPPs, or bioportides, which include protein-mimetic sequences from the interaction domains of sperm proteins. Protocols are included which enable the biological assessment of the impact of bioportides upon the viability and motility of spermatozoa.

Simple Summary Acquired resistance to antiestrogenic therapy remains the major obstacle to curing luminal subtype breast cancer. While current treatment in acquired endocrine-resistant settings includes combined therapy with receptor tyrosine kinase or cyclin-dependent kinase inhibitors, progression to incurable disease remains possible. In recent years, the antioxidant system and the protein quality control network have been associated with the enhanced resistance of breast cancer cells to hormonal therapy. In this work, we raise the hypothesis that antiestrogen treatment induces the accumulation of protein aggregates in sensitive cells, which in turn could hinder the activation of survival pathways. We present evidence concerning a novel way to identify antiestrogen response and disclose a novel protein, RTBC, that controls acquired antiestrogen resistance. This work opens a new avenue for research towards finding breast cancer prognostic markers and therapeutic targets, where the identification of proteins prone to aggregate could help to identify antiestrogen response and understand mechanisms of disease. Abstract The protein quality control network, including autophagy, the proteasome and the unfolded protein response (UPR), is triggered by stress and is overactive in acquired antiestrogen therapy resistance. We show for the first time that the aggresome load correlates with apoptosis and is increased in antiestrogen-sensitive cells compared to endocrine-resistant variants. LC-MS/MS analysis of the aggregated proteins obtained after 4OH-tamoxifen and Fulvestrant treatment identified proteins with essential function in protein quality control in antiestrogen-sensitive cells, but not in resistant variants. These include the UPR modulators RTCB and PDIA6, as well as many proteasome proteins such as PSMC2 and PSMD11. RTCB is a tRNA and XBP1 ligase and its aggregation induced by antiestrogens correlated with impaired XBP1s expression in sensitive cells. Knock down of RTCB was sufficient to restore sensitivity to tamoxifen in endocrine-resistant cells and increased the formation of aggresomes, leading to apoptotic cell death. Analysis of primary human breast cancer samples and their metastases appearing after endocrine treatment showed that RTCB is only localized to aggresomes in the primary tumors, while total aggresomes, including aggregated RTCB, were significantly reduced in the metastases. Therefore, different protein aggregation patterns may indicate loss of function of essential proteins resulting in enhanced protein aggregation that can be used to identify antiestrogen-resistant breast cancer cells and improve the response to antiestrogenic therapy.

Rheumatoid arthritis (RA) is a Th1/Th17-mediated autoimmune disease whose current treatment, consisting in the blockage of inflammatory cytokines by disease-modifying antirheumatic drugs, is not effective for all patients. The therapeutic potential of mesenchymal stromal/stem cells' (MSCs) immunomodulatory properties is being explored in RA. Here, we investigate the effect of human bone marrow (BM)-MSCs on the expression of cytokines involved in RA physiopathology by the distinct functional compartments of CD4+ and CD8+ T cells from RA patients. Peripheral blood mononuclear cells from healthy individuals (n = 6) and RA patients (n = 12) were stimulated with phorbol myristate acetate plus ionomycin and cultured in the presence/absence of BM-MSCs. The expression of (interleukin) IL-2, tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ) was evaluated in naive, central memory, effector memory, and effector CD4+ and CD8+ T cells, whereas IL-6, IL-9, and IL-17 expression was measured in total CD4+ and CD8+ T cells. mRNA expression of IL-4, IL-10, transforming growth factor beta (TGF-β), cytotoxic T-lymphocyte-associated antigen 4, and/or forkhead box P3 was quantified in fluorescence-activated cell sorting-purified CD4+ T cells, CD8+ T cells, and CD4+ Treg. BM-MSCs inhibited the production of TNF-α, IL-17, IL-6, IL-2, IFN-γ, and IL-9 by T cells from RA patients, mainly by reducing the percentage of cells producing cytokines. This inhibitory effect was transversal to all T cell subsets analyzed. At mRNA level, BM-MSCs increased expression of IL-10 and TGF-β by CD4+ and CD8+ T cells. BM-MSCs displayed a striking inhibitory action over T cells from RA patients, reducing the expression of cytokines involved in RA physiopathology. Remarkably, BM-MSC-derived immunomodulation affected either naive, effector, and memory T cells.

Background Evidences from human and animal studies suggest that reproductive function may be affected by mercury. The aim of this review was to explore the mercury influence on human fertility. Methods A systematic search was made in PubMED for papers published between 1975–2017, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Results Increased mercury levels were associated with infertility or subfertility status. Further, infertile subjects with unexplained infertility showed higher levels of mercury in hair, blood and urine than fertile ones. Mercury exposure induced sperm DNA damage and abnormal sperm morphology and motility. Additionally, mercury levels were related with higher incidence of menstrual and hormonal disorders and increased rates of adverse reproductive outcomes. Conclusions Our review showed that mercury negatively impacts human reproduction, affecting the reproductive and endocrine systems in both male and female. However, the molecular mechanisms underlying the mercury-associated decline on fertility remains unknown.

Aim: This work intended to improve the knowledge of the rat model of prostate cancer (PCa) by ultrasonographic monitoring. Materials and Methods: Male Wistar rats were divided into control (n=8) and PCa (n=14) groups. PCa development was induced in the PCa group through the sequential administration of the anti-androgenic drug flutamide, testosterone propionate and the carcinogenic N-methyl-N-nitrosourea. The prostate was evaluated by ultrasonography at five timepoints along 49 weeks of the experimental protocol. Ventral prostate lobes were observed in all ultrasonographic examinations. Results: The ventral prostate area of the control group increased gradually between the first and the last ultrasonographic examination. The ventral prostate area of PCa groups decreased due to flutamide administration and increased after androgen and carcinogen administration. The area of the dorsal prostate lobe increased between the fourth and the fifth ultrasonographic examination. In the last ultrasonographic examination, hypoechoic and anechoic lesions were observed in the PCa group. Conclusion: To our knowledge, this is the first study presenting a follow-up of rat prostatic dimensions by ultrasonography. Ultrasonography is a feasible approach for prostate cancer monitoring in experimental models. published

Submitted by Margarida Fardilha (mfardilha@ua.pt) on 2020-10-02T14:58:22Z No. of bitstreams: 1 manuscript Dias et al_Revised.pdf: 710383 bytes, checksum: f07f2efbd7233a25dfc00fb671d1b1d7 (MD5) Approved for entry into archive by Rita Gonçalves (ritaisabel@ua.pt) on 2020-10-09T11:50:59Z (GMT) No. of bitstreams: 1 manuscript Dias et al_Revised.pdf: 710383 bytes, checksum: f07f2efbd7233a25dfc00fb671d1b1d7 (MD5) Made available in DSpace on 2020-10-09T11:50:59Z (GMT). No. of bitstreams: 1 manuscript Dias et al_Revised.pdf: 710383 bytes, checksum: f07f2efbd7233a25dfc00fb671d1b1d7 (MD5) Previous issue date: 2020-02 published

Germ cell proliferation is epigenetically controlled, mainly through DNA methylation and histone modifications. However, the pivotal epigenetic regulators of germ cell self-renewal and differentiation in postnatal testis are still poorly defined. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2, represses target genes through trimethylation of histone H3 at Lys-27 (H3K27me3), and interacts (in)directly with both protein phosphatase 1 (PP1) and nuclear inhibitor of PP1 (NIPP1). Here, we report that postnatal, testis-specific ablation of NIPP1 in mice results in loss of EZH2 and reduces H3K27me3 levels. Mechanistically, the NIPP1 deletion abrogated PP1-mediated EZH2 dephosphorylation at two cyclin-dependent kinase sites (Thr-345/487), thereby generating hyperphosphorylated EZH2, which is a substrate for proteolytic degradation. Accordingly, alanine mutation of these residues prolonged the half-life of EZH2 in male germ cells. Our study discloses a key role for the PP1:NIPP1 holoenzyme in stabilizing EZH2 and maintaining the H3K27me3 mark on genes that are important for germ cell development and spermatogenesis. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:293 issue:47 pages:18031-18039 ispartof: location:United States status: published

Prostate cancer (PCa) is the second most frequent cancer diagnosed in men worldwide. Among the common treatment options, photodynamic therapy (PDT) is being considered a promising local therapy to treat this cancer. Although PDT is an established treatment modality approved for several types of cancer, the low solubility, the reduced tumor selectivity, the absorption in the therapeutic window and the poor clearance from the body of the currently approved photosensitizers (PS) hampers its wide clinical application. In this regard, herein we synthesized three fluorinated porphyrinoid derivatives and entrapped them into polyvinylpyrrolidone (PVP) to prevent their aggregation and preserve their desirable photophysical properties under the physiological environment. In vitro studies revealed the negligible dark cytotoxicity of all PVP formulations (PS1@PVP, PS2@PVP and PS3@PVP) at the tested concentrations (5.0 to 20 μM), but also confirmed the significant photodynamic effect of PS2@PVP and PS3@PVP towards the PCa cell line PC-3, upon red light irradiation at an irradiance of 17.6 mW.cm−2. To provide insight into the underlying mechanisms of cell death under PDT treatment induced by PS2@PVP and PS3@PVP, their intracellular localization in PC-3 cells was firstly investigated by confocal microscopy. Since both PS2@PVP and PS3@PVP nanoparticles were mainly localized in mitochondria, the involvement of this organelle in PDT-induced apoptosis mediated by both formulations was further explored. Western blot analysis revealed that PDT treatment of PC-3 cells with either PS2@PVP or PS3@PVP resulted in the reduction of the expression level of the anti-apoptotic protein Bcl-2. As the photodamage to Bcl-2 after PDT with PS2@PVP and PS3@PVP was accompanied by the further activation of pro-caspase-3, we assumed that upon irradiation the photogenerated reactive oxygen species (ROS) were able to activate a caspase-dependent apoptotic response as a consequence of a post-mitochondrial event. Taken together, these findings demonstrate that among the tested fluorinated porphyrinoids, PS2@PVP and, particularly, PS3@PVP, are significantly more effective in overall PC-3 cell killing than PS1@PVP, thus highlighting their great potential as therapeutic agents for PCa.

Lung cancer has become the leading killer cancer worldwide, due to late diagnosis and lack of efficient anticancer drugs. We have recently described novel natural-derived tambjamine analogues that are potent anion transporters capable of disrupting cellular ion balance, inducing acidification of the cytosol and hyperpolarization of cellular plasma membranes. Although these tambjamine analogues were able to compromise cell survival, their molecular mechanism of action remains largely unknown. Herein we characterize the molecular cell responses induced by highly active indole-based tambjamine analogues treatment in lung cancer cells. Expression changes produced after compounds treatment comprised genes related to apoptosis, cell cycle, growth factors and its receptors, protein kinases and topoisomerases, among others. Dysregulation of BCL2 and BIRC5/survivin genes suggested the apoptotic pathway as the induced molecular cell death mechanism. In fact, activation of several proapoptotic markers (caspase-9, caspase-3, and PARP) and reversion of the cytotoxic effect upon treatment with an apoptosis inhibitor (Z-VAD-FMK) were observed. Moreover, members of the Bcl-2 protein family suffered changes after tambjamine analogues treatment, with a concomitant protein decrease towards the prosurvival members. Besides this, it was observed cellular accumulation of ROS upon compound treatment and an activation of the stress-kinase p38 MAPK route that, when inhibited, reverted the cytotoxic effect of the tambjamine analogues. Finally, a significant therapeutic effect of these compounds was observed in subcutaneous and orthotopic lung cancer mice models. Taken together, these results shed light on the mechanism of action of novel cytotoxic anionophores and demonstrate the therapeutic effects against lung cancer., Spanish Government and EU funds through the Fondo de Investigaciones Sanitarias (FIS, project PI13/ 00089) and from La Marat o de TV3 Foundation (project 20132730) to R. P erez-Tom as. R. Ramos was supported by the Sociedad Espa~nola de Neumología y Cirugía Tor acica (SEPAR, Project 017/2013), R. Quesada by the Consejería de Educación de la Junta de Castilla y León (project BU340U13) and by the La Marat o de TV3 Foundation (project 20132732) and A. Villanueva by the FIS (project PI13/01339).

HIGHLIGHTS: Male fertility decline has been attributed, in part, to increased oxidative stress. Here we will focus on spermatozoa ROS, namely O2(•−), NO and ONOO(−) and their contribution to protein tyrosine nitration, namely by 3-NT formation. An in depth review will be made on the methods used to detect protein oxidation. Detecting 3-NT in sperm proteins will have a crucial clinical impact, namely on the follow up of anti-oxidant therapies. ABSTRACT: Infertility is a clinical condition that affects around 15% of reproductive-aged couples worldwide. Around half of these cases are due to male factors, the most owing to idiopathic causes. The increase of reactive oxygen species (ROS), which leads to oxidative stress (OS), has been discussed in the last years as a possible cause of male idiopathic infertility. Superoxide anion (O(2)(•−)) and nitric oxide (NO) can react with each other contributing to the formation of peroxynitrite (ONOO(−)). This molecule can then act on spermatozoa proteins, leading to nitration of protein tyrosines – addition of a nitro (NO(2)) group – that is then manifested by the formation of 3-nitrotyrosine (3-NT). In turn, 3-NT may be responsible for the alteration or inactivation of the protein function. This review will focus on the description of spermatozoa ROS, namely O(2)(•−), NO and ONOO(−) and in their contribution to protein tyrosine nitration, namely by 3-NT formation. Previous results about the effect of ONOO(−) and 3-NT in spermatozoa will be presented, as well as, the methods that can be performed to detect the protein oxidation by these species. The impact of measuring, at the clinical level, 3-NT, considered a marker of OS, in spermatozoa will be discussed.

Sulfate-based lipids (SL) have been proposed as players in inflammation, immunity and infection. In spite of the many biochemical processes linked to SL, analysis on this class of lipids has only focused on specific SL sub-classes in individual fluids or cells leaving a range of additional SL in other biological samples unaccounted for. This study describes the mass spectrometry screening of SL in lipid extracts of human fluids (saliva, plasma, urine, seminal fluid) and primary human cells (RBC, neutrophils, fibroblasts and skin epidermal) using targeted precursor ion scanning (PIS) approach. The PIS 97 mass spectra reveal a wide diversity of SL including steroid sulfates, sulfoglycolipids and other unidentified SL, as well as metabolites such as taurines, sulfated polyphenols and hypurate conjugates. Semi-quantification of SL revealed that plasma exhibited the highest content of SL whereas seminal fluid and epithelial cells contained the highest sulphur to phosphorous (S/P) ratio. The complexity of biofluids and cells sulfateome presented in this study highlight the importance of expanding the panel of synthetic sulfate-based lipid standards. Also, the heterogenous distribution of SL provides evidence for the interplay of sulfotransferases/sulfatases, opening new avenues for biomarker discovery in oral health, cardiovascular, fertility and dermatology research areas. published

Introduction: Prostate Cancer (PCa) is the second most common neoplasia in men. Because it is often diagnosed at a late stage, mortality rates remain high. Studying cancer metabolome, which reflects early changes that occur in cells, has gained relevance and may contribute to the identification of early diagnostic biomarkers and understanding tumor biology. Methods: Fourier-transform infrared (FTIR) spectroscopy is a metabolomics technique that probes the biochemical composition of the analyzed samples and allows to discriminate samples with distinct metabolic profiles, allowing the discrimination between cancerous and non-cancerous samples. In this study, FTIR spectra were acquired from PCa and normal prostate cell lines and analyzed by Principal Component Analysis (PCA). Results & Conclusion: Our results indicate a clear discrimination between the different cell lines, meaning at they exhibit distinct metabolic profiles. This discrimination can be attributed to an altered lipid metabolism (3000-2800 cm-1, 1800-1700 cm-1 and 1500-1400 cm-1) and changes in protein con-formation (1700-1600 cm-1). These results suggest that studying cancer metabolome with FTIR spec-troscopy not only allows the understanding of tumor metabolic behavior and may be useful to the de-velopment of new therapeutic targets. published

Sertoli cells are crucial for germ cell support, create a suitable environment for spermatogenesis, and integrate information from the nervous system and germ cell line. To fully understand the role of Sertoli cells, it is necessary to characterize the protein-protein interactions. Identifying the interactome of a given protein may provide leads about the role and molecular mechanism of such protein in Sertoli cells. One of the techniques to characterize protein interactomes consists of co-immunoprecipitation followed by mass spectrometry or Western blot. Co-immunoprecipitation enables the isolation of a protein target and interactome under physiological conditions. In this chapter, we described how to perform an interactomic study using the co-immunoprecipitation technique in Sertoli cells. Moreover, we will focus on how to analyze and interpret the results of a co-immunoprecipitation before mass spectrometry analysis.

Most techniques to study protein-protein interactions, gene expression or signal transduction, among others, in Sertoli cells, depend on obtaining a protein extract of such cells. This is accomplished by lysing the Sertoli cells and solubilizing the intracellular proteins. Depending on the purpose of your study, the technique used to lyse and consequently obtain protein extracts from Sertoli cells must be considered. In this chapter, we will focus on how to obtain a protein Sertoli cell extract suitable for co-immunoprecipitation technique.

Globally, cancer is one of the leading causes of death, so the development of strategies for an early diagnosis of cancer is of great importance. Biochemical alterations precede morphological changes in cells and tissues, so studying cancer metabolome seems like a reasonable approach for early diagnosis, prognosis and to follow treatment progression. Fourier-transform infrared (FTIR) spectroscopy is a valuable tool for studying the metabolome of biological samples, such as cancer cell lines. Unlike staining procedures and other histopathologic approaches, this technique is rapid, non-destructive and does not require reagents. The spectral differences that result from probing the biochemical composition of cancer and normal cells are indicative of distinct metabolic profiles, which allows the discrimination of different cells. Using FTIR spectroscopy and multivariate statistical analysis, several alterations concerning the content of lipids, proteins, nucleic acids and carbohydrates have been identified in cancer cells, some of which can be regarded as potential biomarkers. This review focuses on FTIR spectroscopy as a metabolomics tool to study and characterize cancer cell lines. published

Galactosylceramide (GalCer) and lactosylceramide (LacCer) are structural and signaling lipids, playing important roles in signal transduction and cell adhesion. They are especially abundant in the nervous system and in important components of the myelin sheath. Although neurodegenerative disorders are associated with increased oxidative stress and lipid oxidation, the connection between oxidative stress and glycosphingolipid modification has been scarcely addressed. In this study, we aimed to characterize the structural changes caused by the hydroxyl radical to GalCer and LacCer molecular species using electrospray ionization mass spectrometry (ESI-MS and MS/MS) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS(n)). ESI-MS and LC-MS spectra of 24:1GalCer and 24:1LacCer after free radical oxidation showed the formation of new species, which were identified as keto, hydroxyl and hydroperoxy derivatives, arising from modification in the mono unsaturated fatty acyl chain. Formation of ceramide and oxidized ceramides was also observed as a result of 24:1GalCer and 24:1LacCer radical oxidation. 24:1GlcCer (glucosylceramide) was detected after LacCer oxidation, probably due to oxidative cleavage of lactosyl moiety. This study shows that glycosphingolipids are prone to radical induced oxidation, which can be one of the causes of the increased ceramides content and pro apoptotic events during oxidative conditions and neurodegeneration. This MS study will support the future identification of oxidized galactosyl- and lactosylceramide species in sphingolipidomic studies applied to biological samples related with oxidative conditions.

The oocyte's primary function is to be fertilised by a spermatozoon in order to create a viable embryo. Oocyte growth and development are initiated during embryogenesis and occur in parallel to follicular development. Factors produced by the oocyte bind to receptors on follicular cells, ensuring follicular development. Oocytes begin meiosis during foetal development and are arrested in prophase I by elevated levels of cyclic adenosine monophosphate (cAMP). Activation of mitogen-activated protein kinases triggers degradation of cAMP, allowing oocyte maturation to proceed. The production of progesterone and prostaglandins during the ovulation process ultimately activates proteases, whose action helps to release the oocyte into the Fallopian tube. Oocyte activation depends on fertilisation and is induced by changes in intracellular calcium levels. Dysregulation of these pathways is involved in the pathogenesis of several diseases including the syndrome of oocyte maturation failure.

Submitted by Margarida Fardilha (mfardilha@ua.pt) on 2020-09-11T17:08:05Z No. of bitstreams: 1 0001-3765-aabc-90-01-s2-01101.pdf: 3831978 bytes, checksum: eb998954b2360e19d6181b160b7e2acd (MD5) Approved for entry into archive by Rita Gonçalves (ritaisabel@ua.pt) on 2020-09-14T09:20:58Z (GMT) No. of bitstreams: 1 0001-3765-aabc-90-01-s2-01101.pdf: 3831978 bytes, checksum: eb998954b2360e19d6181b160b7e2acd (MD5) Made available in DSpace on 2020-09-14T09:20:58Z (GMT). No. of bitstreams: 1 0001-3765-aabc-90-01-s2-01101.pdf: 3831978 bytes, checksum: eb998954b2360e19d6181b160b7e2acd (MD5) Previous issue date: 2018 published

Many studies have reported a negative impact of lifestyle factors on testicular function, spermatozoa parameters and pituitary-gonadal axis. However, conclusions are difficult to draw, since studies in the general population are rare. In this study we intended to address the early and late short-term impact of acute lifestyle alterations on young men’s reproductive function. Thirty-six healthy male students, who attended the Portuguese academic festivities, provided semen samples and answered questionnaires at three time-points. The consumption of alcohol and cigarette increased more than 8 and 2 times, respectively, during the academic festivities and resulted in deleterious effects on semen quality: one week after the festivities, a decrease on semen volume, spermatozoa motility and normal morphology was observed, in parallel with an increase on immotile spermatozoa, head and midpiece defects and spermatozoa oxidative stress. Additionally, three months after the academic festivities, besides the detrimental effect on volume, motility and morphology, a negative impact on spermatozoa concentration was observed, along with a decrease on epididymal, seminal vesicles and prostate function. This study contributed to understanding the pathophysiology underlying semen quality degradation induced by acute lifestyle alterations, suggesting that high alcohol and cigarette consumption are associated with decreased semen quality in healthy young men.

Extensive exposure to UVA is thought to increase the risk of malignancy and the progression of melanoma, the most serious type of skin cancer. It is well known that alterations in lipid metabolism represent an early event in carcinogenesis, but the impact of UVA exposure on the lipid composition of cancer cells is still largely unknown. In this study we aimed at investigating lipid remodeling in human melanoma cells in response to UVA exposure. After UVA irradiation, lipid extracts were either immediately collected from SK-MEL-28 cells or collected after a recovery period of 2 h or 24 h. The lipid profiles for each event were determined by liquid chromatography or gas chromatography coupled to mass spectrometry. UVA exposure led to major alterations in both fatty acids (FA) and phospholipid profiles. An increase of monounsaturated FA (MUFA) and FA18:0, as well as a decrease of FA16:0, were observed 24 h after irradiation. Moreover, phosphatidylcholine (PC) decreased and phosphatidylinositol (PI) increased after UVA exposure. Molecular alterations in the PC, lysoPC, PI, phosphatidylethanolamine (PE), ether-linked PE and phosphatidylglycerol (PG) profiles were also observed. The absence of cleaved caspase-3 after 2 h and 24 h of re-incubation is correlated with impairment of apoptosis. Overall, these data showed changes in membrane lipids, which may be associated with lipogenesis after UVA exposure which, in turn, is usually a determinant for cell survival.

The use of the chemotherapeutic drug doxorubicin (DOX) is limited by its toxicity in several organs such as testes. So, we analyzed the effect of endurance treadmill exercise training (EX) performed before sub-chronic DOX treatment on sperm count and motility, testes markers of oxidative damage and apoptosis. Tissue profiling of proteins more susceptible to oxidation was made to identify the molecular pathways regulated by oxidative modifications, as nitration and carbonylation. Twenty-four adult male rats were divided into four groups (n=6/group): sedentary saline (SED+SAL), sedentary sub-chronically injected with DOX (2mg-kg-1 per week, during 7 weeks; SED+DOX), 12 weeks trained saline (EX+SAL) and trained treated with DOX (EX+DOX). DOX treatment started 5 weeks after the beginning of the exercise program. Testes caspase-3, -8 and -9, as well as aconitase activities, the content of malondialdehyde (MDA), sulfhydryl groups (-SH), carbonyl and nitrotyrosine derivatives were determined. Modified proteins were identified by 2D-Western blot followed by MALDI-TOF/TOF mass spectrometry, and bioinformatic analysis was performed to assess the biological processes regulated by these chemical modifications. The decreased sperm motility induced by DOX was not modified by exercise. Significant increases in MDA content in SED+DOX and in caspase-3 and -9 activities in EX+DOX were found. Despite no significant differences in the levels of carbonylated and nitrated proteins, exercise modulated testis proteome susceptibility to oxidation in DOX-treated group, with less modified proteins identified. Zinc finger Ran-binding domain-containing protein 2 (ZRAB2) and AN1-type zinc finger protein 3 (ZFAN3) were among the proteins found oxidativelly modified. Although no marked alterations in testes oxidative damage were noticed, proteomic analysis of oxidativelly modified proteins highlighted the protective role of exercise against oxidative damage of some proteins involved in metabolism and stress response against DOX.

The coordination of ruthenium(II) complexes to anionic oxygen-based donors is very rare. This study describes a simple, one-pot method for obtaining [ruthenium(II)(trithiacyclononane)(curcumin)(S-DMSO)]Cl (1) in 37% yield. The structural characterization of complex 1 by elemental analysis, FT-IR, 1-D and 2-D NMR, ESI+-MS as well as UV-vis and fluorescence spectroscopies is presented. The DNA-melting temperature (Tm) assay shows that salmon sperm DNA (smDNA) in the presence of complex 1 has a higher melting temperature, with ΔTm = 7.4 °C, while in the presence of curcumin the melting temperature remains unaltered. The in vitro cytotoxic activities of curcumin and complex 1 were investigated using the tumor human prostate cell line, PC-3, and the healthy cell line, PNT-2. Complex 1 is innocuous towards normal prostate epithelial cells and, whereas curcumin is toxic, with inhibition rates of ca. 35% and 65% at 50 and 80 μM, respectively. On the tumor cell line PC-3, complex 1 did not cause viability changes whereas curcumin exhibited dose-dependent inhibition, with ca. 73% inhibition at the highest concentration tested, i.e. 80 μM. This study suggests that coordination with the trithiacyclononane ruthenium(II) scaffold stabilizes the photochemical properties of curcumin and strongly changes its biologic activity.

Proteolytic processing of the amyloid-β protein precursor (AβPP) occurs via alternative pathways, culminating with the production of the AβPP intracellular domain (AICD). AICD can translocate to the nucleus and regulate transcription, but its activity is modulated by interactions with other proteins. In the nucleus, AICD, FE65, and Tip60 associate into AFT complexes, which are targeted to nuclear spots which correspond to transcription factories. Here we report that RanBP9 interacts with the cytoplasmic domain of AβPP, through the NPXY internalization motif. Moreover, RanBP9 interaction with Tip60 is also described. The RanBP9-Tip60 interaction dramatically relocated RanBP9 from a widespread cellular distribution to nuclear speckles. AβPP processing is a central aspect in determining the protein's function and that of its resulting proteolytic fragments, among them AICD. The latter results from the amyloidogenic pathway and is the peptidic species predominantly involved in nuclear signaling. Of note RanBP9 transfection was previously demonstrated to increase amyloid-β generation. Here we show that RanBP9 relocates AICD to the Tip60-enriched nuclear speckles, and prevented the formation of nuclear spots formation, having therefore a negative effect on AICD mediated nuclear signaling and consequently AFT complex formation. Furthermore, by transfecting cells with increasing amounts of RanBP9, the expression of AICD-regulated genes, including AβPP itself, was reduced. Given the data presented, one can deduce that RanBP9 has an inhibitory regulatory effect on AICD-mediated transcription and the effect is mediated by relocating AICD away from transcription factories.

Resumo O espermatozoide e o gâmeta masculino e, como tal, a sua principal funcao e fecundar o oocito. Aquando da ejaculacao, esta celula ainda nao se encontra madura, pelo que nao consegue realizar a sua funcao. Ao entrar em contato com o trato reprodutor feminino, o espermatozoide sofre a capacitacao. Este processo e caracterizado por alteracoes bioquimicas e funcionais. A reacao acrossomica e o processo final para a fecundacao e caracteriza‐se pela libertacao das enzimas de proteoliticas que hidrolisam a zona pelucida. Todos estes processos dependem de vias de sinalizacao. A maioria das vias de sinalizacao descritas para celulas somaticas ja foram identificadas no espermatozoide, contudo, os seus efeitos nao sao completamente conhecidos. Nesta revisao serao descritas as principais vias de sinalizacao dos espermatozoides, nomeadamente PPP1CC2, cAMP/PKA, fosfolipase C, PI3K‐AKT e ROS.

Phosphorylation is a major regulatory mechanism in eukaryotic cells performed by the concerted actions of kinases and phosphatases (PPs). Protein phosphorylation has long been relevant to sperm physiology, from acquisition of motility in the epididymis to capacitation in the female reproductive tract. While the precise kinases involved in the regulation of sperm phosphorylation have been studied for decades, the PPs have only recently received research interest. Tyrosine phosphorylation was first implicated in the regulation of several sperm-related functions, from capacitation to oocyte binding. Only afterwards, in 1996, the inhibition of the serine/threonine-PP phosphoprotein phosphatase 1 (PPP1) by okadaic acid and calyculin-A was shown to initiate motility in caput epididymal sperm. Today, the current mechanisms of sperm motility acquisition based on PPP1 and its regulators are still far from being fully understood. PPP1CC2, specifically expressed in mammalian sperm, has been considered to be the only sperm-specific serine/threonine-PP, while other PPP1 isoforms were thought to be absent from sperm. This article examines the “Omics” of human sperm, and reports, for the first time, the identification of three new serine/threonine-protein PPs, PPP1CB, PPP4C, and PPP6C, in human sperm, together with two tyrosine-PPs, MKP1 and PTP1C. We specifically localized in sperm PPP1CB and PPP1CC2 from the PPP1 subfamily, and PPP2CA, PPP4C, and PPP6C from the PPP2 subfamily of the serine/threonine-PPs. A semi-quantitative analysis was performed to determine the various PPs' differential expression in sperm head and tail. These findings contribute to a comprehensive understanding of human sperm PPs, and warrant further research for their clinical and therapeutic significance.

The amyloid-β protein precursor (AβPP) binds several proteins determining metabolism, processing, and the physiological fate of the former. Among these is Fe65, a protein with specific functional significance for AβPP, in particular conferring stability when the latter is dephosphorylated on Thr668. Thus, it follows that phosphatases like protein phosphatase 1 (PP1) are relevant to AβPP processing. Consequently, the identification of AβPP binding proteins, which can be modulated directly or indirectly by PP1, take on added relevance in terms of biological significance. Using the yeast tri-hybrid system and co-immunoprecipitation assays, we describe a novel tri-complex comprising AβPP, Fe65 and PP1. We show that the trimeric complex (AβPP:Fe65:PP1γ) occurs in COS-7 cells, rat hippocampal and cortical primary neurons, and in adult rat hippocampus and cortex. Using overlay assays, we demonstrate that Fe65 is in fact the bridging protein in the complex formed and thus we simultaneously describe another PP1 binding protein. This is singularly important given that PP1 binding proteins determine and confer subcellular localization, as well as substrate specificity, thus regulating the phosphatase activity and subsequent intracellular events. Additionally, we show that this interaction correlates with AβPP Thr668 phosphorylation state, consistent with the role of protein (de)phosphorylation as a key mechanism in regulating cellular events.

Objectives Infertility is a clinical disorder affecting approximately 15% of reproductive-aged couples worldwide. Recently, the influence of oxidative stress (OS) in decreased semen quality has been discussed. OS corresponds to an imbalance between oxidants and antioxidants defenses, present in the organism. High levels of reactive oxygen species (ROS) damage biomolecules present in sperm cells and may lead to the loss of membrane integrity, DNA fragmentation or even to death by apoptosis. This study aimed to evaluate the correlation between human semen clinic parameters and parameters that assessed the presence of OS. Material and methods A total of 32 semen samples, obtained from a randomized group of donors, were included in this study. Basic semen parameters were analyzed according to the WHO's guidelines. The total antioxidant capacity of sperm cells was measured as well as the expression of certain antioxidant proteins, namely superoxide dismutase (SOD) and glutathione peroxidase 4 (GPx4), by colorimetric techniques and immunoblotting, respectively. The effect of ROS in spermatozoa protein oxidation was analyzed by determining the presence of 3-nitrotyrosine and carbonyl groups, by slot blot. Lipid peroxidation was evaluated, by performing the thiobarbituric acid reactive substances (TBARS) assay with colorimetric tests. Results The results indicated that SOD was negatively correlated with viscosity ( p = 0.035), volume ( p = 0.004) and carbonyl groups presence ( p = 0.005). This protein also showed a positive correlation with the presence of tail defects in sperm cells ( p = 0.044). In turn, GPx4 showed a negative correlation with the presence of non-progressive motile spermatozoa ( p = 0.012). TBARS assay revealed a negative correlation with the concentration of sperm cells ( p = 0.000) and the total number of spermatozoa ( p = 0.026), but, in turn, this assay showed a positive correlation with the volume of semen ( p = 0.038). Conclusion It was concluded that the seminal quality is affected by the oxidative balance in sperm cells. The obtained results suggest that lipid peroxidation leads to a reduction in sperm concentration; antioxidant proteins protect the spermatozoa against protein oxidation and contribute to an increased sperm motility and normal semen viscosity. Thus, evaluation of oxidative parameters may be a useful tool for male infertility diagnosis and follow-up of antioxidant treatments.

Objective To investigate the impact of acute lifestyle changes on human sperm functional quality. Methods In the academic festivities week, young and apparently healthy male students who voluntarily submit themselves to acute lifestyle alterations (among the potentially important variations are increase in alcohol, caffeine, and tobacco consumption and circadian rhythm shifts) were used as a model system. Sperm samples were obtained before and after the academic week and compared by traditional semen analysis ( n =54) and also tested for cleaved Poly ADP-ribose polymerase (PARP) protein, an apoptotic marker ( n =35). Results Acute lifestyle changes that occurred during the academic week festivities (the study model) resulted both in a significant reduction in sperm quality, assessed by basic semen analysis (decrease in sperm concentration, total number of spermatozoa, progressive and non-progressive motility and increase in sperm morphological abnormalities) and by an increase in the expression of the apoptotic marker, cleaved PARP, in the ejaculate. Conclusions Acute lifestyle changes have clear deleterious effects on sperm quality. We propose cleaved PARP as a novel molecular marker, valuable for assessing sperm quality in parallel with the basic semen analysis method.

Objective To study the effects of an acute lifestyle change in human semen oxidative stress (OS) by applying seminal parameters and OS markers and to study the feasibility of mid-infrared spectroscopy with Fourier transform infrared spectroscopy (FT-IR) as a complementary tool to evaluate the effects of OS on human sperm samples. Material and methods Sperm samples were collected from healthy male students (n=8) who voluntarily submitted themselves to acute lifestyle changes during academic festivities. The samples were obtained before and after the academic festivities and were compared by basic semen analyses and OS markers, namely with thiobarbituric acid reactive species (TBARS) and total thiol (SH) groups by spectrophotometric assays and carbonyl (CO) groups by slot blot. The samples were also submitted for spectroscopic analysis to evaluate the feasibility of FT-IR coupled with multivariate analysis to calibrate OS biomarkers. Statistical analysis was performed applying paired Wilcoxon tests. Results Acute lifestyle alterations during academic week festivities were associated with a significant decrease in the percentage of normal spermatozoa in the ejaculate (p=0.011) and a decrease in sperm concentration and in semen volume. Regarding OS, acute lifestyle changes promoted a significant increment of TBARS (p=0.018) and an increasing trend in the SH group. With FT-IR and multivariate analysis, it was possible to develop calibration models to the following protein OS biomarkers: SH groups and CO. Conclusions Acute lifestyle changes during academic festivities have negative effects on sperm quality, in both conventional seminal parameters and OS markers. The evaluation of OS biomarkers and FT-IR could improve andrology diagnosis and therapeutic follow-up. published

Production of the amyloid beta (Abeta) peptide via altered metabolism of the amyloid beta-Protein Precursor (AbetaPP) appears to be a key event in the pathology of Alzheimer Disease (AD). Accordingly, altered processing of AbetaPP was observed under conditions of abnormal cellular stress induced by sodium azide in the presence of 2-deoxy-D-glucose (2DG). As previously reported, the production of sAbetaPP (the secreted fragment of AbetaPP) was inhibited. However, our data further suggests that 2DG alone can account for most of the observed effects on AbetaPP processing in COS-1 cells and PC12 cells. It appears that 2DG interferes with the normal glycosylation of AbetaPP and its maturation process, having direct consequences on sAbetaPP production. Interestingly, PMA (phorbol 12-myristate 13-acetate)-induced sAbetaPP production was maintained under the stress conditions used, suggesting that potential non-amyloidogenic AbetaPP processing can still be favoured. This is of potential therapeutic interest, since it indicates that even under adverse stress conditions drugs such as PMA can affect AbetaPP processing, leading to increased sAbetaPP production and a concomitant reduction in Abeta production. However, the induction of sAbetaPP production was not identical when the phosphatase inhibitor OA (okadaic acid) was used. In fact, the typical OA-induced increase in sAbetaPP production could be abolished under specific conditions. This constitutes an interesting precedent for the possible dissociation of the PMA and OA responses in terms of sAbetaPP production. The involvement of protein phosphatases, which are inhibited by OA, inbetaPP processing, was reinforced by the increased co-localization of AbetaPP and PP1alpha (protein phosphatase 1alpha) at the cell surface upon exposure to OA and PMA. Overall, our results support the notion that signal transduction processes may be of particular relevance for our understanding of the molecular basis of AD, and for the design of rational signal transduction therapeutics.

Protein phosphatase 1 (PPP1) is the PPP most ubiquitous and each isoform interact with regulatory subunits that may be responsible for their subcellular localization. We identified PPP1R15B, C1ORF71 as novel regulators and the aim of this study was their further characterization in carcinoma cells. We analysed localization of each regulator in MDA-MB-468 cells and we transfected with constructs that we made with each as a GFP-fusion protein. For PPP1 cellular localization we used specific antibodies for each isoform. We observed the cells under a fluorescent microscope and LSM and we quantified co-localization. We found a high overlap coefficient of both the novel proteins with PPP1alpha and PPP1gamma1. We propose a model in which PPP1 regulator interacts with one or two regulatory subunits that may be used as target for therapeutic strategies.