P–042 Impact of semen parameters, sperm DNA fragmentation and sperm aneuploidy in male infertility

Study question. Should sperm aneuploidies and sperm DNA fragmentation (sDNAfrag) be included as valid tests in the routine investigation of male infertility? Summary answer. Sperm DNA fragmentation was associated with male age, oligozoospermia (OZ), oligoteratozoospermia (OT), astenoteratozoospermia (AT) and oligoastenoteratozoospermia (OAT). Sperm aneuploidies were associated with OT and OAT. What is known already. Semen parameters assist male infertility diagnosis and treatment, but sDNAfrag and aneuploidy analysis could add useful information, as abnormal values compromise fertility. To include these tests in the routine diagnosis it should be determined if behave as informative parameter and add information regarding the fertility status. For that, further studies comparing these tests to semen parameters are needed, since previous results are not consensual. Additionally, standardization of a sDNAfrag cut-off is needed, as different sample sizes and techniques originate distinct results. Also, until a standardization of the protocol is missing, a cut-off value should be defined for each laboratory. Study design, size, duration. A retrospective and prospective investigation was performed, within a 12 years period (April 2007-December 2019). A total of 835 infertile males with a normal karyotype (46,XY) were included. Karyotyping and evaluation of sDNAfrag and sperm aneuploidies were made at a public Genetic unit. All normozoospermic (NZ) patients with a born child and patients whose infertility treatments were done due to female factors were selected from our database and used as controls (60 individuals). Participants/materials, setting, methods. Semen analysis followed WHO–2010 guidelines. sDNAfrag was evaluated using the TUNEL assay. Sperm aneuploidies were detected using FISH (chromosomes 13, 18, 21, X, Y). Several tests were applied: correlations for linear associations between numerical variables, ANOVA for comparisons between means, Dunn-test for post-hoc comparisons. To determine the sDNAfrag cut-off value, the area under the ROC curve, sensitivity and specificity, were calculated, with the Youden-Index used to find a threshold that maximizes both sensitivity and specificity. Main results and the role of chance. Regarding male age, it was observed a positive correlation with sperm concentration, a negative correlation with sperm vitality (VT) and hypoosmolality, and a positive correlation with sDNAfrag. Regarding sDNAfrag, it was observed negative correlation with sperm concentration, total progressive motility (TPM), morphology, VT and hypoosmolality. Regarding sperm aneuploidies, both total sperm aneuploidy and total sperm disomy exhibited a negative association with sperm concentration, TPM and morphology. It was also investigated whose groups of individuals could be indicated for sDNAfrag or sperm aneuploidy testing. The NZ group evidenced significant lower sDNAfrag, total sperm aneuploidy and total sperm disomy in relation to the non-NZ group. In the NZ group, sDNAfrag was significantly lower in relation to the OZ, OT, AT and OAT groups. The NZ group presented significant lower percentages of sperm aneuploidy in relation to the OT and OAT groups, and significant lower percentages of sperm disomy in relation to the OAT group. Additionally, sDNAfrag was positively correlated with total sperm aneuploidy and total sperm disomy. From the present large population, ROC curve analysis allowed estimating a cut-off value of 18.8% for the TUNEL-assay (sDNAfrag), with 0.658 of area under the curve, 53.9% sensitivity and 76.7% specificity. Limitations, reasons for caution. Although presenting a high number of cases and strict controls, the present study was unable to include as controls healthy men with proven fertility. Additionally, the present study did not take into account life-style factors and male associated pathologies besides infertility. Wider implications of the findings: Semen parameters were shown to be negatively correlated with sDNAfrag and sperm aneuploidies. As sDNAfrag testing and sperm aneuploidy testing were associated with semen abnormalities and male age, it is suggested their inclusion in the routine evaluation of infertile men, thus adding important complementary information about the fertility status

Chitosan/virgin-coconut-oil-based system enriched with cubosomes: a 3D drug-delivery approach

Emulsion-based systems that combine natural polymers with vegetable oils have been identified as a promising research avenue for developing structures with potential for biomedical applications. Herein, chitosan (CHT), a natural polymer, and virgin coconut oil (VCO), a resource obtained from coconut kernels, were combined to create an emulsion system. Phytantriol-based cubosomes encapsulating sodium diclofenac, an anti-inflammatory drug, were further dispersed into CHT/VCO- based emulsion. Then, the emulsions were frozen and freeze-dried to produce scaffolds. The scaffolds had a porous structure ranging from 20.4 to 73.4 µm, a high swelling ability (up to 900%) in PBS, and adequate stiffness, notably in the presence of cubosomes. Moreover, a well-sustained release of the entrapped diclofenac in the cubosomes into the CHT/VCO-based system, with an accumulated release of 45 ± 2%, was confirmed in PBS, compared to free diclofenac dispersed (80 ± 4%) into CHT/VCO-based structures. Overall, the present approach opens up new avenues for designing porous biomaterials for drug delivery through a sustainable pathway.The authors especially acknowledge the financial support from the Portuguese FCT (grants CEECIND/01306/2018, SFRH/BPD/93697/2013, and SFRH/BPD/85790/2012). This work was also financially supported by the FCT R&D&I project, with reference PTDC/BII-BIO/31570/2017, and the R&D&I Structured Projects, with reference NORTE-01-0145-FDER-000021. We also acknowledge the financial support from São Paulo Research Foundation (FAPESP) in Brasil through projects 2015/25406-5 and 2021/12071-6, and for the postdoctoral grant to D.G.V., 2019/12665-3. The project 2018/08045-7 is part of a bilateral agreement between FAPESP and the FCT (Portugal), involving the project Nature4Health

A large-scale study of a poultry trading network in Bangladesh: implications for control and surveillance of avian influenza viruses

Since its first report in 2007, avian influenza (AI) has been endemic in Bangladesh. While live poultry marketing is widespread throughout the country and known to influence AI dissemination and persistence, trading patterns have not been described. The aim of this study is to assess poultry trading practices and features of the poultry trading networks which could promote AI spread, and their potential implications for disease control and surveillance. Data on poultry trading practices was collected from 849 poultry traders during a cross-sectional survey in 138 live bird markets (LBMs) across 17 different districts of Bangladesh. The quantity and origins of traded poultry were assessed for each poultry type in surveyed LBMs. The network of contacts between farms and LBMs resulting from commercial movements of live poultry was constructed to assess its connectivity and to identify the key premises influencing it

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 μmol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .−) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ρ0 strain, lacking mitochondrial DNA, was used. The ρ0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 .− precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013

Economic factors influencing zoonotic disease dynamics: demand for poultry meat and seasonal transmission of avian influenza in Vietnam

While climate is often presented as a key factor influencing the seasonality of diseases, the importance of anthropogenic factors is less commonly evaluated. Using a combination of methods-wavelet analysis, economic analysis, statistical and disease transmission modelling-we aimed to explore the influence of climatic and economic factors on the seasonality of H5N1 Highly Pathogenic Avian Influenza in the domestic poultry population of Vietnam. We found that while climatic variables are associated with seasonal variation in the incidence of avian influenza outbreaks in the North of the country, this is not the case in the Centre and the South. In contrast, temporal patterns of H5N1 incidence are similar across these 3 regions: periods of high H5N1 incidence coincide with Lunar New Year festival, occurring in January-February, in the 3 climatic regions for 5 out of the 8 study years. Yet, daily poultry meat consumption drastically increases during Lunar New Year festival throughout the country. To meet this rise in demand, poultry production and trade are expected to peak around the festival period, promoting viral spread, which we demonstrated using a stochastic disease transmission model. This study illustrates the way in which economic factors may influence the dynamics of livestock pathogens

The transcriptome analysis of early morphogenesis in Paracoccidioides brasiliensis mycelium reveals novel and induced genes potentially associated to the dimorphic process

BACKGROUND: Paracoccidioides brasiliensis is a human pathogen with a broad distribution in Latin America. The fungus is thermally dimorphic with two distinct forms corresponding to completely different lifestyles. Upon elevation of the temperature to that of the mammalian body, the fungus adopts a yeast-like form that is exclusively associated with its pathogenic lifestyle. We describe expressed sequence tags (ESTs) analysis to assess the expression profile of the mycelium to yeast transition. To identify P. brasiliensis differentially expressed sequences during conversion we performed a large-scale comparative analysis between P. brasiliensis ESTs identified in the transition transcriptome and databases. RESULTS: Our analysis was based on 1107 ESTs from a transition cDNA library of P. brasiliensis. A total of 639 consensus sequences were assembled. Genes of primary metabolism, energy, protein synthesis and fate, cellular transport, biogenesis of cellular components were represented in the transition cDNA library. A considerable number of genes (7.51%) had not been previously reported for P. brasiliensis in public databases. Gene expression analysis using in silico EST subtraction revealed that numerous genes were more expressed during the transition phase when compared to the mycelial ESTs [1]. Classes of differentially expressed sequences were selected for further analysis including: genes related to the synthesis/remodeling of the cell wall/membrane. Thirty four genes from this family were induced. Ten genes related to signal transduction were increased. Twelve genes encoding putative virulence factors manifested increased expression. The in silico approach was validated by northern blot and semi-quantitative RT-PCR. CONCLUSION: The developmental program of P. brasiliensis is characterized by significant differential positive modulation of the cell wall/membrane related transcripts, and signal transduction proteins, suggesting the related processes important contributors to dimorphism. Also, putative virulence factors are more expressed in the transition process suggesting adaptation to the host of the yeast incoming parasitic phase. Those genes provide ideal candidates for further studies directed at understanding fungal morphogenesis and its regulation

Celecoxib exerts protective effects in the vascular endothelium via COX-2-independent activation of AMPK-CREB-Nrf2 signalling

Although concern remains about the athero-thrombotic risk posed by cyclo-oxygenase (COX)-2-selective inhibitors, recent data implicates rofecoxib, while celecoxib appears equivalent to NSAIDs naproxen and ibuprofen. We investigated the hypothesis that celecoxib activates AMP kinase (AMPK) signalling to enhance vascular endothelial protection. In human arterial and venous endothelial cells (EC), and in contrast to ibuprofen and naproxen, celecoxib induced the protective protein heme oxygenase-1 (HO-1). Celecoxib derivative 2,5-dimethyl-celecoxib (DMC) which lacks COX-2 inhibition also upregulated HO-1, implicating a COX-2-independent mechanism. Celecoxib activated AMPKα(Thr172) and CREB-1(Ser133) phosphorylation leading to Nrf2 nuclear translocation. Importantly, these responses were not reproduced by ibuprofen or naproxen, while AMPKα silencing abrogated celecoxib-mediated CREB and Nrf2 activation. Moreover, celecoxib induced H-ferritin via the same pathway, and increased HO-1 and H-ferritin in the aortic endothelium of mice fed celecoxib (1000 ppm) or control chow. Functionally, celecoxib inhibited TNF-α-induced NF-κB p65(Ser536) phosphorylation by activating AMPK. This attenuated VCAM-1 upregulation via induction of HO-1, a response reproduced by DMC but not ibuprofen or naproxen. Similarly, celecoxib prevented IL-1β-mediated induction of IL-6. Celecoxib enhances vascular protection via AMPK-CREB-Nrf2 signalling, a mechanism which may mitigate cardiovascular risk in patients prescribed celecoxib. Understanding NSAID heterogeneity and COX-2-independent signalling will ultimately lead to safer anti-inflammatory drugs

The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals toxic effects observed.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes" (NORTE-07-0124-FEDER-000028), Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010). Vanessa A. Mesquita gratefully acknowledges the grant from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES). The authors also thank to Doctor Rosane Freitas Schwan to offer the yeast strain and to Doctor Helena M.V.M. Soares, from the Faculty of Engineering of Porto University, for the use of analytical facilities (AAS with flame atomization and AAS with electrothermal atomization)