Extracellular cAMP and MRP4 activity influence in vitro capacitation and fertilizing ability of pig spermatozoa.

cAMP has been reported to be an essential driver of sperm capacitation. In bovine sperm cAMP efflux through multidrug resistance-associated protein 4 (MRP4) has been suggested to maintain intracellular cAMP homeostasis and generate extracellular signaling able to regulate capacitation. The aim of this work was to determine whether extracellular cAMP may influence in vitro pig sperm capacitation and acquisition of fertilizing ability and to evaluate the role of MRP4. In vitro sperm capacitation and gamete coincubation were performed in Brackett and Oliphant's medium (BO) in presence of caffeine (Ctr+) or in BO without caffeine (Ctr-) supplemented with 0, 8, 9, 10 mM cAMP. Despite the percentage of capacitated sperm, assayed by immunolocalization of tyrosine-phosphorylated proteins, was significantly lower in Ctr- compared to Ctr+, it increased supplementing 10 mM cAMP to Ctr- reaching values similar to Ctr+. The absence of caffeine during gamete coincubation reduced the fertilization rate compared to Ctr+, while 10 mM cAMP supplementation to Ctr- increased the fertilization rate reaching values similar to Ctr + . The presence of MRP4 in pig spermatozoa was detected for the first time by western blot and immunohistochemistry assays. To evaluate MRP4 role on pig sperm capacitation, in vitro capacitation and gamete coincubation were performed in Ctr + in presence of MK571, a MRP4 selective inhibitor. MK571 reduced the percentage of capacitated cells and the fertilization rate, while cAMP addition fully reversed MRP4 blockade consequences. Present findings suggest that, under our in vitro conditions, extracellular cAMP and MRP4 activity influence pig sperm capacitating events.

Cell bioenergetics and ATP production of boar spermatozoa.

Cellular metabolism is an important feature of spermatozoa that deserves more insights to be fully understood, in particular in porcine semen physiology. The present study aims to characterize the balance between glycolytic and oxidative metabolism in boar sperm cells. Agilent Seahorse technology was used to assess both oxygen consumption rate (OCR), as an oxidative metabolism index, and extracellular acidification rate (ECAR), as a glycolytic index. Different metabolic parameters were studied on freshly ejaculated sperm cells (identified as day zero sample, d0) and after one day of storage at 17 °C in Androhep extender (d1). Mitochondrial ATP production rate (MitoATP) was higher than the glycolytic ATP production rate (glycoATP) at both d0 and d1 while at d1 the amount of ATP production decreased, in particular, due to OXPHOS reduction. Conversely, glycoATP was not significantly different between d0 and d1. Interestingly, OCR profile showed no different bioenergetic parameters (i.e. ATP turnover, basal or maximal respiration, and spare respiration) between d0 and d1, thus indicating that sperm cell metabolism was reversibly decreased by preservation conditions. Other metabolic parameters showed the same trend, irrespective of the storage time: under stressed conditions (oligomycin plus FCCP), spermatozoa showed an increase in mitochondrial respiration while the metabolic potential of glycolysis did not undergo variations when compared to baseline metabolism. The rate of oxidation of fuel substrates - glucose, fatty acids, and glutamine - showed that sperm reliance on glucose oxidation to maintain baseline respiration was higher than fatty acids or glutamine. Interestingly spermatozoa demonstrated to have a low "capacity" parameter, which indicates that they cannot use only a single fuel substrate to produce energy. This feature of sperm metabolism to be unable to increase oxidation of a particular fuel to compensate for inhibition of alternative fuel pathway(s) was demonstrated by the negative value of "flexibility". Our results showed that ATP production in boar sperm cells relied on mitochondrial oxidative metabolism in freshly ejaculated cells, while, under liquid storage conditions, their oxidative metabolism decreased while the glycolysis remained constant. These results open new fields of research in the preservation techniques of boar sperm cells.

Study of mitochondrial function in thawed bull spermatozoa using selective electron transfer chain inhibitors.

Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the present work was to delineate the mitochondrial activity of bull spermatozoa after incubation with specific inhibitors of the different mitochondrial complexes and evaluate their ROS production. Thawed bull sperm cells (30 × 106 mL-1 in Tyrode's extender) were incubated 1 and 3h at 37 °C with rotenone 5 µM (ROT), complex I inhibitor; dimethyl-malonate 10 mM (DMM), complex II inhibitor; carbonyl cyanide m-chlorophenyl hydrazine 5 µM (CCCP), uncoupling agent; antimycin A 1 µg/mL (ANTI), complex III inhibitor; oligomycin 5 µM (OLIGO), ATP synthase inhibitor, and 0.5% DMSO, vehicle (CTR). Sperm motility and kinematics were assessed by Hamilton Thorn IVOS 12.0. Mitochondrial membrane potential, mitochondrial O2•- production and H2O2 intracellular content were evaluated by BD FACSCalibur flow cytometer, and sperm viability (SYBR-14/PI) and mitochondrial activity (JC-1/SYBR-14/PI) were assessed by epifluorescence microscopy. A multivariate analysis was performed on the results. In addition, sperm kinematic features, registered for each motile spermatozoon, were studied by cluster analysis. The incubation during 1 or 3 h in presence of the inhibitors of mitochondrial functionality only had a minor effect on motility parameters, decreasing the proportion of the SP1 (fast progressive) subpopulation after 3 h of incubation with ROT, ANTI or OLIGO. The percentage of live spermatozoa with active mitochondria was reduced under the effect of ANTI and CCCP both at 1 and 3 h. In conclusion, mitochondrial function is somehow impaired in frozen thawed bull sperm as not all live cells showed active mitochondria. These results support the findings that bull spermatozoa can alternatively rely on oxidative phosphorylation or glycolysis for energy obtainment and that their mitochondria are less affected by ETC inhibitors.

Effects of cryopreservation on the mitochondrial bioenergetics of bovine sperm.

This study evaluated the bioenergetic map of mitochondria metabolism in cryopreserved bovine sperm. The detected oligomycin-sensitive basal respiration supported ATP production; frozen-thawed spermatozoa were found to have a coupling efficiency higher than 0.80. Cell respiration, however, was not stimulated by the protonophoric action of FCCP, as its titration with 1, 2, 4 and 6 µM did not stimulate the uncoupling activity on oxidative phosphorylation as highlighted by unresponsive oxygen consumption. The unusual effect on the stimulation of maximal respiration was not related to fibronectin- or PDL-coated plates used for cellular metabolism analysis. Conversely, irradiation of frozen-thawed bovine sperm with the red light improved mitochondrial parameters. In effect, the maximal respiration of red-light-stimulated sperm in PDL-coated plates was higher than the non-irradiated. In spite of this, red-light irradiation had no impact on membrane integrity and mitochondrial activity evaluated by epifluorescence microscopy.

Assessment of an open-access CASA software for bovine and buffalo sperm motility analysis.

The aim of this study was to verify the reliability of an open access CASA software (BGM) to evaluate the sperm motility of cattle and buffalo, comparing motility and kinematic parameters to those of a commercial one (HTM). Thirty frozen-thawed samples for each species were analyzed with both HTM and BGM, after 1 h of incubation at 37 °C. Sperm viability and mitochondrial membrane potential (MMP) were evaluated through flow cytometric analysis. Agreement of all motility variables between the two systems was assessed. Correlation analysis was performed to identify relationships between motion parameters and sperm viability and MMP. Bland Altman analysis showed good agreement between methods for all motility parameters except for curvilinear velocity (VCL) in cattle, and for average path (VAP), VCL and (amplitude of lateral head displacement) ALH in buffalo, that showed a proportional bias (P > 0.05). In both systems, positive correlation between both viability and high MMP and total and progressive motility of cattle spermatozoa were found; viability and the sperm with high MMP were positive correlated only with VAP, straight-line (VSL), VCL and ALH evaluated with HTM system. Different results were found for buffalo sperm motility parameters, since viability had positive correlations and mitochondrial activity negative ones. Results suggested that motility assessment performed by these two systems are comparable. The discrepancy of VCL, VAP, and ALH could be due to the difference in the algorithms between software. The open-access CASA plug-in is a reliable alternative to the expensive commercial CASA system for sperm motility assessment in cattle and buffalo.

The effects of red LED light on pig sperm function rely upon mitochondrial electron chain activity rather than on a PKC-mediated mechanism.

While irradiation with red LED light has been reported to modulate sperm function in different mammalian species, the mechanisms underlying their response are poorly understood. This work sought to provide new insights into whether this effect relies on a direct action upon mitochondrial electron chain and/or on PKC-linked mechanisms such as those related to opsins. For this purpose, pig semen was light-stimulated for 1, 5 or 10 min in the presence/absence of antimycin A, an inhibitor of the mitochondrial electron chain, or PKC 20-28® (PKCi), a PKC inhibitor. Antimycin A completely blocked the effects of light at all the performed irradiation patterns. This effect was linked to a complete immobility of sperm, which was accompanied with a significant (p < 0.05) drop in several markers of mitochondrial activity, such as JC-1 staining and O2 consumption rate. Antimycin A, however, did not affect intracellular ATP levels, intramitochondrial calcium, total ROS, superoxides or cytochrome C oxidase (CCO) activity. In the case of PKCi, it did also counteract the effects of light on motility, O2 consumption rate and CCO activity, but not to the same extent than that observed for antimycin A. Finally, the effects observed when sperm were co-incubated with antimycin A and PKCi were similar to those observed with antimycin A alone. In conclusion, red LED light acts on sperm function via a direct effect on mitochondrial electron chain. Additionally, light-activated PKC pathways have a supplementary effect to that observed in the electron chain, thereby modulating sperm parameters such as motility and CCO activity.

Assessment of sperm mitochondrial activity by flow cytometry and fluorescent microscopy: a comparative study of mitochondrial fluorescent probes in bovine spermatozoa.

CONTEXT: While conventional semen analysis is a simple, time-saving, and economical means to evaluate sperm quality, it leaves biochemical and metabolic characteristics of spermatozoa aside. To address this issue, the use of fluorescent probes assessing functional sperm parameters, such as JC-1, DiOC6 (3) and MitoTracker, has increased over the last decades. Apparently contradictory observations have nevertheless fostered an ongoing debate on their sensitivity and ability to evaluate the mitochondrial membrane potential (MMP) of sperm cells, thus warranting a re-examination of these probes. AIMS: The present study aims to elucidate the suitability and sensitivity of each probe to evaluate the MMP of bovine spermatozoa by flow cytometry. METHODS: Cryopreserved spermatozoa from ten bulls were thawed, stained with JC-1/SYTOXRed, DiOC6 (3)/propidium iodide (PI) or MitoTracker Deep Red (MTDR)/PI, and evaluated with flow cytometry and fluorescence microscopy. KEY RESULTS: DiOC6 (3), JC-1 and MTDR can be simultaneously co-stained with a viability marker. The results of the present study support the ability of DiOC6 (3)/PI and JC-1/SYTOXRed, but not that of MTDR/PI, to monitor the MMP of spermatozoa. CONCLUSIONS: JC-1/SYTOXRed assessed by flow cytometry was found to be the most sensitive and robust fluorescent probe to assess MMP. Moreover, DiOC6 (3)/PI could be a suitable alternative when the flow cytometer is not equipped with a red laser and/or an adequate optical filter. IMPLICATIONS: Both DiOC6 (3) and JC-1, but not MTDR, could be used as probes to assess the mitochondrial membrane potential of bovine spermatozoa.

Impact of glyphosate and its formulation Roundup® on stallion spermatozoa.

The growing and widespread use of glyphosate-based herbicides (GBHs) has raised an intense public debate about the impact of environmental contamination on animal and human health, including male fertility. The aim of this study was to deepen the impact of glyphosate (Gly) and GBHs on mammalian sperm investigating the effect of in vitro exposure of stallion spermatozoa to Gly and to its commercial formulation Roundup® (R). Spermatozoa were incubated at 37 °C with different Gly or R concentrations (from 0.5 to 720 µg/mL Gly or R at the same Gly-equivalent concentrations). After 1 h of incubation motility, viability, acrosome integrity, mitochondrial activity and ROS production were assessed. Gly, at all the concentrations tested, did not induce any detrimental impact on the sperm quality parameters evaluated. Conversely, R starting from 360 µg/mL (Gly-equivalent dose) significantly (P < 0.05) decreased total and progressive motility, viability, acrosome integrity, mitochondrial activity and the percentage of live spermatozoa with intact mitochondria not producing ROS. Our results indicate that the commercial formulation R is more toxic than its active molecule Gly and that the negative impact on stallion sperm motility might be likely due to a detrimental effect mainly at membrane and mitochondrial level and, at least in part, to redox unbalance. Moreover, based on the data obtained, it can be hypothesized a species-specificity in sperm sensitivity to Gly and GBHs as horse spermatozoa were negatively influenced at higher concentrations of R compared to those reported in literature to be toxic for human and swine male germ cells.

Exogenous Albumin Is Crucial for Pig Sperm to Elicit In Vitro Capacitation Whereas Bicarbonate Only Modulates Its Efficiency.

This work sought to address whether the presence of exogenous bicarbonate is required for pig sperm to elicit in vitro capacitation and further progesterone-induced acrosome exocytosis. For this purpose, sperm were either incubated in a standard in vitro capacitation medium or a similar medium with different concentrations of bicarbonate (either 0 mM, 5 mM, 15 mM or 38 mM) and BSA (either 0 mg/mL or 5 mg/mL). The achievement of in vitro capacitation and progesterone-induced acrosomal exocytosis was tested through the analysis of sperm motility, plasma membrane integrity and lipid disorder, acrosome exocytosis, intracellular calcium levels, mitochondria membrane potential, O2 consumption rate and the activities of both glycogen synthase kinase 3 alpha (GSK3α) and protein kinase A (PKA). While sperm incubated in media without BSA or BSA/bicarbonate, they did not achieve in vitro capacitation; those incubated in media with BSA achieved the capacitated status under any bicarbonate concentration, even when bicarbonate was absent. Moreover, there were differences related to the concentration of bicarbonate, since sperm incubated in media with BSA and with no bicarbonate or 5 mM bicarbonate showed lower overall efficiency in achieving in vitro capacitation than those incubated in the presence of BSA and 15 mM or 38 mM bicarbonate. Additionally, at the end of the experiment, sperm incubated in the presence of BSA and 38 mM bicarbonate showed significantly (p < 0.05) lower values of motility and plasma membrane integrity than those incubated in media with BSA and lower concentrations of bicarbonate. In conclusion, BSA is instrumental for pig sperm to elicit in vitro capacitation and trigger the subsequent progesterone-induced acrosome exocytosis. Furthermore, while exogenous bicarbonate does not seem to be essential to launch sperm capacitation, it does modulate its efficiency.

The role of endogenous antioxidants in male animal fertility.

Mammalian semen is a physiological fluid composed of a cellular fraction (spermatozoa), and a liquid fraction (seminal plasma). Once delivered to the female genital tract, spermatozoa should be able to capacitate; a process which involves a plethora of biochemical and physiological changes required to fertilize the oocyte. Sperm production (spermatogenesis) occurs in the testes, whereby pluripotent spermatogonia differentiate to form the most morphologically specialized cells in the body. Further maturation of spermatozoa occurs in the epididymis, where they are stored prior to ejaculation. During this whole process, spermatozoa are exposed to different environments and cellular processes which may expose them to substantial levels of oxidative stress. To avoid damage associated with the unchecked production of reactive oxygen species (ROS), both spermatozoa, and the parts of the male genital tract in which they reside, are furnished with a suite of antioxidant molecules which are able to provide protection to these cells, thereby increasing their chance of being able to fertilize the oocyte and deliver an intact paternal genome to the future offspring. However, there are a host of reasons why these antioxidant systems may fail, including nutritional deficiencies, genetics, and disease states, and in these situations, a reduction or abolition of fertilizing capacity may result. This review paper focuses on the endogenous antioxidant defences available to spermatozoa during spermatogenesis and sperm maturation, the site of their production and their physiological role. Furthermore, we revised the causes and effects of antioxidant deficiencies (congenital or acquired during the animal's adulthood) on reproductive function in different animal species.

Red LED Light Acts on the Mitochondrial Electron Chain of Donkey Sperm and Its Effects Depend on the Time of Exposure to Light.

This work aimed to investigate how stimulation of donkey sperm with red LED light affects mitochondrial function. For this purpose, freshly diluted donkey semen was stimulated with red light for 1, 5, and 10 min, in the presence or absence of oligomycin A (Omy A), a specific inhibitor of mitochondrial ATP synthase, or FCCP, a specific disruptor of mitochondrial electron chain. The results obtained in the present study indicated that the effects of red LED light on fresh donkey sperm function are related to changes in mitochondria function. In effect, irradiation of donkey sperm resulted in an increase in mitochondrial membrane potential (MMP), the activity of cytochrome C oxidase and the rate of oxygen consumption. In addition, in the absence of oligomycin A and FCCP, light-stimulation augmented the average path velocity (VAP) and modified the structure of motile sperm subpopulations, increasing the fastest and most linear subpopulation. In contrast, the presence of either Omy A or FCCP abolished the aforementioned effects. Interestingly, our results also showed that the effects of red light depend on the exposure time applied, as indicated by the observed differences between irradiation protocols. In conclusion, our results suggest that exposing fresh donkey sperm to red light modulates the function of their mitochondria through affecting the activity of the electron chain. However, the extent of this effect depends on the irradiation pattern and does not exclude the existence of other mechanisms, such as those related to thermotaxis.

Red LED Light Acts on the Mitochondrial Electron Chain of Mammalian Sperm via Light-Time Exposure-Dependent Mechanisms.

This work analyzes the effects of red LED light on mammalian sperm mitochondrial function, using the pig as an animal model. Liquid-stored pig semen was stimulated with red-light for 1, 5 and 10 min in the presence or absence of oligomycin A, a specific inhibitor of mitochondrial ATP synthase, or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific disruptor of mitochondrial electron chain. Whereas exposure for 1 and 5 min significantly (p < 0.05) decreased total motility and intracellular ATP levels, irradiation for 10 min induced the opposite effect. Oligomycin A abolished the light-effects on intracellular ATP levels, O2 consumption and mitochondrial membrane potential, whereas compared to non-irradiated samples, FCCP significantly (p < 0.05) increased O2 consumption when sperm were irradiated for 1 min. Both oligomycin A and FCCP significantly (p < 0.05) decreased total motility. Red-light increased cytochrome c oxidase activity with a maximal effect after 5 min of irradiation, which was abolished by both oligomycin A and FCCP. In conclusion, red-light modulates sperm mitochondrial function via electron chain activity in an exposition, time-dependent manner.

Medium-term effects of the diluted pig semen irradiation with red LED light on the integrity of nucleoprotein structure and resilience to withstand thermal stress.

This study sought to evaluate the effects of irradiating pig seminal doses with red LED light irradiation on their quality and longevity over liquid-storage at 17 °C. For this purpose, boar ejaculates were diluted in a commercial extender at a final concentration of 3 × 107 sperm/mL and stored at 17 °C for 96 h. Upon arrival to our laboratory (5-6 h within collection), 1.5 mL-aliquots were subjected to irradiation with a temperature-controlled red light-emitting diode (LED) for 1 min, 5 min or 10 min. Controls consisted of non-irradiated spermatozoa. Aliquots were then stored at 17 °C for 96 h, and plasma membrane and acrosome integrity, motility and free cysteine radicals of sperm head proteins were evaluated every 24 h. In addition, the sperm resilience to withstand thermal stress following irradiation was evaluated at 24 h, 48 h, 72 h and 96 h by incubating stored seminal doses at 37 °C for 120 min. In our experimental conditions, light-stimulation for 5 min and 10 min counteracted the decrease in thermal stress observed in non-irradiated samples during the first 48 h of storage. Moreover, all irradiation protocols counteracted the decrease in percentages of spermatozoa with altered acrosomes observed in non-irradiated samples after 72 h of storage. The effects of light-stimulation upon sperm motility parameters were less consistent. While liquid-storage also led to an increase in the free cysteine levels of sperm head proteins, this increment was partially mitigated through light-stimulation for 5 min and 10 min. Our results suggest that effects linked with red LED light irradiation would be consistently maintained in our experimental conditions for the first 48 h. Finally, the maintenance of light effect appears to depend upon the specific experimental design, the analyzed sperm parameters and the utilized irradiation patterns.

El derecho a la salud y su impacto sanitario en Sudamérica. 2014-2015 / The right to health and its impact on health in South America. 2014-2015

Resumen Introducción. El derecho a la salud es reconocido constitucionalmente en Sudamérica; sin embargo, se desconoce si el respeto a este derecho tiene relación directa con los indicadores básicos de salud en la población. Objetivo. Hacer una aproximación al derecho a la salud en los países de Sudamérica. Materiales y métodos. A partir de la indagación del derecho a la salud en el marco constitucional y tratados internacionales adoptados en cada país, relacionándolo con algunos indicadores básicos en salud y teniendo en cuenta los conceptos de derecho constitucional y salud como servicio público, se realizó una revisión narrativa de tipo cualitativo de fuente secundaria indexada sobre las categorías "Derecho en salud dentro del marco legal en cada país" e "Indicadores Básicos en Salud". Resultados. Los indicares básicos en salud comparados en los países de estudio son un reflejo de las políticas adoptadas que muestran su relación con el derecho a la salud en su contexto legislativo. Conclusiones. Las tendencias de los comportamientos sanitarios permitieron asociar los impactos encontrados con un avance positivo en el ejercicio del derecho sanitario.

Abstract Introduction: The right to health is constitutionally recognized in South America; however, it is not known whether respect for this right is directly related to the basic health indicators of the population. Objective: To approach to the right to health in South American countries. Materials and methods: A qualitative review of secondary sources was carried out based on a research on the right to health as addressed in the constitutions and international treaties adopted by each country. This was done associating the right to health with some basic health indicators and taking into account the concepts of constitutional right and health as a public service. The following concepts were considered for conducting the review: "Right to health within the legal framework in each country" and "Basic Indicators on Health". Results: The basic health indicators compared in the study countries reflect the adopted policies by country that are associated with the right to health in their legislative context. Conclusions: Trends in health behavior allowed associating the impacts found as a positive progress regarding the practice of health law.

"In vitro" capacitation and further progesterone-induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa.

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone-induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono- and bi-dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono-dimensional Western blot in non-capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi-dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non-capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.

Red-light stimulation of boar semen prior to artificial insemination improves field fertility in farms: A worldwide survey.

A survey of in vivo fertility data from 31 pig farms distributed worldwide was conducted to determine whether stimulating boar semen with LED-based red light increases its reproductive performance following artificial insemination (AI). Red-light stimulation with MaXipig® was found to increase farrowing rates (mean ± SEM, control: 87.2% ± 0.4% vs. light stimulation 90.3% ± 0.5%) and the number of both total and live newborn piglets. Red-light stimulation increased farrowing rates in 27 farms, with an increase ranging from 0.2% to 9.1%. Similar results were observed in litter sizes. Suboptimal management after AI was suggested in those farms with no response to red-light stimulation. Our results indicate that a routine use of red-light stimulation of boar semen can have a positive effect on the reproductive performance. However, the effectiveness of this system appears to highly rely upon proper management of pig farms.

Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes.

Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica.

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a ß-enriched structure (ppEhCP-B9(ß1)) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a ß-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α+ß structure, similar to that of papain but without proteolytic activity (ppEhCP-B9(α+ß1)). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a ß-sheet rich structure (ppEhCP-B9(ß2)) that slowly transforms into a different type of α+ß conformation that exhibited proteolytic activity (ppEhCP-B9(α+ß2)) suggesting that enzymatic activity is gained as slow transformation occurs.

Dual-colour CISH is a reliable alternative to FISH for assessment of topoisomerase 2-alpha amplification in breast carcinomas.

Anthracyclines are among the most powerful antineoplastic drugs available for breast cancer treatment. Although HER2 amplification has been postulated to predict anthracycline benefit, numerous reports have demonstrated that HER2/TOP2A co-amplification is the clinically useful predictive marker of response to anthracyclines. The standard technique to evaluate gene status for target therapy selection is fluorescence in situ hybridization (FISH), but this technique has some disadvantages. Dual-colour chromogenic in situ hybridization (CISH) is an extension of the FISH protocol that allows bright-field microscopy and thus represents a user-friendly alternative to FISH. In order to evaluate whether dual-colour CISH is a reliable alternative to FISH in determining TOP2A gene amplification and to determine the frequency with which TOP2A and HER2 were co-amplified, we analysed 100 invasive breast cancer specimens (70 consecutive and 30 HER2-amplified samples) using tissue microarrays. Thus, a 99 % agreement was found between TOP2A status determined by dual-colour CISH and FISH, as well as a high degree of correlation in TOP2A ratios using both techniques. TOP2A gene amplification was present in 8.6 % of the 70 consecutive samples studied, all of which were HER2-amplified. Co-amplification of TOP2A was observed in 46.5 % of the additional 30 HER2-amplified samples (no TOP2A amplification was seen in non-amplified HER2 samples). We conclude that dual-colour CISH represents an excellent alternative to FISH for determination of TOP2A gene status in invasive breast cancer. Our results showing TOP2A amplification only in HER2-amplified cases also add to the evidence that TOP2A determination should be restricted to those cases.

Preeclampsia leve: cuidados en casa / Home care in mild pre - eclampsia

El presente estudio tiene como objetivo detectar las necesidades de cuidado percibidas por gestantes con diagnóstico de preeclampsia leve que manejan su patología en casa. Se realizó una investigación cualitativa descriptiva exploratoria entrevistando mujeres gestantes con este diagnóstico atendido en el Hospital de San José de Bogotá DC. Para determinar el número que se necesitaba se tuvo en cuenta el principio de saturación, el cual se cumplió con ocho pacientes. El análisis de los datos fue hecho por edición y fundamentado en la teoría de suplencia o ayuda a través del modelo de necesidades humanas para la vida y la salud, como núcleo en la acción de enfermería de Virginia Henderson, en el cual se encontró que estas enfermas requieren un estricto cuidado en el manejo de cuatro necesidades básicas que son fisiológicas, de seguridad, de amor y pertenencia, y de autorrealización. En cada una de ellas las más relevantes fueron: toma de tensión arterial, tranquilidad, afecto de su familia y más información acerca de la patología que padecen. La realidad vista en la presente investigación genera gran expectativa frente a la importancia de la participación del enfermero profesional en el cuidado de la gestante con la patología mencionada, tanto en el área de promoción y prevención como en el diagnóstico precoz, tratamiento oportuno y limitación del daño.

The purpose of this study is to detect the perceptions on required care of women with mild pre-eclampsia who handle their condition at home. A qualitative, descriptive, exploratory research was undertaken surveying women with this diagnosis seen at the San José Hospital in Bogotá D.C. The saturation principle was used to determine the required number, which was met by eight patients. Data analysis was performed by edition and was based on the substitution theory or help through human necessities for life and health, as the fundamentals of the Virginia Henderson theories, which found that this condition requires strict management of four essential necessities, that is, physiological needs, love assurance, emotional bonds (attachment) and self-realization. The most relevant in each of them were: taking of blood pressure, tranquility, receiving love and reassurance from their families and being more informed about their condition. The actual results of this research cause great expectations as to the importance of the nursing professional in the care of these patients, especially in the promotion of health and prevention of disease as well as on timely diagnosis and treatment and limitation of harm.