Effect of the addition of antifreeze protein type I on the quality of post-thawed domestic cat epididymal sperm.

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen-thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 µg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

Occurrence and risk assessment of pesticides in a Mediterranean Basin with strong agricultural pressure (Guadiana Basin: Southern of Portugal).

The study aimed to assess the occurrence and the environmental risk of a group of 51 selected pesticides in the Guadiana Basin (a biodiversity hotspot, in the Mediterranean). The most abundant pesticides were bentazone and 2,4-D, while terbuthylazine together with terbutryn constituted the most ubiquitous pesticides. Eighteen out of the 38 pesticides detected are no longer approved in Europe, and 5 of them are included in the list of priority substances. The risk assessment showed that azinphos ethyl, diflufenican, irganol, imidacloprid, and oxadiazon occurred occasionally, but always in concentrations above their respective ecotoxicological threshold value. Contrary, bentazone, terbuthylazine, and terbutryn presented a high risk in most of the sampled locations and periods. The site-specific risk assessment showed a spatial and temporal pattern, with a higher risk occurring mainly in intermittent streams, in the drought period. The presence of pesticides banned from the EU market since 2009 showed the importance of improving the monitoring process, to identify the main sources of pollution and the fate of these emerging compounds. The results showed the need of implementing actions to improve the sustainable use of pesticides in agricultural areas, working with farmers and management entities to reduce the contamination of aquatic ecosystems. Transboundary water governance is also required to solve potential transboundary contamination problems.

Repercusiones en la salud mental del paciente anciano tras padecer COVID-19: trastorno de estrés postraumático. A propósito de un caso / Impacts on the mental health of the elderly patient after COVID-19: Post-traumatic stress disorder. About clinical case

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Microangiopatía trombótica en el anciano. A propósito de un caso / Thrombotic microangiophaty in elderly. Case report

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Transcriptional profiles of Haloferax mediterranei based on nitrogen availability.

The haloarchaeon Haloferax mediterranei is able to grow in the presence of different inorganic and organic nitrogen sources by means of the assimilatory pathway under aerobic conditions. In order to identify genes of potential importance in nitrogen metabolism and its regulation in the halophilic microorganism, we have analysed its global gene expression in three culture media with different nitrogen sources: (a) cells were grown stationary and exponentially in ammonium, (b) cells were grown exponentially in nitrate, and (c) cells were shifted to nitrogen starvation conditions. The main differences in the transcriptional profiles have been identified between the cultures with ammonium as nitrogen source and the cultures with nitrate or nitrogen starvation, supporting previous results which indicate the absence of ammonium as the factor responsible for the expression of genes involved in nitrate assimilation pathway. The results have also permitted the identification of transcriptional regulators and changes in metabolic pathways related to the catabolism and anabolism of amino acids or nucleotides. The microarray data was validated by real-time quantitative PCR on 4 selected genes involved in nitrogen metabolism. This work represents the first transcriptional profiles study related to nitrogen assimilation metabolism in extreme halophilic microorganisms using microarray technology.

Evaluation of cytotoxicity and genotoxicity of Acacia aroma leaf extracts.

Acacia aroma, native plant from San Luis, Argentina, is commonly used as antiseptic and for healing of wounds. The present study was conducted to investigate the in vitro cytotoxicity and genotoxicity of hot aqueous extract (HAE) and ethanolic extract (EE) of A. aroma. The cytotoxic activity was assayed by neutral red uptake assay on Vero cell. Cell treatment with a range from 100 to 5000 µg/mL of HAE and EE showed that 500 µg/mL and 100 µg/mL were the maximum noncytotoxic concentrations, respectively. The CC50 was 658 µg/mL for EE and 1020 µg/mL for HAE. The genotoxicity was tested by the single-cell gel electrophoresis comet assay. The results obtained in the evaluation of DNA cellular damage exposed to varied concentrations of the HAE showed no significant genotoxic effect at range of 1-20 mg/mL. The EE at 20 mg/mL showed moderate genotoxic effect related to the increase of the DNA percentage contained in tail of the comet; DNA was classified in category 2. At concentrations below 5 mg/mL, the results of cytotoxicity and genotoxicity of aqueous and ethanolic extracts of Acacia aroma guarantee the safety at cell and genomic level. However further studies are needed for longer periods including animal models to confirm the findings.

Effect of cryopreservation and single layer centrifugation on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test.

The aims of this study were: 1) to assess the effect of freezing and thawing on dog sperm DNA fragmentation index (sDFI) using the sperm chromatin dispersion test (SCDt); and 2) to determine whether or not the sperm selection by single layer centrifugation (SLC) using Androcoll-C improves sperm DNA longevity in SLC-selected frozen-thawed dog semen samples. Semen samples were collected from 4 dogs using digital manipulation. After collection, ejaculates were pooled and cryopreserved following a standard protocol. Sperm motility and morphology were assessed before freezing and after thawing as a control for the cryopreservation method used. In experiment 1, sDFI was analyzed immediately before freezing and after thawing (baseline values), showing no significant differences between fresh and frozen-thawed semen samples. In experiment 2, frozen-thawed semen samples were processed or not by SLC using Androcoll-C and longevity of DNA were assessed in terms of sDFI after 24h of in vitro incubation at physiological temperature (38°C). The results showed low values of sDFI in SLC-selected semen in comparison to unselected samples. In conclusion, no effect of cryopreservation was observed on baseline values of dog sperm DNA fragmentation. Additionally, SLC-selection using Androcoll-C improved longevity of frozen-thawed sperm DNA assessed by the SCDt.

A tissue-dependent hypothesis of dental caries.

Current understanding of dental caries considers this disease a demineralization of the tooth tissues due to the acid produced by sugar-fermenting microorganisms. Thus, caries is considered a diet- and pH-dependent process. We present here the first metagenomic analysis of the bacterial communities present at different stages of caries development, with the aim of determining whether the bacterial composition and biochemical profile are specific to the tissue affected. The data show that microbial composition at the initial, enamel-affecting stage of caries is significantly different from that found at subsequent stages, as well as from dental plaque of sound tooth surfaces. Although the relative proportion of Streptococcus mutans increased from 0.12% in dental plaque to 0.72% in enamel caries, Streptococcus mitis and Streptococcus sanguinis were the dominant streptococci in these lesions. The functional profile of caries-associated bacterial communities indicates that genes involved in acid stress tolerance and dietary sugar fermentation are overrepresented only at the initial stage (enamel caries), whereas other genes coding for osmotic stress tolerance as well as collagenases and other proteases enabling dentin degradation are significantly overrepresented in dentin cavities. The results support a scenario in which pH and diet are determinants of the disease during the degradation of enamel, but in dentin caries lesions not only acidogenic but also proteolytic bacteria are involved. We propose that caries disease is a process of varying etiology, in which acid-producing bacteria are the vehicle to penetrate enamel and allow dentin degrading microorganisms to expand the cavity.

Use of single-layer centrifugation with Androcoll-C to enhance sperm quality in frozen-thawed dog semen.

The aim of this study was to investigate whether single-layer centrifugation (SLC) with Androcoll-C could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from five dogs was collected and cryopreserved following a standard protocol. After thawing, the semen samples were divided in two aliquots, one of which was used as a control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining), viability (dual staining with propidium iodine/acridine orange), and acrosome integrity (dual staining with propidium iodine/isothiocyanate-labeled peanut [Arachis hypogaea] agglutinin) were performed on aliquots of fresh semen, frozen-thawed control samples, and frozen-thawed SLC-treated preparations. A multivariate clustering procedure separated 57,577 motile spermatozoa into three subpopulations (sP): sP1 consisted of poorly active and nonprogressive spermatozoa (48.8%), sP2 consisted of moderately slow but progressive spermatozoa (13.3%), and sP3 consisted of highly active and/or progressive spermatozoa (37.8%). SLC with Androcoll-C yielded sperm suspensions with improved motility, viability, and acrosome integrity (P < 0.01). The frozen-thawed SLC-treated samples were enriched in sP3, representing 38.5% of the sperm population. Likewise, sP2 was more frequently observed after SLC, but not significantly so. From these results, we concluded that for dog semen samples selected by SLC with Androcoll-C after thawing, the sperm quality parameters, including motility patterns, are better than in frozen-thawed control samples.

Single-layer centrifugation through PureSperm® 80 selects improved quality spermatozoa from frozen-thawed dog semen.

The aim of this study was to investigate whether single-layer centrifugation (SLC) with PureSperm® 80 could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were divided into two aliquots: one of them was used as control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, frozen-thawed control and frozen-thawed SLC treated samples. A multivariate clustering procedure separated 26,051 motile spermatozoa into three subpopulations (sP): sP1 consisting of highly active but non-progressive spermatozoa (40.3%), sP2 consisting of spermatozoa with high velocity and progressive motility (30.0%), and sP3 consisting of poorly active and non-progressive spermatozoa (29.7%). SLC with PureSperm® 80 yielded sperm suspensions with improved motility, morphology, viability and acrosome integrity (P<0.001). The frozen-thawed SLC treated samples were enriched in sP2, reaching a proportion of 44.1% of the present spermatozoa. From these results, we concluded that SLC with PureSperm® 80 may be an alternative and successful method for improving the quality of frozen-thawed dog spermatozoa. Moreover, sP2 (high-speed and progressive spermatozoa) was more frequently observed after SLC. Finally, this study also demonstrated that the general motile sperm structure present in dogs remained constant despite the effect caused by either cryopreservation or separation by SLC through PureSperm® 80.

Identifying a healthy oral microbiome through metagenomics.

We present the results of an exploratory study of the bacterial communities from the human oral cavity showing the advantages of pyrosequencing complex samples. Over 1.6 million reads from the metagenomes of eight dental plaque samples were taxonomically assigned through a binning procedure. We performed clustering analysis to discern if there were associations between non-caries and caries conditions in the community composition. Our results show a given bacterial consortium associated with cariogenic and non-cariogenic conditions, in agreement with the existence of a healthy oral microbiome and giving support to the idea of dental caries being a polymicrobial disease. The data are coherent with those previously reported in the literature by 16S rRNA amplification, thus giving the chance to link gene functions with taxonomy in further studies involving larger sample numbers.

Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm(®) gradient.

The aims of the present study were to: (1) determine if discrete motile sperm subpopulations exist and their incidence in fresh dog ejaculates, (2) evaluate the effects of cryopreservation on the distribution of spermatozoa within the different subpopulations, and (3) determine the effect of the discontinuous PureSperm(®) gradient on the sperm subpopulation structure of frozen-thawed dog spermatozoa. Semen from 5 dogs were collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm(®). Sperm motility (assessed by computerized-assisted semen analysis, CASA) was assessed before freezing, just after thawing and after preparation on the PureSperm(®) gradients. Cryopreservation had a significant (P<0.001) effect on CASA-derived parameters. PureSperm(®) centrifugation yielded sperm suspensions with improved motility (P<0.01). A multivariate clustering procedure separated 19414 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (20.97%), Subpopulation 2 consisting of slow and low-linear spermatozoa (18.24%), Subpopulation 3 consisting of highly active but non-progressive spermatozoa (20.75%), and Subpopulation 4 consisting of high speed and progressive spermatozoa (40.03%). Although, cryopreservation had a significant (P<0.001) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after freezing and thawing. The selected sperm samples was enrich in Subpopulation 4, reaching a proportion of 31.9% of the present spermatozoa, in contrast with the unselected sperm samples, where this sperm subpopulation accounted for 24.9% of the total. From these results, we concluded that four well-defined motile sperm subpopulations were present either in fresh semen, in unselected sperm samples or in selected preparations from dogs. The discontinuous PureSperm(®) gradient is a simple method to improve the quality of canine frozen-thawed semen samples, since Subpopulation 4 (high-speed and progressive spermatozoa) was more frequently observed after preparation on the gradient. Finally, this study also demonstrated that the general motile sperm structure present in dog remains constant despite the effect caused by either cryopreservation or separation on PureSperm(®) gradient.

Centrifugation on PureSperm(®) density-gradient improved quality of spermatozoa from frozen-thawed dog semen.

The main objective of this study was to investigate if centrifugation through PureSperm(®) density-gradient can improve the post-thaw semen quality of dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm(®). Assessments of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of Propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, unselected samples and selected preparations. Cryopreservation had a significant (P < 0.001) effect on all studied semen parameters. PureSperm(®) centrifugation yielded sperm suspensions with improved motility and viability (P < 0.001). The washing step significantly reduced (P < 0.001) all of the kinematics parameters evaluated as well as reduced the proportion of viable spermatozoa with intact acrosomes (P < 0.05). We concluded that PureSperm(®) centrifugation is a successful method for improving the quality of frozen-thawed dog spermatozoa. However, washing after density-gradient centrifugation dramatically reduces the post-thaw semen quality, indicating that the inclusion of such a washing step is unnecessary.

Comunicación de la noticia del nacimiento de un niño o niña con Síndrome de Down: el efecto de una predicción desalentadora / Communication from the news of the birth of a child with Down Syndrome: the effect of a discouraging prediction

OBJETIVO: conocer la forma como los trabajadores de la salud comunican la noticia del nacimiento de un niño o niña con Síndrome de Down (SD). METODOLOGIA: estudio cualitativo, de corte etnográfico. Participaron 20 madres y una abuela, se realizaron entrevistas semiestructuradas y observaciones en el campo. Resultados: la noticia del nacimiento del niño o niña con SD se da en forma fría y deshumanizada, deSDe una mirada de la discapacidad sin solución. DISCUSION: las madres perciben, en los trabajadores de la salud, desinterés por sus circunstancias. La mayoría les trasmiten la idea de un niño o niña anormal y con una discapacidad que generará muchos problemas. La manera como reciben la noticia no favorece el encuentro con el hijo, y pone en incertidumbre la visión de futuro de madre-niño. CONCLUSIONES: el personal de salud carece de preparación académica para dar la noticia del nacimiento de un niño o niña con SD y de la sensibilidad para apoyar a la madre en el encuentro con su hijo. RECOMENDACIONES: preparar a los estudiantes de las áreas de la salud para dar los diferentes tipos de noticia dentro de un ambiente humanizante en el cual tenga tanto valor la dimensión biológica como la social y psicoafectiva.

OBJECTIVE: to know how health workers give the news of the birth of a child with Down syndrome (DS). METHODOLOGY: qualitative, ethnographic. Involved 20 mothers and one grandmother were semistructured interviews and field observations. RESULTS: the news of the birth of the child with ds occurs in a cold, dehumanized, from an unresolved disability. DISCUSSIONS: mothers perceived health workers disregard for their circumstances. Most will convey the idea of an abnormal child with a disability that will create many problems. The way that mother receive the news is not conducive to meeting with the child, and puts in uncertainty the future vision of mother-child relationship. CONCLUSIONS: the health academically untrained to break the news of the birth of a child with ds and sensitivity to support her in meeting her son. Recommendations: to prepare students in the areas of health to provide different types of news in humanizing environment in which it has much value as the biological and social psicoafective.

Antibacterial activity of extracts of Acacia aroma against methicillin-resistant and methicillin-sensitive Staphylococcus

Antibacterial activity of organic and aqueous extracts of Acacia aroma was evaluated against methicillin-resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus epidermidis. Inhibition of bacterial growth was determined using agar diffusion and bioautographic methods. Among all assayed organic extracts only ethanolic and ethyl acetate extracts presented highest activities against all tested Staphylococcus strains with minimal inhibitory concentration (MIC) values ranging from 2.5 to 10 mg/ml and from 2.5 to 5 mg/ml respectively. The aqueous extracts show little antibacterial activity against Staphylococcus strains. The bioautography assay demonstrated well-defined growth inhibition zones against S. aureus in correspondence with flavonoids and saponins. A. aroma would be an interesting topic for further study and possibly for an alternative treatment for skin infections.