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We have evaluated the Sysmex UF-5000 cytometer use in microbiology for the screening of negative urines, looking for cut-off points to detect bacteria and leukocytes. The number of processed urines was 3569, the highest to date in these studies. The best general cut-off point has been 100 bact/µl, giving an area under the ROC curve of 0.868, a sensitivity of 96%, a specificity of 50%, 1.17% of false negatives, and saving 40% of cultures. The PPV and NPV have been 35.5 and 95.4 respectively. The leukocyte count has not been useful. Finally, we have evaluated urine screening usefulness, concluding that in laboratories such as ours (284 urines/working day) or smaller, it is not cost-effective.

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The two-step protocol (2 S) is currently used for boar semen cryopreservation. In this method, the cryoprotectant penetrant agents (CPAs) are added at 5 °C to reduce the toxicity of CPAs. An alternative is the one-step protocol (1 S), which is easier, cheaper, and reduces the necessity of equipment, but could increase the toxicity of CPAs. Currently, there are no studies that compared both protocols for boar semen cryopreservation. This experiment aimed to study the effect of cryopreservation protocol (1 S vs 2 S) on boar spermatozoa. In the one-step protocol, after centrifugation, the spermatozoa pellet was resuspended at 17 °C in the extender containing CPAs to achieve a concentration of 1 × 109 spermatozoa/mL and then submitted to cryopreservation. For the two-step protocol, the sperm pellet was resuspended in fraction A at 17 °C to achieve a concentration of 1.5 × 109 spermatozoa/ mL, and then allowed to cool to 5º C before fraction B with CPA was added to the sample to achieve a final concentration of 1 × 109 spermatozoa/mL and followed by freezing. The cryopreservation protocol did not impact total motility at 5 °C (1 S: 78.5 % vs 2 S: 79 %, p > 0.05). After thawing, the two-step protocol improved (p < 0.05) total (1 S: 18.2 % vs 2 S: 29.5 %) and progressive motility (1 S: 9 % vs 2 S: 15%). Further, the 2 S protocol increased (p < 0.05) the percentage of rapid spermatozoa (1 S: 8.7 % vs 2 S: 14.6 %) and spermatozoa with intact plasma and acrosomal membrane (IAIP) (1 S: 40.5 % vs 2 S: 61.5 %), and increased (p < 0.05) live sperm cells with high mitochondrial potential (MHIP) (1 S: 42.9 % vs 2 S: 60 %). The boar semen cryopreservation method (TRT) did not (p > 0.05) alter membrane lipid disorder, lipid peroxidation, and superoxide anion. Thus, the best method for boar semen cryopreservation is the two-step protocol.

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BACKGROUND: Melanoma is a malignant cancer that affects melanocytes and is considered the most aggressive skin-type cancer. The prevalence for melanoma cancer for the last five year is about one million cases. The impact caused of this and other types of cancer, revel the importance of research into potential active compounds. The natural products are an important source of compounds with biological activity and research with natural products may enable the discovery of compounds with potential activity in tumor cells. METHODS: The Sulforhodamine B was used to determine cell density after treatment with lawsone derivatives. Apoptosis and necrosis were analyzed by flow cytometer. Morphological changes were observed by fluorescence using the Phalloidin/FITC and DAPI stains. The clonogenic and wound healing assays were used to analyze reduction of colonies formation and migratory capacity of melanoma cells, respectability. RESULTS: In pharmacological screening, seven compounds derived from lawsone were considered to have high cytotoxic activity (GI > 75%). Three compounds were selected to assess the inhibitory concentration for 50% of cells (IC50), and the compound 9, that has IC50 5.3 µM in melanoma cells, was selected for further analyses in this cell line. The clonogenic assay showed that the compound is capable of reducing the formation of melanoma colonies at 10.6 µM concentration. The compound induced apoptotic morphological changes in melanoma cells and increased by 50% the cells dying from apoptosis. Also, this compound reduced the migratory capacity of melanoma cells. CONCLUSIONS: The results of this study showed that the evaluated lawsone derivatives have potential activity on tumor cells. The compound 9 is capable of inducing cell death by apoptosis in melanoma cells (B16F10).

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The combination of hyperthermia and chemotherapy has a potential synergic effect in antitumor activity. The development of new biocompatible and biodegradable polymers to simultaneously encapsulate magnetic nanoparticles (MNPs) and antitumoral drugs offer new cancer treatment opportunities. Here, biodegradable and biocompatible poly(thioether-ester) (PTEe) was used to encapsulate MNPs and 4-nitrochalcone (4NC) using miniemulsification and solvent evaporation. The resulting hybrid particles (MNPs-4NC-PTEe) had nanometer-scale diameters, spherical morphology, negative surface charge, high encapsulation efficiency, and superparamagnetic properties. Results showed that 4NC release occurred through diffusion. Free 4NC and MNPs + 4NC-PTEe did not have any cytotoxic effect on erythrocytes and mouse embryonic fibroblast (NIH3T3) cells. 4NC antitumor activity was verified on human cervical cancer (HeLa) and melanoma (B16F10) cells. Cellular uptake of MNPs + 4NC-PTEe nanoparticles was higher in HeLa cells compared to B16F10 and NIH3T3 cells. The hyperthermia application (115 kHz-500 Oe) potentiated the 4NC effects on HeLa and B16F10 cells when MNPs + 4NC-PTEe nanoparticles were used, indicating more effective antitumor activity. We concluded that the use of MNPs + 4NC-PTEe nanoparticles associated with hyperthermia is a promising form of treatment for some types of cancers.

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This study aimed to assess characteristics of bovine cryopreserved sperm and evaluate its relation to field fertility in fixed-time artificial insemination (FTAI). Semen samples of 16 bulls were used to inseminate 811 Nellore cows, and four of these bulls were also used to inseminate 101 Nellore heifers. Samples of the same ejaculate used for FTAI from each bull were analysed in the laboratory after thawing. Sperm motility and vigour were subjectively assessed by light microscope, and integrity of the plasma and acrosome membranes, and H2 O2 production were evaluated by flow cytometer. Relation among sperm characteristics and pregnancy rate of cows and heifers were evaluated by univariate and multivariate logistic regression. Subjective sperm motility and vigour did not affect the probability of pregnancy in cows or heifers. In univariate analysis for pregnancy in cows, sperm traits related to acrosome injury positively affected probability of pregnancy mainly when associated with plasma membrane integrity; H2 O2 production seems to be less important than plasma membrane integrity in affecting probability of pregnancy. In multivariate analysis, sperm traits related to injured acrosome positively affected probability of cow and heifer pregnancies while intact acrosome was negatively related to cow pregnancy. Intact plasma membrane and high H2 O2 production were positively related to cow pregnancy but negatively related to heifer pregnancy. Results suggest that a capacitation-like status of the acrosome may benefit probability of pregnancy in cows.

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Resumen OBJETIVO: Evaluar si la manipulación de gametos con sorter de citometría de flujo repercute negativamente en los indicadores clave de rendimiento de un laboratorio de reproducción asistida. MATERIALES Y MÉTODOS: Estudio descriptivo y retrospectivo, llevado a cabo en parejas a quienes se efectuó fecundación in vitro mediante inyección intracitoplasmática de espermatozoides (ICSI), con selección espermática, mediante un sorter de citometría de flujo, para selección de sexo. El estudio se efectuó en el New Hope Fertility Center de Guadalajara y Ciudad de México, de junio de 2014 a agosto de 2017. Los resultados se compararon con un grupo control seleccionado al azar. Se evaluaron los indicadores decisivos de rendimiento (KPI´s); tasa de fecundación normal, anormal (1PN, ≥ 3 PN) y fallida; tasa de degeneración posterior a ICSI; tasas de segmentación o división, blastocisto, implantación (segmentación y blastocisto) y recién nacido. Se utilizó la prueba t de Student para dos muestras y se consideró estadísticamente significativo el valor de p < 0.05. RESULTADOS: Se evaluaron 150 ciclos. Grupo 1: ICSI con selección espermática y sorter de citometría de flujo (n = 40); Grupo 2: ICSI sin sorter de citometría de flujo (n = 110). Los indicadores clave de rendimiento del grupo 1 disminuyeron; se reportaron tasas de fecundación fallida de 1.6%, blastocisto 17.4%, implantación en la segmentación 10%, implantación en blastocisto 14.2% y de recién nacido 14.5%. CONCLUSIONES: La manipulación de gametos con sorter de citometría de flujo reportó un efecto negativo en los indicadores clave de rendimiento del laboratorio de reproducción asistida, específicamente en las tasas de blastocisto, implantación de blastocisto y de recién nacido.

Abstract OBJECTIVE: To evaluate if the manipulation of gametes with a flow cytometry sorter has a negative effect on the key performance indicators (KPI´s). MATERIALS AND METHOD: Descriptive and retrospective analysis, in couples undergoing In a Vitro Fertilization (IVF) by ICSI, with sperm selection, using a flow cytometry sorter for sex selection. The study was conducted at the New Hope Fertility Center in Guadalajara and Mexico City, from June 2014 to August 2017. The results were compared with a randomly group without a flow cytometry sorter. KPI´s were evaluated; normal fertilization rate, abnormal (1PN, ≥3 PN), failed fertilization, ICSI damage rate, cleavage rate, blastocyst development rate, implantation rate (cleavage and blastocyst-stage) and live birth rate. A Student's t-test was made for two samples considering significant differences with p < 0.05. RESULTS: 150 cycles were evaluated. Group 1: ICSI with sperm selection by a flow cytometry sorter (n = 40); Group 2: ICSI without sperm selection (n = 110). Observing with statistical significance a decreased of the KPI´s of Group 1: failed fertilization rate (1.6%), blastocyst development rate (17.4%), implantation rate (cleavage-stage) (10%), implantation rate (blastocyst-stage) (14.2%) and live birth rate (14.5%). CONCLUSIONS: The manipulation of gametes with the flow cytometry sorter, has a negative effect on the assisted reproductive technology KPI´s; specifically, in the blastocyst rate, blastocyst implantation rate and live birth rate.

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A apoptose de leucócitos polimorfonucleares (PMN) é um evento central no processo de resolução da inflamação. Sendo a contagem de células somáticas (CCS) um indicador da situação imunológica da glândula mamária, o presente estudo buscou esclarecer a influência que esses fatores têm um sobre o outro e sobre a evolução do processo inflamatório. Marcaram-se as amostras de leite com anexina-V, iodeto de propídeo (PI), anticorpo anti-CH138A. Encontrou-se correlação negativa entre apoptose de PMN e CCS, além de diferença estatística entre um grupo de alta CCS e um grupo de baixa CCS quanto à taxa de PMN viáveis, em apoptose, em necrose e em necrose e/ou apoptose. De modo geral, o grupo de alta celularidade apresentou menos CH138+ em apoptose e mais células em necrose ou viáveis do que o grupo de baixa celularidade. Conclui-se que apoptose de PMN e CCS estão relacionados, e que em mamas com CCS elevada este evento está diminuído. Apesar de haver maior disponibilidade de fagócitos para a defesa nessa situação, os efeitos anti-inflamatórios da apoptose também estão diminuídos, enquanto os efeitos pró-inflamatórios da necrose estão aumentados, o que pode colaborar com a cronificação da inflamação.

Polymorphonuclear leukocyte (PMNL) apoptosis is central to the successful resolution of inflammation. Since Somatic Cell Count (SCC) is an indicator of the mammary gland's immune status, this study sought to clarify the influence that these factors have on each other and on the evolution of the inflammatory process. Milk samples were stained with annexin-V, propidium iodide (PI), primary antibody anti-CH138A. Negative correlation between SCC and PMNL apoptosis was found, and a statistical difference between high SCC group and low SCC group was observed concerning the rate of viable PMNL, apoptotic PMNL, necrotic PMNL and necrotic and/or apoptotic PMNL. Overall, the high cellularity group presented lower proportions of CH138 + cells undergoing apoptosis and higher proportions of viable and necrotic CH138+ cells. Thus, it can be concluded that PMNL apoptosis and SCC are related factors, and that in high SCC, milk apoptosis is delayed. Although there is a greater amount of active phagocytes in this situation, apoptosis' anti-inflammatory effects are decreased, while necrosis' pro-inflammatory effects are increased, which can contribute to chronic inflammation.

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A apoptose de leucócitos polimorfonucleares (PMN) é um evento central no processo de resolução da inflamação. Sendo a contagem de células somáticas (CCS) um indicador da situação imunológica da glândula mamária, o presente estudo buscou esclarecer a influência que esses fatores têm um sobre o outro e sobre a evolução do processo inflamatório. Marcaram-se as amostras de leite com anexina-V, iodeto de propídeo (PI), anticorpo anti-CH138A. Encontrou-se correlação negativa entre apoptose de PMN e CCS, além de diferença estatística entre um grupo de alta CCS e um grupo de baixa CCS quanto à taxa de PMN viáveis, em apoptose, em necrose e em necrose e/ou apoptose. De modo geral, o grupo de alta celularidade apresentou menos CH138+ em apoptose e mais células em necrose ou viáveis do que o grupo de baixa celularidade. Conclui-se que apoptose de PMN e CCS estão relacionados, e que em mamas com CCS elevada este evento está diminuído. Apesar de haver maior disponibilidade de fagócitos para a defesa nessa situação, os efeitos anti-inflamatórios da apoptose também estão diminuídos, enquanto os efeitos pró-inflamatórios da necrose estão aumentados, o que pode colaborar com a cronificação da inflamação.(AU)

Polymorphonuclear leukocyte (PMNL) apoptosis is central to the successful resolution of inflammation. Since Somatic Cell Count (SCC) is an indicator of the mammary gland's immune status, this study sought to clarify the influence that these factors have on each other and on the evolution of the inflammatory process. Milk samples were stained with annexin-V, propidium iodide (PI), primary antibody anti-CH138A. Negative correlation between SCC and PMNL apoptosis was found, and a statistical difference between high SCC group and low SCC group was observed concerning the rate of viable PMNL, apoptotic PMNL, necrotic PMNL and necrotic and/or apoptotic PMNL. Overall, the high cellularity group presented lower proportions of CH138 + cells undergoing apoptosis and higher proportions of viable and necrotic CH138+ cells. Thus, it can be concluded that PMNL apoptosis and SCC are related factors, and that in high SCC, milk apoptosis is delayed. Although there is a greater amount of active phagocytes in this situation, apoptosis' anti-inflammatory effects are decreased, while necrosis' pro-inflammatory effects are increased, which can contribute to chronic inflammation.(AU)

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