The potential effects and interactions of oxidative stress and trace minerals on fresh and frozen semen in bulls - a review.

Reproduction is one of the most important factors determining successful cattle farming systems. Management practices, such as nutritional supplementation, can influence the reproductive performance of cattle. The objective of this literature review is to determine the potential value of injectable trace mineral administration on fresh and cryopreserved semen quality of bulls. A search of keywords related to the topic was performed on published articles and textbooks. The search was narrowed to the 40 most relevant references. Several studies have demonstrated a positive association between trace mineral supplementation and bull semen quality. Moderate amounts of reactive oxygen species (ROS) play an important role in normal spermatogenesis, but oxidative stress (OS), as experienced with adverse environmental conditions or disease, can contribute to idiopathic male infertility by negatively impacting spermatogenesis. Trace minerals such as selenium, copper, zinc, and manganese have been demonstrated to have antioxidant effects in mammals. Due to the complexity of oral ingested trace mineral bioavailability, injectable trace mineral supplementation prior to physiological periods with known deficiencies or increased requirement can benefit the animal. The potential benefits of injectable trace mineral supplementation to minimise oxidative damage to spermatogenesis in breeding bulls need further investigation. Positive results from such studies can lead to the implementation of injectable trace mineral supplementation strategies prior to the breeding season to minimise the detrimental effects of OS and can improve semen quality.

Somatic homozygous loss of SH2B3, and a non-Robertsonian translocation t(15;21)(q25.3;q22.1) with NTRK3 rearrangement, in an adolescent with progenitor B-cell acute lymphoblastic leukemia with the iAMP21.

Intrachromosomal amplification of chromosome 21 (iAMP21) occurs in ∼2% of B-cell acute lymphoblastic leukemia (ALL) and is considered to confer a poor prognosis. The relapse risk is associated with therapy intensity, suggesting that other somatic mutations may influence iAMP21-ALL prognosis. This abnormality is characterized by multiple copies of the RUNX1 gene in chromosome 21 and appears to arise through multiple breakage-fusion bridge cycles and chromothripsis. Rob(15;21) or a ring chromosome 21 have been associated with an increased risk for iAMP21-ALL, suggesting that constitutional genetic abnormalities may also drive leukemogenesis. Here we describe homozygous deletion of the SH2B3 gene, chromothripsis of chromosome 21, and a non-Robertsonian somatic t(15;21)(q25.3;q22.1) with NTRK3 gene rearrangement in an adolescent with iAMP21-B-ALL. Molecular cytogenetic studies detected iAMP21 with aCGH analysis revealing further genomic imbalances. The RT-qPCR analysis detected elevated expression levels of RUNX1 (68-fold) and reduced expression of CDK6 (0.057-fold). Studies with constitutive cells collected from mouth swabs showed that SH2B3 biallelic deletion was a somatic alteration occurring during clonal evolution. The identification of novel secondary genetic changes was valuable to discuss sporadic iAMP21 leukemogenic mechanisms. For the first time, we show a t(15;21)(q25.3;q22.1) with NTRK3 rearrangement in an adolescent with iAMP21-ALL.

Functional analysis of ldlr genetic variants identified in hypercholesterolemic patients in brazil, USING CRISPR/CAS9

BACKGROUND AND AIMS: Introduction: The familial hypercholesterolemia (FH) is one of the main causes of cardiovascular diseases, and it is mainly caused by genetic variants at the low-density lipoprotein receptor (LDLR). Although ultrasequencing technology has allowed the identification of several genetic variants, few of them was functional analyzed. The CRISPR/ Cas9 tool promotes precise genetic editing and allows the creation of experimental models, therefore contributing to the functional validation process. Aim: To use the CRISPR/Cas9 tool to perform in vitro functional analysis of LDLR variants identified in FH patients. METHODS: Two missense LDLR variants were selected within a group of variants identified in FH patients, based on in silico data, the affected protein domain and MAF. Three sgRNAs were designed for each of the variants c.551G>A and c.1118G>A, to analyze the accuracy of the sgRNAs. The sgRNAs were inserted on PX458 plasmid, cloned, purified in E. coli DH5a, and then co-transfected with the DNA template at HepG2 cells. The DNAs templates were designed to contain the selected variants. RESULTS: HepG2 cells co-transfected with PX458 constructs and DNA templates showed considerably transfection rate, being possible to visualize it at fluorescence microscopy. However, it was noted that single transfection of sgRNAs showed a higher transfection efficiency than cotransfection. CONCLUSIONS: We designed sgRNA for c.551G>A and c.1118G>A variants, being able to analyze the transfection efficiency. In further steps, we will select new sgRNAs for LDLR variants that have not been described yet, and functional analysis will be performed to determine the clinical relevance of these variants.

Análise funcional in silico das variantes no gene da APOB em pacientes com hipercolesterolemia familial / In silico functional analysis of variants in the APOB gene in patients with familial hypercholesterolemia

INTRODUÇÃO: O diagnóstico molecular da hipercolesterolemia familial (HF) é atribuído principalmente as variantes nos genes LDLR, LDLRAP1, APOB e PCSK9. O objetivo deste estudo foi realizar análise in silico para investigar o impacto de variantes sem descrição na literatura no gene APOB observado em pacientes com HF. MÉTODOS: Foram selecionados 141 indivíduos com diagnóstico clínico de HF. As variantes no gene da APOB foram selecionadas após sequenciamento dos éxons de 61 genes utilizando a plataforma MiSeq (Illumina). Os dados foram analisados nos programas Real Time Analysis, MiSeq Reporter, BaseSpace Sequence Hub e VariantStudio. Para a análise in silico, as sequências molde das moléculas da apoB-100 e o LDLr foram selecionadas por modelagem comparativa considerando o maior grau de identidade. As sequências proteicas foram alinhadas e os modelos 3D foram construídos utilizando os programas SEAVIEW e MODELLER v9.21. O gráfico de Ramachandran do modelo de menor energia apresenta 0,5% de outliers e análise de regiões de desordem, como principal validação. Os resultados das conformações de ancoragem foram analisados no software PyMol 2.1. Os estudos de docking molecular foram realizados para identificar o melhor complexo de conformação usando o servidor web clusPRO. RESULTADOS: Após a análise molecular dos 141 pacientes foram identificadas 7 variantes missenses sem descrição na literatura no gene APOB (c.433C>T, c.2630C>T, c.2950G>A, c.5743G>A, c.7367C>A, c.9880T>C e c.10780T>C). Os estudos de docking das variantes demonstraram uma maior afinidade entre o LDLr e a apoB-100 (c.2630C> T; Pro877Leu) em comparação com a proteína não mutada. A troca dos resíduos permaneceu como propriedade físico-química, e comparando as distâncias de ligação das proteínas não-mutadas (5Å) e mutadas (3,5Å), sugere-se uma maior afinidade do complexo (LDLr-apoB-100) para a leucina, tal fato é afirmado pela análise da região de desordens da apoB-100, onde a posição 877 está em uma região desorganizada e flexível. Esta maior afinidade poderia levar a uma menor dissociação intracelular deste complexo, resultando em uma alta taxa de degradação do LDLr pelas enzimas lisossômicas, levando ao aumento da concentração plasmática de LDLc. Para as outras variantes não houve alterações significativas. CONCLUSÃO: Os resultados sugerem que estudos in silico baseados na ferramenta de docking molecular podem melhorar o conhecimento da contribuição genética no desenvolvimento da doença HF. Além disso, a variante APOB c.2630C> T deve ser avaliada in vitropara validação do mecanismo proposto. (AU)

Depletion interactions and modulation of DNA-intercalators binding: Opposite behavior of the "neutral" polymer poly(ethylene-glycol).

In this work we have investigated the role of high molecular weight poly(ethylene-glycol) 8000 (PEG 8000) in modulating the interactions of the DNA molecule with two hydrophobic compounds: Ethidium Bromide (EtBr) and GelRed (GR). Both compounds are DNA intercalators and are used here to mimic the behavior of more complex DNA ligands such as chemotherapeutic drugs and proteins whose domains intercalate DNA. By means of single-molecule stretching experiments, we have been able to show that PEG 8000 strongly shifts the binding equilibrium between the intercalators and the DNA even at very low concentrations (1% in mass). Additionally, microcalorimetry experiments were performed to estimate the strength of the interaction between PEG and the DNA ligands. Our results suggest that PEG, depending on the system under study, may act as an "inert polymer" with no enthalpic contribution in some processes but, on the other hand, it may as well be an active (non-neutral) osmolyte in the context of modulating the activity of the reactants and products involved in DNA-ligand interactions.

Blockade of proteinase-activated receptor-4 inhibits the eosinophil recruitment induced by eotaxin-1 in the pleural cavity of mice.

OBJECTIVE AND DESIGN: Although proteinase-activated receptor (PAR)-4 has been implicated in inflammation, its role in regulating eosinophil recruitment in response to chemoattractants has not yet been demonstrated. To investigate the contribution of proteinases and PAR-4 activation to eosinophil migration in response to eotaxin-1 or leukotriene B(4) (LTB(4)), the effects of aprotinin or PAR-4 antagonist trans-cinnamoyl-YPGKF-NH(2) (tcY-NH(2)) on eosinophil migration induced by these chemoattractants were investigated. METHODS: BALB/c mice were pretreated with aprotinin or tcY-NH(2) (30 µg/mouse) prior to intrapleural injection of LTB(4) or eotaxin-1 and the number of infiltrating eosinophils was determined 48 h later. RESULTS: Aprotinin (1 mg/kg) inhibited eosinophil recruitment induced by eotaxin-1 (p < 0.01), but not that induced by LTB(4). Moreover, tcY-NH(2) treatment inhibited eosinophil recruitment in response to eotaxin-1 (p < 0.01 by ANOVA/Tukey post-test). CONCLUSION: These data suggest that aprotinin-inhibited proteinases participate in eosinophil migration induced by eotaxin-1 and that PAR-4 activation plays an important role in regulating this migration.

Occurrence of antimicrobial residues in pasteurized milk commercialized in the state of Paraná, Brazil.

Milk containing antimicrobial residues presents a health risk to the human population. The objective of this study was to use an enzyme-immunoassay technique to determine the occurrence of antimicrobial residues in 151 samples of pasteurized milk sold in cities of the State of Paraná, Brazil, from March 2005 to April 2006. Fifty-nine (41.3%) of the 151 samples contained antimicrobial residues. Residues of neomycin, streptomycin, and/or dihydrostreptomycin and chloramphenicol were found in three, two, and four, respectively. None of the samples with neomycin residues had levels above the maximum residue limit (MRL) permitted in this country, which is 500 microg/kg. Only one sample had a higher level of streptomycin-dihydrostreptomycin (260 microg/kg) than the MRL (200 microg/kg). The four samples positive for chloramphenicol had levels above the zero tolerance level. In the qualitative analysis, 41 of 151 samples contained tetracyclines (tetracycline, chlortetracycline, and/or oxytetracycline), 4 of 82 samples contained gentamicin, and 5 of 151 samples contained beta-lactams (amoxicillin, ampicillin, ceftiofur, cephapirin, and/or penicillin G). It was not possible to determine whether the levels of the antimicrobials found in the qualitative analyses (tetracyclines, gentamicin, and beta-lactams) were above the MRLs because the detection limits were below the MRLs in Brazil. In nine samples, two or more antimicrobial residues were found. The results demonstrate the need for monitoring various antimicrobial residues in pasteurized milk to ensure safety, quality, and integrity and to protect the health of the Brazilian population.

O efeito da cafeína no desempenho de ciclistas sob condçoes de alto risco térmico / Effect of caffeine in the performance of cyclists under high thermal risk

PURPOSE: To verify the effect of caffeine on yield time, the tympanic temperature and body weight with the administration of 5 and 9 mg/kg doses of caffeine and placebo, in cycling races under high thermal risk conditions. METHODS: Eight highly-trained cyclists were studied in 3 races of 45 km using the experimental model and double-blind with intra-subjects randomized. RESULTS: Air temperature ranged from 28.,5 and 32 degrees C and humidity between 71 e 78% with an index of WBGT varying between 24.5 degrees and 27 degrees C, figures that indicate high thermal risk. No significant differences were observed between variables assessed, yet yield time was lower with doses of 5 and 9 mg/kg caffeine than with placebo. CONCLUSION: These data indicate that heat and humidity conditions may be sufficient to mask the ergogenic benefit of caffeine in cycling races of prolonged duration. Therefore, isn't justifiable it's utilization in high thermal risk conditions.

Application of Plackett-Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium.

Here we report the successful implementation of the Plackett-Burman multifactorial design to screen the limiting components for growth and subsequent use of the response surface methodology (RSM) to design a medium that supported exponential growth of the aggregated morphology of the shipworm bacterium, Teredinobacter turnirae. The results obtained with the help of Plackett-Burman design indicated limitations of three components in the growth medium, MnCl2.4H2O, Na2CO3, and K2HPO4. The concentrations of these three components were further optimized using RSM. By increasing the concentrations of the above-mentioned components by 4-fold, 12-fold, and 12-fold, respectively, it became possible to achieve exponential growth of the culture.

Production of an endoglucanase by the shipworm bacterium, Teredinobacter turnirae.

The nutritional behavior of a cellulolytic nitrogen-fixing shipworm bacterium, Teredinobacter turnirae, is described, with respect to various carbon and nitrogen sources, in terms of endoglucanase production. Also, the effects of various surfactants on enzyme production are reported. Among the carbon sources, sucrose results in the maximum enzyme production, followed by cellulose. Ammonium phosphate proves to be the best nitrogen source for endoglucanase production. Various surfactants enhance the enzyme titers, with Triton X-100 yielding the best results. A combination of the above-mentioned components improves the enzyme production by 3.6-fold.

Pleomorphism of the marine bacterium Teredinobacter turnirae.

AIMS: A morphology transition for the marine bacterium, Teredinobacter turnirae is reported. METHODS AND RESULTS: When grown in the rod-shaped morphology, the cells require high concentrations of NaCl (0.3 mol x l(-1)) and secrete extracellular protease and endoglucanase activity. When this bacterium is grown in a medium containing casein as a sole carbon and nitrogen source, a major change in morphology to a stable aggregated form is obtained. CONCLUSION: In the aggregated morphology, much higher protease production rates (170 Units x ml(-1) x d-1 for aggregates vs. 15 Units x ml(-1) x d(-1) for rods, for the same initial biomass) and negligible endoglucanase titres are obtained. In addition, the aggregated morphology does not require sodium chloride for growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The phenomenon reported here describes a novel relationship between the cell morphology and the biochemical characteristics of the bacterium.

The prevalence of bovine viral diarrhoea antibodies in selected South African dairy herds, and control of the disease.

The prevalence of bovine viral diarrhoea (BVD) serologically positive animals in 18 dairy herds with clinical and pathological lesions suggestive of BVD infection, the post-vaccinal seroconversion rates in negative animals vaccinated twice with an inactivated BVD vaccine, and the control measures taken, are described. The pathological and histopathological findings in 6 necropsies performed on animals that died in 5 separate herds closely resembled published descriptions. Positive immunohistochemistry results in 3 cases confirmed the diagnosis in those animals. In 1 herd the prevalence of prevaccinal BVD antibodies was only 36.8%, while the prevalence varied from 79.85 to 100% in the remainder. Control measures taken included immunoprophylaxis with an inactivated vaccine, culling animals that were serologically negative after vaccination that were regarded as probably persistently infected (PI) and the implementation of additional biosecurity measures. The prevalence of serologically negative PI animals in 10 herds varied from 0.38 to 4.04%, with 8 herds less than 1% and 2 herds at 2.79% and 4.04%, respectively. Methods based on vaccinating the herd, followed by serological testing and culling cattle that did not develop an antibody titre, are not reliable. The identification of PI animals should be confirmed by isolation of the virus or identification of the antigen.

Problems associated with the study of cellular oscillations.

Data from the article by Brodsky et al. (2000) have been examined in order to confirm that the fluctuations they report are not random but have a rhythmic basis, thereby verifying their conclusions regarding the existence of periodicity in protein synthesis. Reasons are outlined why oscillations should be expected in all cellular biochemical studies. Associated practical problems are briefly discussed together with comments on the validity and value of less rigorous methods of analysis. The subject of aliasing is raised as justification for doubting the validity of much published data, especially where periodicity has not been suspected. The existence of oscillations indicates the need for a thorough re-evaluation of our understanding of cell biochemistry.

Transient chaos in intracellular dynamics?

Periodogram analyses of the temporal variations of several cellular oscillations occasionally reveal the existence of short duration bands containing a wide range of frequencies. The possibility is considered that these are due to the transient compliance with chaotic conditions.

High frequency oscillations in the activity of phosphotyrosine phosphatase in murine erythroleukaemic cells: action of insulin and hexamethylene bisacetamide.

We have previously reported oscillations in the activities of the phosphoamino acid phosphatases in murine erythroleukaemic cells. In keeping with our predictions we now show that the phosphotyrosine phosphatase activity rhythm has a much shorter period than originally seemed the case, being of the order of 10 min and probably less. The periodic changes show evidence of rhythmic modulation of mean, period and amplitude as with all other cellular oscillations studied. Insulin decreases the frequency of the rhythm while the inducer of differentiation, hexamethylene bisacetamide (HMBA) decreases its amplitude. Current ideas on phosphorylation dynamics in relation to metabolism and mitosis may need to be revised in the light of the observations.

Independent high-frequency oscillations in the amounts of individual isozymes of lactate dehydrogenase in HL60 cells.

Early studies, using kinetic methods, suggested that the isozyme pattern of lactate dehydrogenase in various cells oscillated with time. More recent electrophoretic studies on murine erythroleukaemic cells (which exhibit only one isozyme) indicated very high frequency variations (period 2 min or less) in the amount of the lone active isozyme. We now show that in HL60 cells, the activity stain intensities of the two major isozyme bands both oscillate but the temporal variations are distinct. As with other cellular rhythms, each of the two periodicities seem to be modulated in cyclic fashion with respect to period, amplitude and mean levels, the periods of both the primary and modulating rhythms being of the order of 10-15 min or probably much less.

Distinct, very high frequency oscillations in the activity and amount of active isozyme of lactate dehydrogenase in murine erythroleukaemic cells and a cell-free system.

Electrophoretic and kinetic determinations of the activity of lactate dehydrogenase in murine erythroleukaemic (MEL) cells, sampled at 1 min intervals, reveal distinct oscillations in the activity and amount of active isozyme. Both oscillations have periods in the range of 2-6 min (probably less) and both appear to be rhythmically modulated with respect to period, amplitude and mean. The oscillations also occur in cell-free systems, a fact which throws doubt on the value of studies where it is assumed that such preparations have constant composition.

Insulin stimulation of high frequency phosphorylation dynamics in murine erythroleukemia cells.

The phosphorylation potentials of two proteins (M(r) 81 kDa and 63 kDa) in extracts of murine erythroleukemic (MEL) cells both vary in an oscillatory manner, sometimes changing by as much as 100-fold in 10 min. Direct analysis of the temporal changes indicates the existence of periodic modulation of the frequencies, amplitudes and mean levels of the two rhythms. In both cases, periodogram analyses, by two methods, confirm the presence of several oscillations having periods in the range 20-100 min which tend to occur in (pseudo) periodic bursts. Insulin has been found to enhance these oscillations in a manner comparable with its effect on rhythmic variations in cell morphology. Despite the marked similarity in the behaviour of the two proteins, no particular phase relationship existed between the two temporal variations, suggesting differences between the underlying driving forces.

Oscillatory variations in the amount of protein extractable from murine erythroleukemia cells: stimulation by insulin.

The amount of protein extractable from murine erythroleukemia (MEL) cells varies in an oscillatory manner at high frequency and high amplitude as it does for several other cell lines. Moreover, the rhythm appears to be modulated in periodic fashion with respect to the mean, period and amplitude. The phenomenon thus seems to be universal and fundamental. Time series analyses support the view that several periodicities contribute to the observed protein rhythm. Insulin affects the dynamics as it does for both morphological and phosphorylation oscillations. Caution is necessary in the interpretation of 'specific activities' of cellular components and in electrophoretic studies wherein equal amounts of protein are applied to the wells in an effort to correct for random errors.

[Evaluation of the therapeutic effect of albendazole on experimental and human infections by Hymenolepis nana]. / Avaliação da atividade terapêutica do albendazol sobre infecções experimentale e humana pela Hymenolepis nana.

With the objective of knowing adequately the spectrum of activity of albendazole against intestinal helminthiases, we made observations regarding hymenolepiasis caused by Hymenolepis nana. Two series of investigations were carried out: a) treatment of mice with single doses of either 25 or 50 mg/kg, repeated after ten days, using as controls animals treated with 25 mg/kg of praziquantel or not treated with any antiparasitic drugs; b) treatment of adults and children with 400 mg daily for three consecutive days, repeated after ten days.