Hematological and biochemical parameters of dairy calves submitted to pegbovigrastim administration.

This study was designed to evaluate the response of hematological and biochemical parameters submitted to pegbovigrastim administration and postchallenge with lipopolysaccharide (LPS). In experiment 1, 20 newborn Holstein calves were divided into 2 groups: the Imrestor (Elanco Saúde Animal, São Paulo, Brazil) group (IMR, n = 10), which received a 25 µg/kg of body weight (BW) subcutaneous administration of pegbovigrastim, and the control group (CTR, n = 10), which received a subcutaneous administration of 0.9% saline solution. Blood samples were collected on d 0, 10, 12, and 14 relative to birth to analyze the biochemical and hematological parameters. Moreover, growth measurements were taken on d 0, 7, 14, 21, and 60 relative to birth. The number of total leukocytes in the IMR group increased on d 12 and 14 in comparison to the CTR group, as well as the counts of segmented neutrophils, band cells, and monocytes. No differences were observed in the other hematological, biochemical, and growth parameters. In experiment 2, 20 Holstein calves from 30 to 60 d old were divided into 4 groups: group 1 (LPS, n = 5) received a 0.25 µg/kg of BW single intravenous dose of Escherichia coli LPS at d 0; group 2 (IMR, n = 5) received a 25 µg/kg of BW subcutaneous dose of pegbovigrastim at d 1; group 3 (IMR + LPS, n = 5) received a 0.25 µg/kg of BW intravenous LPS dose at d 0 and a 25 µg/kg of BW subcutaneous dose of pegbovigrastim at d 1; and group 4 (CTR, n = 5) received an intravenous dose of 0.9% sodium chloride at d 0 and a subcutaneous dose of 0.9% sodium chloride at d 1. For the analysis of biochemical and hematological parameters, blood samples were collected on d -1, 0, 1, 2, 3, 4, 8, 14, and 21 relative to LPS administration. An increase in the number of total leukocytes was observed in the IMR, IMR + LPS, and LPS groups, and the IMR group remained as the highest from d 2 to 21. The levels of paraoxonase 1 were higher in the IMR group compared with all the others. The administration of pegbovigrastim in the dairy calves increased the number of circulating leukocytes, especially neutrophils, with an increase in paraoxonase 1, without altering the metabolites for the hepatic function.

The combined and individual impact of diabetes and smoking on key subgingival periodontal pathogens in patients with chronic periodontitis.

BACKGROUND AND OBJECTIVE: Comprehension of the similarities and differences in the composition of the subgingival microbiota of patients with diabetes mellitus (DM), smokers or smokers with DM is an important step in developing therapies specific for these groups at risk for periodontitis. Therefore, the aim of this study was to compare the combined and individual effects of DM and smoking on the levels and prevalence of key subgingival periodontal pathogens in patients with chronic periodontitis. MATERIAL AND METHODS: One hundred patients with generalized chronic periodontitis were allocated into one of the following groups: DM (n = 25, non-smokers with type 2 DM); S (n = 25, non-diabetic smokers); SDM (n = 25, smokers with type 2 DM); and control (n = 25, non-diabetic non-smokers). Two subgingival biofilm samples from healthy sites (probing depth and clinical attachment level ≤3 mm and no bleeding) and 2 from diseased sites (probing depth and clinical attachment level ≥5 mm and bleeding on probing) were analyzed by quantitative polymerase chain reaction for Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Eubacterium nodatum, Parvimonas micra, Fusobacterium nucleatum ssp. and Prevotella intermedia. RESULTS: There were no differences among groups in the mean counts of the bacterial species studied, considering all sampled sites (healthy plus diseased sites). There were also no differences among groups regarding the prevalence of any bacteria species in healthy and diseased sites (P > .05). The mean P. micra count was significantly higher in the healthy sites of both smoking groups, than in those of the control group (P < .05). CONCLUSION: The subgingival levels and prevalence of the bacterial species studied are not significantly different in subjects with chronic periodontitis presenting DM, smokers or smokers with DM. In addition, DM and smoking, jointly and individually, do not considerably affect the subgingival levels of target periodontal pathogens in patients with chronic periodontitis.

Clock genes expression and locomotor activity are altered along the light-dark cycle in transgenic zebrafish overexpressing growth hormone.

In the present work it was demonstrated that transgenic Danio rerio overexpressing growth hormone (GH-transgenic) present either altered gene expression at a determined time point, or different expression pattern along the LD cycle, when compared with non-transgenic (NT) animals, in the positive and negative loops of the circadian system. Gene expression of clock paralogs was reduced in GH fish at the beginning of the dark phase, leading to diminished expression amplitude along the LD cycle. Furthermore, although no differences were observed between NT and GH animals for bmal1a and cry2b expression at each time point, only GH fish presented amplitude along the LD cycle. Also, the locomotor activity behavior was evaluated for both groups. GH-transgenic animals presented higher locomotor activity along the whole LD cycle when compared with NT animals. These data suggest that alterations in the gene expression patterns along the LD cycle of the positive and negative loops of the circadian system, could lead to altered locomotor activity behavior in GH-transgenic fish, and GH overexpression could be responsible for these alterations, either affecting the pathways involved in the expression of genes from the circadian system or altering the metabolism.

Diagnostic Accuracy of Transient Elastography for Detecting Liver Fibrosis After Liver Trannsplantation: A Specific Cut-Off Value Is Really Needed?

INTRODUCTION: Liver transplant recipients often perform liver biopsy (LB), specially in the context of potentially recurring diseases, such as hepatitis C infection. However, the LB has risks of complications, despite being the gold standard. Transient elastography (TE) is a noninvasive method comparable to the LB to evaluate liver fibrosis in various settings, but its accuracy among transplant recipients is not fully understood. OBJECTIVE: To determine the accuracy of TE in liver transplant recipients compared with LB to successfully predict liver fibrosis. PATIENTS AND METHODS: Patients who underwent liver transplantation at Hospital Israelita Albert Einstein from 2010 to 2012 and presented with LB indication were also subjected to TE at the time of LB. The medium value of ten successful measurements was kept as a representative of the liver stiffness. The definition of cut-off points was made to ensure specificity of ≥90 % for all fibrosis stages (F0-F4). RESULTS: LB was performed in 267 patients. TE was not analyzed in only 8 (3 %) due to an elevated body mass index. The optimal liver stiffness cut-off value and diagnostic performance were 8.1 kPa for F ≥ 1, 12.3 kPa for F ≥ 2, 15.1 for F ≥ 3, and 16.7 for F = 4 for all patients and were 8.1 kPa for F ≥ 1, 12.3 kPa for F ≥ 2, 16.5 for F ≥ 3, and 17.6 for F = 4 for patients with hepatitis C. CONCLUSIONS: TE demonstrated good performance in defining cut-off points for fibrosis on liver histology observed in transplant recipients. The TE can be considered an alternative to LB in post-liver transplantation.

Improving the PCR protocol to amplify a repetitive DNA sequence.

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence.

Characterization of glucose-tolerant ß-glucosidases used in biofuel production under the bioinformatics perspective: a systematic review.

ß-glucosidases are enzymes that catalyze the hydrolysis of oligosaccharides and disaccharides, such as cellobiose. These enzymes play a key role in cellulose degrading, such as alleviating product inhibition of cellulases. Consequently, they have been considered essential for the biofuel industry. However, the majority of the characterized ß-glucosidases is inhibited by glucose. Hence, glucose-tolerant ß-glucosidases have been targeted to improve the production of second-generation biofuels. In this paper, we proceeded a systematic literature review (SLR), collected protein structures and constructed a database of glucose-tolerant ß-glucosidases, called betagdb. SLR was performed at PubMed, ScienceDirect and Scopus Library databases and conducted according to PRISMA framework. It was conducted in five steps: i) analysis of duplications, ii) title reading, iii) abstract reading, iv) diagonal reading, and v) full-text reading. The second, third, fourth, and fifth steps were performed independently by two researchers. Besides, we performed bioinformatics analysis on the collected data, such as structural and multiple alignments to detect the most conserved residues in the catalytic pocket, and molecular docking to characterize essential residues for substrate recognizing, glucose tolerance, and the ß-glucosidase activity. We selected 27 papers, 23 sequences, and 5 PDB files of glucose-tolerant ß-glucosidases. We characterized 11 highly conserved residues: H121, W122, N166, E167, N297, Y299, E355, W402, E409, W410, and F418. The presence of these residues may be essential for ß-glucosidases. We also discussed the importance of residues W169, C170, L174, H181, and T226. Furthermore, we proposed that the number of contacts for each residue in the catalytic pocket might be a metric that could be used to suggest mutations. We believe that the herein propositions, together with the sequence and structural data collection, might be helpful for effective engineering of ß-glucosidases for biofuel production and may help to shed some light on the degradation of cellulosic biomass.

Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus.

Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/10(6) cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa; controlling this activity improved the efficiency of SMGT in fish.

Metabolic rate and reactive oxygen species production in different genotypes of GH-transgenic zebrafish.

Growth hormone overexpression increases growth and consequently increases the metabolic rate in fishes. Therefore, the objective of this study was to evaluate the effects of growth hormone overexpression in zebrafish Danio rerio in terms of growth, oxygen consumption, reactive oxygen species production, lipid hydroperoxide content, antioxidant enzyme activity and glutamate-cysteine ligase catalytic subunit gene expression. The employed models were wild type and transgenic (hemizygous and homozygous) zebrafish expressing the Odonthestes argentinensis growth hormone gene directed by the Cyprinus carpio beta-actin promoter. Higher growth parameters were observed in the hemizygous group. The homozygous group possessed higher oxygen consumption and reactive oxygen species production. Growth hormone transgenesis causes a decrease in glutamate-cysteine ligase catalytic subunit expression, an enzyme responsible for glutathione synthesis. Although the lipid hydroperoxide content was similar between groups, we demonstrate that growth hormone overexpression has the potential to generate oxidative stress in fishes.

Topographic trace-elemental analysis in the brain of Wistar rats by X-ray microfluorescence with synchrotron radiation.

Knowledge about the spatial distribution and the local concentration of trace elements in tissues is of great importance, since trace elements are involved in many biological functions of living organisms. However, there are few methods available to measure the spatial (two (three)-dimensional) elemental distribution in animal brain. X-ray microfluorescence with synchrotron radiation is a multielemental mapping technique, which was used in this work to determine the topographic of iron, zinc and copper in coronal sections of female Wistar rats of different ages. Young (14 days old) and middle-aged (20 months old) rats (n = 8) were analyzed. The measurements were carried out at the XRF beam line at the Synchrotron Light National Laboratory (Campinas, Brazil). Two-dimensional scanning was performed in order to study the tendency of elemental concentration variation. The acquisition time for each pixel was 10 s/step and the step size was 300 microm/step in both directions. It was observed that the iron distribution was more conspicuous in the cortical area, thalamus and bellow the thalamus. On the other hand, the zinc distribution was more pronounced in the hippocampus. The iron, copper and zinc levels increased with advancing age. Therefore, this study reinforces the idea that these elements are involved in the chemical mechanisms of the brain that induce some neurological diseases, since they are also present in high levels in specific areas of the brain, such as the hippocampus and the substantia nigra of patients with these disorders.

Gene structure and promoter function of a teleost ribosomal protein: a tilapia (Oreochromis mossambicus) L18 gene.

We have cloned and characterized a tilapia (Oreochromis mossambicus) L18 ribosomal protein gene, including the complete transcribed region and 488 bp of upstream regulatory sequences. We have also isolated two L18 cDNAs from another tilapia (Oreochromis niloticus) with a few conservative nucleotide differences. Our results suggest the presence of two genes in both species. Reporter constructs were tested for transient expression in CV1 cells and in microinjected zebrafish and tilapia embryos. The tilapia L18 promoter was able to drive expression of the reporter gene in all three experiments, with no apparent preference for a particular tissue. The tilapia L18 promoter is therefore likely to be a powerful tool to drive tissue-independent gene expression in fish.

Integrated biological responses of zebrafish (Danio rerio) to analyze water quality in regions under anthropogenic influence.

This study analyzed water quality in regions around Patos lagoon (Southern Brazil) that are under anthropogenic pressure. Water samples were collected from five different sites, including one used as a source for human consumption (COR) and others known to be influenced by human activities (IP). Danio rerio (Teleostei, Cyprinidae) organisms were exposed for 24h to these water samples, plus a control group. It was observed that: (1) reactive oxygen species levels were lower in COR and IP than in the control group; (2) glutamate-cysteine ligase (catalytic subunit) expression was higher in COR than in other sites; (3) exposure to all water samples affected long-term memory (LTM) when compared to control group. Thus, some water samples possess the ability to modulate the antioxidant system and to induce a decline in cognitive functions, as measured by LTM. The obtained results indicate that a combination of variables of different organization level (molecular, biochemical and behavioral) can be employed to analyze water quality in impacted regions.

Mitochondrial control region haplotypes of the South American sea lion Otaria flavescens (Shaw, 1800).

The South American sea lion, Otaria flavescens, is widely distributed along the Pacific and Atlantic coasts of South America. However, along the Brazilian coast, there are only two nonbreeding sites for the species (Refúgio de Vida Silvestre da Ilha dos Lobos and Refúgio de Vida Silvestre do Molhe Leste da Barra do Rio Grande), both in Southern Brazil. In this region, the species is continuously under the effect of anthropic activities, mainly those related to environmental contamination with organic and inorganic chemicals and fishery interactions. This paper reports, for the first time, the genetic diversity of O. flavescens found along the Southern Brazilian coast. A 287-bp fragment of the mitochondrial DNA control region (D-loop) was analyzed. Seven novel haplotypes were found in 56 individuals (OFA1-OFA7), with OFA1 being the most frequent (47.54%). Nucleotide diversity was moderate (π = 0.62%) and haplotype diversity was relatively low (67%). Furthermore, the median joining network analysis indicated that Brazilian haplotypes formed a reciprocal monophyletic clade when compared to the haplotypes from the Peruvian population on the Pacific coast. These two populations do not share haplotypes and may have become isolated some time back. Further genetic studies covering the entire species distribution are necessary to better understand the biological implications of the results reported here for the management and conservation of South American sea lions.

Clonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus / Cloning and evaluation of sbGnRH gene expression in juvenile and adult males of Brazilian flounder Paralichthys orbignyanus

Este estudo buscou clonar o cDNA do sbGnRH, identificar sua expressão em diferentes tecidos do linguado, bem como avaliar possíveis diferenças no RNA mensageiro (RNAm) desse gene no cérebro de linguados machos juvenis e adultos. Por meio da RT-PCR, demonstrou-se pela primeira vez, a clonagem da região codificadora do sbGnRH contendo 297 nucleotídeos do cérebro do linguado. A expressão do sbGnRH foi detectada em vários tecidos periféricos. Foram detectados níveis mais elevados de RNAm do sbGnRH no hipotálamo dos animais adultos. Estes resultados sugerem que o sbGnRH está envolvido na puberdade do linguado.

The objectives of this study were to clone sbGnRH cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain sbGnRH expression in juvenile and adult males. Using RT-PCR the cloning of a 297 nucleotides coding region of sbGnRH from Brazilian flounder brain was demonstrated for the first time. Expression of sbGnRH was detected in several peripheral tissues. Brain gene expression in the adult flounder was higher than those found in juvenile. These results suggest that sbGnRH is involved on the Brazilian flounder puberty.

Mitochondrial control region haplotypes of the South American sea lion Otaria flavescens (Shaw, 1800)

The South American sea lion, Otaria flavescens, is widely distributed along the Pacific and Atlantic coasts of South America. However, along the Brazilian coast, there are only two nonbreeding sites for the species (Refúgio de Vida Silvestre da Ilha dos Lobos and Refúgio de Vida Silvestre do Molhe Leste da Barra do Rio Grande), both in Southern Brazil. In this region, the species is continuously under the effect of anthropic activities, mainly those related to environmental contamination with organic and inorganic chemicals and fishery interactions. This paper reports, for the first time, the genetic diversity of O. flavescens found along the Southern Brazilian coast. A 287-bp fragment of the mitochondrial DNA control region (D-loop) was analyzed. Seven novel haplotypes were found in 56 individuals (OFA1-OFA7), with OFA1 being the most frequent (47.54 percent). Nucleotide diversity was moderate (π = 0.62 percent) and haplotype diversity was relatively low (67 percent). Furthermore, the median joining network analysis indicated that Brazilian haplotypes formed a reciprocal monophyletic clade when compared to the haplotypes from the Peruvian population on the Pacific coast. These two populations do not share haplotypes and may have become isolated some time back. Further genetic studies covering the entire species distribution are necessary to better understand the biological implications of the results reported here for the management and conservation of South American sea lions.