Sex differences in the orofacial antinociceptive effect of metformin and the role of transient receptor potential channels.

Metformin is classified as a biguanide and is used in the treatment of type 2 diabetes. It is used worldwide and has been investigated in drug repositioning. The present study aims to investigate whether there is sexual dimorphism in the orofacial antinociceptive effect of metformin and the participation of TRP channels. Acute nociceptive behavior was induced by administering cinnamaldehyde or capsaicin to the upper lip. Nociceptive behavior was assessed through orofacial rubbing, and the effects of pre-treatment with metformin (125 or 250 mg/Kg) or vehicle (control) were tested on the behavior. Nociceptive behavior was also induced by formalin injected into the temporomandibular joint. The chronic pain model involved infraorbital nerve transection (IONX) was evaluated using Von Frey electronic filaments. Trpv1 gene expression was analyzed in the nerve ganglion. Docking experiments were performed. Metformin, but not the vehicle, produced antinociception (p < 0.0001) in all acute nociceptive behaviors in both sexes, and these effects were attenuated by the TRPV1 antagonist capsazepine and the TRPA1 antagonist HC-030031. In IONX with better (**p < 0.01, ****p < 0.0001 vs. control) results in females. TRPV1 gene expression was observed in the metformin treated group (*p < 0.05 vs. control). Docking experiments revealed that metformin may interact with TRPV1 and TRPA1 channels. Metformin promotes orofacial antinociception in both sexes in acute pain and is more effective in chronic pain in females than in males, through the modulation of TRPV1 and TRPA1 channels. These preclinical findings suggest a potential repositioning of metformin as an analgesic agent in acute and chronic orofacial pain states.

Analysis of physical traits, clinical parameters, and energy metabolism of in vivo- and in vitro-derived Flemish newborn calves during the first day of life.

Studies investigating physiological deviations from normality in newborn calves derived from in vitro fertilization procedures remain important for the understanding of factors that reduce calf survival after birth. The aim of this study was to investigate parameters affecting health and welfare of newborn Flemish calves derived from in vitro embryo production (IVP) in the first hours of life in comparison to in vivo-derived calves. Physical traits of newborn calves and fetal membranes (FM) were recorded soon after birth. Newborn venous blood samples were collected at several time points within the first 24 h of life for analyses of energy substrates, electrolytes, blood gases, acid-base balance, blood chemistry, and haematology. A liver biopsy was taken within the first hour after birth for analysis of gene expression of key enzymes of the fructolytic and glycolytic pathways. Newborn IVP calves were heavier and larger at birth, which was associated with heavier FM. At several time points during the first 24 h of life, IVP-derived calves had altered rectal temperature, blood gases, electrolyte concentrations, blood parameters for liver, kidney and muscle function, and acid-base balance, plasma lipid metabolism, and hemogram parameters. The relative mRNA abundances for triokinase and lactate dehydrogenase-B were greater in IVP calves. In summary, IVP-derived newborn calves were at higher risk of clinical problems after birth, which was markedly greater in heavier and larger calves. Such animals take longer to adapt to extrauterine life and should receive a special attention during the immediate neonatal period.

Pharmacological Potential of cis-jasmone in Adult Zebrafish (Danio rerio).

This study evaluates the pharmacological potential of cis-jasmone (CJ) in adult zebrafish (Danio rerio; aZF). Initially, aZF (n = 6/group) were pretreated (20 µL; p. o.) with CJ (0.1 or 0.3 or 1.0 mg/mL) or vehicle (0.5% Tween 80). The animals were submitted to acute toxicity and locomotion tests, pentylenetetrazole-induced seizure, carrageenan-induced abdominal edema, and cinnamaldehyde-, capsaicin-, menthol-, glutamate-, and acid saline-induced orofacial nociception. The possible mechanisms of anticonvulsant, anxiolytic, and antinociceptive action were evaluated. The involvement of central afferent fibers sensitive to cinnamaldehyde and capsaicin and the effect of CJ on the relative gene expression of TRPA1 and TRPV1 in the brain of aZF were also analyzed, in addition to the study of molecular docking between CJ and TRPA1, TRPV1 channels, and GABAA receptors. CJ did not alter the locomotor behavior and showed pharmacological potential in all tested models with no toxicity. The anticonvulsant effect of CJ was prevented by flumazenil (GABAergic antagonist). The anxiolytic-like effect of CJ was prevented by flumazenil and serotonergic antagonists. The antinociceptive effect was prevented by TRPA1 and TRPV1 antagonists. Chemical ablation with capsaicin and cinnamaldehyde prevented the orofacial antinociceptive effect of CJ. Molecular docking studies indicate that CJ interacted with TRPA1, TRPV1, and GABAA receptors. CJ inhibited the relative gene expression of TRPA1 and TRPV1. CJ has pharmacological potential for the treatment of seizures, anxiety, inflammation, and acute orofacial nociception. These effects are obtained by modulating the GABAergic and serotonergic systems, as well as the TRPs and ASIC channels.

Development of a Nanoformulation for Oral Protein Administration: Characterization and Preclinical Orofacial Antinociceptive Effect.

Nanoencapsulation is a valid alternative for the oral administration of peptide drugs and proteins, as nanoparticles protect them from proteolytic degradation in the gastrointestinal tract and promote the absorption of these macromolecules. The orofacial antinociceptive effect of frutalin (FTL), through the intraperitoneal route, has already been proven. This study aimed to develop, characterize, and evaluate the orofacial antinociceptive activity of an oral formulation containing FTL in acute and neuropathic preclinical tests. Nanoencapsulated FTL was administered by oral route. The acute nociceptive behavior was induced by administering capsaicin to the upper lip and NaCl to the right cornea. The nociceptive behavior was also induced by formalin injected into the temporomandibular joint. The neuropathic pain model involved infraorbital nerve transection (IONX), which induced mechanical hypersensitivity and was assessed by von Frey stimulation. Trpv1 gene expression was analyzed in the trigeminal ganglion. The analyzed sample did not show any cytotoxicity; 52.2% of the FTL was encapsulated, and the size of the nanocapsule was less than 200 nm, the polydispersion was 0.361, and the zeta potential was - 5.87 and - 12.8 mV, with and without FTL, respectively. Nanoencapsulated FTL administered by oral route had an orofacial antinociceptive effect in acute and neuropathic rodent models. The antinociceptive effect of FTL was prevented by ruthenium red, but not by camphor. FTL reduced Trpv1 gene expression. FTL promotes orofacial antinociception, probably due to the antagonism of TRPV1 channels, and the nanoformulation represents an effective method for the oral administration of this protein. HIGHLIGHTS: • Nanoformulation for oral protein administration. • Nanocapsule containing FTL prevents orofacial nociceptive acute and neuropathic pain. • Frutalin promotes orofacial antinociception behavior antagonism of TRPV1 channels.

CRISPR/Cas9 small promoter deletion in H19 lncRNA is associated with altered cell morphology and proliferation.

The imprinted H19 long non-coding RNA, a knowing oncofetal gene, presents a controversial role during the carcinogenesis process since its tumor suppressor or oncogenic activity is not completely elucidated. Since H19 lncRNA is involved in many biological pathways related to tumorigenesis, we sought to develop a non-cancer lineage with CRISPR-Cas9-mediated H19 knockdown (H19-) and observe the changes in a cellular context. To edit the promoter region of H19, two RNA guides were designed, and the murine C2C12 myoblast cells were transfected. H19 deletion was determined by DNA sequencing and gene expression by qPCR. We observed a small deletion (~ 60 bp) in the promoter region that presented four predicted transcription binding sites. The deletion reduced H19 expression (30%) and resulted in increased proliferative activity, altered morphological patterns including cell size and intracellular granularity, without changes in viability. The increased proliferation rate in the H19- cell seems to facilitate chromosomal abnormalities. The H19- myoblast presented characteristics similar to cancer cells, therefore the H19 lncRNA may be an important gene during the initiation of the tumorigenic process. Due to CRISPR/Cas9 permanent edition, the C2C12 H19- knockdown cells allows functional studies of H19 roles in tumorigenesis, prognosis, metastases, as well as drug resistance and targeted therapy.

Physiological impact of the environment on the welfare of transgenic goats raised in a tropical climate.

The present study evaluated the general welfare state of two strains of transgenic goats bred in a region with a hot and humid tropical climate. Nine females were used, being three transgenic for human lysozyme (hLZ group), three transgenic for human glucocerebrosidase (hGCase group), and three non-transgenic (control group). The temperature and humidity index (THI) were recorded during the morning, afternoon, and evening. The physiological parameters measured were respiratory rate, heart rate, and rectal and vaginal temperatures. Venous blood samples were collected using Vacutainer® tubes containing 10% ethylenediaminetetraacetic acid (EDTA). Also, analysis of erythrogram, leukogram, and some biochemical parameters of serum was performed. It was observed that the afternoon shift presented the largest THI, being potentially more impactful on the physiology of animals. In general, respiratory and heart rates were higher in transgenic animals, especially in the hLZ group compared to the control group (P < 0.05). Regarding the hematological parameters, the quantification of red blood cells, hemoglobin, and hematocrit was significantly lower (P < 0.05) in the hGCase group compared to that in the hLZ and control. The leukocyte count was considerably lower (P < 0.05) in the hLZ group compared to that in the hGCase and control. Correlation analysis showed that the increase in THI was associated with a change in physiological parameters normally used as indicators of thermal stress. Despite the differences found among the experimental groups, all the physiological parameters remained within the normal limits recommended for the goat species. Further studies involving a larger number of animals from different categories should be carried out to elucidate the impacts that transgenesis can have on animal welfare under different THI conditions.

In silico analysis of mismatches in RT-qPCR assays of 177 SARS-CoV-2 sequences from Brazil.

INTRODUCTION: Quantitative reverse transcription polymerase chain reaction (RT-qPCR) can detect the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) in a highly specific manner. However, a decrease in the specificity of PCR assays for their targets may lead to false negative results. METHODS: Here, 177 high-coverage complete SARS-CoV-2 genome sequences from 13 Brazilian states were aligned with 15 WHO recommended PCR assays. RESULTS: Only 3 of the 15 completely aligned to all Brazilian sequences. Ten assays had mismatches in up to 3 sequences and two in many sequences. CONCLUSION: These results should be taken into consideration when using PCR-based diagnostics in Brazil.

Application of a cost-effective DNA extraction protocol for screening transgenic and CRISPR-edited primary goat cells.

The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells-GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A-0%, protocol B-93%, protocol C-13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.

Association of the ABCB1 C3435T gene polymorphism (SNPs) with the response to neoadjuvant chemotherapy in women with breast cancer in northeastern Brazil / Associação do polimorfismo (SNPs) do gene ABCB1 C3435T, com a resposta à quimioterapia neoadjuvante, em mulheres com câncer de mama do Nordeste do Brasil

Introduction: breast cancer (BC) is the most common tumor and the leading cause of cancer-related death among the female population worldwide. Polymorphisms genetics of ABCB1 gene contributed to breast cancer susceptibility and interindividual differences in chemotherapy response. Objectives: to evaluate the association between the ABCB1 C3435T gene polymorphism (SNPs) with the response to neoadjuvant chemotherapy in women with breast cancer. Methodology: this study included 32 female patients who received neoadjuvant chemotherapy. The polymorphisms were genotyped through real-time allele-specific polymerase chain reaction (PCR). The statistical analysis was performed using the Fisher's exact test or Pearson's chi-square test in the Statistical Package for Social Sciences (SPSS) version 20.0 software. Results: the genotypes found for the C3435T polymorphism were in Hardy-Weinberg equilibrium and their genotypic distributions were CC= 10 (31.1%), CT= 14 (43.8%), and TT= 08 (25.0%) with χ2: 0.86 and p-value > 0.05. Allele frequencies were C = 0.54 and T = 0.46. There were no significant statistical differences between genotypes considering the response to neoadjuvant chemotherapy and immunohistochemistry; the presence of the T allele was associated with worsen axillary status response to neoadjuvant chemotherapy. Conclusion: no definite association between the presence of C3435T polymorphism and the response to neoadjuvant chemotherapy was observed. Further studies in Brazil involving larger samples will contribute to validating the results of this study.

Introdução: o câncer de mama (CM) é o tumor mais comum e a principal causa de morte relacionada ao câncer na população feminina em todo o mundo. Polimorfismos genéticos do gene ABCB1 contribuem para a suscetibilidade ao câncer de mama e diferenças interindividuais na resposta à quimioterapia. Objetivos: avaliar a associação entre o polimorfismo (SNPs) do gene ABCB1 C3435T com a resposta à quimioterapia neoadjuvante em mulheres com câncer de mama. Metodologia: estudo com 32 pacientes do sexo feminino, que utilizaram quimioterapia neoadjuvante. A genotipagem dos polimorfismos foi feita por reação da polimerase em cadeia (PCR) em tempo real alelo específica. A análise estatística foi realizada mediante o teste exato de Fisher ou Qui-quadrado de Pearson, utilizando o software SPSS (Statistical Package for the Social Sciences) vs20.0. Resultados: os genótipos encontrados para o polimorfismo C3435T estavam em equilíbrio de Hardy-Weinberg e suas distribuições genotípicas foram respectivamente CC= 10 (31,1%), CT= 14 (43,8%), TT= 08 (25,0%) sendo, X2: 0.86 e p-value> 0,05. As frequências alélicas foram de C= 0,54 e T= 0,46. Não ocorreram diferenças estatísticas entre os genótipos, considerando a resposta a quimioterapia neoadjuvante e a imunohistoquímica, sendo a presença do alelo T associada a pior resposta do status axilar à quimioterapia neoadjuvante. Conclusão: não foi possível correlacionar a presença do polimorfismo C3435T com a resposta à quimioterapia neoadjuvante, sendo necessária a realização de novos estudos no Brasil envolvendo casuísticas maiores para a validação dos resultados.

In silico analysis of mismatches in RT-qPCR assays of 177 SARS-CoV-2 sequences from Brazil

Abstract INTRODUCTION: Quantitative reverse transcription polymerase chain reaction (RT-qPCR) can detect the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) in a highly specific manner. However, a decrease in the specificity of PCR assays for their targets may lead to false negative results. METHODS: Here, 177 high-coverage complete SARS-CoV-2 genome sequences from 13 Brazilian states were aligned with 15 WHO recommended PCR assays. RESULTS: Only 3 of the 15 completely aligned to all Brazilian sequences. Ten assays had mismatches in up to 3 sequences and two in many sequences. CONCLUSION: These results should be taken into consideration when using PCR-based diagnostics in Brazil.

Expression of a recombinant bacterial L-asparaginase in human cells.

OBJECTIVE: L-Asparaginase (ASNase) is an enzyme used in the treatment of acute lymphoblastic leukemia (ALL). As the therapeutic ASNases has bacterial origin, severe side effects are associated with its use, among them hypersensitivity and inactivation of the enzyme. In this context, the objective of this work was to produce a recombinant ASNase of bacterial origin in human cells in order to determine the presence and consequences of potential post-translational modifications on the enzyme. RESULTS: Recombinant ASNase was expressed in human cells with a molecular weight of 60 kDa, larger than in Escherichia coli, which is 35 kDa. N-glycosylation analysis demonstrated that the increased molecular weight resulted from the addition of glycans to the protein by mammalian cells. The glycosylated ASNase presented in vitro activity at physiological pH and temperature. Given that glycosylation can act to reduce antigenicity by masking protein epitopes, our data may contribute to the development of an alternative ASNase in the treatment of ALL in patients who demonstrate side effects to currently marketed enzymes.

Effect of aquaporin 3 knockdown by RNA interference on antrum formation in sheep secondary follicles cultured in vitro.

SummaryThe objectives were to develop an effective protocol for transfection of ovine secondary follicles and to assess the effect of attenuating aquaporin 3 (AQP3) using a small interfering RNA (siRNA-AQP3) on antrum formation and follicular growth in vitro. Various combinations of Lipofectamine® volumes (0.5, 0.75 or 1.0 µl), fluorescent oligonucleotide (BLOCK-iT ™) concentrations (3.18, 27.12 or 36.16 nM) and exposure times (12, 14, 16, 18 or 20 h) were tested. The BLOCK-iT™ was replaced by siRNA-AQP3 in the transfection complex. Ovine secondary follicles were isolated and cultured in vitro for 6 days using standard protocols. Follicles were transfected on day 0 or 3 or on both days (0 and 3) and then cultured for an additional 3 or 6 days. As revealed by the fluorescence signal, the Lipofectamine®/BLOCK-iT™ complex (0.75 µl + 27.12 nM by 12 h of incubation) crossed the basement membrane and granulosa cell and reached the oocytes. In general, the rate of intact follicles was higher and the rate of antrum formation was lower in transfected follicles compared with control follicles. In conclusion, ovine secondary follicles can be successfully transfected during in vitro culture, and siRNA-mediated attenuation of AQP3 gene reduced antrum formation of secondary follicles.

ATP-binding cassette (ABC) transporters in caprine preantral follicles: gene and protein expression.

The multidrug resistance proteins ABCB1, ABCC2 and ABCG2 are an energy-dependent efflux pump that functions in systemic detoxification processes. Physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier and various stem cells, until now, these pumps were not identified in goat ovarian tissue. Therefore, the aim of this study is to analyze ABCB1, ABCC2, and ABCG2 mRNA and protein expression in goat preantral follicles. Fragments (3 × 3 × 1 mm) from five pairs of ovary (n = 10) obtained from five goat were collected and immediately submitted to qPCR, Western blot, and immunofluorescence assay for mRNA detection and identification and localization of the ABC transporters, respectively. mRNA for ABCB1, ABCC2, and ABCG2 and the presence of their proteins were observed on ovarian tissue samples. Positive marks were observed for the three transport proteins in all follicular categories studied. However, the marks were primarily localized in the oocyte of primordial, transition and primary follicle categories. In conclusion, goat ovarian tissue expresses mRNA for the ABCB1, ABCC2 and ABCG2 transporters and the expression of these proteins in the preantral follicles is a follicle-dependent stage.

Developmental Outcome and Related Abnormalities in Goats: Comparison Between Somatic Cell Nuclear Transfer- and In Vivo-Derived Concepti During Pregnancy Through Term.

Cloning by somatic cell nuclear transfer (SCNT) is characterized by low efficiency and the occurrence of developmental abnormalities, which are rather poorly studied phenomena in goats. This study aimed at comparing overall SCNT efficiency in goats by using in vitro-matured (IVM) or in vivo-matured oocytes and fibroblast donor cells (mock transfected, transgenic, or wild type), also characterizing symptoms of the Abnormal Offspring Syndrome (AOS) in development, comparing results with pregnancies produced by artificial insemination (AI) and in vivo-derived (IVD) embryos. The SCNT group had lower pregnancy rate (18.3%, 11/60), total number of concepti (20.0%, 12/60), term births (3.3%, 2/60), and live births (1.7%, 1/60) than both the IVD (77.8%, 7/9; 155.5%, 14/9; 122.2%, 11/9; 88.8%, 8/9) and the AI (71.4%, 10/14; 121.4%, 17/14; 100%, 14/14; 78.5%, 11/14) groups, respectively (p < 0.05). No SCNT pregnancies reached term using IVM oocytes, but in vivo-matured oocytes resulted in two term transgenic cloned kids. The proportion fetal membrane (FM) weight/birth weight reflected an increase in FM size and cotyledonary enlargement in clones, for disproportionally bigger newborns in relation to cotyledonary numbers. Overall, goat cloning showed losses and abnormality patterns similar to the AOS in cloned cattle and sheep, which have not been previously well recognized in goats.

Transient Expression of Functional Glucocerebrosidase for Treatment of Gaucher's Disease in the Goat Mammary Gland.

Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.

Comparative expression profiles of genes related to cellular stress and development in oocyte of goats fed with distinct concentrations of lipids

Background: Lipotoxicity is characterized by an excess of saturated fatty acids in the blood stream, in which other nonadipose cells begin to store them, thereby altering the expression of genes related to endoplasmic reticulum stress, whoseeffects have been associated with decreased oocyte quality in several species, decreased mitochondrial activity, and increasedapoptosis. The present study was conducted to investigate the lipotoxicity effect of diets with increasing fat levels on geneexpression in goats oocyte and granulosa cells.Materials, Methods & Results: Thirty does were divided into three groups of 10 animals each, which received forage andconcentrate to provide respectively 2.7% of lipids (LL group), 3.9% lipids (LI group) and 5.1% lipids (LH group) for 28days. Three days before oocyte harvest, follicular wave was synchronized by 1 mL PGF2α intramuscularly, followed bythe insertion of an intravaginal progesterone release device. Viable oocytes and granulosa cells were subjected to qRT-PCRto determine the expression of PLIN2, ATF4, CHOP10, BAX, BCL2, HSP70 genes, were checked, evaluated using thedissociation curve analysis, which obtained the following values: 77.8, 80.2, 83.6, 78.0, 81.0 and 82.0ºC, respectively. Thesteps of qPCR thermal cycle was: denaturation and polymerase activation, annealing and final extension.Throughout theexperimental period, blood samples were taken for cholesterol, triglycerides and total lipids measurements. Total plasmalipids determination was calculated with the following equation: 2 x (cholesterol + triglycerides) × 1.1 with sensitivity ofthe assay for cholesterol was 1.472 mg/dL and for triglycerides, 2.845 mg/dL. In LH group, it was recorded a more pronounced increase in total lipids (+ 60.1%) (P < 0.05), cholesterol (+ 18.8%) and triglycerides (+ 8.5%). These increaseswere three times higher than that in LL and LI groups. All genes were expressed in oocytes...(AU)

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...]

Comparative expression profiles of genes related to cellular stress and development in oocyte of goats fed with distinct concentrations of lipids

Background: Lipotoxicity is characterized by an excess of saturated fatty acids in the blood stream, in which other nonadipose cells begin to store them, thereby altering the expression of genes related to endoplasmic reticulum stress, whoseeffects have been associated with decreased oocyte quality in several species, decreased mitochondrial activity, and increasedapoptosis. The present study was conducted to investigate the lipotoxicity effect of diets with increasing fat levels on geneexpression in goat’s oocyte and granulosa cells.Materials, Methods & Results: Thirty does were divided into three groups of 10 animals each, which received forage andconcentrate to provide respectively 2.7% of lipids (LL group), 3.9% lipids (LI group) and 5.1% lipids (LH group) for 28days. Three days before oocyte harvest, follicular wave was synchronized by 1 mL PGF2α intramuscularly, followed bythe insertion of an intravaginal progesterone release device. Viable oocytes and granulosa cells were subjected to qRT-PCRto determine the expression of PLIN2, ATF4, CHOP10, BAX, BCL2, HSP70 genes, were checked, evaluated using thedissociation curve analysis, which obtained the following values: 77.8, 80.2, 83.6, 78.0, 81.0 and 82.0ºC, respectively. Thesteps of qPCR thermal cycle was: denaturation and polymerase activation, annealing and final extension.Throughout theexperimental period, blood samples were taken for cholesterol, triglycerides and total lipids measurements. Total plasmalipids determination was calculated with the following equation: 2 x (cholesterol + triglycerides) × 1.1 with sensitivity ofthe assay for cholesterol was 1.472 mg/dL and for triglycerides, 2.845 mg/dL. In LH group, it was recorded a more pronounced increase in total lipids (+ 60.1%) (P < 0.05), cholesterol (+ 18.8%) and triglycerides (+ 8.5%). These increaseswere three times higher than that in LL and LI groups. All genes were expressed in oocytes...

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...](AU)

Clonagem do cDNA e sequenciamento parcial do gene que codifica a enzima glutamato desidrogenase hepática de ovino / Cloning of the cDNA and partial sequencing of the gene that codifi es the sheep hepatic enzyme glutamate dehydrogenase

A enzima mitocondrial glutamato desidrogenase (GDH: EC 1.4.1.2) catalisa a desaminação reversível do L-glutamato para 2-oxoglutarato (α-cetoglutarato) usando o NAD+ e NADP+ como coenzimas. É uma das mais importantes enzimas hepáticas encontradas em hepatócitos de bovinos, ovinos e caprinos. Infecções por Fasciola spp., intoxicação grave aguda por toxinas de plantas, tais como Xanthium spp. e Senecio spp. e intoxicação por cobre resultam na liberação dessa enzima no sangue. O aumento da GDH indica danos ou necrose hepática em bovinos e ovinos. Esta é a enzima de escolha para avaliar a função hepática dos ruminantes. No presente trabalho o cDNA que codifica a enzima GDH do hepatócito de ovino foi sintetizado por meio de RT-PCR utilizando mRNA extraído do fígado de ovino. Parte da região de codificação do cDNA da GDH de ovino foi amplificada por PCR usando oligonucleotídeos iniciadores sintetizados a partir do alinhamento de sequências de ORFs de Ovis aries, Bos taurus, Homo sapiens, Rattus norvegicus e Mus musculus disponíveis em banco de dados. O cDNA foi clonado no vetor pGEM® -T Easy (Promega) e inserido em células cálcio-competentes de Escherichia coli DH10B através de choque térmico. O DNA plasmidial foi purificado e após o sequenciamento a presença de um inserto de 1292 pb foi confirmado. O alinhamento da sequência deduzida de aminoácidos com outras espécies revelou alta homologia entre as GDH.

The mitochondrial enzyme Glutamate Dehydrogenase (GDH: EC 1.4.1.2) catalyzes the reversible deamination of the L-glutamate for 2-oxoglutarate (α-ketoglutarate) using NAD+ and NADP+ as coenzymes. It is one of the most important liver enzymes found in hepatocytes of cattle, sheep and goats. Infections by Fasciola spp., severe acute intoxication by toxins of plants such as Xanthium spp. and Senecio spp. as well as intoxication by copper result in the release of this enzyme in blood. The increase of the GDH indicates damage or hepatic necrosis in cattle and sheep. This is an enzyme of choice to evaluate the function of the ruminants. In the present study the cDNA, that codifies the GDH enzyme of the hepatocyte of sheep, was synthesized by means of RT-PCR making use of mRNA extracted from the liver of sheep. Part of the region where the cDNA of the GDH of the ovine is codified was amplified by PCR from primers synthesized through the comparison of the aligned sequences of Ovis aries, Bos taurus, Homo sapiens, Rattus norvegicus and Mus musculus available in the database. The cDNA was cloned in the vector pGEM®-T Easy (Promega) and inserted in Escherichia coli DH10B calcium competent cells by heat shock procedure. The plasmid DNA was purified and after sequencing, the presence of 1292 pb was confirmed. The alignment of the sequence deduced of amino acid with other species revealed high homology among the GDHs.