An indirect ELISA for detecting anti-SARS-CoV-2 antibodies in human sera using a baculovirus-expressed recombinant nucleocapsid antigen.

This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.

Use of anvils by the Great Kiskadee (Aves, Tyrannidae): a description based on citizen science data.

The use of anvils to strike prey is a behavior observed in some species of birds around the world. Here, I investigated the use of anvils by the Great Kiskadee (Pitangus sulphuratus). The study was carried out through the analysis of citizen science photographs and comments made by the authors of these records. In the 365 records analyzed, vertebrates were the predominant prey (n = 213; 58.35%) and Hemidactylus mabouia was the most frequent species. Tree branches were the most used anvil category (n = 199; 54.52%), and in 12.87% of the photographic records the authors described in their comments the behavior of the bird beating the prey before feeding on it. The use of anvils allows birds to strike down different types of prey and expand their food niche. It, thus, favors the establishment of their populations. However, these relationships still require further investigation. By the observation and registration of birds in natural environments, citizen science has become an important research tool for ornithologists.

A novel picorna-like virus identified in the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil (CBW; Anthonomus grandis; Coleoptera: Curculionidae) is considered the major insect pest of cotton, causing considerable losses in yield and fiber quality. An increase in the boll weevil population due to increasingly inefficient chemical control measures is of great concernamong cotton producers. The absence of conventional or transgenic cultivars with minimal resistance to CBW has stimulated the search for new molecular and biological tools for efficient control of this insect pest. In this study, we used a metagenomic approach based on RNA deep sequencing to investigate the presence of viruses and coding viral RNA in apparently healthy native adult CBW insects collected from cotton crops in Mato Grosso state, Brazil. Using an Illumina HiSeq 2000 paired-end platform, 138,798 virus-related reads were obtained, and a consensus sequence of a putative new virus, 10,632 nucleotides in length, was assembled. The sequences of the 5' and 3' untranslated regions (UTRs) were determined by rapid amplification of cDNA ends (RACE), followed by Nanopore sequencing. The complete genome sequence included a 5'-UTR (1,158 nucleotides), a 3'-UTR (561 nucleotides), and a single ORF of 8,913 nucleotides encoding a large polyprotein. Sequence analysis of the putative polyprotein showed several regions with high sequence similarity to structural and non-structural proteins of viruses of the family Iflaviridae. Pairwise alignments of polyprotein amino acid sequences showed the highest sequence identity (32.13%) to a partial polyprotein sequence of a putative iflavirus (QKN89051.1) found in samples from wild zoo birds in China. Phylogenetic analysis based on full polyprotein sequences of different iflaviruses indicated that this new picorna-like virus is most closely related to iflaviruses found in lepidopteran insects, and it was therefore tentatively named "Anthonomus grandis iflavirus 1" (AgIV-1). This is, to our knowledge, the first complete viral genome sequence found in CBW, and it could provide a basis for further studies about the infectivity and transmission of this virus and its possible association with symptoms or acute disease. AgIV-1 could potentially be used to develop biological or molecular tools, such as a viral vector to carry interfering RNA molecules for CBW control.

Expression profiling of Clostridium thermocellum B8 during the deconstruction of sugarcane bagasse and straw.

The gram-positive bacterium Clostridium thermocellum contains a set of carbohydrate-active enzymes that can potentially be employed to generate high-value-added products from lignocellulose. In this study, the gene expression profiling of C. thermocellum B8 was provided during growth in the presence of sugarcane bagasse and straw as a carbon source in comparison to growth using microcrystalline cellulose. A total of 625 and 509 genes were up-regulated for growth in the presence of bagasse and straw, respectively. These genes were mainly grouped into carbohydrate-active enzymes (CAZymes), cell motility, chemotaxis, quorum sensing pathway and expression control of glycoside hydrolases. These results show that type of carbon source modulates the gene expression profiling of carbohydrate-active enzymes. In addition, highlight the importance of cell motility, attachment to the substrate and communication in deconstructing complex substrates. This present work may contribute to the development of enzymatic cocktails and industrial strains for biorefineries based on sugarcane residues as feedstock.

A time series analysis of detection and mortality of hepatitis C in Brazil, 2008-2018.

BACKGROUND: The 69th World Health Assembly approved the Global Health Sector Strategy to eliminate hepatitis C virus (HCV) infection by 2030. In Brazil, efforts have been undertaken to achieve this goal; there are, however, great challenges. It is important to understand the disease profile in different regions of the country in order to design strategies to fight the disease nationwide. The objective of this study was to analyse the time trend of the incidence and mortality of hepatitis C in Brazil during the period from 2008 to 2018 according to sociodemographic and clinical characteristics. METHODS: All newly diagnosed cases of hepatitis C reported between 2008 and 2018, in all regions of Brazil, were included. The indicators were obtained from the databases of the Brazilian Ministry of Health. For the time series analysis, a joinpoint regression model was used. RESULTS: Between 2008 and 2018, 136,759 newly diagnosed cases of hepatitis C were reported considering anti-HCV and HCV RNA positivity, and 271,624 newly diagnosed cases were reported considering one or another positive test. The majority of the records were concentrated in the Southeast (61%) and South (26.2%) Regions. The joinpoint regression model indicated an increasing trend in the detection rate of hepatitis C in Brazil, but there was a decreasing trend in the mortality rate during the period analysed. CONCLUSIONS: Differences were observed in the time trend of hepatitis C and in the sociodemographic and clinical characteristics in different regions of Brazil. These data can provide support to design strategies for the elimination of hepatitis C in Brazil, according to regional particularities.

Quantum mechanical, molecular docking, molecular dynamics, ADMET and antiproliferative activity on Trypanosoma cruzi (Y strain) of chalcone (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one derived from a natural product.

Chagas disease is a leading public health problem. More than 8 million people are affected by the disease, which is endemic in 21 countries in Latin America, generating an average annual cost of 7.2 billion dollars per year. The conventional treatment of Chagas disease is carried out by administering the drug benznidazole (BZN), which has caused numerous adverse reactions. Hence, the search for new, more efficient, and less toxic anti-chagasic agents is essential. Recently, chalcones have been researched to propose new therapies against neglected diseases, mainly Trypanosoma cruzi. The objective of this work was to evaluate for the first time the antiproliferative potential of chalcone derived from the natural product on T. cruzi strain Y. The molecular structure of the chalcone was confirmed by spectrometric data. The toxicity of chalcone in LLC-MK2 cells indicated that a concentration of 514.10 ± 62.40 µM was able to reduce cell viability by 50%. Regarding the effect of chalcone on epimastigote forms, an IC50 value of 46.57 ± 9.81 µM was observed; 45.92 ± 8.42 and 16.32 ± 3.41 µM at times of 24, 48 and 72 hours, respectively. The chalcone was able to eliminate trypomastigote forms at all concentrations tested, except for 31.25 µM, with LC50 values of 117.90 ± 12.60 µM, lower than the reference drug BZN (161.40 ± 31. 80 µM). The mechanism of action may be related to the membrane damage provoked by reduction of the mitochondrial potential. The anti-T. cruzi effect can be assigned through some structural aspects of the chalcone as the nitro group (NO2) is present, which can be enzymatically reduced forming a nitro radical, and the presence of methoxyl groups in the A ring of the chalcone. In silico studies showed that the chalcone had a higher affinity for cruzain when compared to BZN and the co-crystallized inhibitor KB2, as it presented a more thermodynamically stable complex in the order of -6.9 kcal mol-1. The pharmacokinetic prediction showed a significant probability of antiprotozoal activity, a good volume of distribution after being absorbed in the intestine, and a low chance of activity in the central nervous system. Therefore, these results suggest that the chalcone can become a potential cruzain enzyme inhibitor with trypanocidal activity.

Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature.

Asparaginases (ASNases) are a large and structurally diverse group of enzymes ubiquitous amongst archaea, bacteria and eukaryotes, that catalyze hydrolysis of asparagine to aspartate and ammonia. Bacterial ASNases are important biopharmaceuticals for the treatment of acute lymphoblastic leukemia, although some patients experience adverse allergic side effects during treatment with these protein therapeutics. ASNases are currently divided into three families: plant-type ASNases, Rhizobium etli-type ASNases and bacterial-type ASNases. This system is outdated as both bacterial-type and plant-type families also include archaeal, bacterial and eukaryotic enzymes, each with their own distinct characteristics. Herein, phylogenetic studies allied to tertiary structural analyses are described with the aim of proposing a revised and more robust classification system that considers the biochemical diversity of ASNases. Accordingly, based on distinct peptide domains, phylogenetic data, structural analysis and functional characteristics, we recommend that ASNases now be divided into three new distinct classes containing subgroups according to structural and functional aspects. Using this new classification scheme, 25 ASNases were identified as candidates for future new lead discovery.

Nutritional status, health risk behaviors, and eating habits are correlated with physical activity and exercise of brazilian older hypertensive adults: a cross-sectional study.

BACKGROUND: Nutritional status, health risk behaviors, eating habits, and other comorbidities (such as diabetes) may be associated with recommended amounts of physical activity (PA) and exercise (EX) in healthy older adults. However, these associations are still unclear for older hypertensive adults, who require greater care from health professionals. The purpose of this study was to associate the nutritional status, health risk behaviors, eating habits, and the presence of diabetes with recommended amounts of physical activity and exercise practice of older hypertensive adults. METHODS: Ten thousand seven hundred eighty-nine older hypertensive adults (70.9 ± 7.4 years) from the VIGITEL telephone survey were classified according to PA levels (insufficiently active/sufficiently active) and EX practice (non-practitioners/practitioners). Binary logistic regression was used to observe the odds ratio (OR) between independent variables (nutritional status [body mass index], sociodemographic characteristics [age/sex/years of study], risk behaviors [screen time/alcohol/tobacco consumption], eating habits [minimally/ultra-processed foods consumption score], and the presence of diabetes) with recommended amounts of PA/EX (dependent variable). RESULTS: Highest nutritional status (ORPA = 0.975 [95%-CI: 0.965 - 0.985]; OREX = 0.981[95%-CI: 0.972 - 0.991]), age (ORPA = 0.955 [95%-CI: 0.949 - 0.961]; OREX = 0.980[95%-CI: 0.975 - 0.986]), screen time (ORPA = 0.909[95%-CI: 0.835 - 0.990]), alcohol consumption (ORPA = 0.683[95%-CI: 0.621 - 0.758]; OREX = 0.702[95%-CI: 0.637 - 0.779]), tobacco (ORPA = 0.601 [95%-CI: 0.492 - 0.736]; OREX = 0.464[95%-CI: 0.384 - 0.562]) ultra-processed foods consumption score (ORPA = 0.896[95%-CI: 0.871 - 0.921]; OREX = 0.886[95%-CI: 0.863 - 0.909]) and having diabetes (ORPA = 0.780[95%-CI: 0.708 - 0.859]; OREX = 0.831[95%-CI: 0.759 - 0.909]) reduced the odds of being sufficiently active/practicing exercise (p < 0.05). Male sex (ORPA = 1.633[95%-CI: 1.491 - 1.789]; OREX = 1.247[95%-CI: 1.140 - 1.363]), years of study (ORPA = 1.026[95%-CI: 1.018 - 1.035]; OREX = 1.050[95%-CI: 1.041 - 1.058]), and minimally processed foods consumption score increased the odds of being sufficiently active/practicing exercise (ORPA = 1.132[95%-CI: 1.109 - 1.155]; OREX = 1.167[95%-CI: 1.145 - 1.191], respectively; p < 0.05). CONCLUSION: Nutritional status, health risk behaviors, eating habits, and the presence of diabetes were associated with the odds of older hypertensive adults complying with PA and EX recommendations. The results may help health professionals understand how these factors are associated with the changes of older hypertensive adults participating in physical activity and exercise.

Apparent thermal diffusivity of soil in ice-free areas of Keller peninsula in maritime Antarctica.

Heat transfer process in the soil active layer is important for the knowledge of its thermal properties linked with climate issues. The objective of this work was to analyze the energy flux in different soil profiles by estimating the apparent soil thermal diffusivity ($ATD$). The study was carried out in Keller Peninsula, located at King George Island in four different sites differing by soil characteristics, as well as vegetation coverage and landscape setting. The $ATD$ was estimated in function of the long-term hourly temperature records at different soil depths. In addition, we estimated the seasonal mean of the $ATD$ and the freezing $N$-factor. Results showed that $ATD$ values were smaller at shallow depths and increased with depth. The diffusivity values presented lower variability in colder conditions, especially at deeper soil layers. Water content was the main factor affecting soil thermal diffusivity at sites $1$ and $3$ (more than $70$ and $63\%$ of probability). At sites $3$ and $4$ lower $N$-factors were observed, suggesting higher snow pack and permafrost closer to the soil surface. Hence, positive $ATD$ appears in the summer due to thawing increases soil moisture, while negative $ATD$ appears during the freeze of the snow pack and precipitation.

High-sodium maltobionate production by immobilized Zymomonas mobilis cells in polyurethane.

The purpose of this study was the production of maltobionic acid, in the form of sodium maltobionate, by Z. mobilis cells immobilized in polyurethane. The in situ immobilized system (0.125-0.35 mm) was composed of 7 g polyol, 3.5 g isocyanate, 0.02 g silicone, and 7 g Z. mobilis cell, at the concentration of 210 g/L. The bioconversion of maltose to sodium maltobionate was performed with different cell concentrations (7.0-9.0 gimobilized/Lreaction_medium), temperature (30.54-47.46 °C), pH (5.55-7.25), and substrate concentration (0.7-1.3 mol/L). The stability of the immobilized system was evaluated for 24 h bioconversion cycles and storage of 6 months. The maximum concentration of sodium maltobionate was 648.61 mmol/L in 34.34 h process (8.5 gdry_cell/Lreaction_medium) at 39 °C and pH 6.30. The immobilized system showed stability for 19 successive operational cycles of 24 h bioconversion and 6 months of storage, at 4 °C or 22 °C.

Chikungunya virus produced by a persistently infected mosquito cell line comprises a shorter genome and is non-infectious to mammalian cells.

Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work, Aedes aegypti Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in Ae. albopictus C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the nsP3 gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.

Real-world data on neoadjuvant endocrine therapy in ER-positive/HER2-negative breast cancer.

PURPOSE: Neoadjuvant endocrine therapy (NET) has been shown to be effective in ER-positive/HER2-negative breast cancer in clinical trials. However, adoption in clinical practice is still limited. Real-world data may provide useful insights into effectiveness, toxicities and quality of care, potentially rendering clinical trial results to the real-world setting. Our purpose was to report real-world data of a cohort of postmenopausal patients submitted to NET. METHODS: This prospective cohort study evaluated 146 postmenopausal female patients with ER-positive/HER2-negative breast cancer treated with NET at three tertiary hospitals between 2016 and 2018. Clinicopathological information were collected prospectively. Preoperative Endocrine Prognostic Index (PEPI) score was calculated for tumors submitted to at least 16 weeks of NET. RESULTS: Median age was 67 years old, and 87.8% had stage I-II disease. Most tumors had histological grade II (76.1%). Median pretreatment Ki67 expression was 10%. Aromatase inhibitor was used in 99.5% of patients, and median treatment duration was 21.0 weeks. No tumor progressed during NET. Breast-conserving surgery was performed in the majority of patients (63.0%), as well as sentinel lymph-node biopsy (76.7%). Pathological complete response rate was 1.0%. 43 patients (29.5%) had PEPI score 0, and 26% had PEPI scores 4-5. Posttreatment Ki67 median expression was 3.0%, and only five tumors (3.4%) showed marked increase in Ki67 expression during treatment. Seven patients (4.8%) had HER2-positive residual disease, and were treated with adjuvant chemotherapy plus trastuzumab. CONCLUSIONS: Our real-world data shows that NET is effective and safe in postmenopausal patients with ER-positive/HER2-negative breast cancer. Postmenopausal status and low-risk luminal tumor features (luminal A-like) should be used as selection criteria to ensure the best results with NET.

Molecular characterization and sequence analysis of four Brazilian rice stripe necrosis virus isolates.

Rice (Oryza sativa L.) is an important food crop for humanity, being cultivated in tropical and temperate regions of the world. This study reports the nearly complete genome sequences of four Brazilian rice stripe necrosis virus (RSNV) isolates. The nucleotide sequences of the RNA1 and RNA2 genome segments of these Brazilian isolates were 96.5 to 99.9% identical, indicating their close phylogenetic relationship to each other. Phylogeny and recombination analysis indicated that the genome of one of the isolates consisted of RNA segments of different origins, suggesting that a reassortment event had occurred.

DPYSL3 modulates mitosis, migration, and epithelial-to-mesenchymal transition in claudin-low breast cancer.

A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (RNA/protein/phosphoprotein) expression outlier specific to the claudin-low (CLOW) subset of triple-negative breast cancers. A PubMed informatics tool indicated a paucity of data in the context of breast cancer, which further prioritized DPYSL3 for study. DPYSL3 knockdown in DPYSL3-positive ([Formula: see text]) CLOW cell lines demonstrated reduced proliferation, yet enhanced motility and increased expression of epithelial-to-mesenchymal transition (EMT) markers, suggesting that DPYSL3 is a multifunctional signaling modulator. Slower proliferation in DPYSL3-negative ([Formula: see text]) CLOW cells was associated with accumulation of multinucleated cells, indicating a mitotic defect that was associated with a collapse of the vimentin microfilament network and increased vimentin phosphorylation. DPYSL3 also suppressed the expression of EMT regulators SNAIL and TWIST and opposed p21 activated kinase 2 (PAK2)-dependent migration. However, these EMT regulators in turn induce DPYSL3 expression, suggesting that DPYSL3 participates in negative feedback on EMT. In conclusion, DPYSL3 expression identifies CLOW tumors that will be sensitive to approaches that promote vimentin phosphorylation during mitosis and inhibitors of PAK signaling during migration and EMT.

Residual malaria of Atlantic Forest systems and the influence of anopheline fauna.

In Brazil, the Amazon region comprises 99.5% of the reported malaria cases. However, another hotspot of the disease is the Atlantic Forest regions, with the sporadic occurrence of autochthonous human cases. In such context, this study sought to investigate the role of anopheline mosquitoes (Diptera: Culicidae) in the residual malaria transmission in Atlantic Forest areas. Two rural areas in the Espírito Santo state were the surveyed sites. Mosquitoes were captured using Shannon trap and CDC light traps and identified into species based on morphological characters. Ecological indexes (Shannon-Wiener diversity, Simpson's dominance, Pielou equability, and Sorensen similarity) were the tools used in the anopheline fauna characterization and comparison along with the two explored areas. The assessment of the sampling adequacy in the studied areas was possible through the generation of a species accumulation curve. A correlation test verified the influence of climatic variables on the anopheline species abundance. A total of 1471 female anopheline mosquitoes were collected from May 2019 to April 2020, representing 13 species. The species richness was higher in Valsugana Velha (hypo-endemic) than in Alto Caparaó (non-endemic). There was a significant variation in the species abundance between Valsugana Velha (n = 1438) and Alto Caparaó (n = 33). The most abundant species was Anopheles (Kerteszia) cruzii complex Dyar and Knab, 1908 representing 87% of the total anophelines collected. These results suggest that the Plasmodium spp. circulation in Brazilian Atlantic Forest areas occurs mainly due to the high frequency of Anopheles (K.) cruzii complex, considered the principal vector of simian and human malaria in the region.

The influence of distinct techniques of local dental anesthesia in 9- to 12-year-old children: randomized clinical trial on pain and anxiety.

OBJECTIVES: To evaluate pain, disruptive behavior, and anxiety in children undergoing different local dental anesthetic techniques. METHODS: This randomized/parallel clinical trial analyzed three groups of patients (9-12 years old) (n = 35) who received infiltrative anesthesia using conventional (CA), vibrational (VBA), and computer-controlled techniques (CCLAD). The outcomes were pain self-perception (Wong-Baker Faces Pain Rating Scale (WBF); Numerical Ranting Scale (NRS)), disruptive behavior (Face, Legg, Activity, Crying, Consolability Scale (FLACC)), anxiety (Corah's Dental Anxiety Scale; modified Venham Picture test (VPTm)), and physiological parameters (systolic (SBP)/diastolic pressure (DBP); heart rate (HR); oxygen saturation (SpO2); respiratory rate (RR)). Statistical analysis was accomplished using Kruskall-Wallis test and ANOVA for repeated measures (α = 0.05). RESULTS: Dental anxiety levels at the baseline were similar for all patients. CA promoted less pain than VBA in WBF (p = 0.018) and NRS (p = 0.006) and CCLAD in WBF (p = 0.029). There were no differences in disruptive behavior (FLACC p = 0.573), anxiety (VPTm p = 0.474), blood pressure (SBP p = 0.954; DBP p = 0.899), heart rate (p = 0.726), oxygen saturation (p = 0.477), and respiratory rate (p = 0.930) between anesthetic techniques. CONCLUSION: Conventional technique resulted in less pain perception for dental local anesthesia. CLINICAL RELEVANCE: Conventional technique reduces the self-reported pain in children 9-12 years old, and therefore, the use of additional devices or different anesthetic techniques is not justified.

Understanding the dairy production systems in rural settlements in the Brazilian semi-arid region: characterization, typology, and holistic perception.

The objective of this study was to characterize and typify dairy farms and to evaluate society's perception of dairy production in rural settlements in the Brazilian semi-arid region. A questionnaire containing questions related to socioeconomic, livestock, physical, herd management, and production indicators was applied to 17 milk farmers for typological analysis. A second questionnaire was subsequently applied to the same 17 farmers for the holistic perception of dairy farming in rural settlements, in addition to 47 technicians in the area of animal science and 140 laypeople. Milk samples were collected in the settlement's community cooling tank, while the water used for milking management was collected in the milking parlor at the 17 farms. Multivariate analysis techniques and thematic analysis were performed. Three dairy production systems were identified: low production group (0-25 L), medium production group (25-50 L), and high production group (50-100 L). The physicochemical properties (composition, density, and cryoscopy) of milk attended the Brazilian regulation values. The microbiological parameters of milk and water used for milking are at odds with regulations. Regarding the typology, the indicators, livestock, and productivity showed discriminatory power (P < 0.05) between the productive systems. The responses from the segments of society showed different views on dairy production in rural settlements. It is concluded that the dairy farmers have low education and the cleaning product and water used in milking are out of regulation. The livestock and production indicators typified mainly dairy farms, while the holistic perception showed a lack of efficiency in rural extension, ignorance of the potential of the productive system. Finally, the creation of public policies focusing on the development of the milk production system in settlements is recommended.

Identification of ribosomal sites and karyotype analysis in Festuca ulochaeta Steud. and Festuca fimbriata Ness., grasses native to Brazil.

Festuca L. has more than 600 perennial species described, which makes it the largest genus within the family Poaceae. In Brazil, only two native species of Festuca have been described, for which cytogenetic studies need to be strengthened: Festuca ulochaeta and Festuca fimbriata. The aim of this study was to characterize the karyotypes of F. ulochaeta and F. fimbriata based on the mapping of rDNA sites. The FISH was performed with 35S and 5S rDNA probes. Both species have 42 chromosomes, of which 36 were metacentric and six were submetacentric. Festuca fimbriata has two pairs of 35S rDNA sites, one located on the metacentric pair 4, in an interstitial position, and one at the submetacentric pair 14 in the proximal position. Festuca ulochaeta has one pair of 35S rDNA in interstitial-proximal position in the metacentric pair 3. Both species showed 5S rDNA sites only on chromosome pair 21 in the terminal position of the short arm. The analysis of the chromosomal characteristics indicates that these species have a symmetrical karyotype and allopolyploid origin.

Dengue and Zika virus multi-epitope antigen expression in insect cells.

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients' sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

Evaluation of pain, disruptive behaviour and anxiety in children aging 5-8 years old undergoing different modalities of local anaesthetic injection for dental treatment: a randomised clinical trial.

Objective: To evaluate the influence of different local anaesthetic techniques in pain, disruptive behaviour and anxiety in children´s dental treatment.Material and methods: This was a randomised and parallel clinical trial. The sample consisted of 105 children (5-8 years old) that were divided into three groups (n = 35) according to the anaesthetic technique: conventional anaesthesia (CA); vibrational anaesthesia (VBA); computer-controlled local anaesthesia delivery (CCLAD). The outcomes were self-perception of pain (Wong-Baker Faces Pain Rating Scale - WBF; Numerical Rating Scale - NRS); disruptive behaviour (Face, Legg, Activity, Cry, Consolability Scale - FLACC); anxiety (Corah's Dental Anxiety Scale; modified Venham Picture test - VPTm) and physiological parameters (blood pressure - systolic - SBP and diastolic - DBP; heart rate - HR; oxygen saturation - SpO2; respiratory rate - RR). Data were statistically analysed with Kruskall-Wallis test and ANOVA for repeated measures with Tukey post hoc test (α = 0.05).Results: All the patients exhibited the same level of dental anxiety at baseline (Corah's Dental Anxiety Scale). There was no difference in self-perception pain, irrespective the evaluation tool used (WBF - p = .864; VAS - p = .761). No differences were detected in disruptive behaviour (FLACC - p = .318); anxiety (VPTm - p = .274); blood pressure (SBP - p = .239; DBP - p = .512); heart rate (p = .728); oxygen saturation (p = .348) and respiratory rate (p = .238) between anaesthetic techniques.Conclusion: Different anaesthetic dental local techniques do not affect the levels of pain, disruptive behaviour, anxiety and physiological parameters in children aged 5-8 years old.