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An unprecedented outbreak of rabies occurred in Rio Grande do Sul state (RS) from 2012 onward, resulting in thousands of bovine deaths, important economic losses, and posing risk to human health. This article describes a genetic analysis of 145 rabies viruses (RABV) recovered from herbivorous from RS between 2012 and 2017, based on partial sequence analysis of the nucleoprotein (N) gene. High nucleotide ( nt ) identity (95.5 to 100%) and amino acid ( aa ) similarity (96.7 to 100%) were observed among the analyzed sequences. These sequences displayed a high sequence nt identity/ aa similarity with bovine RABV sequences (96.4–97.9%; 98.1–100%, respectively) and vampire bat RABV sequences (96.3–97.5%; 97.8–99.5%). Phylogenetic analyzes based on the N sequence allowed for the segregation of viruses into two distinct clusters . Cluster 1 comprised RABV sequences covering the whole studied period, whereas cluster 2 grouped a lower number of viruses from 2013, 2014, 2015, to 2017. In some cases, viruses obtained from the same region within a short period of time grouped to distinct clusters or sub-clusters, indicating the co-circulation of distinct virus lineages in these outbreaks. The segregation into sub-clusters was also observed for viral sequences obtained from the same region at different times, indicating the involvement of distinct viruses. In summary, partial sequence analyses revealed a high conservation of N protein and the circulation of two lineages and different sublineages of RABV in the region. In addition, our results confirm the suitability of N gene to study the genetic relationships among RABV isolates.

Discussion section, 9th paragraph.

Bats and dogs are the main reservoirs of rabies virus (RABV) in Latin America and are responsible for the maintenance of different cycles of infection. In the two neighbour and most southern Brazilian states of Rio Grande do Sul (RS) and Santa Catarina (SC), rabies in dogs has been successfully controlled for more than 30 years. However, rabies associated to the rural cycle remains endemic, with a significant, though oscillating—annual incidence of rabies in cattle. Despite the plethora of studies on genetic analyses of Brazilian RABV, isolates from southern Brazil have only scarcely been investigated. This work was performed to identify the genetic lineages of RABVs circulating in states of RS and SC. Fifty-nine RABV cattle isolates from RS and SC were selected and submitted to reverse transcription/polymerase chain reaction (RT-PCR) followed by sequencing of the nucleoprotein gene. In RS, the circulation of two sublineages (1A and 1B) of RABV was detected, both with characteristics of lineages usually detected in vampire bats (Desmodus rotundus). In SC, only one sublineage of RABV (1B) was detected. Nevertheless, the findings reported here are expected to contribute to the understanding of the biology of the virus in the region and its interactions with the natural host D. rotundus.

Background L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this “L” enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana . Results Plasmids were constructed and modified such that the cloned plant genes were targeted to the K. lactis LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis, of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30 mg.L -1 of L-ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose. Conclusions This work is the first attempt to engineer K. lactis cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of “L” isomers precursors in the culture medium. We have engineered K. lactis strains capable of converting lactose and D-galactose into L-galactose, by the integration of the genes from the A. thaliana L-galactose pathway. L-galactose is a rare sugar, which is one of the main precursors for L-AA production.

AbstractObjectiveTo determine whether tocilizumab improves clinical outcomes for patients with severe or critical coronavirus disease 2019 (covid-19).DesignRandomised, open label trial.SettingNine hospitals in Brazil, 8 May to 17 July 2020.ParticipantsAdults with confirmed covid-19 who were receiving supplemental oxygen or mechanical ventilation and had abnormal levels of at least two serum biomarkers (C reactive protein, D dimer, lactate dehydrogenase, or ferritin). The data monitoring committee recommended stopping the trial early, after 129 patients had been enrolled, because of an increased number of deaths at 15 days in the tocilizumab group.InterventionsTocilizumab (single intravenous infusion of 8 mg/kg) plus standard care (n=65) versus standard care alone (n=64).Main outcome measureThe primary outcome, clinical status measured at 15 days using a seven level ordinal scale, was analysed as a composite of death or mechanical ventilation because the assumption of odds proportionality was not met.ResultsA total of 129 patients were enrolled (mean age 57 (SD 14) years; 68% men) and all completed follow-up. All patients in the tocilizumab group and two in the standard care group received tocilizumab. 18 of 65 (28%) patients in the tocilizumab group and 13 of 64 (20%) in the standard care group were receiving mechanical ventilation or died at day 15 (odds ratio 1.54, 95% confidence interval 0.66 to 3.66; P=0.32). Death at 15 days occurred in 11 (17%) patients in the tocilizumab group compared with 2 (3%) in the standard care group (odds ratio 6.42, 95% confidence interval 1.59 to 43.2). Adverse events were reported in 29 of 67 (43%) patients who received tocilizumab and 21 of 62 (34%) who did not receive tocilizumab.ConclusionsIn patients with severe or critical covid-19, tocilizumab plus standard care was not superior to standard care alone in improving clinical outcomes at 15 days, and it might increase mortality.Trial registrationClinicalTrials.gov NCT04403685.

This study aimed to (i) synthesize and characterize magnetic poly(styrene-co-ethylene glycol dimethacrylate) particles (STY-EGDMA-M) as immobilization support for Candida antarctica lipase B (CALB), (ii) determine kinetic and thermodynamic parameters of the immobilized biocatalyst in emollient ester synthesis, and (iii) develop a mathematical model for the bioprocess. The enzyme support was characterized by optical microscopy, hysteresis analysis, Fourier-transform infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. Magnetic copolymer particles were found to have spherical shape, ideal for use as immobilization support. The results confirmed that magnetic particles were incorporated into the polymer matrix, whose saturation magnetization was about 7 emu g−1. Immobilized CALB showed an activity of 570 ± 48 U g−1 in the hydrolysis of butyl butyrate. The derivative was used to synthesize octyl oleate by esterification of oleic acid with octanol. The influence of temperature on the esterification reaction was analyzed, and a mathematical model was developed to determine apparent kinetic and thermodynamic parameters. The highest ester conversion (56%) was obtained at 55 °C, and chemical equilibrium was achieved in 12 h of reaction. Under these conditions, the reaction was spontaneous, with negative Gibbs energy (− 4.73 kJ mol−1 K−1), positive entropy (+ 0.463 kJ mol−1 K−1), endothermic nature (+ 147.53 kJ mol−1), and low activation energy (61.71 kJ mol−1). A second-order reversible kinetic model provided a satisfactory fit to the data (R2 = 0.9965), within the 95% confidence interval. In an operational stability test, conducted for five consecutive cycles of 24 h, the biocatalyst had a half-life of 315.07 h.

Given the importance of menstrual blood in the pathogenesis of endometriosis and the multifunctional roles of menstrual mesenchymal stem cells (MenSCs) in regenerative medicine, this issue has gained prominence in the scientific community. Moreover, recent reviews highlight how robust the integrated assessment of omics data are for endometriosis. To our knowledge, no study has applied the multi-omics approaches to endometriosis MenSCs. This is a case-control study at a university-affiliated hospital. MenSCs transcriptome and proteome data were obtained by RNA-seq and UHPLC-MS/MS detection. Among the differentially expressed proteins and genes, we emphasize ATF3, ID1, ID3, FOSB, SNAI1, NR4A1, EGR1, LAMC3, and ZFP36 genes and MT2A, TYMP, COL1A1, COL6A2, and NID2 proteins that were already reported in the endometriosis. Our functional enrichment analysis reveals integrated modulating signaling pathways such as epithelial–mesenchymal transition (↑) and PI3K signaling via AKT to mTORC1 (↓ in proteome), mTORC1 signaling, TGF beta signaling, TNFA signaling via NFkB, IL6 STAT3 signaling, and response to hypoxia via HIF1A targets (↑ in transcriptome). Our findings highlight primary changes in the endometriosis MenSCs, suggesting that the chronic inflammatory endometrial microenvironment can modulate these cells, providing opportunities for endometriosis etiopathogenesis. Moreover, they identify challenges for future research leveraging knowledge for regenerative and precision medicine in endometriosis.

The Late Triassic (Carnian) upper Santa Maria Formation of south Brazil has yielded some of the oldest unequivocal records of dinosaurs. Here, we describe a new saurischian dinosaur from this formation, Nhandumirim waldsangae, gen. et sp. nov., based on a semiarticulated skeleton, including trunk, sacral, and caudal vertebrae, one chevron, right ilium, femur, partial tibia, fibula, and metatarsals II and IV, as well as ungual and non-ungual phalanges. The new taxon differs from all other Carnian dinosauromorphs through a unique combination of characters, some of which are autapomorphic: caudal centra with sharp longitudinal ventral keels; brevis fossa extending for less than three-quarters of the ventral surface of the postacetabular ala of the ilium; dorsolateral trochanter ending well distal to the level of the femoral head; distal part of the tibia with a mediolaterally extending tuberosity on its cranial surface and a tabular caudolateral flange; conspicuous, craniomedially oriented semicircular articular facet on the distal fibula; and a straight metatarsal IV. This clearly distinguishes Nhandumirim waldsangae from both Saturnalia tupiniquim and Staurikosaurus pricei, which were collected nearby and at a similar stratigraphic level. Despite not being fully grown, the differences between Nhandumirim waldsangae and those saurischians cannot be attributed to ontogeny. The phylogenetic position of Nhandumirim waldsangae suggests that it represents one of the earliest members of Theropoda. Nhandumirim waldsangae shows that some typical theropod characters were already present early in dinosaur evolution, and it represents possibly the oldest record of the group known in Brazil.

Polycyclic aromatic hydrocarbons (PAHs) play a central role in materials science due to their extended π-conjugated systems, with their stability and reactivity depending critically on their aromatic character. In this work, we systematically investigated the aromaticity and stability of a broad range of linear (acenes, phenacenes, biphenylenes, and cyclobuta-acenes) and belt-like (cyclacenes, cyclophenacenes, and cyclobiphenylenes) PAHs containing five to twelve benzene rings. A diverse set of aromaticity descriptors was employed, including geometric (HOMA), electronic (MCI, FLU) and magnetic (NICS) descriptors, plus the recently developed Q2 indices, based on the components of the distributed multipole analysis (DMA) electric quadrupole tensor. These data were complemented by stability analyses using singlet–triplet energy splitting (ΔES–T) and fractional occupation number-weighted densities (NFOD) values. Our results indicate that acenes and phenacenes follow a comparable aromatic trend, with inner rings possessing lower aromaticity and the edge rings showing a more pronounced aromatic character. A subtle difference is observed in the position of the most aromatic ring, which lies slightly closer to the interior in acenes. Phenacenes, however, exhibit greater overall stability, attributed to their armchair edges. For biphenylenes and cyclobuta-acenes, the antiaromatic cyclobutadiene moiety perturbs the aromaticity only in its direct neighborhood and preserves the aromaticity in the remaining chains. In belt-like systems, cyclacenes exhibit strong radical character and low stability, consistent with longstanding synthetic challenges, whereas cyclophenacenes display enhanced aromaticity and stability with extending size. Cyclobiphenylenes combine localized antiaromatic centers with preserved benzene-like aromaticity in rings distant from the cyclobutadiene unit.

The estrogen-metabolizing activities of cytochrome P450 (CYP) enzymes have been implicated in endometriosis. However, their regulation in various sources of endometrial tissue under different hormonal conditions has not been clarified. Our objective was to study the hormone regulation of a specific CYP enzyme, namely CYP3A4, in control ( n  = 15) and endometriosis patients ( n  = 42). To this end, we evaluated mRNA expression (using real-time PCR) of CYP3A4 in tissue samples classified according to the phase of menstrual cycle at which they were obtained as confirmed by the related circulating hormone levels. Protein expression was also evaluated by Western Blot. In order to further investigate the hormonal regulation of CYP3A4, stromal cells from ovarian endometriotic lesions were cultured with the prevailing hormones of the distinct phases of the menstrual cycle. We observed that all control and endometriosis tissues express CYP3A4. Nevertheless, changes in CYP3A4 gene expression related to cycle phase were only seen in the control eutopic endometrium and not in samples from endometriosis patients, with an increase in the luteal phase. Stromal cells isolated from ovarian endometriotic lesions expressed CYP3A4 and their exposure to luteal phase-mimicking hormones (estradiol + progesterone) reduced CYP3A4 mRNA in parallel with a diminished expression of the corresponding receptors, estrogen receptor alpha and progesterone receptor. Our findings suggest that steroid hormones are able to regulate CYP3A4 mRNA expression, although the circulating levels of these hormones can only regulate control endometrium and not endometriosis tissues, probably because of dysregulated local steroid concentration in these latter samples.