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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.

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.

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.

A species’ diet is highly dependent on the availability of food resources in space and time, as well as on intrinsic factors such as sex or age. Accurate assessments of variations in the diet composition of bird populations across spatial scales, seasons and demographic groups are essential not only for understanding the basic ecology of species, but also for the conservation of endangered ones. However, our current knowledge about how birds’ diet change according to spatio-temporal variations or intrinsic factors is very limited. Here, we used a multi-marker metabarcoding approach to characterize the diet of a declining shrub-steppe passerine, the Dupont’s Lark (Chersophilus duponti), throughout a large part of its global distribution range. We also investigated spatial, phenological and sexual variations in its diet. Using markers from two genomic regions (18S and COI), we analyzed fecal samples from 303 adult Dupont’s larks from Morocco and Spain during the breeding and non-breeding seasons. Overall, arthropods from the orders Coleoptera, Lepidoptera, Julida and Orthoptera were the main prey consumed by Dupont’s Larks. We found that Dupont’s Lark diet varied spatially, as well as temporally, reflecting dietary plasticity in response to changes in prey availability across landscapes and the species’ phenological periods. High dietary overlap and no differences between sexes were observed, suggesting similar foraging behavior and nutritional requirements in both sexes. This is the first study providing detailed information on Dupont’s Lark food ecology over much of its distribution, which is fundamental for the management and conservation of this declining steppe species.

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.

Upon infection, Mycobacterium leprae , an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae -containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae . We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3β-hydroxysteroid dehydrogenase (3β-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae . The 3β-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3β-HSD activity with the 17β-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3β-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

Treating sarcopenia in older individuals remains a challenge, and nutritional interventions present promising approaches in individuals that perform limited physical exercise. We assessed the efficacy of leucine administration to evaluate whether the regular intake of this essential amino acid can improve muscle mass, muscle strength and functional performance and respiratory muscle function in institutionalized older individuals. The study was a placebo-controlled, randomized, double-blind design in fifty participants aged 65 and over (ClinicalTrials.gov identifier NCT03831399). The participants were randomized to a parallel group intervention of 13 weeks’ duration with a daily intake of leucine (6 g/day) or placebo (lactose, 6 g/day). The primary outcome was to study the effect on sarcopenia and respiratory muscle function. The secondary outcomes were changes in the geriatric evaluation scales, such as cognitive function, functional impairment and nutritional assessments. We also evaluated whether leucine administration alters blood analytical parameters and inflammatory markers. Administration of leucine was well-tolerated and significantly improves some criteria of sarcopenia in elderly individuals such as functional performance measured by walking time (p = 0.011), and improved lean mass index. For respiratory muscle function, the leucine-treated group improved significantly (p = 0.026) in maximum static expiratory force compared to the placebo. No significant effects on functional impairment, cognitive function or nutritional assessment, inflammatory cytokines IL-6, TNF-alpha were observed after leucine administration compared to the placebo. The use of l-leucine supplementation can have some beneficial effects on sarcopenia and could be considered for the treatment of sarcopenia in older individuals.

Background/Objectives: Oreganum vulgare essential oil (OEO) is safe, effective, multifunctional, and widely used. This study aimed to evaluate OEO’s chemical composition and antimicrobial activity in vitro against S. aureus and E. coli. Methods: The composition of OEO was determined by gas chromatography–mass spectrometry (GC-MS). Results: Compounds included monoterpenes with known antimicrobial activity, such as 2-menthen-1-ol (36.33%), linalyl acetate (9.26%), terpinene-4-ol (9.01%), 4-thujanol (6.33%), menthen (5.81%), sabinene (5.18%), and carvacrol methyl ether (5.14%). Conclusions: OEO had a strong antimicrobial activity with a minimum inhibitory concentration (MIC) of 1.90 mg/mL for S. aureus and 0.49 mg/mL for E. coli after 18 h incubation. The minimum bactericidal concentration (MBC) was 7.9 mg/mL against S. aureus and 0.99 mg/mL against E. coli. Thus, OEO could be used as a natural antimicrobial against S. aureus and E. coli infections.

It has been suggested that menstrual blood-derived mesenchymal stem/stromal cells (MenMSCs) are associated with the etiopathogenesis of endometriosis and considerable effort has been invested in searching for target genes and deciphering associated molecular pathways. However, reference gene stability for proper reproducible normalization in the analyses of the expression data validation is still unexplored in this experimental context. Therefore, in this exploratory study, we used stringent case and control selection criteria and collected menstrual blood from women with a laparoscopic diagnosis of advanced endometriosis and from fertile women without endometriosis. We tested for the first time the stability of 32 candidate reference genes to achieve increased accuracy and reliable results in the quantification of gene expression and direct future experiments using reverse transcription-quantitative PCR (RT-qPCR) in MenMSCs for endometriosis studies. Using the RefFinder web tool, we recommend the EIF2B1 and POP4 reference genes for the normalization of RT-qPCR data in study designs similar to ours. Furthermore, we suggest avoiding the commonly used GAPDH and ACTB reference genes as they are unstable. This high-visibility study is capable of directing different experimental designs as MenMSCs are derived from a minimally invasive tissue source with multifunctional roles in regenerative medicine.