Leprosy in children under fifteen years of age in the most hyperendemic municipality in Brazil / Hanseníase em menores de quinze anos no município mais hiperendêmico do Brasil

ABSTRACT Objective: To describe leprosy involvement and physical disability profiles in children and adolescents under 15 years old. Methods: Ecological time series study, based on data from the Brazilian Notifiable Diseases Information System, including new cases of leprosy residing in Palmas (TO), from 2001 to 2020. Results: A total of 471 notified cases in children and adolescents under 15 years of age were evaluated, resulting in a detection coefficient of 26.5 per 100,000 inhabitants. Of these, 52% (n=243) were women, 5% (n=24) corresponded to grade two disability, and 36% (n=168) were diagnosed through spontaneous demand. The temporal trend analysis showed a 0.5% reduction in the detection coefficient. There was a significant decrease in the diagnosis of the undetermined and tuberculoid clinical forms and a significant increase in the dimorphous form. Diagnosis through contact examination increased significantly by 13.1% and that through spontaneous demand decreased by 4.9%. The detection coefficient of cases with grade two disability reduced significantly by 7.4% while those with grade one increased by 16.8%. Conclusions: Despite the downward trend in the detection coefficient in children and adolescents under 15 years of age and in cases with grade two disability, other factors indicate failure in the adequate management of leprosy in Palmas.

RESUMO Objetivo: Descrever os perfis de acometimento de hanseníase e incapacidade física em menores de 15 anos. Métodos: Estudo ecológico de série temporal, baseado em dados do Sistema Nacional de Agravos de Notificação, incluindo casos novos de hanseníase residentes em Palmas (TO), no período de 2001 a 2020. Resultados: Foram avaliados 471 casos notificados em crianças e adolescentes menores de 15 anos, resultando em um coeficiente de detecção de 26,5 por cem mil habitantes. Destes, 52% (n=243) eram do gênero feminino, 5% (n=24) correspondiam ao grau dois de incapacidade física, e 36% (n=168) foram diagnosticados por demanda espontânea. A análise de tendência temporal mostrou queda do coeficiente de detecção em 0,5%. Houve queda significativa no diagnóstico das formas clínicas indeterminada e tuberculoide e aumento significativo da dimorfa. O diagnóstico por exame de contato teve um aumento significativo de 13,1% e o por demanda espontânea, queda significativa de 4,9%. O coeficiente de detecção de casos com grau dois de incapacidade apresentou uma queda significativa de 7,4%, enquanto o de casos com grau um, apresentou um aumento de 16,8%. Conclusões: Apesar da tendência de queda do coeficiente de detecção em menores de 15 anos e do coeficiente de detecção de casos com grau dois de incapacidade, outros fatores indicam falha no manejo adequado da hanseníase em Palmas.

Ethanol stress responses of Kluyveromyces marxianus CCT 7735 revealed by proteomic and metabolomic analyses.

Kluyveromyces marxianus CCT 7735 offers advantages to ethanol production over Saccharomyces cerevisiae, including thermotolerance and the ability to convert lactose to ethanol. However, its growth is impaired at high ethanol concentrations. Herein we report on the protein and intracellular metabolite profiles of K. marxianus at 1 and 4 h under ethanol exposure. The concentration of some amino acids, trehalose and ergosterol were also measured. We observed that proteins and metabolites from carbon pathways and translation were less abundant, mainly at 4 h of ethanol stress. Nevertheless, the concentration of some amino acids and trehalose increased at 8 and 12 h under ethanol stress, indicating an adaptive response. Moreover, our results show that the abundance of proteins and metabolites related to the oxidative stresses responses increased. The results obtained in this study provide insights into understanding the physiological changes in K. marxianus under ethanol stress, indicating possible targets for ethanol tolerant strains construction.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress.

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure.

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes ß-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three ß-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).

The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 µg mL(-1) Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.

Metabolic engineering of Kluyveromyces lactis for L-ascorbic acid (vitamin C) biosynthesis.

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.