Genetic basis for hyper production of hyaluronic acid in natural and engineered microorganisms

Hyaluronic acid, or HA, is a rigid and linear biopolymer belonging to the class of the glycosaminoglycans, and composed of repeating units of the monosaccharides glucuronic acid and N-acetylglucosamine. HA has multiple important functions in the human body, due to its properties such as bio compatibility, lubricity and hydrophilicity, it is widely applied in the biomedical, food, health and cosmetic fields. The growing interest in this molecule has motivated the discovery of new ways of obtaining it. Traditionally, HA has been extracted from rooster comb-like animal tissues. However, due to legislation laws HA is now being produced by bacterial fermentation using Streptococcus zooepidemicus, a natural producer of HA, despite it being a pathogenic microorganism. With the expansion of new genetic engineering technologies, the use of organisms that are non-natural producers of HA has also made it possible to obtain such a polymer. Most of the published reviews have focused on HA formulation and its effects on different body tissues, whereas very few of them describe the microbial basis of HA production. Therefore, for the first time this review has compiled the molecular and genetic bases for natural HA production in microorganisms together with the main strategies employed for heterologous production of HA

Physiological and proteomic analyses of Saccharum spp. grown under salt stress

Sugarcane (Saccharum spp.) is the world most productive sugar producing crop, making an understanding of its stress physiology key to increasing both sugar and ethanol production. To understand the behavior and salt tolerance mechanisms of sugarcane, two cultivars commonly used in Brazilian agriculture, RB867515 and RB855536, were submitted to salt stress for 48 days. Physiological parameters including net photosynthesis, water potential, dry root and shoot mass and malondialdehyde (MDA) content of leaves were determined. Control plants of the two cultivars showed similar values for most traits apart from higher root dry mass in RB867515. Both cultivars behaved similarly during salt stress, except for MDA levels for which there was a delay in the response for cultivar RB867515. Analysis of leaf macro- and micronutrients concentrations was performed and the concentration of Mn2+ increased on day 48 for both cultivars. In parallel, to observe the effects of salt stress on protein levels in leaves of the RB867515 cultivar, two-dimensional gel electrophoresis followed by MS analysis was performed. Four proteins were differentially expressed between control and salt-treated plants. Fructose 1,6-bisphosphate aldolase was down-regulated, a germin-like protein and glyceraldehyde 3 phosphate dehydrogenase showed increased expression levels under salt stress, and heat-shock protein 70 was expressed only in salt-treated plants. These proteins are involved in energy metabolism and defense-related responses and we suggest that they may be involved in protection mechanisms against salt stress in sugarcane

Novel homologous lactate transporter improves l‑lactic acid production from glycerol in recombinant strains of Pichia pastoris

Background: Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous l-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for l-lactic acid production. Results: Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. Conclusions: We showed that P. pastoris has a great potential as a fermentative organism for producing l-lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half

Host-defense peptides AC12, DK16 and RC11 with immunomodulatory activity isolated from Hypsiboas raniceps skin secretion

Inflammation is a natural defense mechanism of the immune system; however, when unregulated, it can lead to chronic illness. Glucocorticoids are the most commonly used agents to effectively treat inflammatory conditions, including autoimmune diseases, however these substances can trigger a number of side effects. Thus, viable alternatives to the use of these drugs would be advantageous. In this study, we have analyzed the anti-inflammatory profile of three synthetic peptides first identified in skin secretion of the tree frog Hypsiboas raniceps. Structural characterization was performed using NMR spectroscopy and Mass Spectrometry, and the peptides were tested in vitro in RAW 264.7 cells and in vivo in Balb/c mice for their functional properties. The samples did not show a significant antimicrobial profile. NMR spectroscopy indicated that AC12 (ACFLTRLGTYVC) has a disulfide bond between C2 and C11 and a β-sheet-turn-β-sheet conformation in aqueous solution. This peptide showed no cytotoxic effect in mammalian cells and it was the most effective in reducing anti-inflammatory markers, such as NO, TNF-α and IL-12. Peptide DK16 (DKERPICSNTFRGRKC) demonstrated anti-inflammatory properties in vitro, while RC11 (RCFRRRGKLTC) significantly altered the cell viability in RAW 264.7 but was shown to be safe in Balb/c erythrocytes. Our results indicate that, of the three peptides studied, AC12 is the most efficient in reducing anti-inflammatory markers, and it could be a potential agent for the treatment of inflammatory diseases.publishe

ASPECTOS GENÉTICOS DA DISTROFIA ENDOTELIAL DE FUCHS

Fuchs Endothelial Dystrophy (FED) is an inherited corneal disease characterized by progressive degeneration of the corneal endothelium, leading to edema and loss of visual function. Although the exact pathogenesis is not yet fully elucidated, increasing evidence suggests a significant genetic component in susceptibility to DEF. Previous studies have identified several genetic variants associated with the disease, highlighting the complexity of genetic inheritance and the possible interaction between different genetic and environmental factors. Objectives: Investigate the association between specific genetic variations and the predisposition to Fuchs Endothelial Dystrophy (FED), aiming to identify genetic markers that can contribute to early diagnosis and the development of new therapeutic strategies. Methodology: The research was carried out through online access to the National Library of Medicine (MEDLINE), Scientific Electronic Library Online (SCIELO), Latin American and Caribbean Literature in Health Sciences (LILACS) databases in June 2024. Results and Discussions:  Significant associations between specific genetic variants and predisposition to Fuchs Endothelial Dystrophy (FED). In particular, we identified that variants in genes related to the structure and function of the corneal endothelium, such as TCF4 and COL8A2, are strongly associated with the disease. These findings corroborate previous studies and suggest that the integrity of the corneal endothelium may be influenced by specific genetic alterations. Furthermore, analysis of genetic interactions revealed possible networks of genes that can modulate the FED phenotype, highlighting the genetic complexity underlying the disease. These interactions may provide important insights into the molecular mechanisms involved in the pathogenesis of DEF and guide the development of targeted therapies. Conclusion: In summary, this study highlights the importance of genetic variants, especially in genes such as TCF4 and COL8A2, in the predisposition to Fuchs Endothelial Dystrophy (FED). The identification of these genetic markers offers perspectives for the development of more accurate diagnostic strategies and potential targeted therapeutic targets.A Distrofia Endotelial de Fuchs (DEF) é uma doença corneana hereditária caracterizada pela degeneração progressiva do endotélio corneano, levando a edema e perda da função visual. Embora a patogênese exata ainda não esteja completamente elucidada, evidências crescentes sugerem um componente genético significativo na suscetibilidade à DEF. Estudos anteriores identificaram várias variantes genéticas associadas à doença, destacando a complexidade da herança genética e a possível interação entre diferentes fatores genéticos e ambientais. Objetivos: Investigar a associação entre variações genéticas específicas e a predisposição à Distrofia Endotelial de Fuchs (DEF), visando identificar marcadores genéticos que possam contribuir para o diagnóstico precoce e o desenvolvimento de novas estratégias terapêuticas. Metodologia: A pesquisa foi realizada através do acesso online nas bases de dados National Library of Medicine (MEDLINE), Scientific Electronic Library Online (SCIELO), Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS) no mês de junho de 2024. Resultados e Discussões: Associações significativas entre variantes genéticas específicas e a predisposição à Distrofia Endotelial de Fuchs (DEF). Em particular, identificamos que variantes em genes relacionados à estrutura e função do endotélio corneano, como TCF4 e COL8A2, estão fortemente associadas com a doença. Essas descobertas corroboram estudos anteriores e sugerem que a integridade do endotélio corneano pode ser influenciada por alterações genéticas específicas. Além disso, a análise de interações genéticas revelou possíveis redes de genes que podem modular o fenótipo da DEF, destacando a complexidade genética subjacente à doença. Essas interações podem oferecer insights importantes sobre os mecanismos moleculares envolvidos na patogênese da DEF e orientar o desenvolvimento de terapias direcionadas. Conclusão: Em síntese, este estudo destaca a importância das variantes genéticas, especialmente em genes como TCF4 e COL8A2, na predisposição à Distrofia Endotelial de Fuchs (DEF). A identificação desses marcadores genéticos oferece perspectivas para o desenvolvimento de estratégias diagnósticas mais precisas e potenciais alvos terapêuticos direcionados

Shoot dry mass for cultivars(A) RB855536 and (B) RB867515; and root dry mass for cultivars (C) RB855536 and (D) RB867515 after being subjected to 48 days of salt stress (100 mM NaCl).

<p>Lipid peroxidation levels (MDA) in sugarcane leaves during 48 days of salt stress (100 mM NaCl) for (E) cultivar RB855536 and (F) RB867515. Values are presented as mean ± SD (n = 6 plants). "•" are control plants and "▪" are salt-treated plants. *Significant at p≤0.05.</p

Net CO₂-exchange (µmol.m⁻²s⁻¹) for (A) cultivar RB855536 and (B) cultivar RB867515 over time.

<p>Leaf water potential (MPa) in sugarcane leaves during 48 days of salt treatment for (C) cultivar RB855536; and (D) cultivar RB867515. Values are presented as mean ± SD (n = 6). "•" are control plants and "▪" are salt-treated plants. *Significant at p≤0.05.</p

Cultivar RB867515 sugarcane leaf proteins differentially expressed between water-treated and salt-treated plants.

<p>Proteins were trypsin digested and identified by MALDI-ToF/ToF MS. “1-4” are differentially expressed proteins and “a-h” are proteins with no difference in protein expression.</p