Bioengineered xylanase from Misiones Argentina rainforest: A bakery enhancement approach.

In this study, we pursued the heterologous expression of the xylanase gene from Trichoderma atroviride, a native fungus in the province of Misiones, and used it to enhance the textural properties of baked goods through varying enzymatic concentrations. This marks the inaugural exploration into its functionality in the context of bread production. The recombinant xylanase exhibited improved activity, reaching 36,292 U L-1, achieved by supplementing the culture medium with dextrose. Following the optimization of recombinant xylanase concentration, promising results emerged, notably reducing hardness and chewiness parameters of bread significantly. Our findings underscore the potential of this native fungal enzyme for industrial processes, offering a sustainable and efficient means to enhance the quality of baked goods with broad implications for the food industry. No prior research has been documented on the heterologous expression of the xylanase gene derived from T. atroviride, from the Misiones rainforest, expressed in Kluyveromyces lactis. PRACTICAL APPLICATION: This research, focusing on the isolation and cloning of xylanase enzyme from Trichoderma atroviride, a native fungus in the province of Misiones, offers a valuable tool for improving the texture of bakery products. By optimizing enzyme concentrations, our findings present a practical approach for the food industry, offering a viable solution to improve the overall quality and consumer satisfaction of bakery products.

Ilex paraguariensis Hosts Root-Trichoderma spp. with Plant-Growth-Promoting Traits: Characterization as Biological Control Agents and Biofertilizers.

In this study, the effect of native plant-growth-promoting microorganisms (PGPM) as bio-inoculants was assessed as an alternative to improve Ilex paraguariensis Saint Hilaire growth in the nursery. Fourteen Trichoderma strains isolated from yerba mate roots were evaluated in vitro for their potential as biological control agents (BCA) and PGPM. The PGPM properties were evaluated through the strain's antagonistic activity against three fungal pathogens (Alternaria sp., F. oxysporum, and F. solani) plus the production of extracellular cell-wall-degrading enzymes such as chitinase, ß-1,3-glucanase, and cellulase. These results were used to calculate different PGPM indices to select the strains with the optimal properties. Four Trichoderma strains: T. asperelloides LBM193, LBM204, LBM206, and Trichoderma sp. LBM202, were selected based on their indirect and direct PGPM properties used in an inoculation assay on yerba mate plants in greenhouse conditions. A highly significant positive effect of bio-inoculation with these Trichoderma strains was observed in one-year-old yerba mate seedlings. Inoculated plants exhibited a greater height, chlorophyll content, and dry weight than un-inoculated plants; those treated with LBM193 manifested the best results. Yerba mate plants treated with LBM202 exhibited a healthy appearance and were more vigorous, showing potential for biocontrol agent. In conclusion, yerba mate seedlings in the Misiones region were found to have a reservoir of Trichoderma species that increases the yield of this crop in the nursery and protects them from adverse biotic and abiotic agents.

Purification and characterization of a fungal laccase expressed in Kluyveromyces lactis suitable for baking.

Laccase enzyme can replace chemical additives to improve texture properties and the volume of bread. Laccase encoding gene from Phlebia brevispora, a native fungus from Misiones, Argentina, was expressed in the generally recognized as safe yeast Kluyveromyces lactis. To improve laccase activity, medium conditions were optimized. The use of iron sulfate at a concentration of 1 mM led to optimum laccase activity (1289 U·L-1 ) on the fourth day of incubation. SDS-PAGE analysis revealed that the molecular mass of purified laccase was about 180 kDa. Optimum pH for the enzyme was 4 and optimum temperature was 40°C. Laccase exhibited high stability at low pH and high temperature. The application of recombinant laccase to bread decreased hardness, gumminess, and chewiness and increased bread volume. Based on these results, recombinant laccase from P. brevispora with improved yield is a good option for application as an improver of the physicochemical quality of bread at the industrial level. Besides, it will allow us to advance toward our goal of developing healthy alternatives for the bakery industry. No previous work has been reported concerning the heterologous expression of the laccase gene native to the province of Misiones, Argentina, with an aim for application in baking. PRACTICAL APPLICATION: Healthy bakeries became a trend in recent years. The use of the laccase enzyme increases the specific volume and decreases the hardness of bread, being thus an alternative for the replacement of chemical additives in the bakery industry.

Diagrammatic scale for quantification of severity of white thread blight disease in yerba mate (Ilex paraguariensis Saint Hilaire).

The yerba-mate industry is one of the most important economic activities in Misiones, a province in the northeast of Argentina that is the world's leading producer and exporter of this crop. White thread blight disease caused by Ceratobasidium niltonzousanum affects the cultivation reducing its quality and productivity. Due to the lack of a standardized visual method to quantify the severity of this disease in yerba mate, a diagrammatic scale was developed and validated. Yerba-mate branches were collected in a field in the north of Misiones province, and the actual severity was determined digitally. A six-level scale was developed using the DOSLOG software, based on the Weber-Fechner law. The validation was carried out by twenty raters. One evaluation without the diagrammatic scale and two evaluations with the scale were carried out in 14-day intervals. Accuracy, precision, and reproducibility of the scale were evaluated through linear regressions and correlation analysis, obtaining R2 values ranged between 0.70 and 0.94. Using the diagrammatic scale developed in this work, raters enhanced the accuracy and precision of the estimates, and the repeatability of the scale improved by 94.74%. The scale was appropriate to assess the damage of white thread blight in yerba mate.

Exploring novel Penicillium lipolytic activity from the Paranaense rainforest.

In this study, we evaluated the presence of lipases in twenty fungal strains of the genus Penicillium using an efficient and low-cost method with a view to an application in the treatment of cooking oil residues. The Paranaense rainforest is one of the most biodiverse places on the planet, making it the most likely site to find new fungal strains with lipolytic potential. The objective of this study was to determine the lipolytic potential and the isoenzyme profile of fungi belonging to the Penicillium genus isolated from the Paranaense rainforest. Seven fungal strains were selected using qualitative screening. Quantitative analysis revealed that the isolate Penicillium sp. LBM 088 was the best producer of lipase, reaching 1224 U mL-1 of lipolytic activity. Zymogram gels of the seven selected strains showed different enzymatic profiles: In general, the molecular mass of proteins varied from 26 to 42 kDa. Also, proteins from fungi grown on olive oil showed a higher variation in their molecular mass than proteins from fungi grown without the oil. The search for new lipase-secreting organisms should lead to the exploitation of biodiversity in the region.

Isolation and selection of endophytic spore-forming bacteria with plant growth promoting properties isolated from Ilex paraguariensis St. Hil. (yerba mate).

Yerba mate (Ilex paraguariensis St. Hil.) is a species native to the subtropical regions of South America. Despite being an important crop for the region, there are few studies on the use of microorganisms to improve the growth of seedlings in the nursery stage. The objective of this study was to isolate spore-forming endophytic bacteria with plant growth promoting properties associated with yerba mate seedlings and determine their phytobeneficial effect under controlled laboratory conditions. Isolates were selected based on their sporulation capacity and evaluated for in vitro plant growth promoting properties (nitrogen fixation, phosphate solubilization, production of siderophores and synthesis of indolic compounds). Yerba mate seedlings were inoculated with the most promising isolates, which were identified via analyses of the sequence of their 16S rDNA gene as Bacillus circulans (12RS3) and Bacillus altitudinis (19RS3, T5S-T4). After 120 days plants showed higher root dry weight when inoculated with isolate 19RS3 and higher shoot dry weight with 19RS3 and T5S-T4. In conclusion, further studies to determine the ability of these isolates to adapt to the climatic conditions and to survive amidst the native soil microflora in yerba mate cultivated native soils, will be crucial for developing such strains as biofertilizer.

Enzymatic hydrolysis of barley straw for biofuel industry using a novel strain of Trametes villosa from Paranaense rainforest.

Agricultural practices generate lignocellulosic waste that can be bioconverted by fungi to generate value-added products such as biofuels. In this context, fungal enzymes are presented as an alternative for their use in the hydrolysis of cellulose to sugars that can be fermented to ethanol. The aim of this work was to characterize LBM 033 strain and to analyze its efficiency in the hydrolysis of cellulosic substrates, including barley straw. LBM 033 strain was identified as Trametes villosa by molecular techniques, through the use of the ITS and rbp2 markers and the construction of phylogenetic trees. The cell-free supernatant of T. villosa LBM 033 showed high titers of hydrolytic enzymatic activities, necessary for the hydrolysis of the holocellulosic substrates, hydrolyzing pure cellulose to cellobiose and glucose and also degraded the polysaccharides contained in barley straw to short soluble oligosaccharides. These results indicate that macro fungi from tropical soil environments, such as T. villosa LBM 033 can be a valuable resource for in-house, cost effective production of enzymes that can be applied in the hydrolysis stage, which could reduce the total cost of bioethanol production.

Adding value to lignocellulosic wastes via their use for endoxylanase production by Aspergillus fungi.

Agroforestry industries in the world generate lignocellulosic wastes that can be a huge problem of pollution, or the wastes can be used for different biotechonological applications such as substrates for microorganism growth and enzyme production. Fungi such as Aspergillus niger can grow in almost every substrate and produce hydrolytic enzymes such as endoxylanases, giving added value to agroforestry wastes generated by industries in the northeast of Argentina. In this context, the aim of this work was to use agroforestry wastes as substrates for the production of endoxylanases by Aspergillus niger and to optimize nitrogen sources and physical variables for the highest endoxylanase activity. A. niger LBM 055 and A. niger LBM 134 produced high endoxylanase levels when they were grown with sugarcane and cassava bagasses as carbon sources. A. niger LBM 134 reached the highest endoxylanase activity when nitrogen sources and physical variables were optimized. The fungus exhibited up to 110 U mL-1 of endoxylanase activity when it was grown with sugarcane bagasse and more than 160 U mL-1 with cassava bagasse. Therefore, endoxylanase production was optimized using agricultural bagasses and cost 20 times less than enzyme production using synthetic xylan.

Genome Sequence of Kosakonia radicincitans Strain YD4, a Plant Growth-Promoting Rhizobacterium Isolated from Yerba Mate (Ilex paraguariensis St. Hill.).

Kosakonia radicincitans strain YD4 is a rhizospheric isolate from yerba mate (Ilex paraguariensis St. Hill.) with plant growth-promoting effects on this crop. Genes involved in different plant growth-promoting activities are present in this genome, suggesting its potential as a bioinoculant for yerba mate.

Exploring the genes of yerba mate (Ilex paraguariensis A. St.-Hil.) by NGS and de novo transcriptome assembly.

Yerba mate (Ilex paraguariensis A. St.-Hil.) is an important subtropical tree crop cultivated on 326,000 ha in Argentina, Brazil and Paraguay, with a total yield production of more than 1,000,000 t. Yerba mate presents a strong limitation regarding sequence information. The NCBI GenBank lacks an EST database of yerba mate and depicts only 80 DNA sequences, mostly uncharacterized. In this scenario, in order to elucidate the yerba mate gene landscape by means of NGS, we explored and discovered a vast collection of I. paraguariensis transcripts. Total RNA from I. paraguariensis was sequenced by Illumina HiSeq-2000 obtaining 72,031,388 pair-end 100 bp sequences. High quality reads were de novo assembled into 44,907 transcripts encompassing 40 million bases with an estimated coverage of 180X. Multiple sequence analysis allowed us to predict that yerba mate contains ∼ 32,355 genes and 12,551 gene variants or isoforms. We identified and categorized members of more than 100 metabolic pathways. Overall, we have identified ∼ 1,000 putative transcription factors, genes involved in heat and oxidative stress, pathogen response, as well as disease resistance and hormone response. We have also identified, based in sequence homology searches, novel transcripts related to osmotic, drought, salinity and cold stress, senescence and early flowering. We have also pinpointed several members of the gene silencing pathway, and characterized the silencing effector Argonaute1. We predicted a diverse supply of putative microRNA precursors involved in developmental processes. We present here the first draft of the transcribed genomes of the yerba mate chloroplast and mitochondrion. The putative sequence and predicted structure of the caffeine synthase of yerba mate is presented. Moreover, we provide a collection of over 10,800 SSR accessible to the scientific community interested in yerba mate genetic improvement. This contribution broadly expands the limited knowledge of yerba mate genes, and is presented as the first genomic resource of this important crop.

The complete genome of a putative endornavirus identified in yerba mate (Ilex paraguariensis St. Hil.).

We present the first report of a virus infecting the subtropical tree crop yerba mate (Ilex paraguariensis St. Hil.). Total RNA purification, followed by next-generation sequencing, transcripts assembly and annotation, resulted in the identification of a new endornavirus species infecting yerba mate. The complete sequence of the linear dsRNA viral genome is 13,954-nt long, contains a single 13,743 nt ORF, and presents a 149 nt 5'UTR and a 61 nt 3'UTR. The predicted ORF encodes a 4,581 aa polypeptide with a UDP-glucose glycosyl-transferase, a capsular polysaccharide synthesis protein, and a RNA-dependent RNA polymerase domain. The name yerba mate endornavirus is proposed for the identified virus. Due to the intriguing peculiarities of this virus family, and the complete lack of the yerba mate virus literature, we consider that the information reported here will be helpful in leading to a new and needed attention to this important topic and crop.

Dislipidemias e isoformas de apoliproteína E como aporte multidisciplinario en el diagnóstico de la enfermedad de Alzheimer esporádica / Dyslipidemias and apolipoprotein E isoforms as a multidisciplinary contribution for the diagnosis of sporadic Alzheimer’s disease

INTRODUCCION: La apoliproteína E (Apo E) juega un papel muy importante en la distribución y el metabolismo del colesterol y de los triglicéridos en diferentes órganos. El alelo E4 del gen de la Apo E, representa un factor de riesgo genético importante para la enfermedad de Alzheimer esporádica en la población general. La detección temprana es la mejor manera para prevenir o retrasar el desarrollo de la enfermedad de Alzheimer.OBJETIVO: Determinar las diferentes isoformas de Apo E y relacionarlas con el perfil lipídico en pacientes con enfermedad de Alzheimer y grupo control.METODOS: Las isoformas de Apo E se analizaron con los Polimorfismos de Nucleótidos Simples (SNP) C112A y A158C, utilizando la técnica Reacción en cadena de la Polimerasa con Análisis de Fragmentos de Restricción de Longitud Polimórfica (PCR-RFLP), se evaluó el perfil lipídico por métodos enzimático-colorimétrico y se realizaron test neuropsicológicos.RESULTADOS: En esta primera etapa, se analizó el genotipo de 14 individuos del grupo control. El genotipo E3/E3 fue el más frecuente (8 individuos), seguido por el E3/E4 (5 individuos) y por el E2/E3 (1 individuo). La evaluación de siete de los pacientes del grupo control arrojó un valor promedio de 228 mg/dl para colesterol total, 136 mg/dl para triglicéridos y 54,40 mg/dl para C-HDL.CONCLUSIONES: El objetivo planteado no puso ser cumplido debido a la dificultad obtenida en la puesta a punto de la técnica PCR-RFLP.

INTRODUCTION: The apolipoprotein (ApoE) plays an important role in the distribution and metabolism of cholesterol and triglycerides in different organs. E4 allele of the ApoE gene represents an important risk factor for sporadic Alzheimer’s disease in general population. Early detection is the best way to prevent or delay the development of the Alzheimer’s disease.OBJECTIVE: To analyze the different isoforms of ApoE and relate them with the lipid profile in patients with Alzheimer’s disease and a control group.METHODS: ApoE isoforms were analyzed with the single nucleotide polymorphisms (SNP) C112A y A158C, using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The analysis included an evaluation of the lipidic profile by enzymatic-colorimetric methods and neuropsychological tests.RESULTS: In this first stage, the genotype of 14 individuals belonging to the control group was analyzed. The E3/E3 genotype was the most prevalent (8 individuals), followed by E3/E4 (5 individuals) and E2/E3 (1 individual). According to the evaluation of the seven control patients, the average value for total cholesterol was 228 mg/dl, for triglycerides 136 mg/dl and for C-HDL 54.40 mg/dl.CONCLUSIONS: The objective could not be fulfilled fue to the difficulties arisen during the development of the PRC-RFLP technique.

Dislipidemias e isoformas de apoliproteína E como aporte multidisciplinario en el diagnóstico de la enfermedad de Alzheimer esporádica / Dyslipidemias and apolipoprotein E isoforms as a multidisciplinary contribution for the diagnosis of sporadic Alzheimers disease

INTRODUCCION: La apoliproteína E (Apo E) juega un papel muy importante en la distribución y el metabolismo del colesterol y de los triglicéridos en diferentes órganos. El alelo E4 del gen de la Apo E, representa un factor de riesgo genético importante para la enfermedad de Alzheimer esporádica en la población general. La detección temprana es la mejor manera para prevenir o retrasar el desarrollo de la enfermedad de Alzheimer.OBJETIVO: Determinar las diferentes isoformas de Apo E y relacionarlas con el perfil lipídico en pacientes con enfermedad de Alzheimer y grupo control.METODOS: Las isoformas de Apo E se analizaron con los Polimorfismos de Nucleótidos Simples (SNP) C112A y A158C, utilizando la técnica Reacción en cadena de la Polimerasa con Análisis de Fragmentos de Restricción de Longitud Polimórfica (PCR-RFLP), se evaluó el perfil lipídico por métodos enzimático-colorimétrico y se realizaron test neuropsicológicos.RESULTADOS: En esta primera etapa, se analizó el genotipo de 14 individuos del grupo control. El genotipo E3/E3 fue el más frecuente (8 individuos), seguido por el E3/E4 (5 individuos) y por el E2/E3 (1 individuo). La evaluación de siete de los pacientes del grupo control arrojó un valor promedio de 228 mg/dl para colesterol total, 136 mg/dl para triglicéridos y 54,40 mg/dl para C-HDL.CONCLUSIONES: El objetivo planteado no puso ser cumplido debido a la dificultad obtenida en la puesta a punto de la técnica PCR-RFLP.

INTRODUCTION: The apolipoprotein (ApoE) plays an important role in the distribution and metabolism of cholesterol and triglycerides in different organs. E4 allele of the ApoE gene represents an important risk factor for sporadic Alzheimers disease in general population. Early detection is the best way to prevent or delay the development of the Alzheimers disease.OBJECTIVE: To analyze the different isoforms of ApoE and relate them with the lipid profile in patients with Alzheimers disease and a control group.METHODS: ApoE isoforms were analyzed with the single nucleotide polymorphisms (SNP) C112A y A158C, using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The analysis included an evaluation of the lipidic profile by enzymatic-colorimetric methods and neuropsychological tests.RESULTS: In this first stage, the genotype of 14 individuals belonging to the control group was analyzed. The E3/E3 genotype was the most prevalent (8 individuals), followed by E3/E4 (5 individuals) and E2/E3 (1 individual). According to the evaluation of the seven control patients, the average value for total cholesterol was 228 mg/dl, for triglycerides 136 mg/dl and for C-HDL 54.40 mg/dl.CONCLUSIONS: The objective could not be fulfilled fue to the difficulties arisen during the development of the PRC-RFLP technique.

Recent patents related to phosphorylation signaling pathway on cancer.

Phosphorylation and dephosphorylation play an important role in the regulation of growth factor and cytokine signal transduction to modulate cell proliferation, differentiation, survival, and apoptosis. In some cellular systems, the information suggests that EGFR, somatostatin receptors, SHP-1, Akt and PI3K can regulate carcinogenesis implied process through regulated the activity of NF-κB. Current patents related to signaling pathway that includes somatostatin receptors, phosphotyrosine phosphatases, tyrosine kinases, AKT/PKB and PI3K are focusing in diagnosis, prognosis and treatment. Many recent patented techniques include inhibition, antagonism or alternative therapeutic methods. Furthermore, it is necessary to deepen understanding of the molecular mechanisms involved in cancer to develop other alternative therapies focusing not only on new inhibitors.

White rot fungi laccases for biotechnological applications.

White rot fungi have an enzymatic system producing oxidative and hydrolytic enzymes that act on the degradation of certain components of the cell wall. They can be applied in several technological processes, such as paper industry, bio-fuels and environmental pollution. Laccases are multi-copper enzymes of wide substrate specificity and high non-specific oxidation capacity that use molecular oxygen to oxidize various aromatic compounds, and are highly relevant biotechnological applications. In this review, we present some significant patents on laccase production and recombinant DNA technology for diverse biotechnology applications.

Phosphotyrosine phosphatases in cancer diagnostic and treatment.

The activation of proteins by post-translational modification represents an important cellular mechanism for regulating most aspects of biological organization and control, including growth, development, homeostasis, and cellular communication. The complexity of protein modification includes phosphorylation and dephosphorylation, on proteins of different signaling pathways corresponding to growth, development, disease states, and aging. Current patents in phosphotyrosine phosphatases signaling pathway are focusing in diagnosis, prognosis and treatment. Many, new diagnosis techniques detect changes in mRNA expression with microarray technologies and others introduced specific antibodies for detection proteins changes, introducing to Biomedicine at Transcriptomic and Proteomic era. Many recent invent development alternative therapy with antibodies and inhibitors to PTPs that demonstrate the need to deepen understanding of the molecular mechanisms involved in the development of cancer.

Copper inducing effect on laccase production of white rot fungi native from Misiones (Argentina).

Fungi may be selected as models for gene expression studies and further adaptation for biotechnological enzyme production. The aim of this work was to evaluate laccase production and to analyze the effect of Cu(2+) on selected fungi natives of Misiones, Ganoderma applanatum (strain F), Peniophora sp. (BAFC 633), Pycnoporus sanguineus (BAFC 2126) and Coriolus versicolor f. antarcticus (BAFC 266). Fungi secretion system of G. applanatum, Peniophora sp., P. sanguineus and C. versicolor f. antarcticus is sensitive to stimulation by copper. Biomass values of G. applanatum, Peniophora sp. and C. versicolor f. antarcticus did not show differences between treatments. P. sanguineus biomass underwent a dramatic growth inhibition with 1mM Cu(2+) and marked delay in growth with 0.5mM Cu(2+). Proteins were increased with copper in Peniophora sp., C. versicolor and G. applanatum. G. applanatum and Peniophora sp. reached the highest enzyme activity at 10th day equivalent to 49.2-fold and 19.7-fold higher than the control samples, respectively. Copper produced an increase of constitutive laccases in all fungi and an additional inducible isoenzyme in Peniophora sp., C. versicolor f. antarcticus and G. applanatum.