E-Stilbenes: General Chemical and Biological Aspects, Potential Pharmacological Activity Based on the Nrf2 Pathway.

Stilbenes are phytoalexins, and their biosynthesis can occur through a natural route (shikimate precursor) or an alternative route (in microorganism cultures). The latter is a metabolic engineering strategy to enhance production due to stilbenes recognized pharmacological and medicinal potential. It is believed that in the human body, these potential activities can be modulated by the regulation of the nuclear factor erythroid derived 2 (Nrf2), which increases the expression of antioxidant enzymes. Given this, our review aims to critically analyze evidence regarding E-stilbenes in human metabolism and the Nrf2 activation pathway, with an emphasis on inflammatory and oxidative stress aspects related to the pathophysiology of chronic and metabolic diseases. In this comprehensive literature review, it can be observed that despite the broad number of stilbenes, those most frequently explored in clinical trials and preclinical studies (in vitro and in vivo) were resveratrol, piceatannol, pterostilbene, polydatin, stilbestrol, and pinosylvin. In some cases, depending on the dose/concentration and chemical nature of the stilbene, it was possible to identify activation of the Nrf2 pathway. Furthermore, the use of some experimental models presented a challenge in comparing results. In view of the above, it can be suggested that E-stilbenes have a relationship with the Nrf2 pathway, whether directly or indirectly, through different biological pathways, and in different diseases or conditions that are mainly related to inflammation and oxidative stress.

Use of the gonadal structures of the mangrove crab Ucides cordatus as a biomarker for environmental contamination by metals.

This study investigates the concentration of metals in the different developmental phases of the gonads of Ucides cordatus from mangrove areas of Vila do Bacuriteua on the Bragança Peninsula, Caeté-Taperaçu Marine Extractive Reserve, state of Pará, on the Brazilian Amazon coast. Elemental analysis was determined by inductively coupled plasma optical emission spectrometry (ECP-OES). Metal concentration in males ranged from Cd = 0.04 to Cu = 2.27 mg kg-1 ww in the Developing and Developed gonadal stages, respectively. In females it was from Cd = 0.11 to Cu = 8.43 mg kg-1 ww only in the Developed stage). The elements Cr and Pb, for both sexes, presented concentrations above the limits allowed by the Brazilian regulatory body and only Pb by the international agencies, revealing a higher degree of contamination of these metals in the gonads of the mangrove crabs. Increasing mangrove crab consumption amplifies the risks of metal contamination among the human population and may cause public health problems.

Lemon-derived carbon dots as antioxidant and light emitter in fluorescent films applied to nanothermometry.

The design of luminescent nanomaterials for the development of nanothermometers with high sensitivity and free of potentially toxic metals has developed in several fields, such as optoelectronics, sensors, and bioimaging. In addition, luminescent nanothermometers have advantages related to non-invasive measurement, with their wide detection range and high spatial resolution at the nano/microscale. Our study is the first, to our knowledge, to demonstrate a detailed study of a fluorescent film (Film-L) thermal sensor based on carbon dots derived from lemon bagasse extract (CD-L). The CD-L properties were explored as an antioxidant agent; their cytotoxicity was evaluated by using a human non-tumoral skin fibroblast (HFF-1) cell line from an MTT assay. The CD-L were characterized by HRTEM, DLS, FTIR, UV-VIS, and fluorescence spectroscopy. These confirmed their particle size distribution below 10 nm, graphitic structure in the core and surface organic groups, and strong blue emission. The CD-L showed cytocompatibility behavior and scavenging potential reactive species of biological importance: O2•- and HOCl, with IC50 of 276.8 ± 4.0 and 21.6 ± 0.7, respectively. The Film-L emission intensities (I425 nm) are temperature-dependent in the 298 to 333 K range. The Film-L luminescent thermometer shows a maximum relative thermal sensitivity of 2.69 % K-1 at 333 K.

Fuzzy Soft Skills Assessment through Active Learning Sessions.

This research aims to present a Fuzzy Expert System with psychologist expertise that seeks to assist professors, researchers and educational institutions in assessing the level of incorporation of students' Soft Skills while attending Active Learning sessions. The difficulties encountered by higher education institutions, researchers and professors in evaluating subjective and behavioral components, such as Soft Skills, was one of the problems that motivated the undertaking of this research. The theoretical framework on which this work is based includes the development and evaluation of Soft Skills in students, some concepts and characteristics about Active Learning and the main attributes and properties of Fuzzy Logic. This research is of an exploratory applied nature, a qualitative and quantitative approach is proposed, in which the methodological triangulation between the bibliographic analysis, the case study and the modeling and implementation of the expert system called Fuzzy Soft Skills Assessment was used to achieve the objective proposed.

Insights about levels and sources of organic pollution in an urbanized Amazon estuary (Belém, PA, Northern Brazil).

Amazon aquatic systems have been affected by organic pollution from urbanized regions. This study was conducted to determine the levels, sources, and distribution patterns of 16 polycyclic aromatic hydrocarbons (PAHs) and 6 steroid markers in surficial sediments from an important urbanized Amazon estuarine system (Belém, PA, Northern Brazil). Total PAH concentration (∑PAH) ranged from 878.2 to 9905.7 ng g-1, 3295.2 ng g-1 on average, suggesting a highly contaminated environment. PAH molecular ratios and statistical analysis indicated that PAH originated from a mixture of local sources emissions, mainly related to the combustion of fossil fuels and biomass. Coprostanol levels (maximum concentration = 292.52 ng g-1) could be compared to the mid-range reported in the literature. Studied stations, except for one, presented sterol ratio data indicating organic matter related to untreated sewage. Sterols indicative of sewage contamination showed a correlation with pyrogenic PAH amounts which are transported by the same channels where sewage is discharged.

Use of Fungal Mycelium as Biosupport in the Formation of Lichen-like Structure: Recovery of Algal Grown in Sugarcane Molasses for Lipid Accumulation and Balanced Fatty Acid Profile.

In this study, a lichen-like structure was obtained through the production of a unique biomass, formed by algae cells of Scenedesmus obliquus adhering to the mycelium of filamentous fungal Mucor circinelloides. This structure was composed in two steps; in the first one, microalgal cells and spores were incubated separately, and in the second one, after 72 h of growth, isolated, mature mycelium was harvested and added to cell culture. For spores' incubation, a culture medium containing only 2 g·L-1 of glucose and minerals was used. This culture medium, with low sugar content, provided a fungal biomass to the anchorage of microalgae cells. WC medium was used without and with sugarcane molasses supplementation for microalgae cells' incubation. The lichen-type structure that was formed resulted in 99.7% efficiency in the recovery of microalgae cells and in up to 80% efficiency in the recovery of algae biomass in the lichen biomass composition. In addition, the resulting consortium attained a satisfactory lipid accumulation value (38.2 wt%) with a balanced fatty acid composition of 52.7% saturated plus monounsaturated fatty acids and 47.4% polyunsaturated fatty acids. Since fungal species are easy to recover, unlike microalgae, the lichen-like structure produced indicates an efficient low-cost bioremediation and harvesting alternative; in addition, it provides an oleaginous biomass for various industrial applications.

Combined Reconstruction of the Anterior Cruciate Ligament and Anterolateral Ligament Injury Compared to the Isolated Reconstruction of the Anterior Cruciate Ligament: A Meta-Analysis.

The present research aims to compare the outcomes from the combined reconstruction of the anterior cruciate ligament (ACL) and of the anterolateral ligament (ALL) with the standard isolated ACL reconstruction in patients with chronic ACL injury. To do so, a meta-analysis was carried out to determine whether the combined ACL and ALL reconstruction would lead to a significant improvement in knee function according to the International Knee Documentation Committee (IKDC), the Lysholm test and KT-2000 evaluation scores and lower graft rupture rates in comparison with isolated reconstruction. To identify randomized controlled trials (RCTs) comparing the combined ACL and ALL reconstruction with the isolated ACL reconstruction, papers published between 2010 and 2019 were searched in the MEDLINE, EMBASE, SPORTDiscus, LILACS and Cochrane Central Register of Controlled Trials databases, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria. The stability of the knee joint is only marginally improved with the combined reconstruction of ACL and ALL, and both reconstruction techniques show functional results. The main outcomes sought were patient function and graft stability and rupture rates after ACL reconstruction. Out of the 421 studies identified, 6 were included in our meta-analysis. Study quality (internal validity) was assessed using the Cochrane risk-of-bias tool; in general, the studies included presented moderate-quality evidence. The graft rupture rate was higher in patients undergoing isolated ACL reconstruction (relative risk, 0.22; 95% confidence interval, 0.12 to 0.41; p < 0.00001).

Combined Reconstruction of the Anterior Cruciate Ligament and Anterolateral Ligament Injury Compared to the Isolated Reconstruction of the Anterior Cruciate Ligament: A Meta-Analysis / Reconstrução combinada do ligamento cruzado Anterior e lesão do ligamento anterolateral comparada àreconstrução isolada do ligamento cruzado anterior: Uma metanálise

Abstract The present research aims to compare the outcomes from the combined reconstruction of the anterior cruciate ligament (ACL) and of the anterolateral ligament (ALL) with the standard isolated ACL reconstruction in patients with chronic ACL injury. To do so, a meta-analysis was carried out to determine whether the combined ACL and ALL reconstruction would lead to a significant improvement in knee function according to the International Knee Documentation Committee (IKDC), the Lysholm test and KT-2000 evaluation scores and lower graft rupture rates in comparison with isolated reconstruction. To identify randomized controlled trials (RCTs) comparing the combined ACL and ALL reconstruction with the isolated ACL reconstruction, papers published between 2010 and 2019 were searched in the MEDLINE, EMBASE, SPORTDiscus, LILACS and Cochrane Central Register of Controlled Trials databases, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria. The stability of the knee joint is only marginally improved with the combined reconstruction of ACL and ALL, and both reconstruction techniques show functional results. The main outcomes sought were patient function and graft stability and rupture rates after ACL reconstruction. Out of the 421 studies identified, 6 were included in our meta-analysis. Study quality (internal validity) was assessed using the Cochrane risk-of-bias tool; in general, the studies included presented moderate-quality evidence. The graft rupture rate was higher in patients undergoing isolated ACL reconstruction (relative risk, 0.22; 95% confidence interval, 0.12 to 0.41; p < 0.00001).

Resumo O objetivo da presentepesquisa é comparar, por meio de uma metanálise, os resultados da reconstrução combinada do ligamento cruzado anterior (LCA) e do ligamento anterolateral (LLA), comparado com a reconstrução isolada padrão, em pacientes com lesão crônica do ligamento cruzado anterior. Buscando alcançar o objetivo da pesquisa, foi realizada uma meta-análise para determinar se a combinação da reconstrução combinada LCA e LLA levaria àmelhoria significativa da função do joelho, medida pelos escores de avaliação International Knee Documentation Committee (IKDC), Lysholm, KT-2000 e menor taxa de ruptura do enxerto, em comparação com a reconstrução isolada. Para identificar ensaios clínicos randomizados (ECR) comparando a reconstrução combinada do LCA e LLA com a reconstrução isolada do LCA, foram pesquisados artigos publicados entre 2010 e 2019 nas bases MEDLINE, EMBASE, SPORTDiscus, LILACS e Cochrane Central RegisterofControlledTrials e seguiram os critérios de Itens de Relatórios Preferidos para Revisões Sistemáticas e Metanálises (PRISMA). A estabilidade da articulação do joelho é apenas marginalmente aprimorada com a reconstrução combinada de LCA e LLA, e ambas as técnicas de reconstrução mostram resultados funcionais. Os principais desfechos procurados foram a função do paciente e as taxas de estabilidade e ruptura do enxerto após a reconstrução do LCA. Dos 421 estudos identificados, 6estudos foram incluídos em nossa meta-análise. A qualidade do estudo (validade interna) foi avaliada usando o instrumento Cochrane risco-de-viés; em geral, foi encontrada uma qualidade moderada de evidências dos estudos incluídos. Os pacientes submetidos à reconstrução isolada do LCA mostraram maior taxa de ruptura do enxerto (RR 0,22; índice de confiança [IC]95%: 0,12-0,41; p< 0,00001).

Adjustment of the Operational Parameters of an Unconventional Integrated and Illuminated Internally Photobioreactor (ILI-PBR) for the Batch Autotrophic Cultivation of the Chlorella minutissima, Using the Taguchi Method.

Microalgae crops targeting biotechnological applications are conducted in photobioreactors, which allow the adjustment and control of parameters of luminosity, agitation and mixing in the cultivation medium to promote better cell growth and accumulation of metabolites. In this sense, the present work used the Taguchi method to find the best adjustment of the operational parameters of an unconventional photobioreactor denominated internally illuminated integrated photobioreactor (ILI-PBR) with LED light aiming at elevating the biomass concentration, volumetric biomass productivity, and volumetric lipid productivity of Chlorella minutissima microalgae cultivated under autotrophic regime. The effects of the factors were evaluated: illumination (blue, white and red); photoperiod (12 h light:12 h dark, 1 h light:1 h dark, and 24 h clear:0 h dark); aeration (0, 3 vvm, 0.4 vvm, and 0.5 vvm); and recirculation flow rate of cultivation medium (5 L min-1, 6.5 L min-1, and 9 L min-1) on the variable responses: biomass concentration, biomass volumetric productivity, lipid content, and volumetric lipid productivity. The use of the Taguchi method allowed the increase of biomass concentration, volumetric biomass productivity, and volumetric lipid productivity in the biomass of Chlorella minutissimain 8.6%, 42%, and 143%, respectively, with the adjustment of the operational parameters of the photobioreactor used.

The postharvest ripening of strawberry fruits induced by abscisic acid and sucrose differs from their in vivo ripening.

In this study, we compared the chemical composition of strawberry (Fragaria × ananassa) fruits that were ripened in vivo (attached to plant) to those ripened during postharvest storage. The effects of the application of abscisic acid (ABA) and sucrose on the postharvest ripening were also evaluated. The results suggested that the postharvest ripening process was dependent on the signal triggered by ABA and differed from in vivo ripening, resulting in fruits with altered chemical composition and firmness. The application of sucrose in unripe strawberries resulted in the induction of ripening, which is dependent on ABA and its derivatives. This induction was more pronounced during the first days of storage and associated with the application of mannitol rather than water, suggesting that mannitol negatively regulated the postharvest strawberry ripening. These results provide further insights into the role of ABA and sucrose in the regulation of postharvest ripening of strawberry.

Mixture design as a potential tool in modeling the effect of light wavelength on Dunaliella salina cultivation: an alternative solution to increase microalgae lipid productivity for biodiesel production.

For a feasible microalgae biodiesel, increasing lipid productivity is a key parameter. An important cultivation parameter is light wavelength (λ). It can affect microalgal growth, lipid yield, and fatty acid composition. In the current study, the mixture design was used as an alternative to model the influence of the λ on the Dunaliella salina lipid productivity. The illumination was considered to be the mixture of different λ (the light colors blue, red, and green). All experiments were performed with and without sodium acetate (4 g/L), as carbon source, allowing the identification of the impact of the cultivation regimen (autotrophic or mixotrophic). Without sodium acetate, the highest lipid productivity was obtained using blue and red light. The use of mixotrophic cultivations significantly enhanced the results. The optimum obtained result was mixotrophic cultivation under 65% blue and 35% green light, resulting in biomass productivity of 105.06 mgL-1day-1, a lipid productivity of 53.47 mgL-1day-1, and lipid content of 50.89%. The main fatty acids of the oil obtained in this cultivation were oleic acid (36.52%) and palmitic acid (18.31%).

ABA-dependent salt and drought stress improve strawberry fruit quality.

Strawberry crop is very sensitive to osmotic stress conditions. We investigated the effect of the stress induced by mild drought (DS) and salt (SS) stresses, on molecular, physiological, and metabolic processes in the strawberry crop (Fragaria ananassa), cv. Camarosa. The results showed that the fruit yield was not affected. Mild DS and SS also resulted in an increased content of phenolics, anthocyanins, and l-ascorbic acid, and an increased antioxidant activity. These effects were accompanied by increased levels of ABA and its derivatives (phaseic and dehydrophasic acids), alongside the upregulation of several genes involved on their synthesis. Therefore, the results obtained in this study suggest that mild DS and SS improve the functional quality of strawberry fruits through and ABA-dependent mechanism.

Usefulness of in-house PCR methods for hepatitis B virus DNA detection.

The aim of the present study was to evaluate the performance of three in-house PCR techniques for HBV DNA detection and compare it with commercial quantitative methods to evaluate the usefulness of in-house methods for HBV diagnosis. Three panels of HBsAg reactive sera samples were evaluated: (i) 50 samples were examined using three methods for in-house qualitative PCR and the Cobas Amplicor HBV Monitor Assay; (ii) 87 samples were assayed using in-house semi-nested PCR and the Cobas TaqMan HBV test; (iii) 11 serial samples obtained from 2 HBV-infected individuals were assayed using the Cobas Amplicor HBV test and semi-nested PCR. In panel I, HBV DNA was detected in 44 samples using the Cobas Amplicor HBV test, 42 samples using semi-nested PCR (90% concordance with Cobas Amplicor), 22 samples using PCR for the core gene (63.6% concordance) and 29 samples using single-round PCR for the pre-S/S gene (75% concordance). In panel II, HBV DNA was quantified in 78 of the 87 HBsAg reactive samples using Cobas TaqMan but 52 samples using semi-nested PCR (67.8% concordance). HBV DNA was detected in serial samples until the 17th and 26th week after first donation using in-house semi-nested PCR and the Cobas Amplicor HBV test, respectively. In-house semi-nested PCR presented adequate concordance with commercial methods as an alternative method for HBV molecular diagnosis in low-resource settings.

Carotenoid biosynthetic and catabolic pathways: gene expression and carotenoid content in grains of maize landraces.

Plant carotenoids have been implicated in preventing several age-related diseases, and they also provide vitamin A precursors; therefore, increasing the content of carotenoids in maize grains is of great interest. It is not well understood, however, how the carotenoid biosynthetic pathway is regulated. Fortunately, the maize germplasm exhibits a high degree of genetic diversity that can be exploited for this purpose. Here, the accumulation of carotenoids and the expression of genes from carotenoid metabolic and catabolic pathways were investigated in several maize landraces. The carotenoid content in grains varied from 10.03, in the white variety MC5, to 61.50 µg·g⁻¹, in the yellow-to-orange variety MC3, and the major carotenoids detected were lutein and zeaxanthin. PSY1 (phythoene synthase) expression showed a positive correlation with the total carotenoid content. Additionally, the PSY1 and HYD3 (ferredoxin-dependent di-iron monooxygenase) expression levels were positively correlated with ß-cryptoxanthin and zeaxanthin, while CYP97C (cytochrome P450-type monooxygenase) expression did not correlate with any of the carotenoids. In contrast, ZmCCD1 (carotenoid dioxygenase) was more highly expressed at the beginning of grain development, as well as in the white variety, and its expression was inversely correlated with the accumulation of several carotenoids, suggesting that CCD1 is also an important enzyme to be considered when attempting to improve the carotenoid content in maize. The MC27 and MC1 varieties showed the highest HYD3/CYP97C ratios, suggesting that they are promising candidates for increasing the zeaxanthin content; in contrast, MC14 and MC7 showed low HYD3/CYP97C, suggesting that they may be useful in biofortification efforts aimed at promoting the accumulation of provitamin A. The results of this study demonstrate the use of maize germplasm to provide insight into the regulation of genes involved in the carotenoid pathway, which would thus better enable us to select promising varieties for biofortification efforts.

Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains.

KEY MESSAGE: The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification. Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.

Unraveling the structure of sugarcane bagasse after soaking in concentrated aqueous ammonia (SCAA) and ethanol production by Scheffersomyces (Pichia) stipitis.

BACKGROUND: Fuel ethanol production from sustainable and largely abundant agro-residues such as sugarcane bagasse (SB) provides long term, geopolitical and strategic benefits. Pretreatment of SB is an inevitable process for improved saccharification of cell wall carbohydrates. Recently, ammonium hydroxide-based pretreatment technologies have gained significance as an effective and economical pretreatment strategy. We hypothesized that soaking in concentrated aqueous ammonia-mediated thermochemical pretreatment (SCAA) would overcome the native recalcitrance of SB by enhancing cellulase accessibility of the embedded holocellulosic microfibrils. RESULTS: In this study, we designed an experiment considering response surface methodology (Taguchi method, L8 orthogonal array) to optimize sugar recovery from ammonia pretreated sugarcane bagasse (SB) by using the method of soaking in concentrated aqueous ammonia (SCAA-SB). Three independent variables: ammonia concentration, temperature and time, were selected at two levels with center point. The ammonia pretreated bagasse (SCAA-SB) was enzymatically hydrolysed by commercial enzymes (Celluclast 1.5 L and Novozym 188) using 15 FPU/g dry biomass and 17.5 Units of ß-glucosidase/g dry biomass at 50°C, 150 rpm for 96 h. A maximum of 28.43 g/l reducing sugars corresponding to 0.57 g sugars/g pretreated bagasse was obtained from the SCAA-SB derived using a 20% v/v ammonia solution, at 70°C for 24 h after enzymatic hydrolysis. Among the tested parameters, pretreatment time showed the maximum influence (p value, 0.053282) while ammonia concentration showed the least influence (p value, 0.612552) on sugar recovery. The changes in the ultra-structure and crystallinity of native SCAA-SB and enzymatically hydrolysed SB were observed by scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid-state (13)C nuclear magnetic resonance (NMR) spectroscopy. The enzymatic hydrolysates and solid SCAA-SB were subjected to ethanol fermentation under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) by Scheffersomyces (Pichia) stipitis NRRL Y-7124 respectively. Higher ethanol production (10.31 g/l and yield, 0.387 g/g) was obtained through SSF than SHF (3.83 g/l and yield, 0.289 g/g). CONCLUSIONS: SCAA treatment showed marked lignin removal from SB thus improving the accessibility of cellulases towards holocellulose substrate as evidenced by efficient sugar release. The ultrastructure of SB after SCAA and enzymatic hydrolysis of holocellulose provided insights of the degradation process at the molecular level.

The realm of cellulases in biorefinery development.

Geopolitical concerns (unstable supply of gasoline, environmental pollution, and regular price hikes), economic, and employment concerns have been prompting researchers, entrepreneurs, and policy makers to focus on harnessing the potential of lignocellulosic feedstock for fuel ethanol production and its commercialization. The carbohydrate skeleton of plant cell walls needs to be depolymerised into simpler sugars for their application in fermentation reactions as a chief carbon source of suitable ethnologic strains for ethanol production. The role of cellulolytic enzymes in the degradation of structural carbohydrates of the plant cell wall into ready-to-fermentable sugar stream is inevitable. Cellulase synergistically acts upon plant cell wall polysaccharides to release glucose into the liquid media. Cellulase predominantly dominates all the plant cell wall degrading enzymes due to their vast and diverse range of applications. Apart from the major applications of cellulases such as in detergent formulations, textile desizing, and development of monogastric feed for ruminants, their role in biorefinery is truly remarkable. This is a major area where new research tools based upon fermentation based formulations, biochemistry, and system biology to expedite the structure-function relationships of cellulases including cellulosomes and new designer enzymatic cocktails are required. In the last two decades, a considerable amount of research work has been performed on cellulases and their application in biomass saccharification. However, there are still technical and economic impediments to the development of an inexpensive commercial cellulase production process. Advancements in biotechnology such as screening of microorganisms, manipulation of novel cellulase encoding traits, site-specific mutagenesis, and modifications to the fermentation process could enhance the production of cellulases. Commercially, cheaper sources of carbohydrates and modified fermentation conditions could lead to more cost-effective production of cellulases with the goal to reduce the cost of ethanol production from lignocellulosics. Implementation of integrated steps like cellulase production and cellulase mediated saccharification of biomass in conjunction with the fermentation of released sugars in ethanol in a single step so called consolidated bio-processing (CBP) is very important to reduce the cost of bioethanol. This paper aims to explore and review the important findings in cellulase biotechnology and the forward path for new cutting edge opportunities in the success of biorefineries.

Molecular assay optimized by Taguchi experimental design method for venous thromboembolism investigation.

Two mutations - Factor V Leiden (1691G > A) and the 20210G > A on the Prothrombin gene - are key risk factors for a frequent and potentially fatal disorder called Venous Thromboembolism. These molecular alterations can be investigated using real-time Polymerase Chain Reaction (PCR) with Fluorescence Resonance Energy Transfer (FRET) probes and distinct DNA pools for both factors. The objective of this paper is to present an application of Taguchi Experimental Design Method to determine the best parameters adjustment of a Molecular Assays Process in order to obtain the best diagnostic result for Venous Thromboembolism investigation. The complete process contains six three-level factors which usually demands 729 experiments to obtain the final result, if using a Full Factorial Array. In this research, a Taguchi L27 Orthogonal Array is chosen to optimize the analysis and reduce the number of experiments to 27 without degrading the final result accuracy. The application of this method can lessen the time and cost necessary to achieve the best operation condition for a required performance. The results is proven in practice and confirmed that the Taguchi method can really offer a good approach for clinical assay efficiency and effectiveness improvement even though the clinical diagnostics can be based on the use of qualitative techniques.

Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli.

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests.

Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests.