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2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable intermediate that naturally occurs during microbial degradation of lignin by bacteria, represents a promising building block for diverse biomaterials and polyesters such as biodegradable plastics. The lack of a chemical synthesis method has hindered large-scale utilization of PDC and metabolic engineering approaches for its biosynthesis have recently emerged. In this study, we demonstrate a strategy for the production of PDC via manipulation of the shikimate pathway using plants as green factories. In tobacco leaves, we first showed that transient expression of bacterial feedback-resistant 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB) produced high titers of protocatechuate (PCA), which was in turn efficiently converted into PDC upon co-expression of PCA 4,5-dioxygenase (PmdAB) and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (PmdC) derived from Comamonas testosteroni. We validated that stable expression of AroG in Arabidopsis in a genetic background containing the QsuB gene enhanced PCA content in plant biomass, presumably via an increase of the carbon flux through the shikimate pathway. Further, introducing AroG and the PDC biosynthetic genes (PmdA, PmdB, and PmdC) into the Arabidopsis QsuB background, or introducing the five genes (AroG, QsuB, PmdA, PmdB, and PmdC) stacked on a single construct into wild-type plants, resulted in PDC titers of ~1% and ~3% dry weight in plant biomass, respectively. Consistent with previous studies of plants expressing QsuB, all PDC producing lines showed strong reduction in lignin content in stems. This low lignin trait was accompanied with improvements of biomass saccharification efficiency due to reduced cell wall recalcitrance to enzymatic degradation. Importantly, most transgenic lines showed no reduction in biomass yields. Therefore, we conclude that engineering plants with the proposed de-novo PDC pathway provides an avenue to enrich biomass with a value-added co-product while simultaneously improving biomass quality for the supply of fermentable sugars. Implementing this strategy into bioenergy crops has the potential to support existing microbial fermentation approaches that exploit lignocellulosic biomass feedstocks for PDC production.

Background: Nowadays medicines derived from natural sources have drawn much attention as potential therapeutic agents in the suppression and treatment of cancer because of their low toxicity and fewer side effects. Objective: The present review aims to assess the currently available knowledge on the ethnomedicinal uses and pharmacological activities of bioactive compounds obtained from medicinal mushrooms towards cancer treatment. Methods: A literature search has been conducted for the collection of research papers from universally accepted scientific databases. These research papers and published book chapters were scrutinized to retrieve information on ethnomedicinal uses of mushrooms, different factors involved in cancer cell proliferation, clinical and in silico pharmaceutical studies made for possible treatments of cancer using mushroom derived compounds. Overall, 241 articles were retrieved and reviewed from the year 1970 to 2020, out of which 98 relevant articles were finally considered for the preparation of this review. Results: This review presents an update on the natural bioactive substances derived from medicinal mushrooms and their role in inhibiting the factors responsible for cancer cell proliferation. Along with it, the present review also provides information on the ethnomedicinal uses, solvents used for extraction of anti-cancer metabolites, clinical trials, and in silico studies that were undertaken towards anticancer drug development from medicinal mushrooms. Conclusion: The present review provides extensive knowledge on various anti-cancer substances obtained from medicinal mushrooms, their biological actions, and in silico drug designing approaches, which could form a basis for the development of natural anti-cancer therapeutics.

Background:: Aloe vera has been reported as a topical antibiotic and healing agent for wounds, but advantages of oral administration and mechanisms of wound healing have not been reported. Present study focuses on the evaluation of effects of oral administration of Aloe vera for excisional cutaneous wounds in Sprague Dawley rats. Methods:: Sprague Dawley (SD) rats were inflicted with excisional wounds and were either treated with Aloe vera orally (Aloe vera) or kept untreated (wound). In contrast, healthy rats were kept as control group. Wound area was measured from day 7th to day 21st. Collagen content was estimated by hydroxyproline assay. Histology was analysed by hematoxylin and eosin staining. Angiogenesis was observed by indirect ELISA for Insulin like Growth Factor (IGF-1) and Vascular Endothelial Growth Factor (VEGF) protein from skin, serum and bone marrow. Chemotaxis was evaluated by RT-PCR analysis for Stromal cell-Derived Factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR-4) from skin and bone marrow. Results:: Aloe vera healed wounds earlier than untreated rats with gradual improvement in wound areas and collagen content. Aloe vera also improved the expression of IGF-1 and VEGF in skin and bone marrow indicating an improvement in angiogenesis. RT- PCR analysis showed increased expression of genes for chemotaxis (SDF-1 and CXCR-4) in skin and bone marrow. Conclusion: Aloe vera improves healing by increasing collagen content, improving angiogenesis and chemotaxis.

In recent decades, the processing of traditional Chinese medicines has received widespread attention as people's requirements for safety and effectiveness of traditional Chinese medicine have increased. Because of its theory and technical methods, the fermentation processing of traditional Chinese medicines has been distinct from other methods. Based on generalized fermentation, the fermentation of traditional Chinese medicine is based on collection of appropriate traditional Chinese medicinal materials for processing according to the characteristics of the bacteria, so that the original properties of the medicinal materials are modied and the effectiveness is increased. Strong fermentation, liquid fermentation, and two-way solid fermentation are popular fermentation methods. Currently, the study of traditional Chinese medicine's fermentation process has drawn wide interest from academics. In the future, in order to open up new avenues for fermentation and production of traditional Chinese medicine, the selection of fermenting microorganisms and the mode of action of strains in the fermentation phase need to be examined in depth.

2,3-Butanediol (2,3-BDO) is a chemical platform used in several applications, such as cosmetics, medicines, polymers, and fuels. Its levo-isomer is used as an antifreeze agent of industrial interest. Glycerol has been extensively investigated to produce 2,3-BDO by different bacteria. However, few studies involving Paenibacillus polymyxa and glycerol have been conducted so far. In this study, biodiesel-derived glycerol was used for the 2,3-BDO production by P. polymyxa PM 3605. About 14.5 g/L of 2,3-BDO was obtained from 50 g/L of crude glycerol in shaken flasks at 37 °C and 200 rpm. By supplementing the glycerol-containing medium with 10 g/L of sugarcane molasses, the final 2,3-BDO production was enhanced to up to 19 g/L. This was the highest 2,3-BDO titer produced by the P. polymyxa strain from crude glycerol as the main carbon source, and sugarcane molasses as a secondary substrate, until now. The selectivity of levo-2,3-BDO was high, and the production of meso-2,3-BDO and acetoin were not detected. As 2,3-BDO metabolism is dependent on strict oxygen control to ensure a microaerobic condition, the production found here has potential for improvement, indicating that the glycerol use by P. polymyxa PM 3605 is promising for the levo-2,3-BDO synthesis.

Human milk oligosaccharides (HMOs) are potent bioactive compounds that modulate neonatal health and are of interest for development as potential drug treatments for adult diseases. The potential of these molecules, their limited access from natural sources, and difficulty in large-scale isolation of individual HMOs for studies and applications have motivated the development of chemical syntheses and in vitro enzymatic catalysis strategies. Whole cell biocatalysts are emerging as alternative self-regulating production platforms that have the potential to reduce the cost for enzymatic synthesis of HMOs. Whole cell biocatalysts for the production of short-chained, linear and small monofucosylated HMOs have been reported but those for fucosylated structures with higher complexity have not been explored. In this study, we established a strategy for producing a difucosylated HMO, lactodifucotetraose (LDFT), from lactose and L-fucose in Escherichia coli. We used two bacterial fucosyltransferases with narrow acceptor selectivity to drive the sequential fucosylation of lactose and intermediate 2′-fucosyllactose (2′-FL) to produce LDFT. Deletion of substrate degradation pathways that decoupled cellular growth from LDFT production, enhanced expression of native substrate transporters and modular induction of the genes in the LDFT biosynthetic pathway allowed complete conversion of lactose into LDFT and minor quantities of the side product 3-fucosyllactose (3-FL). Overall, 5.1 g/L of LDFT was produced from 3 g/L lactose and 3 g/L L-fucose in 24 h. Our results demonstrate promising applications of engineered microbial biosystems for the production of multi-fucosylated HMOs for biochemical studies.

Background: In fish farming, the plant extracts containing antioxidant compounds have been added to the diet for enhancing pathogen resistance. In vitro studies evaluating the antioxidant effect of herbal extracts on fish cell models have focused on ROS production and the respiratory burst mechanism. However, the effects on enzymatic antioxidant defense on salmon leukocytes have not been evaluated. This study aims to evaluate the enzymatic antioxidant defense and ROS-induced cell damage in Salmon Head Kidney-1 (SHK-1) cell line exposed to polyphenol-enriched extract from Sambucus nigra flowers. Results: Firstly, the Total Reactive Antioxidant Power (TRAP) assay of elderflower polyphenol (EP) was evaluated, showing 459 and 489 times more active than gallic acid and butyl hydroxy toluene (BHT), respectively. The toxic effect of EP on salmon cells was not significant at concentrations below 120 µg/mL and no hemolysis activity was observed between 20 and 400 µg/mL. The treatment of SHK-1 cell line with EP decreased both the lipid peroxidation and protein oxidation induced by H 2 O 2 , which could be associated with decreasing oxidative stress in the SHK-1 cells since the GSH/GSSG ratio increased when only EP was added. Conclusions: These results suggest that plant extracts enriched with polyphenols could improve the enzymatic antioxidant defense of salmon leukocytes and protect the cells against ROS-induced cell damage. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

It has been established that wood decay and cavities in tree stems contribute significantly to tree failures. Several techniques have been reported by researchers for detecting wood decay and cavities in living trees. These techniques are reviewed in this study under two broad categories: invasive and noninvasive methods. The invasive methods include traditional (decay detecting drill, increment borer, and boroscope), radiographic, acoustic, and electrical resistivity techniques. The noninvasive methods comprise microwave scanning, magnetic resonance imaging, X-ray tomography, and traditional techniques involving the use of mallets. Two or more methods are usually combined to investigate the health status of a tree for comparison and validation of results. The prospects and challenges of the various techniques in diagnosing wood decay, cavities, and other structural defects in living trees are reported. This review aims to help researchers in this field identify areas of further work towards the efficient monitoring and management of forest and urban trees.

This study developed a new coupled mathematical model of the extraction processes that occur in a ‘Crown Model’ type industrial extractor. The model incorporates the counter-current crossed flows of the porous medium and miscella, along with the mass transfer between them; the diffusion in the entire extraction field; the miscella transport between the percolation sections; the recirculating flows in the extraction sections; the influence of the loading and drainage zones; and the slug flow regime, which was discovered during experiments on a specially designed laboratory installation. The operating mode of a real extractor was investigated. A comparison between the predicted and experimental data showed satisfactory agreement. The coefficients for controlling oil losses were determined. The effect of varying the number of stages in the extractor with and without changing the total extractor length, as well as the effect of varying the layer height, on the oil yield and oil losses were investigated.

The paper contains data on registration of the SARS-CoV-2 virus in the European mink population. Profound and detailed studies of the virus circulation in the European mink population and the clinical manifestations of the disease, comprehensive approaches to the disease diagnosis, including epidemiological studies, clinical and post-mortem examinations, molecular genetic laboratory diagnostics (polymerase chain reaction and full-genome sequencing) contributed to better understanding of the disease features. The paper presents the data on post-mortem and histological lesions in the European minks infected with the new coronavirus SARS-CoV-2 obtained during the research. All the animals from which the pathological material was collected were infected with SARS-CoV-2, and the diagnosis was made using polymerase chain reaction (RT-PCR). The obtained and presented in the paper data reveal the features and dynamics of pathological processes in the body of infected animals (European mink), demonstrate the characteristics of the lesions in organs and tissues in case of acute and chronic disease, explain the clinical and post-mortem disease pattern and indicate the causes of animal deaths. All this together will allow veterinary specialists not only to quickly and timely diagnose the disease in the population of fur animals (European mink), but also to take necessary therapeutic and preventive measures in a timely manner, to select the most effective means for symptomatic and pathogenetic therapy as well as the most rational and effective substances and disinfection procedures.

This research reports the success of the fabrication of hydrophobically modified chitosan cryogel. Chitosan was modified with alkyl groups through reductive animation. By varying the alkyl chain length and the substitution degree, the resulting cryogel could be optimized for strength, and the adsorption and release of hydrophobic dye, a model for hydrophobic medicines. By optimizing these attributes, the hydrogel was found to have the potential as a biomedical material.

International audience; Acidification is an important process in the production of several dairy products and has an impact in taste, viscosity, and shelf life of the products. This paper deals with the development of an empirical mathematical model to describe milk acidification in terms of pH dynamics. The model is built upon experiments of pasteurized skimmed milk acidification for acid-induced casein precipitation and extended to predict the partitioning of calcium and phosphates in the whey and the dynamics of pH during fermentation of yoghurt manufacture. Furthermore, the model can be used directly or in optimization problems to provide recommendations about product design.

We describe a systematic approach to establish predictive models of CHO cell growth, cell metabolism and monoclonal antibody (mAb) formation during biopharmaceutical production. The prediction is based on a combination of an empirical metabolic model connecting extracellular metabolic fluxes with cellular growth and product formation with mixed Monod-inhibition type kinetics that we generalized to every possible external metabolite. We describe the maximum specific growth rate as a function of the integral viable cell density (IVCD). Moreover, we also take into account the accumulation of metabolites in intracellular pools that can influence cell growth. This is possible even without identification and quantification of these metabolites as illustrated with fed-batch cultures of Chinese Hamster Ovary (CHO) cells producing a mAb. The impact of cysteine and tryptophan on cell growth and cell productivity was assessed, and the resulting macroscopic model was successfully used to predict the impact of new, untested feeding strategies on cell growth and mAb production. This model combining piecewise linear relationships between metabolic rates, growth rate and production rate together with Monod-inhibition type models for cell growth did well in predicting cell culture performance in fed-batch cultures even outside the range of experimental data used for establishing the model. It could therefore also successfully be applied for in silico prediction of optimal operating conditions.

Background: Adipogenesis and fibrogenesis can be considered as a competitive process in muscle, which may affect the intramuscular fat deposition. The CCAAT/enhancer-binding protein beta (C/EBPβ) plays an important role in adipogenesis, which is well-characterized in mice, but little known in bovine so far. Results: In this study, real-time qPCR revealed that the level of C/EBPβ was increased during the developmental stages of bovine and adipogenesis process of preadipocytes. Overexpression of C/EBPβ promoted bovine fibroblast proliferation through mitotic clonal expansion (MCE), a necessary process for initiating adipogenesis, by significantly downregulating levels of p21 and p27 (p

Thermophilic anaerobic bacteria have received increasing interest as a promising bioprocessing platform for bioethanol production due to their broad substrate range and higher operating temperature. However, ethanol concentration is still lower than the commercial requirements on account of the high by-product formation and low ethanol tolerance. Our previous transcriptome analysis showed the gene expression level of pyruvate formate lyase-activating protein (PflA) was significantly down-regulated when Thermoanaerobacterium aotearoense SCUT27/Δldh was stimulated by various concentrations of ethanol. Based on this, we constructed the mutant strain T. aotearoense SCUT27/Δldh/ΔpflA, which not only possesses higher ethanol tolerance and production but also has enhanced ethanol/acetate ratio that is important for economical ethanol production. Compared to T. aotearoense SCUT27/Δldh, the mutant strain could resist to 5% (v/v) exogenous ethanol and produce 49 % more ethanol (38.82 g/L vs. 26.05 g/L) in 5-L bioreactor. In addition, the final concentration of ethanol reached 45.01 g/L by immobilized cell fermentation, which is the highest value obtained in T. aotearoense SCUT27. Simultaneously, the ethanol/acetate ratio of the mutant strain achieved 6.82, which was 2.16-fold higher than that of the parental strain.

The processes of chipless deformation of wood by means of a force field are less laborious and are not as-sociated with significant energy consumption and irrecoverable waste, as in the processing of such wood by chip cutting. To determine the cutting force on a laboratory setup, a factorial experiment was carried out for two main factors: the diameter of the cutting circle and the sharpening angle of the circular blade. Then, to estimate the va-riance characterizing the experimental error, a separate series of 5 experiments was set in the center of the plan, i.e. in conditions where each factor varies at the basic level. A mathematical model in natural values of the factors for the force deformation of wood by a disk has been obtained. Further, the following influencing factors have been investigated: wood thickness and feed rate. The analysis of the obtained regression equation indicates that the thickness of the cut workpiece has a major influence on the cutting power, which imposes a limitation on the feed rate. The next 4-factor experiment made it possible to reveal the combined influence of the named factors, as well as the moisture content of the wood and the length of the cut, on the quality indicators of the cutting process with circular knives. The analysis of the adequacy of the obtained regression equation showed its high accuracy and made it possible to reveal the influence of dominant external factors on the quality of the separated surfaces

Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed.

A method based on fluid dynamic gauging (FDG) was developed to investigate the membrane fouling behavior of streams containing dissolved wood components and small particles extracted using a mild steam explosion pretreatment. Industrially chipped softwood was subjected to saturated steam at 7 bar for 20 min, followed by cross-flow filtration of steam explosion liquors using 10 kDa polysulfone membranes at 2 bar transmembrane pressure. The results showed a severe decline in permeate flux during the initial stages of the cross-flow filtration. The FDG profiles from five filtration experiments revealed that thicker fouling layers were formed during initial fouling on pristine membranes compared to subsequent fouling on non-pristine membranes. The difference in fouling behavior suggests that cake layer formation was dominant during initial fouling, whereas pore blocking was more pronounced during refouling. This study highlights how FDG can be used to gain a better mechanistic understanding of the fouling behavior of extracted wood components.

Predictive microbiology models and 2.8E6 ham temperature measurements quantified absolute (API) and relative preservation indicators (RPI) assessing the performance of a residential refrigerator operating at 5 °C, the conventional setting for energy use assessments. Factors considered were compressor (single/variable speed, SS/VS), ambient temperature (21.1/32.2 °C), food load (22.5/39 kg), doors (closed/opening cycles), and fixed/movable samples (simulating breakfast use). APIT(t) based on 48 h temperature-dependent Listeria monocytogenes exponential growth ranged 0.23–0.36 log CFU/g at 32.2 °C/VS for all sample types and loads. The API value at the recommended 5 °C for ham (API5 °C, 0.155 log CFU/g) defined RPI = APIT(t)/API5 °C with values 1 indicating a better, similar or worse preservation than constant 5 °C storage. The highest RPI value (2.32) was observed for VS/32.2 °C/high load/movable samples; however, it reached 1.10 when the entire ham temperature profile was lowered by 3.7 °C, the maximum possible without risking product freezing. A probabilistic analysis considering the variability of predictive microbiology parameters and temperature measurements, yielded high 95% percentile RPI values for all conditions. Although the SS compressor showed better RPIs, operating strategies for all compressor types should be modified to ensure lower-temperatures and shorter temperature recovery times while still meeting energy use regulations. The higher energy efficiency and multiple adjustments possible of VS compressors suggest that they should deliver better food preservation than SS compressors. This study showed an effective use of product temperature to assess the preservation performance of all cold chain components.

Limited information is available on polysaccharides in Brassica rapa L. (BRP) cultivated at the plateau with an altitude over 3000 m. In the present study, an efficient ultrasonic-assisted extraction (UAE) procedure for polysaccharides from Brassica rapa L. was investigated and optimized by response surface methodology (RSM). Based on the optimal extraction parameters, gradient ethanol precipitation was used as the purification procedure of BRPs, obtaining four fractions named as BRP-20, BRP-40, BRP-60, and BRP-80, respectively. The four fractions presented different physicochemical properties and antioxidant activities. According to the chemical composition detection and monosaccharide compositions analysis, the uronic acids contents of BRP-40 and BRP-60 were higher than the other two fractions. The results of monosaccharide composition suggested that these four fractions were all heteropolysaccharides, consisted of rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, and glucuronic acid. Furthermore, BRP-80 gave the best performance in radical scavenging tests, which could attribute to its lowest molecular weight. What’s more, animal experiment suggested that BRP-80 had anti-fatigue and antioxidant activity in vivo. Overall, polysaccharides from Brassica rapa L. might be developed as a novel source of natural antioxidant and be a potential candidate of anti-fatigue dietary supplements.

Glycosmis stenocarpa is a species of shrub found in the Northern provinces of Vietnam. Its roots contain different carbazolic derivatives, mainly Murrayafoline A (Mu-A), which exhibits valuable biological activities. In this study, we performed an extraction of Mu-A from the roots of G. stenocarpa and optimized this process using response surface methodology (RSM) according to a central composite design, with three independent parameters including extraction time (min), extraction temperature (°C), and solvent/material ratio (mL/g). Two dependent variables were the Mu-A content (mg/g raw materials) and extraction efficiency (%). The optimal conditions to extract Mu-A were found to be as follows: extraction temperature, 67°C; extraction time, 165 min; and solvent/material ratio, 5:1. Under these conditions, the Mu-A content and extraction efficiency were 38.94 ± 1.31 mg/g raw materials and 34.98 ± 1.18%, respectively. Mu-A exhibited antiproliferation and antitumor-promoting activity against the HepG-2 cell line. The present optimization work of Mu-A extraction from G. stenocarpa roots contributed to the attempt of designing a large-scale extraction process for the compound and further exploitation of its potential in vivo applications.

Background: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. Results: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L −1 h −1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L −1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1 , which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. Conclusions: This report describes the first time that alginate production and alginate lyase ( alyA1 ) expression levels have been evaluated in A . vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Achieving a high product titer through pathway optimization often requires screening many combinations of enzymes and genetic parts. Typically, a library is screened in a single chassis that is a model or production organism. Here, we present a technique where the library is first introduced into B. subtilis XPORT, which has the ability to transfer the DNA to many Gram-positive species using an inducible integrated conjugated element (ICE). This approach is demonstrated using a two-gene pathway that converts tyrosine to melanin, a pigment biopolymer that can serve as a protective coating. A library of 18 pathway variants is conjugated by XPORT into 18 species, including those isolated from soil and industrial contaminants. The resulting 324 strains are screened and the highest titer is 1.2 g/L in B. amyloliquefaciens BT16. The strains were evaluated as co-cultures in an industrial process to make mycelia-grown bulk materials, where the bacteria need to be productive in a stressful, spatially non-uniform and dynamic environment. B. subtilis BGSC 3A35 is found to perform well under these conditions and make melanin in the material, which can be seen visually. This approach enables the simultaneous screening of genetic designs and chassis during the build step of metabolic engineering.

Background: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). Results: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. Conclusions: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine–metallo enzyme type.

Elaboration of bio-hydrogels was proposed using autohydrolysis extracts of Hericium erinaceus and Paulownia elongata x fortunei (leaves, bark and petioles). Paulownia extracts have been used as a solvent of H. erinaceus gels to provide an enrichment in phenolic content and confer antioxidant properties. Autohydrolysis under non isothermal heating of H. erinaceus was optimized. Regarding to antioxidant features, 210 °C was established as optimal extraction temperature. At this temperature, the extraction yield (67.4 g/100 g mushroom), the total phenol content (2.2 g GAE/100 g extract) and the antioxidant capacity (5.6 g Trolox eq./100 g extract) were maximum. The extract produced at 210 °C contained 30% oligosaccharides, mostly composed of glucose. Rheological testing indicated that glucan-based hydrogels formulated with selected H. erinaceus extracts in the presence of hydrothermal liquors of Paulownia exhibited stronger viscoelastic properties (around 30-fold) than those prepared with their commercial counterparts. The greatest enhancement of the mechanical properties was identified for hydrogels prepared with hydrothermal H. erinaceus extracts treated at 160 °C enriched with Paulownia petioles liquors (elastic modulus: 7 103 Pa, viscous modulus: 103 Pa), followed by barks and leaves ones. The absence of syneresis was identified for hydrogels prepared with tested hydrothermal extracts.

To utilize the effect of probiotics in the oral cavity, an easily applicable, orally dissolving dosage form of Lactobacillus paracasei was prepared by high-speed electrospinning. The scaled-up electrostatic drying technology resulted in high productivity of submicronic, fast-dissolving fibers. To minimize the viability loss of the bacteria, polyvinyl alcohol-polyethylene oxide (PVA-PEO) based polymer systems combined with different stabilizing excipients (glucose, lactose, mannitol, saccharose, trehalose, inulin, and skim milk) were developed. The use of excipients was successful to decrease osmotic and dehydration stress while increasing bacterial survival during electrospinning and long-term storage. The application of trehalose, saccharose, and skim milk as stabilizing excipient, gave 80% or higher bacterial survival during electrospinning; furthermore, in skim milk and mannitol containing formulations 0.5 log unit and 0.2 log unit viability loss was detected after one year. Skim milk showed the best results considering survival rate and long-term stability: after 1 year of storage, a cell count of 7.4 109 CFU/g was measured at 7 °C and 1.56 1010 CFU/g at −20 °C (initial loading was 2.7 1010 CFU/g). Thus, electrospinning using well-selected excipients provides an adequate system that can sustain high-viability of probiotic cells during long-term storage.

CRISPR-Cas transcriptional programming in bacteria is an emerging tool to regulate gene expression for metabolic pathway engineering. Here we implement CRISPR-Cas transcriptional activation (CRISPRa) in P. putida using a system previously developed in E. coli. We provide a methodology to transfer CRISPRa to a new host by first optimizing expression levels for the CRISPRa system components, and then applying rules for effective CRISPRa based on a systematic characterization of promoter features. Using this optimized system, we regulate biosynthesis in the biopterin and mevalonate pathways. We demonstrate that multiple genes can be activated simultaneously by targeting multiple promoters or by targeting a single promoter in a multi-gene operon. This work will enable new metabolic engineering strategies in P. putida and pave the way for CRISPR-Cas transcriptional programming in other bacterial species.

Biocleaning of cultural heritage items is mainly performed using living microorganisms. Approaches utilizing the enzymes of isolated microorganisms have not been frequently investigated. To find an enzymatic alternative for the removal of an oil-based overpainting, we focused on the characterization and use of a yeast Extracellular Enzymatic Mixture (EEM). A historical silk yeast was selected for its lipolytic properties and its EEM was extracted after cultivation on a medium supplemented with linseed oil. The EEM protein content was visualized by SDS-PAGE, its concentration assessed by fluorimeter and the enzymatic activity evaluated by p-NPP spectrophotometric lipase assay. The yeast growth was suppressed by adding diverse metal ions (Cd, Zn, Cr and Cu) in Reasoner's 2A (R2A) broth, while the quantity and activity of EEM were affected by adding Fe and Pb. Various delivery systems (agar-agar, tylose and klucel G) alone or in a combination with EEM were assayed on the historical painting surface. The colorimetric measurements and the ATR-FTIR analysis indicated that the combinations tylose-EEM and klucel G-EEM can be easily and effectively applied as biocleaning procedures to remove oil-based overpainting from fragile and valuable historical painting surfaces.

Background: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. Results: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. Conclusions: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L -1 , representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

The zein solutions containing different concentrations of cuminaldehyde (5%, 10%, and 20%, w/w) were electrospun. The morphology and average diameter of fibers were evaluated by scanning electron microscopy and the optimized fiber (20% cuminaldehyde) was chosen. Proton nuclear magnetic resonance spectroscopy and encapsulation efficiency showed the presence and desirable content of loaded cuminaldehyde in cuminaldehyde-loaded zein fiber, respectively. Chemical evaluations using Fourier transform infrared spectroscopy, X-ray diffraction, and confocal Raman spectroscopy assigned the domination of helical domains, amorphousness, and acceptive distribution to the resultant fiber, respectively. Modeling of Z19 and Z22 as well as molecular docking confirmed the results of chemical evaluations. Furthermore, the enhanced onset thermostability of cuminaldehyde (from 80 to 99 °C) was observed after the incorporation into zein fiber. Finally, the diffusion controlled-release of cuminaldehyde from a hydrophobic surface, its low-risk toxicity, and potent antibacterial activity of cuminaldehyde-loaded zein fiber against Staphylococcus aureus and Escherichia coli suggested its potentiality to be used in food and therapeutic applications.

Background: Acidithiobacillus ferrooxidans is a facultative anaerobe that depends on ferrous ion oxidation as well as reduced sulfur oxidation to obtain energy and is widely applied in metallurgy, environmental protection, and soil remediation. With the accumulation of experimental data, metabolic mechanisms, kinetic models, and several databases have been established. However, scattered data are not conducive to understanding A. ferrooxidans that necessitates updated information informed by systems biology. Results: Here, we constructed a knowledgebase of iron metabolism of A. ferrooxidans (KIMAf) system by integrating public databases and reviewing the literature, including the database of bioleaching substrates (DBS), the database of bioleaching metallic ion-related proteins (MIRP), the A. ferrooxidans bioinformation database (Af-info), and the database for dynamics model of bioleaching (DDMB). The DBS and MIRP incorporate common bioleaching substrates and metal ion-related proteins. Af-info and DDMB integrate nucleotide, gene, protein, and kinetic model information. Statistical analysis was performed to elucidate the distribution of isolated A. ferrooxidans strains, evolutionary and metabolic advances, and the development of bioleaching models. Conclusions: This comprehensive system provides researchers with a platform of available iron metabolism-related resources of A. ferrooxidans and facilitates its application. How to cite: Zhou Z, Ma W, Liu Y, et al. Potential application of a knowledgebase of iron metabolism of Acidithiobacillus ferrooxidans as an alternative platform. Electron J Biotechnol 2021;51; https://doi.org/10.1016/j.ejbt.2021.04.003

Chinese quince is one kind of crop that has edibility and historical medical applications. However, the high amount of lignin makes processing Chinese quince fruit difficult; on the other hand, this lignin represents a valuable resource if a way of using it in industrial applications could be found. In this study, the structural features and antioxidant activities of Brauns native lignin (BNL) isolated from Chinese quince fruit after oven drying (60 °C, 75 °C, and 90 °C), sun drying, freeze-drying, and subcritical dimethyl ether drying were assessed. The BNL samples had low molecular weights (Mw, 2604–5511 g/mol), high carbohydrate content (6.10–9.95 %), and high phenolic OH content (1.76–2.29 mmol/g). NMR spectra showed that the BNL is of the SGH-type with the relative content of p-hydroxyphenyl units ranging from 13 to 21 %. Oven drying (90 °C) reduced the carbohydrate content and increased the phenolic hydroxyl content. Freeze-drying reduced the relative content of β-O-4′ linkages and increased the H-unit content. In terms of antioxidant activities, all samples possessed high DPPH free radical scavenging capacity (IC50, 0.12−0.22 mg/mL) and strong reducing power. These results indicate that BNL extracted from Chinese quince fruit is a promising source of natural antioxidant and biomass material.

High pressure homogenization (HPH) at increasing intensity (50, 100, 150 MPa and 150 MPa for 5 passes) was combined with an alkaline-acid recovery method to prepare protein-enriched okara powders intended as ingredients with improved techno-functional properties. The treatment allowed obtaining from 4 to 8 g powder per 100 g fresh okara. The intensity of HPH treatment significantly affected amount, composition, particle size, protein structure and techno-functional properties. Treatments at intermediate pressures in the range 50−100 MPa resulted the most feasible from an industrial perspective allowing to obtain a good balance between protein content and the techno-functionalities (e.g. solubility, foaming, gelling and emulsifying properties) of the okara ingredient. Thus, HPH combined with simple pH-driven precipitation can be considered an efficacious and industrially feasible pre-treatment for okara valorisation into innovative ingredients.

Microbial fuel cell (MFC) technology is a promising solution for both organic and inorganic effluent treatment, as it is capable of handling a variety of complex contaminants with simultaneous energy and resource recovery. Unlike conventional treatment technologies, MFCs can achieve removal of organic matter, persistent organic compounds, heavy metals and nutrients from categorically different waste effluents, while recovering energy and valuable substances. This makes the technology economically attractive, and viable for a wide range of industries for their waste treatment applications. However, in order to implement this technology as an on-site treatment unit, limitations pertaining to costs, scale-up and performance have to be addressed, with due emphasis on developing strategies that can be easily applied in industrial settings. This review summarizes the recent progress in the field of waste treatment and environmental remediation using MFCs, and examines these operational challenges. Cost-effective materials that have been implemented on the field and at large scales have also been highlighted. In addition, the review highlights the fact that there is a need to optimize the technology depending on energy or resource recovery. The MFC-based technology utilizes the synergy of bioremediation and bioelectricity production from wastes, offering a sustainable approach to industrial waste management.

Thermal properties are critical parameters to design and implement efficient thermal processes especially for food manufacturing. Current methods to measure temperature-dependent thermal properties are expensive, time consuming and labor intensive with certain methods requiring composition analysis of food. To solve those problems, a novel method and equipment, named TPCell, was developed to estimate temperature-dependent thermal conductivity in a single experiment. For simultaneous estimation of temperature-dependent thermal conductivity and volumetric heat capacity, a new generation of TPCell was designed with complementary experimental profile and parameters were estimated by analyzing the parameter sensitivity coefficients. For different heating profile, the scaled sensitivity coefficients (SSC) of multiple parameters and optimal values were compared to determine the number of parameters that can be estimated. The thermal properties of sweet potato puree were estimated using sequential parameter estimation. The temperature-dependent thermal conductivity was found as 0.516 ± 0.011 W/mK at 26 °C and 0.798 ± 0.030 W/mK at 135 °C. Also, the volumetric heat capacity in the same temperature range was 3.520 × 106 ± 0.124 × 106 J/m3K. Therefore, the complementary experiment was successful in simultaneous estimation of temperature-dependent thermal conductivity and volumetric heat capacity.

The combination of solid phase microextraction (SPME) and gas chromatograph-mass spectrometer (GC-MS) is frequently used for comprehensive analysis of aroma components in foods because it can be used to easily analyze volatile components, allowing saving of the amount of solvent used. In this study, SPME-GC-MS analysis of sake samples before and after sherry cask storage was performed to investigate the special flavor derived from sherry cask storage. A GC column with polyethylene glycol as the stationary phase, which is the first choice for volatile component analysis, was used. However, the peak of the acid having a carbonyl group was tailed due to its bond with the hydroxyl group of the stationary phase. In the analysis of sake samples, a large and tailing peak derived from the large amount of fatty acids in Japanese sake was observed. Additionally, it was not possible to analyze other co-eluting components. To overcome this problem, a novel extraction condition was examined using SPME and tris (hydroxymethyl) aminomethane (Tris). By adding Tris solution to sake, the fatty acid peak was removed successfully, thereby facilitating analysis of the peaks of compounds co-eluting with fatty acids and comprehensive analysis of the aroma components in sake. Furthermore, a comparative analysis of sake before and after storage in sherry cask showed that levels of fatty alcohols, organic acid esters, fatty acid esters, and terpenes increased significantly after storage in sherry cask, suggesting that these ingredients might constitute the special flavor of sherry cask-stored sake.

Anaerobic digestion is a well-established technology for energy production and to diminish the environmental impact of different types of organic residues, which are mainly produced from human activities (processing food) but also from other recent activities related to energy generation processes (biofuel production). In the present study, mango organic residues: mango peel (MP) and mango seed (MS), and microalgal residue biomass (MRB) were co-digested in different proportions using a mixture design. The optimized mixture proportions of 60:20:20% of MP, MS and MRB, respectively, were determined by applying the simplex-centroid method to reach the highest biochemical methane potential (BMP); additionally, the mixture with the lowest BMP was selected (40:20:40%). The effects of thermal, physical and biological pretreatments of the selected mixtures were evaluated in order to improve its BMP. However, any pretreatment showed an improvement for the mixture 60:20:20, whereas the mixture 40:20:40 achieved an increment of 160.1, 72.5 and 96.4%, when the substrates were pretreated by thermal, physical and biological, respectively. The mixture optimization of these substrates shows an alternative way to reduce the environmental impact, due to their potential use in co-digestion process for biogas production.

Saccharopolyspora spinosa is a well-known actinomycete for producing the secondary metabolites, spinosad, which is a potent insecticides possessing both efficiency and safety. In the previous researches, great efforts, including physical mutagenesis, fermentation optimization, genetic manipulation and other methods, have been employed to increase the yield of spinosad to hundreds of folds from the low-yield strain. However, the metabolic network in S. spinosa still remained un-revealed. In this study, two S. spinosa strains with different spinosad production capability were fermented and sampled at three fermentation periods. Then the total RNA of these samples was isolated and sequenced to construct the transcriptome libraries. Through transcriptomic analysis, large numbers of differentially expressed genes were identified and classified according to their different functions. According to the results, spnI and spnP were suggested as the bottleneck during spinosad biosynthesis. Primary metabolic pathways such as carbon metabolic pathways exhibited close relationship with spinosad formation, as pyruvate and phosphoenolpyruvic acid were suggested to accumulate in spinosad high-yield strain during fermentation. The addition of soybean oil in the fermentation medium activated the lipid metabolism pathway, enhancing spinosad production. Glutamic acid and aspartic acid were suggested to be the most important amino acids and might participate in spinosad biosynthesis.

Background A wide variety of bacterial species produces protease enzyme, and the application of the same enzyme has been manipulated precisely and used in various biotechnological areas including industrial and environmental sectors. The main aim of this research study was to isolate, screen, and identify alkaline protease-producing bacteria that were sampled from leather industry effluent present in the outer skirts of Addis Ababa, Ethiopia. Purpose To isolate and characterize the alkaline protease-producing bacteria from leather industrial effluents. Methods Samples are collected from Modji leather industrial effluents and stored in the microbiology lab. After isolated bacteria from effluent using serial dilution and followed by isolated protease-producing bacteria using skim milk agar media. After studying primary and secondary screening using zonal inhibition methods to select potential protease-producing bacteria using skim milk agar media. Finally, to identify the potential bacteria using biochemical methods, bacterial biomass, protease activity, and gene sequencing (16S rRNA) method to finalize the best alkaline protease producing bacteria identified. Results First twenty-eight different bacterial colonies were isolated initially from the leather industry effluent sample situated at the Modjo town of Ethiopia. The isolated bacteria were screened using the primary and secondary screening method with skim milk agar medium. At the primary level, we selected three isolates namely ML5(14 mm), ML12(18 mm), and MS12 (15 mm), showing the highest zone of proteolysis as a result of casein degradation on the agar plates were selected and subjected to primary screening. Further secondary screening confirmed that the zone of inhibition methods ML5 (14.00±0.75 mm), ML12 (19.50±0.66 mm), and MS12 (15.00±1.32 mm) has efficient proteolytic activity and can be considered as effective protease producer. The three isolates were then subjected to morphological and biochemical tests to identify probably bacterial species, and all the three bacterial isolates were found out to be of Bacillus species. The shake flask method was carried out to identify the most potent one having greater biomass production capabilities and protease activity. ML12 isolated from leather effluent waste showed the highest protease activity (19 U/ml), high biomass production, and the same was subjected to molecular identification using 16s sequencing and a phylogenetic tree was constructed to identify the closest neighbor. The isolate ML12 (Bacillus cereus strain -MN629232.1) is 97.87% homologous to Bacillus cereus strain (KY995152.1) and 97.86% homologous to Bacillus cereus strain (MK968813.1). Conclusions This study has exposed that from twenty-eight different bacterial samples isolated from leather industry effluent; further primary and secondary screening methods were selected three potential alkaline protease strains. Finally, based on its biochemical identification, biomass, and protease activity, ML12 (Bacillus cereus strains) is the best strain identified. The alkaline protease has the significant feature of housing potent bacterial species for producing protease of commercial value.

A novel microaerobically inducible pUC18-derived plasmid was produced in Escherichia coli chromosomally expressing Vitreoscilla hemoglobin. The plasmid contains a copy of the positive replication control molecule rnaII under the control of the Pvgb promoter. The plasmid was unstable in the engineered strain. Sequencing showed that the plasmid was fragmented during preculture development, probably due to interactions between the host and vector genetic elements. Therefore, the rnaII copy under the control of the microaerobic promoters Pvgb, Pyfid, or Pyfidm was inserted in the minimal plasmid pUC57mini. Such plasmids were successfully produced in the engineered strain. The maximum plasmid DNA yield from biomass was 16.6 ± 0.1 mg g−1 in microaerobic cultures of the engineered strain using the Pyifd promoter. This was 14-fold greater than that using the original pUC57mini and the wild-type strain. Our results show the importance of considering host-vector interactions during the biodesign process and the potential of microaerobic induction for intensifying pDNA manufacturing.

Valorizing spent coffee grounds (SCG) through hydrothermal liquefaction (HTL) has great potential for biocrude production. To improve its economic viability, an integrated biorefinery route for SCG valorization was proposed in this study. High-value polyphenols and polysaccharides were extracted using water under ultrasound assistance. A central composite design was applied to jointly maximize the yields of both polyphenols and polysaccharides. Under the optimal conditions: a solid:water ratio of 1:23, an ultrasound power of 213 W, and an extraction time of 30 min at the temperature of 68 °C, the polyphenols yield was 1.0 wt.% (10 mg gallic acid equivalent/g SCG) and polysaccharides’ yield was 1.1 wt.% (11 mg glucose equivalent/g SCG). The biochemical compositions of raw and post-extraction SCG were characterized and exhibited appreciable difference, implying the impact of ultrasound-assisted extraction on feedstock composition. HTL of both raw and post-extraction SCG were conducted under three different temperatures (270 °C, 295 °C & 320 °C). It was found that 295 °C was the most desirable temperature, resulting in a biocrude yield of 31.1 wt.% and 27.7 wt.% for raw and post-extraction SCG, respectively. As for the biocrudes’ chemical composition, ester is the main component and increasing HTL temperature favored the fatty acid formation over ester, consequently led to more formation of amides.

Porcine circovirus type 2 (PCV2) is a non-enveloped, icosahedral virus of the Circoviridae family, with a small, circular, single-stranded DNA genome. PCV2 infections cause substantial economic losses in the pig industry worldwide. Currently, commercially produced PCV2 vaccines are expensive, whereas plant-based expression systems can produce recombinant proteins at low cost for use as vaccines. In this study, recombinant PCV2 capsid protein (rCap) was transiently expressed in Nicotiana benthamiana and purified by metal affinity chromatography, with a yield of 102 mg from 1 kg plant leaves. Electron microscopy confirmed that purified rCap self-assembled into virus-like particles (VLPs) at neutral pH. It was shown to provoke a strong immune response in guinea pigs. The results indicate that plant systems can enable production of large amounts of proteins to serve as candidates for subunit vaccines.

The present study is focused on developing microfibers based on the European fish eel skin gelatin (ESG) using the electrospinning process and evaluating its ability to encapsulate European fish eel oil (EO). Based on the scanning electron microscopy images, electrospinning of 15 % (w/v) gelatin solution in ethanol/water 40 % (v/v) was efficient to produce eco-friendly microfibers. Regarding electrospinning parameters, the increase of the voltage from 10 to 17 kV and the flow rate of feed solution from 0.04 to 0.2 mL/h improved the electrospinnability of ESG solution (15 %, w/v). Furthermore, ESG-based microfibers, loaded with EO, were prepared. The effects of the EO/ESG ratios (1/2 and 1:4, w/w) and two emulsification methods, homogenization (H) and homogenization followed by ultrasonication (HS) treatment, on the electrospun microfibers formation were investigated. The success of EO encapsulation was confirmed by FTIR spectroscopy analysis. The data also revealed that the highest microencapsulation efficiency encapsulation (89.79 %) was noted with EO-loaded microfibers prepared at EO/ESG ratio of 1:4 (w/w) by combining the two emulsification processes (HS). These results suggested that EO-loaded microfibers may be promising to be used as active encapsulating materials in food and nutraceutical fields., This study was funded by the Ministry of Higher Education and Scientific Research, Tunisia. Authors would also like to thank the Central Support Service for Experimental Research (SCSIE) of the University of Valencia for the electronic microscopy service.

Silver nanoparticles (AgNPs) have several applications in nanomedicine where the use of bacterial proteins for synthesis and capping of nanoparticles remain unstudied. Therefore, an extracellular protein of Enterobacter cloacae (SMP1) with ∼15.6 kDa size was partially purified and utilized for the synthesis of AgNPs. Synthesized AgNPs were detected by UV–vis spectrophotometer and characterized by dynamic light scattering (DLS), atomic force microscopy (AFM), cryo-scanning transmission electron microscopy (Cryo-STEM), and high-resolution transmission electron microscopy (HR-TEM). These AgNPs were monodispersed, spherical/quasi-spherical with zeta potential (ZP) of -26.9 mV with an average size of 58.75 nm measured by DLS. The encapsulation of AgNPs with protein was confirmed by electron elemental mapping and the potential mechanism of the synthesis of protein encapsulated AgNPs is outlined. These AgNPs showed broad spectrum bactericidal activity to clinical antibiotic-resistant bacteria. This study revealed that the extracellular protein of bacteria play a vital role in the synthesis of protein-encapsulated bactericidal AgNPs.

This paper studies the improvement of freeze–thaw stability of emulsions by incorporating glucosamine into soy protein isolate (SPI) by using transglutaminase. The occurrence of enzymatic glycosylated SPI reaction is confirmed by SDS-PAGE. Through the measurement of creaming index and oiling off, we find that both soy protein isolate-enzymatic without glucosamine (SPI-E) and glycosylated SPI by transglutaminase (SPI-G) can effectively improve the freeze–thaw stability of emulsions. The analysis of particle size, degree of flocculation and coalescence indicates that macromolecular aggregates were formed because transglutaminase enzyme promoted glycosylation and also promoted the cross-linking of protein and protein or the internal cross-linking of protein. Therefore, the particle size of the SPI-G emulsion before freezing and thawing is larger than that of the SPI emulsion. After freeze–thaw cycles, it can be seen that although the particle size of SPI-G is increasing, it is significantly smaller than that of SPI emulsion after freeze–thaw. According to the optical microscope, it can be seen that the SPI emulsion has obvious aggregation of oil droplets after freeze–thawing, and SPI-G effectively improves oil droplet aggregation.

Background: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin γ-thionin ( Capsicum chinense ) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. Results: The combined treatment of γ-thionin (3.5 µM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while γ-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. Conclusions: The γ-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), which is required for various redox systems involving antioxidative stress enzymes, is thus important for stress tolerance mechanisms. Here, we analyzed the stress response of the NADPH-depleted cells of Saccharomyces cerevisiae. A cell viability assay showed that the NADPH depletion induced by disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase, which is the major determinant of the intracellular NADPH/NADP+ ratio, enhanced the tolerance of S. cerevisiae to both oxidative and nitrosative stresses. The subsequent analyses demonstrated that the antioxidative transcriptional factor Yap1 was activated and the cytosolic catalase Ctt1, whose expression is regulated by Yap1, was upregulated in zwf1Δ cells irrespective of the presence or absence of stress stimuli. Moreover, deletion of the YAP1 or CTT1 gene inhibited the increased stress tolerance of zwf1Δ cells, indicating that Ctt1 dominantly contributed to the higher stress tolerance of zwf1Δ cells. Our findings suggest that an NADPH-independent mechanism enhances oxidative and nitrosative stress tolerance in ZWF1-lacking yeast cells.

Imaging flow cytometry (IFC) combines microscopy-based capturing of cell images with conventional flow cytometry-based high-throughput analysis. Thus, IFC approach may limit false-positive results and provide more accurate and unambiguous data. This may be particularly important in the case of the single-cell analysis of relatively small biological objects such as bacteria. The microbial quality control of milk samples is of a special interest during milk processing and several methods have been previously developed to address the presence of bacterial species in milk and other economically important dairy products. However, the vast majority of screening assays is time-consuming and cost-ineffective to be used as a routine test. In the present study, we propose combined acridine orange (AO) staining and IFC-based profiling of microbial species as a novel alternative method for the microbial quality control of milk samples. Eight bacterial profiles (Lactococcus lactis, Lactobacillus rhamnosus GG, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Staphylococcus aureus, Salmonella typhimurium and Sarcina lutea) were characterized using a number of IFC parameters and IFC approach was also validated using commercially available milk samples and probiotic supplement. The obtained results suggest that IFC-based microbial profiling may be considered as a potentially useful method for rapid screening for selected bacterial species in milk samples.

Two novel extracellular keratinases were produced by Actinomadura keratinilytica strain Cpt20. Both enzymes were purified to homogeneity using heat-treatment (60 °C for 30 min) and ammonium sulfate salt fractionation (40 %–70 %), followed by anion-exchange chromatography with fast protein liquid chromatography (FPLC) system. The purified keratinases, designated as KERA-71 and KERB-19, are monomeric and named according to their molecular masses of 71 kDa and 19 kDa, respectively, as estimated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), zymography, and high-performance liquid chromatography (HPLC). N-terminal residues of both enzymes exhibited high identity with other Actinomadura keratinases. Their hydrolytic activities were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF) and di-iodopropyl fluorophosphates (DFP), classifying them in the serine proteases family. While KERA-71 was ideally active at 50 °C and pH 8, KERB-19 illustrated optimum activity at 40 °C and pH 7. The thermo-activity and thermo-stability of both enzymes were improved with 10 mM Ca2+. Interestingly, the KERA-71 displayed broader substrate specificity, higher catalytic efficiency (kcat/Km), and a high degree of hydrolysis (DH) than actinobacterial keratinases, including KERB-19, KERDZ, KERAK-29, Actinase E, and KERAB. Interestingly, both enzymes exhibited effective keratinase activities with high potential, illustrating their possibility to be used in the process of keratin-containing wastes valorization and leather industry.