In this study, the evolution of the yeast microflora present on the berry surface, during the ripening of Barbera grapes, was monitored. Sampling was performed in three vineyards located in the “Nizza” Barbera d’Asti DOC zone and different methodologies have been employed. A culture-dependent method based on the identification of strains grown on solid media by ARDRA (Amplified Ribosomal DNA Restriction Analysis) and the D1-D2 domain of ribosomal 26S DNA capillary sequencing was coupled to NGS (Next Generation Sequencing) targeting ITS (Internal Transcribed Sequence) amplicons with the Illumina MiSeq platform. By using culture-dependent techniques, the most frequently detected species was the yeast-like fungus Aureobasidium pullulans, which was dominant in the culturable fraction. Among yeasts, the presence of oligotrophic basidiomycetes such as Cryptococcus spp., Rhodotorula graminis and Sporidiobolus pararoseus was observed at the beginning of ripening. Afterward, upon approaching th... [more]

Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) have caused severe diseases in swine populations worldwide. Here, a polycistronic baculovirus vector was developed to express a bivalent vaccine, consisting of the CSFV-E2 and PCV2-Cap protein, and an immunomodulator protein derived from the Flammulina velutipes, FVE-FIP, as well as the selection marker, green fluorescent protein. The simultaneous expression of the CSFV-E2 and PCV2-Cap protein was mediated by the 2A-like sequence derived from the Perina nuda virus (PnV), while the expression of the FVE-FIP was driven by the internal ribosome entry site (IRES) element derived from the Rhophalosipum padi virus (RhPV). The Western blot analysis result suggested that the CSFV-E2, PCV2-Cap, and FVE-FIP protein were successfully co-expressed by the infected Spodoptera frugiperda IPBL-Sf21 (Sf21) cell line. The extracted cell lysate containing all three recombinant proteins was administered to Balb/C mice with or without... [more]

This work explored the technical feasibility of using crab shell (CS) as a promising, low-cost biosorbent to individually and simultaneously remove Zn2+ and Ni2+ from aqueous solutions. It was found that in both monometallic and bimetallic systems, Zn2+ and Ni2+ biosorption by CS was strongly dependent on the solution pH, with the optimum biosorption occurring at a pH of 6.0 for both heavy metals. The obtained isotherms for Zn2+ and Ni2+ biosorption onto CS in monometallic and bimetallic systems demonstrated that CS has a higher affinity for Zn2+ than for Ni2+. The experimental equilibrium data for the bimetallic system revealed that when one heavy metal is present in the system, there is a decrease in the equilibrium biosorption capacity for the other heavy metal; therefore, the combined action of Zn2+ and Ni2+ was antagonistic. The Sips and Redlich−Peterson isotherm models best fitted the equilibrium biosorption data for Zn2+ and Ni2+ in the monometallic systems, while the modified S... [more]

Cyclic GMP-AMP (cGAMP) has lately been extensively investigated in cancer immunotherapy due its activation of the innate immunity stimulation of interferon genes (STING) pathway within antigen presenting cells (APC) leading to an increase in tumor specific CD8+ T cells. As negatively charged dinucleotides are prone to enzymatic degradation before being taken up by APC, there is a need for an appropriate carrier. Therefore, polyethylenimine (PEI), a gold standard for oligonucleotide delivery, was selected. Molecular weight, type of PEI and N/P ratio between PEI/cGAMP were investigated in terms of toxicity, efficacy and physicochemical properties of the nanocomplexes (NCs) such as size, zeta potential and shape. Due to lack of nano-medicine regulations and the need for a case-by case assessment, here we examine these parameters by several orthogonal methods, such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and online asymmetric flow field flow fractionation (A... [more]

The main objective is to effectively denature the autolysis enzymes of C. frondosa on the premise of avoiding the quality deterioration caused by overheating. The effects of the different thermal treatments (blanching at 40−80 °C for 45 min, boiling and steaming at 100 °C for 15−120 min) on the cooking yield, moisture content, protein degradation, texture, and enzyme inactivation were studied, and the inner relationship was investigated by multivariate analysis. The autolysis enzymes of C. frondosa were thermally stable and cannot be denatured completely by blanching. Boiling and steaming could efficiently inactivate the enzymes but overheating for 60−120 min reduced the cooking yield and texture quality. Boiling at 100 °C for 45 min was suitable for pre-treatment, with cooking yield of 70.3% and protein content of 78.5%. Steaming at 100 °C for at least 30 min was preferable for long-term storage and instant food, in which the relative activity was only 3.2% with better palatability.

Edible oil extraction is a large and well-developed sector based on solvent assisted extraction using volatile organic compounds such as hexane. The extraction of oil from oilseeds generates large volumes of oilseed by-products rich in proteins, fibres, minerals and secondary metabolites that can be valued. This work reviews the current status and the bio-macro-composition of oilseeds, namely soybean, rapeseed, sunflower and flaxseed, and the refining process, comprising the extraction of oil, the valorisation and separation of valuable secondary metabolites such as phenolic compounds, and the removal of anti-nutritional factors such as glucosinolates, while retaining the protein in the oilseed meal. It also provides an overview of alternative solvents and some of the unconventional processes used as a replacement to the conventional extraction of edible oil, as well as the solvents used for the extraction of secondary metabolites and anti-nutritional factors. These biologically active... [more]

The present study evaluated the effect of multiple variables (drying time, drying temperature, biomass/solvent ratio, glass beads/biomass ratio, extraction time, and extraction speed) in the solubilization of three different phycobiliproteins (C-PC, APC, and PE) from a thermotolerant Oscillatoria sp. The strain was grown in BG11 media (28 °C, light: dark cycle of 12:12 h at 100 µmol·m−2·s−1, 20 days) and the experiments were conducted according to a two-level randomized factorial design with six center points (38 runs). Results show that biomass/solvent ratio, glass beads/biomass ratio, and extraction time, are the most significant variables in the extraction of all three proteins, whereas the glass beads/biomass ratio and extraction time significantly affect their purity. The optimized conditions allow a statistical increase in the concentration of C-PC, APC, and PE extracted from the biomass; however, the purity was lower in comparison with the expected value. The latter occurs due t... [more]

The lipophilic yeast Malassezia furfur, is a member of the cutaneous commensal microbiota and is associated with several chronic diseases such as dandruff, pityriasis versicolor, folliculitis, and seborrheic dermatitis, that are often difficult to treat with current therapies. The development of alternatively effective antifungal therapies is therefore of paramount importance. In this study, we investigated the treatment effect of citral on M. furfur. The minimal inhibitory concentration of citral for M. furfur was 200 μg/mL, and the minimal fungicidal concentration was 300 μg/mL. Citral significantly increased the proportion of yeast cells to mycelial forms 2.6-fold. Phosphatidylserine externalization, DNA fragmentation, and metacaspase activation supported a citral-induced apoptosis in M. furfur. Moreover, citral at sub-minimum inhibitory concentrations reduced the invasion of M. furfur in HaCaT keratinocytes. Finally, we demonstrated that citral inhibited IL-6 and TLR-2 expression a... [more]

Cancer is a leading cause of death worldwide, and most of the currently available drugs for cancer treatment have limited potential. Natural products and their relatives continue to represent a very high percentage of the drugs used for cancer treatment. Curcumin is one of several natural drugs that has recently attracted much attention due to its putative cancer-preventive and anticancer properties. As well, Nitric Oxide (NO) holds a great potential for NO-based treatments for a wide variety of diseases. Here, for the first time, we tested the anti-cancer activities of an NO−Curcumin hybrid, hypothesizing that by joining the effects of curcumin and NO in one compound, the hybrid compound would be more potent than curcumin alone in treating colon cancer. To compare the anti-cancer activities of curcumin and NO−curcumin, we treated different colon cancer cell lines with either curcumin or NO−curcumin and tested their effects on cell proliferation and death. Our results show that NO−curc... [more]

In this study, several covalent methods of immobilization based on acrylic supports, Schiff bases and epoxides have been applied to a commercial cocktail with a high β-glucosidase activity secreted by Aspergillus fumigatus. This cocktail was preliminary compared to a commercial secretome of Aspergillus niger, which was also subjected to the aforementioned immobilization methods. Due to its higher activity, the cocktail from A. fumigatus immobilized on ReliZyme™ HA403 activated with glutaraldehyde was employed for pNPG and cellobiose hydrolysis in diverse operational conditions and at diverse enzyme loadings, showing a very high activity at high enzyme load. A kinetic model based on the Michaelis−Menten hypothesis, in which double inhibition occurs due to glucose, has been selected upon fitting it to all experimentally retrieved data with the lowest-activity immobilized enzyme. This model was compared to the one previously established for the free form of the enzyme, observing that cell... [more]

The high-osmotic-pressure environment of honey is not suitable for the survival of microorganisms, except for osmotic-tolerant fungal and bacterial spores. In this study, shotgun metagenomic sequencing technology was used to identify yeast species present in honey samples. As a result, Zygosaccharomyces spp. yeast, including Zygosaccharomyces rouxii, Z. mellis and Z. siamensis, were isolated. The intracellular trehalose and glycerin concentrations of yeast, as well as the antioxidant-related CAT, SOD and POD enzyme activities, increased under a high-glucose environment (60%, w/v). To learn more about the osmotic resistance of Z. mellis, iTRAQ-based proteomic technology was used to investigate the related molecular mechanism at the protein level, yielding 522 differentially expressed proteins, of which 303 (58.05%) were upregulated and 219 (41.95%) were downregulated. The iTRAQ data showed that the proteins involved in the pathway of the cell membrane and cell-wall synthesis, as well as... [more]

Actinomycetes are natural architects of numerous secondary metabolites including antibiotics. With increased multidrug-resistant (MDR) pathogens, antibiotics that can combat such pathogens are urgently required to improve the health care system globally. The characterization of actinomycetes available in Nepal is still very much untouched which is the reason why this paper showcases the characterization of actinomycetes from Nepal based on their morphology, 16S rRNA gene sequencing, and metabolic profiling. Additionally, antimicrobial assays and liquid chromatography-high resolution mass spectrometry (LC-HRMS) of ethyl acetate extracts were performed. In this study, we employed a computational-based dereplication strategy for annotating molecules which is also time-efficient. Molecular annotation was performed through the GNPS server, the SIRIUS platform, and the available databases to predict the secondary metabolites. The sequencing of the 16S rRNA gene revealed that the isolates BN6... [more]

Edible marine species are valuable sources of bioactive peptides. This study investigated the ACE-inhibitory activity of protein hydrolysates from the red algae Gracilaria tenuistipitata. Fifteen groups of protein hydrolysates were prepared by a two-step enzymatic hydrolysis of G. tenuistipitata: initial hydrolysis with several glycolytic enzymes, followed by three separate proteolytic reactions (Alcalase, Neutrase and Flavourzyme) for 2−10 h. Results showed that the hydrolysate GTN4H had the highest ACE-inhibitory activity in vitro. Furthermore, oral administration of GTN4H significantly reduced systolic blood pressure in spontaneously hypertensive rats. Fraction A derived from GTN4H displayed the highest ACE-inhibitory activity among fractions. Further purification of fraction A by RP-HPLC obtained a purified peptide (MW: 1776 Da) with 17 amino acids and 95.4% ACE-inhibitory activity.

A plant cell-based recombinant glucocerebrosidase was approved by the FDA in 2012 for the treatment of human inherited Gaucher disease, indicating that plant suspension cells have advantages in biosafety and a low production cost as a commercial pharmaceutical recombinant protein expression system. A low allergenic rice suspension cell-based recombinant protein expression system controlled by the αAmy3/RAmy3D promoter has been shown to result in relatively high protein yields in plant cell-based systems. Although several recombinant proteins have been produced in rice suspension cell-based systems, yields must be improved to compete with the current commercial protein expression systems. Different strategies were performed and showed successful improvements in recombinant protein yields in this rice system. The review updates and highlights strategies for potential improvements of the αAmy3-based rice suspension cell-based system.

Bioactive peptides with high potency against numerous human disorders have been regarded as a promising therapy in disease control. These peptides could be released from various dietary protein sources through hydrolysis processing using physical conditions, chemical agents, microbial fermentation, or enzymatic digestions. Considering the diversity of the original proteins and the complexity of the multiple structural peptides that existed in the hydrolysis mixture, the screening of bioactive peptides will be a challenge task. Well-organized and well-designed methods are necessarily required to enhance the efficiency of studying the potential peptides. This article, hence, provides an overview of bioactive peptides with an emphasis on the current strategy used for screening and characterization methods. Moreover, the understanding of the biological activities of peptides, mechanism inhibitions, and the interaction of the complex of peptide−enzyme is commonly evaluated using specific in... [more]

Grape pomace (GP) is a good source of high-value compounds as up to 60% of grape polyphenols remain in it after wine-making. To overcome traditional membrane technologies’ d rawbacks, such as fouling, a novel Zero Head Space extraction (ZHE) procedure was developed. The reaction vessel comprised a filtration device with a nitrocellulose membrane. The separation was performed at 50 lb/in2 and 23 °C, with no headspace during the process. Water and methanol (both acidifie d) were evaluated as solvents during two extraction stages for the recovery and fractionation of polyphenols. Aqueous extract (AE) was mainly constituted by monomeric polyphenols while Methanol extract (ME) presented less soluble compounds, as well as a higher concentration of total anthocyanin content than AE. Additional methanolic (CE) and acetone (CAE) extractions of residual GP showed CE presented a similar profile to ME (at a lower concentration), indicating ZHE efficiency at extracting polyphenols in GP. CAE presen... [more]

Translational medicine, the exchange between laboratory (bench) and the clinic (bedside), is decidedly taking on a vital role. Many companies are now focusing on a translational medicinal approach as a therapeutic strategy in decision making upon realizing the expenses of drug attrition in late-stage advancement. In addition, the utility of biomarkers in clinical decision and therapy guidance seeks to improve the patient outcomes and decrease wasteful and harmful treatment. Efficient biomarkers are crucial for the advancement of diagnoses, better molecular targeted therapy, along with therapeutic advantages in a broad spectrum of various diseases. Despite recent advances in the discovery of biomarkers, the advancement route to a clinically validated biomarker remains intensely challenging, and many of the candidate biomarkers do not progress to clinical applications, thereby widening the innovation gap between research and application. The present article will focus on the clinical vie... [more]

Human bone morphogenetic protein 2 (hBMP2) is routinely used in medical applications as an inducer of osteoformation. The recombinant production of BMP2 is typically performed using stable Chinese hamster ovary (CHO) cell lines. However, this process is inefficient, resulting in low product titers. In contrast, transient gene expression (TGE), which also enables the production of recombinant proteins, suffers from short production times and hence limited total product amounts. Here, we show that TGE-based BMP2 production is more efficient in HEKsus than in CHOsus cells. Independently of the cell lines, a bicistronic plasmid co-expressing EGFP and BMP2 facilitated the determination of the transfection efficiency but led to inferior BMP2 titers. Finally, we used a high cell density transient transfection (HCD-TGE) protocol to improve and extend the BMP2 expression by performing four rounds of serial transfections on one pool of HEKsus cells. This repeated transient transfection (RTT) pro... [more]

The ongoing pandemic of coronavirus disease (COVID-19) stimulated an unprecedented international collaborative effort for rapid diagnosis, epidemiologic surveillance, clinical management, prevention, and treatment. This review focuses on the current and new therapeutical approaches, summarizing the viral structure and life cycle, with an emphasis on the specific steps that can be interfered by antivirals: (a) inhibition of viral entry with anti-spike monoclonal antibodies; (b) inhibition of the RNA genome replication with nucleosidic analogs blocking the viral RNA polymerase; (c) inhibition of the main viral protease (Mpro), which directs the formation of the nonstructural proteins. An overview of the immunomodulatory drugs currently used for severe COVID-19 treatment and future therapeutical options are also discussed.

Oral antidiabetic drugs are usually costly and are associated with several adverse side effects. This has led to the use of medicinal plants that are considered to have multiple therapeutic targets and are readily accessible. In the Eastern Cape province of South Africa, the number of people using medicinal plants for the management of diabetes has been climbing steadily over the past two decades due to their cultural acceptability, accessibility, affordability, efficacy, and safety claims. In this study, a review of antidiabetic medicinal plants used in the Eastern Cape province of South Africa was conducted. A comprehensive literature survey was thoroughly reviewed using several scientific databases, ethnobotanical books, theses and dissertations. About forty-eight (48) plant species were identified as being used to treat diabetes by the people of Eastern Cape province. Among the plant species, only eight (8) species have not been scientifically evaluated for their antidiabetic activ... [more]

We propose a virtual screening method based on imbalanced data mining in this paper, which combines virtual screening techniques with imbalanced data classification methods to improve the traditional virtual screening process. First, in the actual virtual screening process, we apply k-means and smote heuristic oversampling method to deal with imbalanced data. Meanwhile, to enhance the accuracy of the virtual screening process, a particle swarm optimization algorithm is introduced to optimize the parameters of the support vector machine classifier, and the concept of ensemble learning is brought in. The classification technique based on particle swarm optimization, support vector machine and adaptive boosting is used to screen the molecular docking conformation to improve the accuracy of the prediction. Finally, in the experimental construction and analysis section, the proposed method was validated using relevant data from the protein data bank database and PubChem database. The experi... [more]

Bergenin is a phenolic glycoside that has been reported to occur naturally in several plant species, reported as a cardioprotective. However, bergenin, one of the important phytochemicals in these plants, is still not reported as a cardioprotective. The present study was designed to investigate the cardioprotective effects of bergenin on isoproterenol-induced myocardial infarction in rats. Bergenin and atenolol were administered through intraperitoneal (i.p.) injection to Sprague Dawley (SD) rats in separate experiments for five (5) days. At the end of this period, rats were administered isoproterenol (80 mg/kg s.c.) to induce myocardial injury. After induction, rats were anaesthetized to record lead II ECG, then sacrificed, blood was collected to analyze cardiac marker enzymes, and a histopathological study of the heart tissues was also performed. Pretreatment with bergenin showed a significant decrease in ST-segment elevation, deep Q-wave, infarct size, and also normalized cardiac ma... [more]

SARS-CoV-2, the virus that caused the widespread COVID-19 pandemic, is homologous to SARS-CoV. It would be ideal to develop antivirals effective against SARS-CoV-2. In this study, we chose one therapeutic target known as the main protease (Mpro) of SARS-CoV-2. A crystal structure (Id: 6LU7) from the protein data bank (PDB) was used to accomplish the screening and docking studies. A set of phytocompounds was used for the docking investigation. The nature of the interaction and the interacting residues indicated the molecular properties that are essential for significant affinity. Six compounds were selected, based on the docking as well as the MM-GBSA score. Pentagalloylglucose, Shephagenin, Isoacteoside, Isoquercitrin, Kappa-Carrageenan, and Dolabellin are the six compounds with the lowest binding energies (−12 to −8 kcal/mol) and show significant interactions with the target Mpro protein. The MMGBSA scores of these compounds are highly promising, and they should be investigated to det... [more]

Hibiscetin, as one of the main bioactive constituents of Hibiscus sabdariffa, has many pharmacological activities, but its antihyperglycemic activity has not been fully interpreted yet. The current research was developed from this perspective. The study intended to appraise the antidiabetic capability of hibiscetin in a high-fat diet (HFD) and streptozotocin (STZ; 50 mg/kg, intraperitoneally)-induced diabetes in an experimental animal. The efficiency of hibiscetin at 10 mg/kg in an “HFD/STZ model” remedy in rats with experimentally caused diabetes was explored for 42 days. The efficacy of hibiscetin was observed on several diabetes parameters. The average body weight and an array of biochemical markers were determined, including blood glucose, insulin, total protein (TP), lipid profile, aspartate aminotransferase (AST), alanine aminotransferase (ALT), IL-6, IL-1β, tumor necrosis factor-α (TNF-α), adiponectin, leptin, resistin, malondialdehyde (MDA), catalase (CAT), glutathione (GSH), a... [more]

(A. trifoliata) is a significant medicinal and edible fruit crop and has some important bioactivities. However, there are few studies on the bacteriostatic activity of A. trifoliata, and the underlying mechanism of A. trifoliata for antibacterial activity is still unknown. Therefore, the bacteriostatic activity and antibacterial mechanism of A. trifoliata were investigated by a combination of chemical assays, using the UHPLC-TOF-MS/MS technique. The results indicated that alkaloids, triterpenoids, and flavonoids are the major secondary bioactive compounds in A. trifoliata that play a crucial role in antibacterial activity. We found that EEPA exhibited both bacteriostatic and bactericidal effects against all Gram-positive and Gram-negative bacteria tested, with IZDs ranging from 13.80 ± 0.79 to 17.00 ± 0.58 mm. Significant differences in terms of sensitivity between Gram-positive and Gram-negative bacteria were not observed. In contrast, both antibiotics (kanamycin sulfate and ampicilli... [more]