Showing papers in "Molecular Biotechnology in 2021"

Elucidating Methods for Isolation and Quantification of Exosomes: A Review.

Abstract: Exosomes are the smallest extracellular vesicles present in most of the biological fluids. They are found to play an important role in cell signaling, immune response, tumor metastasis, etc. Studies have shown that these vesicles also have diagnostic and therapeutic roles for which their accurate detection and quantification is essential. Due to the complexity in size and structure of exosomes, even the gold standard methods face challenges. This comprehensive review discusses the various standard methods such as ultracentrifugation, ultrafiltration, size-exclusion chromatography, precipitation, immunoaffinity, and microfluidic technologies for the isolation of exosomes. The principle of isolation of each method is described, as well as their specific advantages and disadvantages. Quantification of exosomes by nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing, electron microscopy, dynamic light scattering, and microfluidic devices are also described, along with the applications of exosomes in various biomedical domains.

Benchtop Isolation and Characterisation of Small Extracellular Vesicles from Human Mesenchymal Stem Cells

Abstract: The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs’ size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5 nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.

Electropsun Polycaprolactone Fibres in Bone Tissue Engineering: A Review

Abstract: Regeneration of bone tissue requires novel load bearing, biocompatible materials that support adhesion, spreading, proliferation, differentiation, mineralization, ECM production and maturation of bone-forming cells. Polycaprolactone (PCL) has many advantages as a biomaterial for scaffold production including tuneable biodegradation, relatively high mechanical toughness at physiological temperature. Electrospinning produces nanofibrous porous matrices that mimic many properties of natural tissue extracellular matrix with regard to surface area, porosity and fibre alignment. The biocompatibility and hydrophilicity of PCL nanofibres can be improved by combining PCL with other biomaterials to form composite scaffolds for bone regeneration. This work reviews the most recent research on synthesis, characterization and cellular response to nanofibrous PCL scaffolds and the composites of PCL with other natural and synthetic materials for bone tissue engineering.

Novel Strategies to Combat Bacterial Biofilms.

Abstract: Biofilms are considered as a severe problem in the treatment of bacterial infections; their development causes some noticeable resistance to antibacterial agents. Biofilms are responsible for at least two-thirds of all infections, displaying promoted resistance to classical antibiotic treatments. Therefore, finding new alternative therapeutic approaches is essential for the treatment and inhibition of biofilm-related infections. Therefore, this review aims to describe the potential therapeutic strategies that can inhibit bacterial biofilm development; these include the usage of antiadhesion agents, AMPs, bacteriophages, QSIs, aptamers, NPs and PNAs, which can prevent or eradicate the formation of biofilms. These antibiofilm agents represent a promising therapeutic target in the treatment of biofilm infections and development of a strong capability to interfere with different phases of the biofilm development, including adherence, polysaccharide intercellular adhesion (PIA), quorum sensing molecules and cell-to-cell connection, bacterial aggregation, planktonic bacteria killing and host-immune response modulation. In addition, these components, in combination with antibiotics, can lead to the development of some kind of powerful combined therapy against bacterial biofilm-related infections.

LncRNA CCDC144NL-AS1 Serves as a Prognosis Biomarker for Non-small Cell Lung Cancer and Promotes Cellular Function by Targeting miR-490-3p

Abstract: This study revealed the prognostic significance of long non-coding RNA (lncRNA) CCDC144NL-AS1 in NSCLC patients and discussed the effect and mechanism of proliferation, migration, and invasion of non-small cell lung cancer (NSCLC) cells. 128 pairs of NSCLC tissues and paracancerous tissues were collected, and qRT-PCR was used to detect the differential expression of lncRNA CCDC144NL-AS1 in all tissues and cells lines. Kaplan-Meier analysis and Cox proportional hazards model analysis were used to estimate the prognostic value of lncRNA CCDC144NL-AS1. CCK-8 and Transwell assays confirmed the effect of lncRNA CCDC144NL-AS1 on the proliferation, migration, and invasion of NSCLC. Bioinformatics was used to predict the microRNAs that lncRNA CCDC144NL-AS1 might bind to miR-490-3p. The regulation of lncRNA CCDC144NL-AS1 on miR-490-3p was verified by luciferase activity assay with wide type or mutation. The expression of lncRNA CCDC144NL-AS1 was enhanced in both NSCLC tissues and cell lines. Patients with overexpression of lncRNA CCDC144NL-AS1 have a poor prognosis, and lncRNA CCDC144NL-AS1 is an independent prognostic factor for NSCLC. Increased the relative expression level of lncRNA CCDC144NL-AS1 can promote the proliferation, migration, and invasion of NSCLC cells. LncRNA CCDC144NL-AS1 might target miR-490-3p. LncRNA CCDC144NL-AS1 can be used as an oncogene of NSCLC to predict patient prognosis and promote tumor proliferation, migration, and invasion by targeting miR-490-3p.

Development and Genetic Engineering of Hyper-Producing Microbial Strains for Improved Synthesis of Biosurfactants

Abstract: Current research energies are fixated on the synthesis of environmentally friendly and non-hazardous products, which include finding and recognizing biosurfactants that can substitute synthetic surfactants. Microbial biosurfactants are surface-active compounds synthesized intracellularly or extracellularly. To use biosurfactants in various industries, it is essential to understand scientific engagements that demonstrate its potentials as real advancement in the 21st century. Other than applying a substantial effect on the world economic market, engineered hyper-producing microbial strains in combination with optimized cultivation parameters have made it probable for many industrial companies to receive the profits of 'green' biosurfactant innovation. There needs to be an emphasis on the worldwide state of biosurfactant synthesis, expression of biosurfactant genes in expressive host systems, the recent developments, and prospects in this line of research. Thus, molecular dynamics with respect to genetic engineering of biosynthetic genes are proposed as new biotechnological tools for development, improved synthesis, and applications of biosurfactants. For example, mutant and hyper-producing recombinants have been designed efficaciously to advance the nature, quantity, and quality of biosurfactants. The fastidious and deliberate investigation will prompt a comprehension of the molecular dynamics and phenomena in new microorganisms. Throughout the decade, valuable data on the molecular genetics of biosurfactant have been produced, and this solid foundation would encourage application-oriented yields of the biosurfactant production industry and expand its utilization in diverse fields. Therefore, the conversations among different interdisciplinary experts from various scientific interests such as microbiology, biochemistry, molecular biology, and genetics are indispensable and significant to accomplish these objectives.

Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-COV-2 Main Protease (3CLpro).

Abstract: The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.

CRISPR/Cas-Based Modifications for Therapeutic Applications: A Review

Abstract: The CRISPR-Cas genome editing system is an intrinsic property of a bacteria-based immune system. This employs a guide RNA to detect and cleave the PAM-associated target DNA or RNA in subsequent infections, by the invasion of a similar bacteriophage. The discovery of Cas systems has paved the way to overcome the limitations of existing genome editing tools. In this review, we focus on Cas proteins that are available for gene modifications among which Cas9, Cas12a, and Cas13 have been widely used in the areas of medicine, research, and diagnostics. Since CRISPR has been already proven for its potential research applications, the next milestone for CRISPR will be proving its efficacy and safety. In this connection, we systematically review recent advances in exploring multiple variants of Cas proteins and their modifications for therapeutic applications.

Folic Acid/Peptides Modified PLGA-PEI-PEG Polymeric Vectors as Efficient Gene Delivery Vehicles: Synthesis, Characterization and Their Biological Performance.

Abstract: Polymeric vectors are safer alternatives for gene delivery owing to their advantages as compared to viral vectors. To improve the stability and transfection efficiency of poly(lactic-co-glycolic acid) (PLGA)- and poly(ethylenimine) (PEI)-based vectors, poly(ethylene glycol) (PEG), folic acid (FA), arginylglycylaspartic acid (RGD) peptides and isoleucine-lysine-valine-alanine-valine (IKVAV) peptides were employed and PLGA-PEI-PEG-FA and PLGA-PEI-PEG-RGD copolymers were synthesized. PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA nanocomplexes (NCs) were formed through bulk mixing. The structure and properties, including morphology, particle size, surface charge and DNA encapsulation, of NCs were studied. Robust NCs with spherical shape, uniform size distribution and slightly positive charge were able to completely bind DNA above their respective N/P ratios. The critical N/P ratio for PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA NCs was identified to be 12:1, 8:1 and 10:1, respectively. The covalent modification of PEI through a combination of biodegradable PLGA, hydrophilic PEG and targeting motifs significantly decreased the cytotoxicity of PEI. The developed NCs showed both N/P ratio and cell type-dependent transfection efficiency. An increase in N/P ratio resulted in increased transfection efficiency, and much improved transfection efficiency of NCs was observed above their respective critical N/P ratios. This study provides a promising means to produce polymeric vectors for gene delivery.

Alternatives to Conventional Antibiotic Therapy: Potential Therapeutic Strategies of Combating Antimicrobial-Resistance and Biofilm-Related Infections

Abstract: Antibiotics have been denoted as the orthodox therapeutic agents for fighting bacteria-related infections in clinical practices for decades. Nevertheless, overuse of antibiotics has led to the upsurge of species with antimicrobial resistance (AMR) or multi-drug resistance. Bacteria can also grow into the biofilm, which accounts for at least two-thirds of infections. Distinct gene expression and self-produced heterogeneous hydrated extracellular polymeric substance matrix architecture of biofilm contribute to their tolerance and externally manifest as antibiotic resistance. In this review, the difficulties in combating biofilm formation and AMR are introduced, and novel alternatives to antibiotics such as metal nanoparticles and quaternary ammonium compounds, chitosan and its derivatives, antimicrobial peptides, stimuli-responsive materials, phage therapy and other therapeutic strategies, from compounds to hydrogel, from inorganic to biological, are discussed. We expect to provide useful information for the readers who are seeking for solutions to the problem of AMR and biofilm-related infections.

Comparative Transcriptome Analysis of Rice Resistant and Susceptible Genotypes to Xanthomonas oryzae pv. oryzae Identifies Novel Genes to Control Bacterial Leaf Blight.

Abstract: The bacterial leaf blight in rice caused by Xanthomonas oryzae pv. oryzae (Xoo) affects crop losses worldwide. In spite of developing resistant varieties by introgressing different Xa genes, the occurrence of diseases is evident. Here we report identification of several genes that are associated with improved plant immunity against Xoo in a resistant genotype BPT-5204 in comparison with susceptible genotype TN-1. The RNA sequencing information was developed to identify the genes that could provide durable resistance in rice. Xoo-resistant rice genotype BPT-5204 with Xa 5, 13 and 21 genes is compared with sensitive Taichung Native 1 (TN-1) to identify the genetic pathways and gene networks involved in resistance mechanisms. The higher levels of salicylic acid resulted in upregulation of many pathogenesis-related (PR) and redox protein encoding transcripts which resulted in higher hypersensitive response in BPT-5204. Many Serine/threonine protein kinase, leucine-rich repeat (LRR) transmembrane protein kinase, protein kinase family genes, Wall-associated kinase (WAK) were upregulated that resulted in activation of bZIP, WRKY, MYB, DOF and HSFs transcription factors that are associated with improved plant immunity. The study provided roles of many genes and their associated plant immunity pathways that can be used for developing resistant rice cultivars.

Bioinformatics Analysis of SARS-CoV-2 to Approach an Effective Vaccine Candidate Against COVID-19.

Abstract: The emerging Coronavirus Disease 2019 (COVID-19) pandemic has posed a serious threat to the public health worldwide, demanding urgent vaccine provide. According to the virus feature as an RNA virus, a high rate of mutations imposes some vaccine design difficulties. Bioinformatics tools have been widely used to make advantage of conserved regions as well as immunogenicity. In this study, we aimed at immunoinformatic evaluation of SARS-CoV-2 proteins conservancy and immunogenicity to design a preventive vaccine candidate. Spike, Membrane and Nucleocapsid amino acid sequences were obtained, and four possible fusion proteins were assessed and compared in terms of structural features and immunogenicity, and population coverage. MHC-I and MHC-II T-cell epitopes, the linear and conformational B-cell epitopes were evaluated. Among the predicted models, the truncated form of Spike in fusion with M and N protein applying AAY linker has high rate of MHC-I and MCH-II epitopes with high antigenicity and acceptable population coverage of 82.95% in Iran and 92.51% in Europe. The in silico study provided truncated Spike-M-N SARS-CoV-2 as a potential preventive vaccine candidate for further in vivo evaluation.

Melanin-Decolorizing Activity of Antioxidant Enzymes, Glutathione Peroxidase, Thiol Peroxidase, and Catalase.

Abstract: Melanin is the most important factor to determine skin color. Many research efforts are being undertaken to decompose the already-produced melanin compounds in skin for beauty. This research investigated the effects on reducing melanin color of the three antioxidant enzymes, Glutathione peroxidase (GPX), Thiol peroxidase (TPX), and Catalase, in lysosomal fraction. Melanin solution was treated with the enzymes and hydrogen peroxide, then reacted for 48 h. GPX and TPX decolorized melanin, and between them, GPX was more efficient, but Catalase was not effective. GPX also inhibited the production of melanin in B16F10 melanoma cells. GPX, which is present in almost all microorganisms, plays an important role in the cellular defense mechanism by reactive oxygen species. In addition, it was not cytotoxic, but was significantly effective in decolorizing melanin color. Therefore, in the biological and microbiological field, its possibility of utilization in skin whitening cosmetic is high.

DNA Fingerprinting and Genetic Relationships Similarities Among the Accessions/Species of Ocimum Using SCoT and ISSR Markers System

Abstract: Studies on genetic diversity could enhance taxonomic authentication and evolutionary relationship among the species of Ocimum. Therefore, diversity among 36 Ocimum accessions representing species from different regions of world were analyzed using Start Codon-Targeted Polymorphism (SCoT) and inter-simple sequences repeat (ISSR) marker. Marker systems used in this study was potentially targeted the different regions of the genome and included 18 SCoT and 15 ISSR primers, which showed successful amplification profile for Ocimum. Between these two, SCoT revealed the highest mean value of percentage of Polymorphism (84.6%), polymorphic information content (PIC, 0.65), and resolving power (Rp, 8.80), which were higher than ISSR. A total of 140 and 111 amplicons were obtained with SCoT and ISSR marker. The Mantel test indicted a significant correlation (r2 = 0.44) between ISSR and SCoT, which suggested a common genetical background among the accessions. The principal coordinate study showed the selection of different Ocimum genotypes by the cluster analysis. This study will help and support identification, genetic mapping, and molecular ecology to enhance the breeding program's efficiency for developing elite varieties to meet industrial demand globally. The present study is the first report of the genetic diversity, and relationship determination with SCoT-based molecular marker among Ocimum accessions.

Correction to: Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent.

Abstract: Laccases are polyphenol oxidoreductases used in a number of industrial applications. Due to the increasing demand for these “green catalysis” enzymes, the identification and biochemical characterisation of their novel properties is essential. In our study, cloned Madurella mycetomatis laccase (mmlac) genes were heterologously expressed in the methylotrophic yeast host Pichia pastoris. The high yield of the active recombinant protein in P. pastoris demonstrates the efficiency of a reliably constructed plasmid to express the laccase gene. The optimal biochemical conditions for the successfully expressed MmLac enzyme were identified. Detailed structural properties of the recombinant laccase were determined, and its utility in decolourisation and textile bleaching applications was examined. MmLac demonstrates good activity in an acidic pH range (4.0–6.0); is stable in the presence of cationic metals, organic solvents and under high temperatures (50–60 °C); and is stable for long-term storage at − 20 °C and − 80 °C for up to eight weeks. The structural analysis revealed that the catalytic residues are partially similar to other laccases. MmLac resulted in an increase in whiteness, whilst demonstrating high efficiency and stability and requiring the input of fewer chemicals. The performance of this enzyme makes it worthy of investigation for use in textile biotechnology applications, as well as within environmental and food technologies.

Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach.

Abstract: Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

Molecular Cloning and Differential Gene Expression Analysis of 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) in Andrographis paniculata (Burm. f) Nees

Abstract: Andrographis paniculata 1-deoxy-D-xylulose-5-phosphate synthase (ApDXS) gene (GenBank Accession No MG271749.1) was isolated and cloned from leaves for the first time. Expression of ApDXS gene was carried out in Escherichia coli Rosetta cells. Tissue-specific ApDXS gene expression by quantitative RT-PCR (qRT-PCR) revealed maximum fold expression in the leaves followed by stem and roots. Further, the differential gene expression profile of Jasmonic acid (JA)-elicited in vitro adventitious root cultures showed enhanced ApDXS expression compared to untreated control cultures. A. paniculata 3-hydroxy-3-methylglutaryl-coenzyme A reductase (ApHMGR) gene expression was also studied where it was up-regulated by JA elicitation but showed lower expression compared to ApDXS. The highest expression of both genes was found at 25 µm JA elicitation followed by 50 µm. HPLC data indicated that the transcription levels were correlated with increased andrographolide accumulation. The peak level of andrographolide accumulation was recorded at 25 μM JA (9.38-fold) followed by 50 µM JA (7.58-fold) in elicitation treatments. The in silico generated ApDXS 3D model revealed 98% expected amino acid residues in the favored and 2% in the allowed regions of the Ramachandran plot with 92% structural reliability. Further, prediction of conserved domains and essential amino acids [Arg (249, 252, 255), Asn (307) and Ser (247)] involved in ligand/inhibitor binding was carried out by in silico docking studies. Our present findings will generate genomic information and provide a blueprint for future studies of ApDXS and its role in diterpenoid biosynthesis in A. paniculata.

Revisiting the Characteristics of Testicular Germ Cell Lines GC-1(spg) and GC-2(spd)ts

Abstract: Spermatogenesis is a multifaceted and meticulously orchestrated process involving meiosis, chromatin build up, transcriptional and translational hushing, and spermiogenesis. Male germ cell lines GC-1spg (GC-1) and GC-2(spd)ts (GC-2) provide a useful resource to comprehend the molecular events occurring during such a tightly regulated process. Using cDNA microarray, expression profiling of GC-1 and GC-2 cell lines was done to precisely understand their characteristics and uniqueness. Our observations indicate that whilst both the cell lines are indeed of testicular origin, GC-2 is not haploid as was originally thought. Data analysis of the 23,351 transcripts detected in GC-1 and 20,992 in GC-2 cell lines demonstrates an 80% transcript overlap between GC-1 and GC-2 cells and ~ 40% similarity of both with the primary spermatocyte transcriptome. 3152 and 793 transcripts exclusive to GC-1 and GC-2, respectively, were identified. The presence of transcripts for 36 genes was validated in these cell lines including those showing testis-specific expression, as well as genes not reported previously. Overall, this study provides the transcriptome database of GC-1 and GC-2 cells. Analysis of the data demonstrates the transcriptomic transitions between GC-1 and GC-2 thus providing a glimpse to the process of germ cell differentiation from type B spermatogonium into preleptotene spermatocyte.

Heterologous Expression of Lignocellulose-Modifying Enzymes in Microorganisms: Current Status

Abstract: Heterologous expression of the carbohydrate-active enzymes in microorganisms is a promising approach to produce bio-based compounds, such as fuels, nutraceuticals and other value-added products from sustainable lignocellulosic sources. Several microorganisms, including Saccharomyces cerevisiae, Escherichia coli, and the filamentous fungi Aspergillus nidulans, have unique characteristics desirable for a biorefinery production approach like well-known genetic tools, thermotolerance, high fermentative capacity and product tolerance, and high amount of recombinant enzyme secretion. These microbial factories are already stablished in the heterologous production of the carbohydrate-active enzymes to produce, among others, ethanol, xylooligosaccharides and the valuable coniferol. A complete biocatalyst able to heterologous express the CAZymes of glycoside hydrolases, carbohydrate esterases and auxiliary activities families could release these compounds faster, with higher yield and specificity. Recent advances in the synthetic biology tools could expand the number and diversity of enzymes integrated in these microorganisms, and also modify those already integrated. This review outlines the heterologous expression of carbohydrate-active enzymes in microorganisms, as well as recent updates in synthetic biology.

Xylanolytic Enzymes in Pulp and Paper Industry: New Technologies and Perspectives.

Abstract: The pulp and paper industry discharges massive amount of wastewater containing hazardous organochlorine compounds released during different processing stages. Therefore, some cost-effective and nonpolluting practices such as enzymatic treatments are required for the potential mitigation of effluents released in the environment. Various xylanolytic enzymes such as xylanases, laccases, cellulases and hemicellulases are used to hydrolyse raw materials in the paper manufacturing industry. These enzymes are used either individually or in combination, which has the efficient potential to be considered for bio-deinking and bio-bleaching components. They are highly dynamic, renewable, and high in specificity for enhancing paper quality. The xylanase act on the xylan and cellulases act on the cellulose fibers, and thus increase the bleaching efficacy of paper. Similarly, hemicellulase enzyme like endo-xylanases, arabinofuranosidase and β-d-xylosidases have been described as functional properties towards the biodegradation of biomass. In contrast, laccase enzymes act as multi-copper oxidoreductases, bleaching the paper by the oxidation and reduction process. Laccases possess low redox potential compared to other enzymes, which need some redox mediators to catalyze. The enzymatic process can be affected by various factors such as pH, temperature, metal ions, incubation periods, etc. These factors can either increase or decrease the efficiency of the enzymes. This review draws attention to the xylanolytic enzyme-based advanced technologies for pulp bleaching in the paper industry.

Reviewing Challenges of Predicting Protein Melting Temperature Change Upon Mutation Through the Full Analysis of a Highly Detailed Dataset with High-Resolution Structures

Abstract: Predicting the effects of mutations on protein stability is a key problem in fundamental and applied biology, still unsolved even for the relatively simple case of small, soluble, globular, monomeric, two-state-folder proteins. Many articles discuss the limitations of prediction methods and of the datasets used to train them, which result in low reliability for actual applications despite globally capturing trends. Here, we review these and other issues by analyzing one of the most detailed, carefully curated datasets of melting temperature change (ΔTm) upon mutation for proteins with high-resolution structures. After examining the composition of this dataset to discuss imbalances and biases, we inspect several of its entries assisted by an online app for data navigation and structure display and aided by a neural network that predicts ΔTm with accuracy close to that of programs available to this end. We pose that the ΔTm predictions of our network, and also likely those of other programs, account only for a baseline-like general effect of each type of amino acid substitution which then requires substantial corrections to reproduce the actual stability changes. The corrections are very different for each specific case and arise from fine structural details which are not well represented in the dataset and which, despite appearing reasonable upon visual inspection of the structures, are hard to encode and parametrize. Based on these observations, additional analyses, and a review of recent literature, we propose recommendations for developers of stability prediction methods and for efforts aimed at improving the datasets used for training. We leave our interactive interface for analysis available online at http://lucianoabriata.altervista.org/papersdata/proteinstability2021/s1626navigation.html so that users can further explore the dataset and baseline predictions, possibly serving as a tool useful in the context of structural biology and protein biotechnology research and as material for education in protein biophysics.

Recent Advances in Nano-Bio-Sensing Fabrication Technology for the Detection of Oral Cancer

Abstract: Nanotechnology-based miniaturized devices have been a breakthrough in the pre-clinical and clinical research areas, e.g. drug delivery, personalized medicine. They have revolutionized the discovery and development of biomarker-based diagnostic devices for detection of various diseases such as tuberculosis, malaria and cancer. Nanomaterials (NMs) hold tremendous diagnostic potential due to their high surface-to-volume ratio and quantum confinement phenomenon, improving the detection limit of clinically relevant biomolecules in bio-fluids. Thus, they are helpful in the translation of bench-on platform to point-of-care (POC) screening device. The nanomaterial-based biosensor fabrication technology has also simplified and improved oral cancer (OC) or oral squamous cell carcinomas (OSCC) diagnosis. The fabrication of nano-bio sensors involves application specific modifications of NMs. The unique properties functionalized NMs have augmented their application on the nano-biosensing platform for the detection of clinically relevant biomolecules in bio-fluids. Therefore, this article summarizes the recent advancements in the process of fabrication of nano-biosensors for detection of OC.

Traditional System Versus DNA Barcoding in Identification of Bamboo Species: A Systematic Review

Abstract: Bamboo, a gramineous plant belonging to the family Poaceae, comprises of 1575 species from 116 genera across the globe. It has the ability to grow and evolve on degraded land and hence, can be utilized in the various applications as an alternative for plastic and wood. DNA barcoding, a long genomic sequence, identifies barcode region which shows species-specific nucleotide differences. This technology is considered as advanced molecular technique utilized for characterization and classification of the various species by applying distinctive molecular markers. Recent investigations revealed the potential application of various barcode regions such as matK, rbcL, rpoB, rpoC1, psbA-trnH, and ITS2, in identification of many bamboo species from different genus. In this review we comprehensively discussed the relevance of DNA barcoding as a tool in classification/identification of various bamboo species. We highlighted the methodology, how this advance technology overcomes the challenges associated with traditional methods along with prospects for future research.

METTL14-Mediated miR-30c-1-3p Maturation Represses the Progression of Lung Cancer via Regulation of MARCKSL1 Expression

Abstract: Lung cancer (LC) is a pulmonary malignant tumor with extremely low 5-year survival rate. N6-methyladenosine (m6A) is confirmed to regulate diverse pathophysiological processes including cancers. Methyltransferase-like 14 (METTL14) is an important RNA methyltransferase in m6A modification. However, researches on the regulatory mechanism of METTL14 on LC progression are relatively rare. Tumor xenograft experiment was conducted to investigate the effect of METTL14 on LC in vivo. The relative expression of METTL14, miR-30c-1-3p, and myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1) in LC tissues and/or cell lines was determined using qRT-PCR. Western blot assay was used to measure the protein levels of METTL14 and MARCKSL1 in tumor xenograft model and/or LC cell lines. MTT, wound healing, and transwell assays were performed to detect LC cell viability and metastasis. RNA immunoprecipitation assay and qRT-PCR were used to verify the effects of METTL14 on pri-miR-30c-1-3p. The relationship between miR-30c-1-3p and MARCKSL1 was confirmed by the dual-luciferase reporter assay. METTL14 was remarkably downregulated in LC tissues and cell lines. METTL14 mediated the maturation of miR-30c-1-3p. The overexpressed METTL14 and overexpressed miR-30c-1-3p suppressed the cell viability and metastasis in LC. Meanwhile, the increased METTL14 also repressed the growth of tumor xenograft in vivo. In addition, MARCKSL1 was confirmed to be the target gene of miR-30c-1-3p. High expression of MARCKSL1 and low expression of miR-30c-1-3p reversed the suppressive effects of METTL14 overexpression on cell viability and metastasis. METTL14 promoted the maturation of miR-30c-1-3p and mediated MARCKSL1 expression to inhibit the progression of LC. This study may provide a new insight for the LC clinical therapy.

COVID-19: Structural Considerations for Virus Pathogenesis, Therapeutic Strategies and Vaccine Design in the Novel SARS-CoV-2 Variants Era.

Abstract: COVID-19 pandemic caused by SARS-CoV-2 globally impacted the humanity causing tragic outcomes; costing millions of lives, destroying economies and demolishing public health infrastructures. The emergence of vaccines using various ingenious approaches in less than a year was deemed the light at the end of the tunnel. However, recent emergence of variants of SARS-CoV-2 in several parts of the world revealed that another hurdle is ahead in the fight against COVID-19. This review will highlight how SARS-CoV-2 mutations, creating different virus variants could potentially impact virus pathogenesis as well as different therapy approaches and vaccine design.

Fabrication and Characterization of Nanofibrous Poly (L-Lactic Acid)/Chitosan-Based Scaffold by Liquid-Liquid Phase Separation Technique for Nerve Tissue Engineering.

Abstract: Fabrication method is one of the essential factors which directly affect on the properties of scaffold Several techniques have been well established to fabricate nanofibrous scaffolds such as electrospinning However, preparing a three-dimensional (3-D) interconnected macro-pore scaffold essential for transporting the cell metabolites and nutrients is difficult using the electrospinning method The main aim of this study was developing a highly porous scaffold by poly (L-lactic acid) (PLLA)/chitosan blend using liquid–liquid phase separation (LLPS) technique, a fast and cost–benefit method, in order to use in nerve tissue engineering In addition, the effect of different polymeric concentrations on morphology, mechanical properties, hydrophilicity, in vitro degradation rate and pH alteration of the scaffolds were evaluated Moreover, cell attachment, cell viability and cell proliferation of scaffolds as candidates for nerve tissue engineering was investigated PLLA/chitosan blend not only had desirable structural properties, porosity, hydrophilicity, mechanical properties, degradation rate and pH alteration but also provided a favorable environment for attachment, viability, and proliferation of human neuroblastoma cells, exhibiting significant potential for nerve tissue engineering applications However, the polymeric concentration in blend fabrication had influence on both characteristics and cell responses It concluded that PLLA/chitosan nanofibrous 3-D scaffold fabricated by LLPS method as a suitable candidate for nerve tissue engineering

Fabrication and Evaluation of Layered Double Hydroxide-Enriched ß-Tricalcium Phosphate Nanocomposite Granules for Bone Regeneration: In Vitro Study.

Abstract: One of the most important challenges facing tissue engineering researches is the scaffold design with optimum physical and mechanical properties for growth and proliferation of cells, and tissue formation. The aim of this study was to produce a novel nanocomposite containing β-tricalcium phosphate and layered double hydroxide (β-TCP-LDH) and analyzing the capacity of its osteogenic activity in vitro. In this paper, β-tricalcium phosphate and layered double hydroxide powders were synthesized by co-precipitation processes. Then, the porous nanocomposite granules were prepared by the polyurethane sponge replication method. In this study, four kinds of β-TCP granules containing LDHs nanoparticles (ranging from 0.1 to 10 wt%) have been prepared. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) analyses were selected to study the phase structure, morphology, and phase distribution, respectively. Physicochemical characterizations demonstrated that the granules were synthesized successfully. Interconnected macro pores ranging over 200–500 μm were observed for all kinds of granules. SEM micrographs showed that human mesenchymal stem cells (hMSCs) were attached to the surfaces of the granules and proliferated in good shape. The results warranted that the synthesized granules exhibited good biocompatibility and mineralization. Based on the results of compressive strength and porosity tests, the most suitable type of granule is β-TCP/LDH 10 wt% with 77% porosity and compressive modulus of 231.4 MPa, which can be utilized in bone tissue engineering. To our knowledge, layered double hydroxides have not previously been incorporated into tricalcium phosphate granules for bone grafting. Also, this study is the first report on the effects of LDH on the mechanical properties and porosity of β-TCP granules. Our results demonstrated that β-TCP/LDH nanocomposite granule has a great potential for bone defects regeneration and tissue engineering applications.

Computational Lock and Key and Dynamic Trajectory Analysis of Natural Biophors Against COVID-19 Spike Protein to Identify Effective Lead Molecules.

Abstract: New pandemic infection of coronaviridae family virus spread to more than 210 countries with total infection of 1,136,851 and 62,955 (4.6%) deaths until 5th April 2020. Which stopped the regular cycle of humankind but the nature is consistently running. There is no micro molecule remedy found yet to restore the regular life of people. Hence, we decided to work on natural biophores against the COVID proteins. As a first step, major phytoconstituents of antiviral herbs like Leucas aspera, Morinda citrifolia, Azadirachta indica, Curcuma longa, Piper nigrum, Ocimum tenuiflorum, and Corallium rubrum collected and performed the lock and key analysis with major spike protein of COVID-19 to find the best fitting lead biophore using computational drug design platform. The results of protocol run showed, phytoconstituents of Morinda citrifolia and Leucas aspera were found lower binding energy range of - 55.18 to - 25.34 kcal/mol, respectively and compared with Hydroxychloroquine (HCQ) (- 24.29 kcal/mol) and Remdesivir (- 25.38 kcal/mol). The results conclude that, core skeletons chromen, anthracene 9, 11 dione and long-chain alkyl acids/ester-containing biophores showen high stable antagonistic affinity with S-protein. Which leads the breakdown of spike protein and ACE2 receptor complex formation and host mechanism of corono virus. In addition, the dynamic trajectory analysis confirmed the complete denaturation of spike protein by the molecule 4-(24-hydroxy-1-oxo-5-n-propyltetracosanyl)-phenol from Leucas aspera and stability of spike-ligand complex. These biophores will aid the researcher to fabricate new promising analogue and being recommended to assess its COVID-19 treatment.

Engineering of Plants for Efficient Production of Therapeutics.

Abstract: Plants are becoming useful platforms for recombinant protein production at present time. With the advancement of efficient molecular tools of genomics, proteomics, plants are now being used as a biofactory for production of different life saving therapeutics. Plant-based biofactory is an established production system with the benefits of cost-effectiveness, high scalability, rapid production, enabling post-translational modification, and being devoid of harmful pathogens contamination. This review introduces the main challenges faced by plant expression system: post-translational modifications, protein stability, biosafety concern and regulation. It also summarizes essential factors to be considered in engineering plants, including plant expression system, promoter, post-translational modification, codon optimization, and fusion tags, protein stabilization and purification, subcellular targeting, and making vaccines in an edible way. This review will be beneficial and informative to scholars and readers in the field of plant biotechnology.

MicroRNA miR-29a Inhibits Colon Cancer Progression by Downregulating B7-H3 Expression: Potential Molecular Targets for Colon Cancer Therapy.

Abstract: MiR-29a belongs to one of the subtypes of miRNAs known as non-coding single-stranded RNAs and is preferentially expressed in normal tissues. B7-H3, a member of the B7/CD28 immunoglobulin superfamily, was shown to be overexpressed in several solid malignant tumors, including colon cancer. In addition, it is associated with tumor progression and poor prognosis. We used immunohistochemical and Western blotting to assess B7-H3 protein expression levels in colon cancer and adjacent normal tissues and then compared their relationships with clinicopathological factors. Quantitative real-time reverse-transcription PCR was used to assess B7-H3 and miRNA-29a mRNA expression levels, and then their relationship and clinical significance were evaluated. In addition, colon cancer Caco-2 cells, which constitutively overexpress B7-H3, were transfected with lentivirus particles for miR-29a upregulation. Invasion and migration assays were carried out in vitro along with the establishment of a subcutaneous xenograft model in vivo to determine the role of miRNA-29a in colon cancer progression. The B7-H3 protein showed elevated expression in colon carcinoma and was relevant to TNM staging, lymph node metastasis, and reduced survival. Meanwhile, miR-29a was preferentially expressed in normal colon tissues, while B7-H3 transcript levels had no marked differences between tumor and normal tissue specimens. In vitro, miR-29a upregulation resulted in reduced B7-H3 expression. Furthermore, miR-29a upregulation reduced the invasive and migratory abilities of colon carcinoma cells. In animal models, upregulation of miR-29a slowed down the growth of subcutaneous xenotransplanted tumors and resulted in prolonged survival time. MiR-29a downregulates B7-H3 expression and accordingly inhibits colon cancer progression, invasion, and migration, indicating miR-29a and B7-H3 might represent novel molecular targets for advanced immunotherapy in colon cancer.