A Combined Study on Optimization, In Silico Modeling, and Genetic Modification of Large Scale Microbial Cellulase Production

Cellulase is a biocatalyst that hydrolyzes cellulosic biomass and is considered a major group of industrial enzymes for its applications. Extensive work has been done on microbial cellulase but fungi are considered a novel strain for their maximum cellulase production. Production cost and novel microbial strains are major challenges for its improvement where cheap agro wastes can be essential sources of cellulose as substrates. The researcher searches for more cellulolytic microbes from natural sources but the production level of isolated strains is comparatively low. So genetic modification or mutation can be employed for large-scale cellulase production before optimization. After genetic modification than in silico molecular modeling can be evaluated for substrate molecule’s binding affinity. In this review, we focus not only on the conventional methods of cellulase production but also on modern biotechnological approaches applied to cellulase production by a sequential study on common cellulase-producing microbes, modified microbes, culture media, carbon sources, substrate pretreatment process, and the importance of optimum pH and temperature on fermentation. In this review, we also compare different cellulase activity determination methods. As a result, this review provides insights into the interrelationship between the characteristics of optimizing different culture conditions, genetic modification, and in silico enzyme modeling for the production of cellulase enzymes, which may aid in the advancement of large-scale integrated enzyme manufacturing of substrate-specific enzymes

Molecular Docking and Dynamics Simulation of Natural Compounds from Betel Leaves (<em>Piper betle</em> L.) for Investigating the Potential Inhibition of Alpha-Amylase and Alpha-Glucosidase of Type 2 Diabetes

Piper betle L. is widely distributed and commonly used medicinally important herb. It can also be used as a medication for type 2 diabetes patients. In this study, compounds of P. betle were screened to investigate the inhibitory action of alpha-amylase and alpha-glucosidase against type 2 diabetes through molecular docking, molecular dynamics simulation, and ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis. The molecule apigenin-7-O-glucoside showed the highest binding affinity among 123 (one hundred twenty-three) tested compounds. This compound simultaneously bound with the two-target proteins alpha-amylase and alpha-glucosidase, with high molecular mechanics-generalized born surface area (MM/GBSA) values (ΔG Bind = −45.02 kcal mol−1 for alpha-amylase and −38.288 for alpha-glucosidase) compared with control inhibitor acarbose, which had binding affinities of −36.796 kcal mol−1 for alpha-amylase and −29.622 kcal mol−1 for alpha-glucosidase. The apigenin-7-O-glucoside was revealed to be the most stable molecule with the highest binding free energy through molecular dynamics simulation, indicating that it could compete with the inhibitors’ native ligand. Based on ADMET analysis, this phytochemical exhibited a wide range of physicochemical, pharmacokinetic, and drug-like qualities and had no significant side effects, making them prospective drug candidates for type 2 diabetes. Additional in vitro, in vivo, and clinical investigations are needed to determine the precise efficacy of drugs

Effect of gamma irradiation on chemical composition, antioxidant activity, antibacterial activity, shelf life, and cytotoxicity in the peels of two mango varieties grown in Bangladesh

Mango (Mangifera indica) is perhaps the most notable sound natural product, however short-lived. A few synthetic compounds like calcium carbide, pesticides, formalin and calcium-based arrangements have been utilizing for its protection, notwithstanding, those synthetic compounds are perilous for our human wellbeing. Besides, mango strip is a food-handling by-product that is discarded since it isn't economically reasonable. For this study, the nutritional, antibacterial, and antioxidant properties of peels from two mango cultivars (Amrapali, and BARI-11) were analyzed with four doses (0.5, 1.0, 1.5, and 2 kGy) of gamma irradiation. The impacts of radiation on storage quality and life span of usability were likewise investigated. Mango peels were found to have many phytochemicals. Be that as it may, radiation altogether impacted tested parameters like fat, protein, carb, fiber, sugar, energy, soluble solids, etc. in both of the mango peels. On the other hand, the peel samples showed 50% DPPH scavenging activity at varied dosages. Moreover, samples exposed to 1.5 kGy of radiation showed higher antioxidant activity. At a radiation dose of 1.5 kGy, amrapali peel proved to be less hazardous to brine shrimp lethality assay than other samples. The widest inhibitory zones against Pseudomonas sp. were obtained at a dosage of 150 µg/disc. After two weeks of storage, the 1.5 kGy radiation dosage left the mangoes' texture firm, indicating that it was extending the fruit's shelf life by greatly reducing the microbial attack. Thus mangoes could be preserved using radiation without significantly affecting their nutritional value

Performance Evaluation of Developed Bangasure&trade; Multiplex rRT-PCR Assay for SARS-CoV-2 Detection in Bangladesh: A Blinded Observational Study at Two Different Sites

In this study, we evaluated the performance of the in-house developed rRT-PCR assay for SARS-CoV-2 RNA targeting the envelope (E) and nucleocapsid (N) genes with internal control as human RNase P. A total of 50 positive samples and 50 negative samples of SARS-CoV-2 were tested by a reference kit at site 1 and a subset (30 positives and 16 negatives) of these samples are tested blindly at site 2. The limit of detection (LoD) was calculated by using a replication-deficient complete SARS-CoV-2 genome and known copy numbers, where Pseudo-virus samples were used to evaluate accuracy. On site 1, among the 50 SARS-CoV-2 positive samples 24, 18, and eight samples showed high (Ct &lt; 26), moderate (26 &lt; Ct &le; 32), and low (32 &lt; Ct &le; 38) viral load, respectively, whereas in site 2, out of 30 SARS-CoV-2 positive samples, high, moderate, and low viral loads were found in each of the 10 samples. However, SARS-CoV-2 was not detected in the negative sample. So, in-house assays at both sites showed 100% sensitivity and specificity with no difference observed between RT PCR machines. The Ct values of the in-house kit had a very good correlation with the reference kits. LoD was determined as 100 copies/mL. It also displayed 100% accuracy in mutant and wild-type SARS-CoV-2 virus. This Bangasure&trade; RT-PCR kit shows excellent performance in detecting SARS-CoV-2 viral RNA compared to commercially imported CE-IVD marked FDA authorized kits

<i>Klebsiella pneumoniae</i> Volatile Organic Compounds (VOCs) Protect <i>Artemia salina</i> from Fish Pathogen <i>Aeromonas</i> sp.: A Combined In Vitro, In Vivo, and In Silico Approach

Antibiotic resistance is an alarming threat all over the world, and the biofilm formation efficacy of bacteria is making the situation worse. The antagonistic efficacy of Klebsiella pneumoniae against one of the known fish pathogens, Aeromonas sp., is examined in this study. Moreover, Aeromonas sp.’s biofilm formation ability and in vivo pathogenicity on Artemia salina are also justified here. Firstly, six selected bacterial strains were used to obtain antimicrobial compounds against this pathogenic strain. Among those, Klebsiella pneumoniae, another pathogenic bacterium, surprisingly demonstrated remarkable antagonistic activity against Aeromonas sp. in both in vitro and in vivo assays. The biofilm distrusting potentiality of Klebsiella pneumoniae’s cell-free supernatants (CFSs) was likewise found to be around 56%. Furthermore, the volatile compounds of Klebsiella pneumoniae were identified by GC-MS in order to explore compounds with antibacterial efficacy against Aeromonas sp. through an in silico study, where 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) (PDB: 5B7P) was chosen as a target protein for its unique characteristics and pathogenicity. Several volatile compounds, such as oxime- methoxy-phenyl-, fluoren-9-ol, 3,6-dimethoxy-9-(2-phenylethynyl)-, and 2H-indol-2-one, 1,3-dihydro- showed a strong binding affinity, with free energy of −6.7, −7.1, and −6.4 Kcal/mol, respectively, in complexes with the protein MTAN. Moreover, the root-mean-square deviation, solvent-accessible surface area, radius of gyration, root-mean-square fluctuations, and hydrogen bonds were used to ensure the binding stability of the docked complexes in the atomistic simulation. Thus, Klebsiella pneumoniae and its potential compounds can be employed as an alternative to antibiotics for aquaculture, demonstrating their effectiveness in suppressing Aeromonas sp

Plant-based phytochemical screening by targeting main protease of SARS-CoV-2 to design effective potent inhibitors

Currently, a worldwide pandemic has been declared in response to the spread of coronavirus disease 2019 (COVID-19), a fatal and fast-spreading viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The low availability of efficient vaccines and treatment options has resulted in a high mortality rate, bringing the world economy to its knees. Thus, mechanistic investigations of drugs capable of counteracting this disease are in high demand. The main protease (Mpro) expressed by SARS-CoV-2 has been targeted for the development of potential drug candidates due to the crucial role played by Mpro in viral replication and transcription. We generated a phytochemical library containing 1672 phytochemicals derived from 56 plants, which have been reported as having antiviral, antibacterial, and antifungal activity. A molecular docking program was used to screen the top three candidate compounds: epicatechin-3-O-gallate, psi-taraxasterol, and catechin gallate, which had respective binding affinities of −8.4, −8.5, and −8.8 kcal/mol. Several active sites in the targeted protein, including Cys145, His41, Met49, Glu66, and Met165, were found to interact with the top three candidate compounds. The multiple simulation profile, root-mean-square deviation, root-mean-square fluctuation, radius of gyration, and solvent-accessible surface area values supported the inflexible nature of the docked protein–compound complexes. The toxicity and carcinogenicity profiles were assessed, which showed that epicatechin-3-O-gallate, psi-taraxasterol, and catechin gallate had favorable pharmacological properties with no adverse effects. These findings suggest that these compounds could be developed as part of an effective drug development pathway to treat COVID-19

Use of Next-Generation Sequencing for Identifying Mitochondrial Disorders

Mitochondria are major contributors to ATP synthesis, generating more than 90% of the total cellular energy production through oxidative phosphorylation (OXPHOS): metabolite oxidation, such as the &beta;-oxidation of fatty acids, and the Krebs&rsquo;s cycle. OXPHOS inadequacy due to large genetic lesions in mitochondrial as well as nuclear genes and homo- or heteroplasmic point mutations in mitochondrially encoded genes is a characteristic of heterogeneous, maternally inherited genetic disorders known as mitochondrial disorders that affect multisystemic tissues and organs with high energy requirements, resulting in various signs and symptoms. Several traditional diagnostic approaches, including magnetic resonance imaging of the brain, cardiac testing, biochemical screening, variable heteroplasmy genetic testing, identifying clinical features, and skeletal muscle biopsies, are associated with increased risks, high costs, a high degree of false-positive or false-negative results, or a lack of precision, which limits their diagnostic abilities for mitochondrial disorders. Variable heteroplasmy levels, mtDNA depletion, and the identification of pathogenic variants can be detected through genetic sequencing, including the gold standard Sanger sequencing. However, sequencing can be time consuming, and Sanger sequencing can result in the missed recognition of larger structural variations such as CNVs or copy-number variations. Although each sequencing method has its own limitations, genetic sequencing can be an alternative to traditional diagnostic methods. The ever-growing roster of possible mutations has led to the development of next-generation sequencing (NGS). The enhancement of NGS methods can offer a precise diagnosis of the mitochondrial disorder within a short period at a reasonable expense for both research and clinical applications

Efficacy of phytochemicals derived from Avicennia officinalis for the management of COVID-19: a combined In silico and biochemical study

The recent coronavirus disease 2019 (COVID-19) pandemic is a global threat for healthcare management and the economic system, and effective treatments against the pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for this disease have not yet progressed beyond the developmental phases. As drug refinement and vaccine progression require enormously broad investments of time, alternative strategies are urgently needed. In this study, we examined phytochemicals extracted from Avicennia officinalis and evaluated their potential effects against the main protease of SARS-CoV-2. The antioxidant activities of A. officinalis leaf and fruit extracts at 150 µg/mL were 95.97% and 92.48%, respectively. Furthermore, both extracts displayed low cytotoxicity levels against Artemia salina. The gas chromatography–mass spectroscopy analysis confirmed the identifies of 75 phytochemicals from both extracts, and four potent compounds, triacontane, hexacosane, methyl linoleate, and methyl palminoleate, had binding free energy values of −6.75, −6.7, −6.3, and −6.3 Kcal/mol, respectively, in complexes with the SARS-CoV-2 main protease. The active residues Cys145, Met165, Glu166, Gln189, and Arg188 in the main protease formed non-bonded interactions with the screened compounds. The root-mean-square difference (RMSD), root-mean-square fluctuations (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), and hydrogen bond data from a molecular dynamics simulation study confirmed the docked complexes′ binding rigidity in the atomistic simulated environment. However, this study′s findings require in vitro and in vivo validation to ensure the possible inhibitory effects and pharmacological efficacy of the identified compounds