A Combined Experimental and Computational Study of Novel Benzotriazinone Carboxamides as Alpha-Glucosidase Inhibitors.

Diabetes is a chronic metabolic disorder of the endocrine system characterized by persistent hyperglycemia appears due to the deficiency or ineffective use of insulin. The glucose level of diabetic patients increases after every meal and medically recommended drugs are used to control hyperglycemia. Alpha-glucosidase inhibitors are used as antidiabetic medicine to delay the hydrolysis of complex carbohydrates. Acarbose, miglitol, and voglibose are commercial drugs but patients suffer side effects of flatulence, bloating, diarrhea, and loss of hunger. To explore a new antidiabetic drug, a series of benzotriazinone carboxamides was synthesized and their alpha-glucosidase inhibition potentials were measured using in vitro experiments. The compounds 14k and 14l were found to be strong inhibitors compared to the standard drug acarbose with IC50 values of 27.13 ± 0.12 and 32.14 ± 0.11 µM, respectively. In silico study of 14k and 14l was carried out using molecular docking to identify the type of interactions developed between these compounds and enzyme sites. Both potent compounds 14k and 14l exhibited effective docking scores by making their interactions with selected amino acid residues. Chemical hardness and orbital energy gap values were investigated using DFT studies and results depicted affinity of 14k and 14l towards biological molecules. All computational findings were found to be in good agreement with in vitro results.

Selection, identification and crystal structure of shark-derived single-domain antibodies against a green fluorescent protein.

Shark variable domain of new antigen receptors (VNARs) are the smallest naturally occurring binding domains with properties of low complexity, small size, cytoplasmic expression, and ease of engineering. Green fluorescent protein (GFP) molecules have been analyzed in conventional microscopy, but their spectral characteristics preclude their use in techniques offering substantially higher resolution. Besides, the GFP molecules can be quenched in acidic environment, which makes it necessary to develop anti-GFP antibody to solve these problems. In view of the diverse applications of GFP and unique physicochemical features of VNAR, the present study aims to generate VNARs against GFP. Here, we identified 36 VNARs targeting eCGP123, an extremely stable GFP, by phage display from three immunized sharks. These VNARs bound to eCGP123 with affinity constant KD values ranging from 6.76 to 605 nM. Among them, two lead VNARs named aGFP-14 and aGFP-15 with nanomolar eCGP123-binding affinity were selected for in-depth characterization. aGFP-14 and aGFP-15 recognized similar epitopes on eCGP123. X-ray crystallography studies clarified the mechanism by which aGFP14 interacts with eCGP123. aGFP-14 also showed cross-reaction with EGFP, with KD values of 47.2 nM. Finally, immunostaining analyses demonstrated that aGFP-14 was able to bind effectively to the EGFP expressed in both cultured cells and mouse brain tissues, and can be used as a fluorescence amplifier for EGFP. Our research demonstrates a feasible idea for the screening and production of shark-derived VNARs. The two high-affinity VNARs developed in the study contribute to the diversity of GFP sdAbs and may enhance the applications of GFP.

Wastewater treatment by using microalgae: Insights into fate, transport, and associated challenges.

The remediation of wastewater with microalgae is a new topic that concentrates on devising a cost-effective and environmentally beneficial method. Multiple microalgae and bacterial consortiums have recently been evaluated to determine if they can purify effluent from various sources. Critical to a system's efficacy is its ability to remove nutrients such as nitrogen (N) and phosphorus (P) and heavy metals such as arsenic (As), lead (Pb), and copper (Cu). This study compared traditional wastewater treatment systems to microalgae-based systems for treating different types of wastewater. The research investigates the potential for microalgae to cleanse wastewater. The research also evaluates wastewater parameters, methods, and scientific techniques for extracting nutrients and heavy metals from polluted water. According to the literature, Microalgae can remove between 98.7% and 100% of nitrogen (N), phosphorous (P), and heavy metals from various effluents. The paper concludes by discussing the difficulties of using microalgae to remediate wastewater. The elimination of nutrients from the effluent is influenced by biomass production, osmotic capacity, temperature, pH, and O2 concentration. Therefore, a "pilot" study is recommended to investigate contaminants.

In silico Screening of Potential SARS-CoV-2 Main Protease Inhibitors from Thymus schimperi.

Background: COVID-19 is still instigating significant social and economic chaos worldwide; however, there is no approved antiviral drug yet. Here, we used in silico analysis to screen potential SARS-CoV-2 main protease (Mpro) inhibitors extracted from the essential oil of Thymus schimperi which could contribute to the discovery of potent anti-SARS-CoV-2 phytochemicals. Methods: The absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles of compounds were determined through SwissADME and ProToxII servers. AutoDock tools were used for molecular docking analysis studies, while Chimera, DS studio, and LigPlot were used for post-docking studies. Molecular dynamic simulations were performed for 200 ns under constant pressure. Results: All compounds exhibited a bioavailability score of ≥0.55 entailing that at least 55% of the drugs can be absorbed unchanged. Only five (9%), nine (16%) and two (3.6%) of the compounds showed active hepatotoxicity, carcinogenicity, and immunotoxicity, respectively. Except for flourazophore P, which showed a little mutagenicity, all other compounds did not show mutagenic properties. On the other hand, only pinene beta was found to have a little cytotoxicity. Five compounds demonstrated effective binding to the catalytic dyad of the SARS-CoV-2 Mpro substrate binding pocket, while two of them (geranylisobutanoate and 3-octane) are found to be the best hits that formed hydrogen bonds with Glu166 and Ser144 of SARS-CoV-2 Mpro. Conclusion: Based on our in silico analysis, top hits from Thymus schimperi may serve as potential anti-SARS-CoV-2 compounds. Further in vitro and in vivo studies are recommended to characterize these compounds for clinical applications.

Corrigendum: Identification of novel therapeutic candidates against SARS-CoV-2 infections: An application of RNA sequencing toward mRNA based nanotherapeutics.

[This corrects the article DOI: 10.3389/fmicb.2022.901848.].

Experimental and Computational Analysis of Newly Synthesized Benzotriazinone Sulfonamides as Alpha-Glucosidase Inhibitors.

Diabetes mellitus is a chronic metabolic disorder in which the pancreas secretes insulin but the body cells do not recognize it. As a result, carbohydrate metabolism causes hyperglycemia, which may be fatal for various organs. This disease is increasing day by day and it is prevalent among people of all ages, including young adults and children. Acarbose and miglitol are famous alpha-glucosidase inhibitors but they complicate patients with the problems of flatulence, pain, bloating, diarrhea, and loss of appetite. To overcome these challenges, it is crucial to discover new anti-diabetic drugs with minimal side effects. For this purpose, benzotriazinone sulfonamides were synthesized and their structures were characterized by FT-IR, 1H-NMR and 13C-NMR spectroscopy. In vitro alpha-glucosidase inhibition studies of all synthesized hybrids were conducted using the spectrophotometric method. The synthesized compounds revealed moderate-to-good inhibition activity; in particular, nitro derivatives 12e and 12f were found to be the most effective inhibitors against this enzyme, with IC50 values of 32.37 ± 0.15 µM and 37.75 ± 0.11 µM. In silico studies, including molecular docking as well as DFT analysis, also strengthened the experimental findings. Both leading compounds 12e and 12f showed strong hydrogen bonding interactions within the enzyme cavity. DFT studies also reinforced the strong binding interactions of these derivatives with biological molecules due to their lowest chemical hardness values and lowest orbital energy gap values.

Identification of Novel Therapeutic Candidates Against SARS-CoV-2 Infections: An Application of RNA Sequencing Toward mRNA Based Nanotherapeutics.

Due to fast transmission and various circulating SARS-CoV-2 variants, a significant increase of coronavirus 2019 infection cases with acute respiratory symptoms has prompted worries about the efficiency of current vaccines. The possible evasion from vaccine immunity urged scientists to identify novel therapeutic targets for developing improved vaccines to manage worldwide COVID-19 infections. Our study sequenced pooled peripheral blood mononuclear cells transcriptomes of SARS-CoV-2 patients with moderate and critical clinical outcomes to identify novel potential host receptors and biomarkers that can assist in developing new translational nanomedicines and vaccine therapies. The dysregulated signatures were associated with humoral immune responses in moderate and critical patients, including B-cell activation, cell cycle perturbations, plasmablast antibody processing, adaptive immune responses, cytokinesis, and interleukin signaling pathway. The comparative and longitudinal analysis of moderate and critically infected groups elucidated diversity in regulatory pathways and biological processes. Several immunoglobin genes (IGLV9-49, IGHV7-4, IGHV3-64, IGHV1-24, IGKV1D-12, and IGKV2-29), ribosomal proteins (RPL29, RPL4P2, RPL5, and RPL14), inflammatory response related cytokines including Tumor Necrosis Factor (TNF, TNFRSF17, and TNFRSF13B), C-C motif chemokine ligands (CCL3, CCL25, CCL4L2, CCL22, and CCL4), C-X-C motif chemokine ligands (CXCL2, CXCL10, and CXCL11) and genes related to cell cycle process and DNA proliferation (MYBL2, CDC20, KIFC1, and UHCL1) were significantly upregulated among SARS-CoV-2 infected patients. 60S Ribosomal protein L29 (RPL29) was a highly expressed gene among all COVID-19 infected groups. Our study suggested that identifying differentially expressed genes (DEGs) based on disease severity and onset can be a powerful approach for identifying potential therapeutic targets to develop effective drug delivery systems against SARS-CoV-2 infections. As a result, potential therapeutic targets, such as the RPL29 protein, can be tested in vivo and in vitro to develop future mRNA-based translational nanomedicines and therapies to combat SARS-CoV-2 infections.

RP-HPLC Method Development, Validation, and Drug Repurposing of Sofosbuvir Pharmaceutical Dosage Form: A Multidimensional Study.

A smooth, exceptionally sensitive, correct, and extra reproducible RP-HPLC technique was developed and demonstrated to estimate Sofosbuvir (SOF) in pharmaceutical dosage formulations. This process was carried out by Agilent High-Pressure Liquid Chromatograph 1260 with GI311C Quat. Pump, Phenomenex Luna C-18 (150 mm × 4.6 mm × 5 µm) (USA), and Photodiode Array Detector (PDA) G1315D. The cell section, including acetonitrile and methanol with 80:20 v/v and solution (B) 0.1% phosphoric acid (40:60), was used for the study. However, 10 µL of the sample was injected with a drift flow of 1 mL/min. The separation occurred at a column temperature of 30 °C, and the eluents used PDA set at 260 nm. The retention time of SOF was 5 min. The calibration curve was modified linearly within the range of 0.05-0.15 mg/mL with a correlation coefficient of 0.99 and genuine linear dating among top vicinity and consciousness in the calibration curve. The detection and quantification restrictions were 0.001 and 0.003 mg/mL, respectively. SOF recovery from pharmaceutical components ranged from 98% to 99%. The percentage assay of SOF was 99%. Analytical validation parameters, such as specificity, linearity, precision, accuracy, and selectivity, were studied, and the percentage relative standard deviation (%RSD) was less than 2%. All other key parameters were observed within the desired thresholds. Hence, the proposed RP-HPLC technique was proven effective for developing SOF in bulk and pharmaceutical pill dosage forms. SOF was found to interact with SARS-COV-2 nsp12, and molecular docking results revealed its high affinity and firm binding within the active site groove of nsp12. The key interacting residues include; LYS-72, GLN-75, MET-80 ALA-99, ASN-99, TRP-100, TYR-101 with ASN-99 and TRP-100 forming hydrogen bonds. Molecular Dynamics simulation of SOF and nsp12 complex elucidated that the system was stable throughout 20ns. Therefore, this drug repurposing strategy for SOF can be used for treating COVID-19 infections by performing animal experiments and accurate clinical trials in the future.

Determining the level of essential elements in patients with Ewing Sarcoma: A correlation.

BACKGROUND: Metal ion aberrant metabolism is essential for health and disease, and its research has sparked a lot of interest. This study aims to compare the critical mineral resources-magnesium (Mg), sodium (Na), calcium (Ca) and potassium (K)-in biological materials (scalp hair, blood, and serum) of 87 Ewing Sarcoma (ES) hypertensive men and women, age range 31-60 years, in an urban area, with 62 nonhypertensive subjects from the same age range and living area. METHODS: An atomic-absorption spectrophotometer was used after microwave-induced acid digestion to determine elemental concentrations. The results' authenticity and precision were verified using a traditional wet acid digestion procedure and accredited reference materials. The average convalesces from all elements have been within the 99.2%-99.7% of certified values. RESULTS: In the biological samples from patients with ES hypertension, the amount of Na was found to be higher than in controls. Patients with ES hypertension had lower Mg, K, and Ca levels in their biological samples (scalp hair and blood) than healthy controls of both genders. CONCLUSIONS: Ca, Mg, and K deficiency can work with other ES hypertension risk factors. These findings will help physicians and other healthcare professionals determine the depletion of essential micronutrients in the biological samples (blood and scalp hair) of patients with hypertension. After microwave-induced acid digestion, the elemental concentration was determined using an atomic absorption spectrophotometer. The results' authenticity and precision were confirmed using a traditional wet-acid digestion procedure and accredited oriented materials. The average recoveries from all elements have been within the 99.2%-99.7% of certified values.

IgNAR antibody: Structural features, diversity and applications.

In response to the invasion of exogenous microorganisms, one of the defence strategies of the immune system is to produce antibodies. Cartilaginous fish is among those who evolved the earliest humoral immune system that utilizes immunoglobulin-type antibodies. The cartilaginous fish antibodies fall into three categories: IgW, IgM, and IgNAR. The shark Immunoglobulin Novel Antigen Receptor (IgNAR) constitutes disulfide-bonded dimers of two protein chains, similar to the heavy chain of mammalian IgGs. Shark IgNAR is the primary antibody of a shark's adaptive immune system with a serum concentration of 0.1-1.0 mg/mL. Its structure comprises of one variable (V) domain (VNAR) and five constant (C1 -C5) domains in the secretory form. VNARs are classified into several subclasses based on specific properties such as the quantity and position of additional non-canonical cysteine (Cys) residues in the VNAR. The VDJ recombination in IgNAR comprises various fragments; one variable component, three diverse sections, one joining portion, and a solitary arrangement of constant fragments framed in each IgNAR gene cluster. The re-arrangement happens just inside this gene cluster bringing about a VD1D2D3J segment. Therefore, four re-arrangement procedures create the entire VNAR space. IgNAR antibody can serve as an excellent diagnostic, therapeutic, and research tool because it has a smaller size, high specificity for antigen-binding, and perfect stability. The domain characterization, structural features, types, diversity and therapeutic applications of IgNAR molecules are highlighted in this review. It would be helpful for further research on IgNAR antibodies acting as an essential constituent of the adaptive immune system and a potential therapeutic agent.

Genome re-seqeunce and analysis of Burkholderia glumae strain AU6208 and evidence of toxoflavin: A potential bacterial toxin.

Burkholderia glumae, the primary causative agent of bacterial panicle blight in rice, has been reported as an opportunistic pathogen in patients with chronic infections. This study aimed to re-sequence the clinical isolate B. glumae strain AU6208 and comparatively analyze its genome using B. glumae strain BGR1 from rice plant as the reference. Re-sequencing results revealed that the genome of strain AU6208 comprised 96 contigs corresponding to a 6.1 Mbp genome of the strain AU6208, with 5322 coding sequences and 68.2 % GC content; this is much larger compared to the genome previously sequenced by us and described by Seo et al (2015), which was reported to be 4.1 Mbp comprising >1200 contigs, 4361 coding sequences, and 67.31 % GC content. Moreover, this updated genome shares >80 % identity to the 7.2 Mbp genome of BGR1, which encodes 6491 coding sequences and has 68.3 % GC content. Further computational analysis revealed that the strain AU6208 encodes several bacteriocin biosynthesis genes, antibiotic, as well as virulent genes such as toxoflavin genes, which included 425 specialty genes and 12 toxoflavin genes. Upon further characterization, 12 toxoflavins (ToxA, B, C, D, E, F, G, H, I, J, TofI, and TofR) were found in AU6208 with 70-100 % sequence, family, and domain similarity with that of BGR1. Upon comparison with BGR1, the structural characterizations of selected toxoflavin genes (ToxB, ToxC, ToxG, H, and TofI) revealed variations in 2D and 3D structures such as differences in α-helix, ß-sheets, loops, physiological properties of proteins, RMSD values, etc. These variations may play significant role in different mode of action in different hosts thereby indicating that in addition to their respective hosts, toxoflavins could also contribute to exploit other hosts across the kingdom. In addition to understanding the epidemiology of strain AU6208, this updated genomics data will also unfold the pathogenicity of bacteria in diversity of various hosts and anti-virulence.

Anti-HIV activity of new pyrazolobenzothiazine 5,5-dioxide-based acetohydrazides.

A series of fifteen new 2-[3-(3-chlorophenyl)-5,5-dioxidobenzo[e]pyrazolo[4,3-c][1,2]thiazin-4(2H)-yl]-N'-arylmethyleneacetohydrazides (5a-o) were synthesized and screened for their anti-HIV-1 and cytotoxicity activity. Out of fifteen pyrazolobenzothiazine-based hydrazones, thirteen were found to be active inhibitors of HIV with EC50 values <20 µM. Moreover, the cytotoxicity results showed that most of the compounds were toxic to PBM, CEM and Vero cell lines. This information could be used for structural modifications to acquire good candidates of HIV drugs.

2-[2-(3-Chloro-phen-yl)-2-oxoeth-yl]-1,2-benzisothia-zol-3(2H)-one 1,1-dioxide.

In the title compound, C(15)H(10)ClNO(4)S, the benzothia-zole ring system is essentially planar [maximum deviation = 0.0382 (13) Šfor the N atom] and forms a dihedral angle of 74.43 (6)° with the chloro-substituted benzene ring. In the crystal structure, weak inter-molecular C-H⋯O hydrogen bonds form R(2) (2)(10) and R(2) (2)(16) ring motifs.

3-(3-Chloro-benzo-yl)-4-hydr-oxy-2H-1,2-benzothia-zine 1,1-dioxide.

In the title compound, C(15)H(10)ClNO(4)S, the heterocyclic thia-zine ring adopts a half-chair conformation with the S and N atoms displaced by 0.476 (5) and 0.227 (5) Å, respectively, on opposite sides of the mean plane formed by the remaining ring atoms. The structure is stabilized by inter-molecular N-H⋯O and C-H⋯O hydrogen bonds. In addition, intra-molecular O-H⋯O and C-H⋯N inter-actions are also present.