Discovery of azaleatin as a potential allosteric inhibitor for dengue NS2B-NS3 protease using in vitro and in silico studies.

The rise in dengue cases in tropical and sub-tropical areas has become a significant health concern. At present, there is no definitive cure for dengue fever, which underscores the importance of identifying potent inhibitors. Dengue NS2B-NS3 protease is the prime drug target due to its vital function for replication. Quercetin, a flavone, has anti-dengue virus properties but is limited by low bioavailability. Previous studies have shown that methoxy substitution in flavones improves bioavailability and metabolic stability. Azaleatin is a derivative of quercetin with a methoxy substitution at the C5 position, however its ability to inhibit dengue is unknown. In this study, azaleatin was investigated for its inhibition against dengue NS2B-NS3 protease using in vitro and in silico techniques. The fluorescence assay was used to determine the IC50 value and inhibition kinetics. The molecular interaction between azaleatin and NS2B-NS3 was studied using CB-Dock2 and AutoDock Vina. The complex's stability was then analysed using GROMACS. Besides, the ADMETlab 2.0 was utilized to predict pharmacokinetic of the azaleatin. Results showed that azaleatin inhibits dengue NS2B-NS3 protease non-competitively with a Ki of 26.82 µg/ml and an IC50 of 38 µg/ml. Molecular docking indicated binding of the azaleatin to the allosteric pocket of NS2B-NS3 with a docking score of -8.2 kcal/mol. Azaleatin was found stable in the pocket along 100 ns, supporting its inhibitory mode. The compound has favourable pharmacokinetic profiles and conformed to Lipinski's Rule of Five. Taken together, azaleatin inhibits NS2B-NS3 protease in a non-competitive mode, suggesting its potential as safer anti-dengue compound.Communicated by Ramaswamy H. Sarma.

Exploring a novel GH13_5 α-amylase from Jeotgalibacillus malaysiensis D5T for raw starch hydrolysis.

α-Amylase plays a crucial role in the industrial degradation of starch. The genus Jeotgalibacillus of the underexplored marine bacteria family Caryophanaceae has not been investigated in terms of α-amylase production. Herein, we report the comprehensive analysis of an α-amylase (AmyJM) from Jeotgalibacillus malaysiensis D5T (= DSM28777T = KCTC33550T). Protein phylogenetic analysis indicated that AmyJM belongs to glycoside hydrolase family 13 subfamily 5 (GH13_5) and exhibits low sequence identity with known α-amylases, with its closest counterpart being the GH13_5 α-amylase from Bacillus sp. KSM-K38 (51.05% identity). Purified AmyJM (molecular mass of 70 kDa) is stable at a pH range of 5.5-9.0 and optimally active at pH 7.5. The optimum temperature for AmyJM is 40 °C, where the enzyme is reasonably stable at this temperature. Similar to other α-amylases, the presence of CaCl2 enhanced both the activity and stability of AmyJM. AmyJM exhibited activity toward raw and gelatinized forms of starches and related α-glucans, generating a mixture of reducing sugars, such as glucose, maltose, maltotriose, maltotetraose, and maltopentaose. In raw starch hydrolysis, AmyJM exhibited its highest efficiency (51.10% degradation) in hydrolyzing raw wheat starch after 3-h incubation at 40 °C. Under the same conditions, AmyJM also hydrolyzed tapioca, sago, potato, rice, and corn raw starches, yielding 16.01-30.05%. These findings highlight the potential of AmyJM as a biocatalyst for the saccharification of raw starches, particularly those derived from wheat.

Draft genome sequence of Vibrio sp. PBL-C16, isolated from Batu Laut Beach in Selangor, Malaysia.

Vibrio sp. PBL-C16 is a bacterium that was isolated from Batu Laut Beach in Selangor, Malaysia. Here, we present a high-quality annotated draft genome of strain PBL-C16 and suggest its potential glycoside hydrolase enzymes for polysaccharide degradation.

Synthesis and biological activity of imidazole phenazine derivatives as potential inhibitors for NS2B-NS3 dengue protease.

A series of new imidazole-phenazine derivatives were synthesized via a two-step process. The condensation of 2,3-diaminophenazine and benzaldehyde derivatives proceeds with intermediate formation of an aniline Schiff base, which undergoes subsequent cyclodehydrogenation in situ. The structures of the synthesized compounds were characterized by 1D and 2D NMR, FTIR and HRMS. A total of thirteen imidazole phenazine derivatives were synthesized and validated for their inhibitory activity as anti-dengue agents by an in vitro DENV2 NS2B-NS3 protease assay using a fluorogenic Boc-Gly-Arg-Arg-AMC substrate. Two para-substituted imidazole phenazines, 3e and 3k, were found to be promising lead molecules for novel NS2B-NS3 protease inhibitors with IC50 of 54.8 µM and 71.9 µM, respectively, compared to quercetin as a control (IC50 104.8 µM). The in silico study was performed using AutoDock Vina to identify the binding energy and conformation of 3e and 3k with the active site of the DENV2 NS2B-NS3 protease Wichapong model. The results indicate better binding properties of 3e and 3k with calculated binding energies of -8.5 and -8.4 kcal mol-1, respectively, compared to the binding energy of quercetin of -7.2 kcal mol-1, which corroborates well with the experimental observations.

Phytochemical profiling of the essential oils from three Curcuma species and their in vitro and in silico dengue protease inhibition activity.

The chemical compositions, in vitro and in silico anti-dengue activity of the essential oils of the rhizomes of Curcuma longa Linn., C. aeruginosa Roxb., and C. xanthorrhiza Roxb. had been investigated. The C. longa oil was mainly composed of ar-turmerone (54.0%) and curlone (17.7%), while the C. aeruginosa oil was rich in curzerenone (23.4%), 1,8-cineole (21.2%), and camphor (7.1%). Xanthorrhizol (21.6%), ß-curcumene (19.5%), ar-curcumene (14.2%), and camphor (9.2%) were the major compounds in the C. xanthorrhiza oil. Among the oils, the C. longa oil was found to be the most active NSB-NS3 protease inhibitor (IC50 1.98 µg/mL). PLS biplot disclosed that the essential oils were classified into three separated clusters based on their characteristic chemical compositions, with C. longa positioned closest to the in vitro anti-dengue activity. Four compounds from the C. longa oil have both hydrogen and hydrophobic bonds that could be responsible for the DENV-2 NS2B-NS3 inhibitory effect.

Computational study of nitro-benzylidene phenazine as dengue virus-2 NS2B-NS3 protease inhibitor.

According to the World Health Organisation (WHO), as of week 23 of 2022, there were more than 1,311 cases of dengue in Malaysia, with 13 deaths reported. Furthermore, there was an increase of 65.7% during the same period in 2021. Despite the increase in cumulative dengue incidence, there is no effective antiviral drug available for dengue treatment. This work aimed to evaluate several nitro-benzylidene phenazine compounds, especially those that contain 4-hydroxy-3,5-bis((2-(4-nitrophenyl)hydrazinylidene)-methyl)benzoate through pharmacophore queries selection method as potential dengue virus 2 (DENV2) NS2B-NS3 protease inhibitors. Herein, molecular docking was employed to correlate the energies of selected hits' free binding and their binding affinities. Pan assay interference compounds (PAINS) filter was also adopted to identify and assess the drug-likeness, toxicity, mutagenicity potentials, and pharmacokinetic profiles to select hit compounds that can be considered as lead DENV2 NS2B-NS3 protease inhibitors. Molecular dynamics assessment of two nitro-benzylidene phenazine derivatives bearing dinitro and hydroxy groups at the benzylidene ring showed their stability at the main binding pocket of DENV2 protease, where their MM-PBSA binding energies were between -22.53 and -17.01 kcal/mol. This work reports those two nitro-benzylidene phenazine derivatives as hits with 52-55% efficiency as antiviral candidates. Therefore, further optimisation is required to minimise the lead compounds' toxicity and mutagenicity.

Draft genome sequence of Thermovorax subterraneus 70BT, a thermophile isolated from a geothermally active underground mine that produces hydrogen.

Thermovorax subterraneus 70BT is a thermophile found in a geothermically active underground mine. The strain 70BT belongs to the class of Clostridia, order of Thermosediminibacterales, and family of Thermosediminibacteraceae. Strain 70BT was the only type strain since the genus was discovered >10 years ago. Strain 70BT was compared to strains from other genera in terms of its phenotypics, chemotaxonomics, and phylogenetics (16S rRNA gene) in previous studies. However, the genome sequence of this strain has not been described. We herein described the genome sequence of strain 70BT. In total, the assembled genome of strain 70BT has a size of 2,451,552 bp, contributed by 44 contigs, with a coverage of 445X, a N50 of 86,294 bp, and a GC% of 43.8. A total of 2,540 genes were encoded in the genome, including 2,431 protein-coding sequences, 46 pseudogenes, and 63 RNA genes. Through the Cluster of Orthologous Groups (COGs) analysis, a total of 2,404 protein-coding genes were functionally assigned to COGs in the genome of strain 70BT. Among the members of Thermosediminibacteraceae family, strain 70BT has the closest relationship to Caldanaerovirga acetigignens JW/SA-NV4T based on the genome-to-genome comparison indexes (i.e., ANI, dDDH, AAI, and POCP). An earlier study reported that strain 70BT could produce hydrogen. We discovered genes encoding [FeFe] hydrogenase through gene mining analysis. For future research, this genome data will be used as a reference for all matters pertaining to the genus Thermovorax and family Thermosediminibacteraceae.

Draft Genome Sequence of Microaerobacter geothermalis Nad S1T, a Microaerophilic Bacterium Isolated from Tenusia Hot Spring.

Microaerobacter geothermalis Nad S1T is a rare Bacillaceae thermophile that grows optimally at 55°C and circumneutral pH. Although strain Nad S1T was discovered >10 years ago, its genome is yet to be described. The release of the Nad S1T genome sequence serves as reference genetic information for subsequent use.

Genome Analysis of Celeribacter sp. PS-C1 Isolated from Sekinchan Beach in Selangor, Malaysia, Reveals Its ß-Glucosidase and Licheninase Activities.

A halophilic marine bacterial strain, PS-C1, was isolated from Sekinchan beach in Selangor, Malaysia. The 16S rRNA gene sequence analysis indicated that strain PS-C1 was associated with the genus Celeribacter. To date, there have been no reports on enzymes from the genus Celeribacter. The present study reports on the cellular features of Celeribacter sp. PS-C1, its annotated genome sequence, and comparative genome analyses of Celeribacter glycoside hydrolase (GH) enzymes. The genome of strain PS-C1 has a size of 3.87 Mbp and a G+C content of 59.10%, and contains 3739 protein-coding genes. Detailed analysis using the Carbohydrate-Active enZYmes (CAZy) database revealed that Celeribacter genomes harboured at least 12 putative genes encoding industrially important GHs that are grouped as cellulases, ß-glucanases, hemicellulases, and starch-degrading enzymes. Herein, the potential applications of these enzymes are discussed. Furthermore, the activities of two types of GHs (ß-glucosidase and licheninase) in strain PS-C1 were demonstrated. These findings suggest that strain PS-C1 could be a reservoir of novel GH enzymes for lignocellulosic biomass degradation.

Designing DNA probe from HPV 18 and 58 in the E6 region for sensing element in the development of genosensor-based gold nanoparticles.

The E6 region has higher protuberant probability annealing than consensus probe focusing on another region in the human papillomavirus (HPV) genome in terms of detection and screening method. Here, we designed the first multiple virus single-stranded deoxyribonucleic acid (ssDNA) for multiple detections in an early phase of screening for cervical cancer in the E6 region and became a fundamental evolution of detection electrochemical HPV biosensor. Gene profiling of the virus ssDNA sequences has been carried by high-end bioinformatics tools such as GenBank, Basic Local Alignment Searching Tools (BLAST), and Clustal OMEGA in a row. The output from bioinformatics tools resulted in 100% of similarities between our virus ssDNA probe and HPV complete genome in the databases. The cross-validation between HPV genome and our designed virus ssDNA provided high specificity and selectivity during screening methods compared with Pap smear. The DNA probe for HPV 18, 5' COOH-GAT CCA GAA GGT ACA GAC GGG GAG GGC ACG 3', while 5'COOH-GGG CGC TGT GCA GTG TGT TGG AGA CCC CGA3' as DNA probe for HPV 58 designed with 66.77% guanine (G) and cytosine (C) content for both. Our virus ssDNA probe for the HPV biosensor promises high sensitivity, specificity, selectivity, repeatability, low fluid consumption, and will be useful in mini-size diagnostic devices for cervical cancer detection.

MicroRNA of N-region from SARS-CoV-2: Potential sensing components for biosensor development.

An oligonucleotide DNA probe has been developed for the application in the DNA electrochemical biosensor for the early diagnosis of coronavirus disease (COVID-19). Here, the virus microRNA from the N-gene of severe acute respiratory syndrome-2 (SARS-CoV-2) was used for the first time as a specific target for detecting the virus and became a framework for developing the complementary DNA probe. The sequence analysis of the virus microRNA was carried out using bioinformatics tools including basic local alignment search tools, multiple sequence alignment from CLUSTLW, microRNA database (miRbase), microRNA target database, and gene analysis. Cross-validation of distinct strains of coronavirus and human microRNA sequences was completed to validate the percentage of identical and consent regions. The percent identity parameter from the bioinformatics tools revealed the virus microRNAs' sequence has a 100% match with the genome of SARS-CoV-2 compared with other coronavirus strains, hence improving the selectivity of the complementary DNA probe. The 30 mer with 53.0% GC content of complementary DNA probe 5' GCC TGA GTT GAG TCA GCA CTG CTC ATG GAT 3' was designed and could be used as a bioreceptor for the biosensor development in the clinical and environmental diagnosis of COVID-19.

Draft Genome Sequence of Cellulomonas sp. PS-H5, Isolated from Sekinchan Beach in Selangor, Malaysia.

Cellulomonas sp. PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation method. The present work reports a high-quality draft annotated genome sequence of this strain and suggests its potential glycoside hydrolase enzymes for cellulose, hemicellulose, and starch degradations.

Draft Genome Sequence of Roseovarius sp. PS-C2, Isolated from Sekinchan Beach in Selangor, Malaysia.

Roseovarius sp. PS-C2 is a bacterium that was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation technique. Here, we present a high-quality annotated draft genome of strain PS-C2 and suggest potential applications of this bacterium.

Identification of potential dual -targets anti- toxoplasma gondii compounds through structure-based virtual screening and in-vitro studies.

Toxoplasma gondii is the etiologic agent of toxoplasmosis, a disease which can lead to morbidity and mortality of the fetus and immunocompromised individuals. Due to the limited effectiveness or side effects of existing drugs, the search for better drug candidates is still ongoing. In this study, we performed structure-based screening of potential dual-targets inhibitors of active sites of T. gondii drug targets such as uracil phosphoribosyltransferase (UPRTase) and adenosine kinase (AK). First screening of virtual compounds from the National Cancer Institute (NCI) was performed via molecular docking. Subsequently, the hit compounds were tested in-vitro for anti- T. gondii effect using cell viability assay with Vero cells as host to determine cytotoxicity effects and drug selectivities. Clindamycin, as positive control, showed a selectivity index (SI) of 10.9, thus compounds with SI > 10.9 specifically target T. gondii proliferation with no significant effect on the host cells. Good anti- T. gondii effects were observed with NSC77468 (7-ethoxy-4-methyl-6,7-dihydro-5H-thiopyrano[2,3-d]pyrimidin-2-amine) which showed SI values of 25. This study showed that in-silico selection can serve as an effective way to discover potentially potent and selective compounds against T. gondii.

Current Status and Potential Applications of Underexplored Prokaryotes.

Thousands of prokaryotic genera have been published, but methodological bias in the study of prokaryotes is noted. Prokaryotes that are relatively easy to isolate have been well-studied from multiple aspects. Massive quantities of experimental findings and knowledge generated from the well-known prokaryotic strains are inundating scientific publications. However, researchers may neglect or pay little attention to the uncommon prokaryotes and hard-to-cultivate microorganisms. In this review, we provide a systematic update on the discovery of underexplored culturable and unculturable prokaryotes and discuss the insights accumulated from various research efforts. Examining these neglected prokaryotes may elucidate their novelties and functions and pave the way for their industrial applications. In addition, we hope that this review will prompt the scientific community to reconsider these untapped pragmatic resources.

Raw oil palm frond leaves as cost-effective substrate for cellulase and xylanase productions by Trichoderma asperellum UC1 under solid-state fermentation.

Production of cellulases and xylanase by a novel Trichoderma asperellum UC1 (GenBank accession no. MF774876) under solid state fermentation (SSF) of raw oil palm frond leaves (OPFL) was optimized. Under optimum fermentation parameters (30 °C, 60-80% moisture content, 2.5 × 106 spores/g inoculum size) maximum CMCase, FPase, ß-glucosidase and xylanase activity were recorded at 136.16 IU/g, 26.03 U/g, 130.09 IU/g and 255.01 U/g, respectively. Cellulases and xylanase were produced between a broad pH range of pH 6.0-12.0. The enzyme complex that comprised of four endo-ß-1,4-xylanases and endoglucanases, alongside exoglucanase and ß-glucosidase showed thermophilic and acidophilic characteristics at 50-60 °C and pH 3.0-4.0, respectively. Glucose (16.87 mg/g) and fructose (18.09 mg/g) were among the dominant sugar products from the in situ hydrolysis of OPFL, aside from cellobiose (105.92 mg/g) and xylose (1.08 mg/g). Thermal and pH stability tests revealed that enzymes CMCase, FPase, ß-glucosidase and xylanase retained 50% residual activities for up to 15.18, 4.06, 17.47 and 15.16 h of incubation at 60 °C, as well as 64.59, 25.14, 68.59 and 19.20 h at pH 4.0, respectively. Based on the findings, it appeared that the unique polymeric structure of raw OPFL favored cellulases and xylanase productions.

Synthesis of geranyl propionate in a solvent-free medium using Rhizomucor miehei lipase covalently immobilized on chitosan-graphene oxide beads.

The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan-graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box-Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76 ± 0.02% of recovered protein had yielded 7.2 ± 0.04 mg g-1 and 211 ± 0.3% U g-1 of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20 hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70 rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.

Enzymatic properties and mutational studies of chalcone synthase from Physcomitrella patens.

PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant's active site.

Bacteriocin release protein-mediated secretory expression of recombinant chalcone synthase in Escherichia coli.

Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.