Investigation on plasmid DNA separation under vacuum suction using cellulose generated and polyethersulfone ultra filters

E. coli DH5α harbouring pET28a+ plasmid vector was lysed by alkaline lysis and the clarified plasmid solutions were subjected to ultrafiltration experiments using two types of ultrafiltration membrane; i.e., cellulose generated (Ultracel) and polyethersulfome PES (Biomax). Transmembrane pressures (TMP) through vacuum suction of 0.1 to 0.7 bar were applied to the plasmid sample, and the corresponding flow rates and fluxes for both filters were investigated. Even though, these two filters showed a slight different in the flux, a marked different in DNA transmission were observed. DNA transmission were generally higher with cellulose generated filters, whereby DNA transmission by Ultracel filter was at44.3, 63.9, 74.1, 55.6 and 54.5% at TMP of 0.1, 0.18, 0.39, 0.6 and 0.7 bar, respectively. Meanwhile, for PES filters the DNA transmission was at 22.3, 38.1, 39.1, 38.0 and 37.5% at TMP of 0.1, 0.3, 0.4, 0.6 and 0.7 bar, respectively. With the cellulose generated filter, DNA transmission reached an optimum (~70%) at about 0.6 bar after which the transmission depleted at higher TMP of 0.7 bar. Throughout all of the TMP, DNA transmissions observed were generally lower with PES filter. The properties of the filter material could have contributed to the differences in DNA permeatio

In silico screening of aptamers configuration against hepatitis B surface antigen

Aptamer has been long studied as a substitute of antibodies for many purposes. However, due to the exceeded length of the aptamers obtained in vitro, difficulties arise in its manipulation during its molecular conjugation on the matrix surfaces. Current study focuses on computational improvement for aptamers screening of hepatitis B surface antigen (HBsAg) through optimization of the length sequences obtained from SELEX. Three original aptamers with affinity against HBsAg were truncated into five short hairpin structured aptamers and their affinity against HBsAg was thoroughly studied by molecular docking, molecular dynamics (MD) simulation, and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method. The result shows that truncated aptamers binding on HBsAg “a” determinant region are stabilized by the dynamic H-bond formation between the active binding residues and nucleotides. Amino acids residues with the highest hydrogen bonds hydrogen bond interactions with all five aptamers were determined as the active binding residues and further characterized. The computational prediction of complexes binding will include validations through experimental assays in future studies. Current study will improve the current in vitro aptamers by minimizing the aptamer length for its easy manipulation

Isolation of moderately halophilic lipase producing bacteria from sponges in Pahang coastal water, Malaysia

Sponges (Porifera) harbour diverse microorganisms which can be the potential source for microbial enzymes such as lipase. In this study, moderately halophilic lipase producing bacteria were isolated from sponges tissues collected near Balok, at Pahang coastal water. Out of 70 isolates that grew on tributyrin agar plate, only 7 isolates had produced clear zones surrounding their colonies. Out of these, 5 isolates appeared to be gram-positive rod; meanwhile, the other 2 isolates were gram-negative rod in morphologies. These isolates were subjected to several biochemical tests i.e., oxidase, gelatin hydrolysis, lactose fermentation, citrate and motility test, and 16S rRNA gene amplification and sequencing. The results from 16S rRNA sequencing showed that 2 isolates (NHTH 6B and NHTH 28A) were highly similar (>97%) with Paenibacillus illinoisensis; isolate NHTH 26A with Stenotrophomonas pavanii; and isolate NHTH 29A with Enterobacter aerogenes. Phylogenetic analysis on selected isolates (NHTH 6B, NHTH 26A, NHTH 28A and NHTH 29A) with other species from the database showed high bootstrap values of above 50%. This showed that diverse phyla of lipase producing bacteria were isolated from the sponge collected from Pahang coastal water. In the isolation of industrial important species, the presence of pathogenic group of microorganism in this sponge could indicate issues on water quality and safety in this area

Identifying potential inhibitors of human hexokinase II for the development of anti-dengue therapeutics via virtual screening approaches

The human hexokinase II (HK2) has been suggested as a potential therapeutic target for the development of drugs against dengue virus (DENV) infection. In this paper, compounds with potential HK2 inhibitory activity have been identified using ligandbased and structure-based virtual screening approaches. Ligand-based drug design was performed by using Ultra-Fast Shape Recognition with Atom Types (UFSRAT) and Ultrafast Shape Recognition with CREDO Atom Types (USRCAT) programmes by utilising 2-Deoxyglucose (2-DG) as the query molecule, which is a known HK2 inhibitor. The molecules identified from the programmes showed great similarity to 2-DG with scores ranged from 0.78-0.85 and 0.88-0.97 for UFSRAT and USRCAT, respectively. The analogues were docked against the crystal structure of HK2 (PDB ID: 2NZT) in complex with alpha-D-glucose (GLC) and beta-D-glucose-6-phosphate (BG6) by using AutoDock Vina programme, on both A and B chains where the active sites were located. The docking hits for molecules from UFSRAT showed binding energies ranged from -7.1 to -4.8 kcal/mol when docked on chain A, while the hits for chain B showed scores ranged from -6.7 to -4.8 kcal/mol. On the other hand, the binding energies for molecules from USRCAT when docked on both A and B chains were similar, which ranged from -7.0 to - 5.2 kcal/mol. The hits bind firmly at the cavities, where both GLC and BG6 were oriented towards the active sites of HK2. Taken together, this study has successfully discovered compounds which have potentials as potent inhibitors of HK2, thus pave the path towards the development of dengue therapeutics

COVID19: bahaya varian Delta dan bagaimana ia menakluki dunia

Hampir dua tahun lamanya kita bergelut dengan pandemik COVID-19 yang berpunca dari coronavirus bernama SARS-CoV-2. Secara kronologi, virus ini dipercayai berasal dari sebuah pasar di Wuhan, China. Ia kemudiannya merebak ke seluruh China dan kemudiannya ke seluruh dunia. Akibat interaksi virus dengan persekitaran, termasuk populasi manusia serata dunia telah menyebabkan berlakunya mutasi kepada genom virus untuk membolehkan ia semakin serasi dengan manusia. Dari varian asal Wuhan kepada varian-varian baharu yang diberi nama dari Alpha, Beta, Gamma dan yang terkini dan paling ditakuti adalah varian Delta. Mengapa varian Delta lebih bahaya? Untuk menjawab persoalan ini, kita perlu memahami biologi virus serta mekanisma jangkitannya terlebih dahulu. Coronavirus ini adalah virus yang membawa jujukan genom dalam format kimia mRNA. Rantaian genom tersebut dibaluti oleh struktur yang terdiri dari protein envelop (E-Protein), protein membrane (M-Protein) dan protein deduri (Spike protein) (Rajah 1) .Secara asasnya, virus ini berbentuk sfera dan mempunyai struktur deduri yang terbina dari protein yang dinamakan ‘Spike’. Protein ‘Spike’ inilah menjadi perhatian saintis dunia kerana peranannya yang signifikan dalam proses jangkitan sel perumah

In Silico development of CRISPR/Cas9 Construct for Oryza sativa subsp. indica

Oryza sativa, which is commonly known as rice, is one of the staple foods consumed by Asian. There are three subspecies of O. sativa that are different in their geographical adaptations which are Japonica, Javanica and Indica. Climate change causes increasing greenhouse gas emissions fluctuating rainfall, and drought which is the limiting factors of rice production in Malaysia. In rice, SUMO E2-Conjugating Enzyme (OsSCE1) gene plays a role as a negative regulator in the drought stress response. OsSCE1 gene is a type of small ubiquitin-like modifier (SUMO)-conjugating enzyme involved in a few regulatory processes such as SUMOylation. The crop improvement can be made possible through a genome editing approach which enables the manipulation of targeted genetic traits and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is one of the genomes editing technologies that are accessible. In this study, the CRISPR construct of drought tolerance of Oryza sativa subsp. indica was developed. The CRISPR construct development involved several stages; OsSCE1 gene prediction, sgRNA design, and vector construction. FGENESH and GeneMarkS were used for OsSCE1 gene prediction. The predicted OsSCE1 gene has been validated using Polymerisation Chain Reaction (PCR) and sequencing. The BLASTN result shows high similarity with Oryza sativa in chromosome 10 with 99% identities. This step followed by sgRNA design which was carried out manually with the help of gRNA prediction tools such as WU-CRISPR, CCTop, Benchling, and CRISPR-P. Lastly, vector construction was executed virtually using Benchling. Overall, the OsSCE1 gene was successfully characterized and validated, and the in silico CRISPR construct for Oryza sativa subsp. indica was developed. To ensure a good outcome, in silico CRISPR construct should be verified through in vivo studies in further

Microbial hydrolytic enzymes: In silico studies between polar and tropical regions

Enzyme is important as biocatalyst for industrial and biotechnological applications. Cold active enzymes have showed many advantages compare to mesophilic enzymes. Their cold active and thermolabile characteristics have shown potential benefits in many industries. In silico characterization of hydrolytic enzymes originated from polar and tropical regions was conducted. Amino acid sequences and molecular structures of mesophilic and psychrophilic bacterial enzyme homologues were compared thoroughly. Amino acid sequences from these two homologs do not showed any extraordinary differences. Overall, protein folds were highly similar when psychrophilic homologies were matched with mesophilic homologies. Active site residues located in catalytic domain of both psychrophilic and mesophilic enzymes were highly conserved. Thus, the hydrolytic mechanisms of these cold active enzymes still remain similar to mesophilic enzymes. However, the differences were indicated between these two enzymes at the substrate-binding sites. Cold active enzymes showed unique loop conformations and smaller side chains at the entrances. These characteristics provided larger active site of the enzymes. Larger entrance of active site was expected to stipulate substrates binding and products exiting with lower energy consumption. This part of the enzymes indicated one of the important features of cold active enzyme to work efficiently at lower temperature. Based on this in silico study, cold active enzymes have much more advantages compared to the mesophilic enzymes that made them valuable to be further researched and applied at industrial level

Basic analysis of L-specific microbial dehalogenases

Enzymes are macromolecular biological catalysis that is important to speed up reaction. Enzymes can be found naturally from the microorganism such as bacteria, fungi and yeast which may not be genetically modified. In this perspectives, resorting to bioremediation to clean-up such environment may prove feasible and beneficial as microorganisms effective in degrading such substances. The liberation of excess halogenated compounds into the environment is becoming a major global issue, since the toxic contaminants tend to accumulate and persist in the biosphere. Some of the microbes that able to utilise these toxic compounds have been successfully isolated from the soil contaminated environment and were further charcterised. Apart from pollutant degradation, commercial enzyme like protease will be discussed that has potential for commercialisation in many manufacturing processes