METHODS: The main aim of this paper is to review the available techniques in gene knockout strategies for microbial cells. The review is done in terms of their methodology, recent applications in microbial cells. In addition, the advantages and disadvantages of the techniques are compared and discuss and the related patents are also listed as well.
RESULTS: Traditionally, gene knockout is done through wet lab (in vivo) techniques, which were conducted through laboratory experiments. However, these techniques are costly and time consuming. Hence, various dry lab (in silico) techniques, where are conducted using computational approaches, have been developed to surmount these problem.
CONCLUSION: The development of numerous techniques for gene knockout in microbial cells has brought many advancements in the study of gene functions. Based on the literatures, we found that the gene knockout strategies currently used are sensibly implemented with regard to their benefits.
MATERIALS AND METHODS: Biofilm yield of 32 Helicobacter pylori strains (standard strain and 31 clinical strains) were determined by crystal-violet assay and grouped into poor, moderate and good biofilm forming groups. Whole genome sequencing of these 32 clinical strains was performed on the Illumina MiSeq platform. Annotation and comparison of the differences between the genomic sequences were carried out using RAST (Rapid Annotation using Subsystem Technology) and SEED viewer. Genes identified were confirmed using PCR.
RESULTS: Genes identified to be associated with biofilm formation in H. pylori includes alpha (1,3)-fucosyltransferase, flagellar protein, 3 hypothetical proteins, outer membrane protein and a cag pathogenicity island protein. These genes play a role in bacterial motility, lipopolysaccharide (LPS) synthesis, Lewis antigen synthesis, adhesion and/or the type-IV secretion system (T4SS). Deletion of cagA and cagPAI confirmed that CagA and T4SS were involved in H. pylori biofilm formation.
CONCLUSIONS: Results from this study suggest that biofilm formation in H. pylori might be genetically determined and might be influenced by multiple genes. Good, moderate and poor biofilm forming strain might differ during the initiation of biofilm formation.
MATERIALS AND METHODS: The OncoCarta(™) panel v1.0 assay was used to characterize oncogenic mutations. In addition, exons 4-11 of the TP53 gene were sequenced. Statistical analyses were conducted to identify associations between mutations and selected clinico-pathological characteristics and risk habits.
RESULTS: Oncogenic mutations were detected in PIK3CA (5.7%) and HRAS (2.4%). Mutations in TP53 were observed in 27.7% (31/112) of the OSCC specimens. Oncogenic mutations were found more frequently in non-smokers (p = 0.049) and TP53 truncating mutations were more common in patients with no risk habits (p = 0.019). Patients with mutations had worse overall survival compared to those with absence of mutations; and patients who harbored DNA binding domain (DBD) and L2/L3/LSH mutations showed a worse survival probability compared to those patients with wild type TP53. The majority of the oncogenic and TP53 mutations were G:C > A:T and A:T > G:C base transitions, regardless of the different risk habits.
CONCLUSION: Hotspot oncogenic mutations which are frequently present in common solid tumors are exceedingly rare in OSCC. Despite differences in risk habit exposure, the mutation frequency of PIK3CA and HRAS in Asian OSCC were similar to that reported in OSCC among Caucasians, whereas TP53 mutations rates were significantly lower. The lack of actionable hotspot mutations argue strongly for the need to comprehensively characterize gene mutations associated with OSCC for the development of new diagnostic and therapeutic tools.
OBJECTIVE: This study investigates the antidiabetic and antioxidant effects of M. latifolia bark extracts, fractions, and isolated constituents.
MATERIALS AND METHODS: Melicope latifolia extracts (hexane, chloroform, and methanol), fractions, and isolated constituents with varying concentrations (0.078-10 mg/mL) were subjected to in vitro α-amylase and dipeptidyl peptidase-4 (DPP-4) inhibitory assay. Molecular docking was performed to study the binding mechanism of active compounds towards α-amylase and DPP-4 enzymes. The antioxidant activity of M. latifolia fractions and compounds were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and β-carotene bleaching assays.
RESULTS: Melicope latifolia chloroform extract showed the highest antidiabetic activity (α-amylase IC50: 1464.32 μg/mL; DPP-4 IC50: 221.58 μg/mL). Fractionation of chloroform extract yielded four major fractions (CF1-CF4) whereby CF3 showed the highest antidiabetic activity (α-amylase IC50: 397.68 μg/mL; DPP-4 IC50: 37.16 μg/mL) and resulted in β-sitosterol (1), halfordin (2), methyl p-coumarate (3), and protocatechuic acid (4). Isolation of compounds 2-4 from the species and their DPP-4 inhibitory were reported for the first time. Compound 2 showed the highest α-amylase (IC50: 197.53 μM) and β-carotene (88.48%) inhibition, and formed the highest number of molecular interactions with critical amino acid residues of α-amylase. The highest DPP-4 inhibition was exhibited by compound 3 (IC50: 911.44 μM).
DISCUSSION AND CONCLUSIONS: The in vitro and in silico analyses indicated the potential of M. latifolia as an alternative source of α-amylase and DPP-4 inhibitors. Further pharmacological studies on the compounds are recommended.