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Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection

Khairol Mokhtar NHI, Hussin A, Hamid AA, Zainal Ariffin SH, Shahidan MA

Comb Chem High Throughput Screen, 2021 Aug 02.
PMID: 34342257 DOI: 10.2174/1386207324666210802122538

AIMS: We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on prostate-specific antigen (PSA).
BACKGROUND: High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based enzyme-linked lectin sorbent assays (ELLSAs), as they can mask specific binding signals and cause false-positive results.
METHODS: In this study, we constructed an ELLSA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELLSA.
RESULTS: The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELLSA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM.
CONCLUSION: The ELLSA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.
Fulltext Nutritional Composition, in vitro Antioxidant Activity and Artemia salina L. Lethality of Pulp and Seed of Tamarindus indica L. Extracts

Khairunnuur FA, Zulkhairi A, Azrina A, Moklas MM, Khairullizam S, Zamree MS, et al.

Malays J Nutr, 2009 Mar;15(1):65-75.
PMID: 22691806 MyJurnal

This study was designed to examine the nutritional composition, antioxidant activity and medium lethal concentration (LC50 value) of Tamarindus indica L. pulp and seed extracts in vitro. The extraction was set at 40◦C, 60◦C and 100◦C for 12 hours, 6 hours and 15 minutes respectively to determine the optimum extraction parameter whereas the anti-oxidant activity of the extracts was measured using iron (III) reduction (FRAP) assay. Total phenolic content (TPC) of the extracts was estimated as gallic acid equivalent by Folin-Ciocalteau method. Toxicity potential of the extract was assessed in vitro by Artemia salina lethality test both in seed and pulp samples. The results showed that tamarind seed contained a higher percentage of carbohydrate, protein, fat and energy (15%, 82%, 95% and 33.13% respectively) than the pulp. On the other hand, the pulp demonstrated a high moisture (51.1%) and ash (34.84%) content than the seed. For the mineral analysis, tamarind seed contained higher Ca and C (1.0% and 50.73% respectively) than the pulp (0.27% and 40.40% respectively). No heavy metals were detected in both samples. Seed extracted at 60◦C/6 hours and 100◦C/15 minutes showed the highest TPC value and were significantly different (p<0.05) than the seed extracted at 40◦C/12 hours. Anti-oxidant activity is positively correlated to the TPC value of the extracts (R=0.991). The pulp and seed extracted at 100◦C/15 minutes showed the highest FRAP value among its groups (216.17 ± 14.06 μmol (Fe)/g and 659.74 ± 16.40 μmol (Fe)/g respectively). This study indicates that tamarind pulp and seed extracts possess beneficial antioxidant properties and the optimum extraction parameter is 100◦C for 15 minutes. In Artemia salina lethality test, tamarind pulp caused significant mortality of the crustacean larvae with LC50 in the range of 26-28 μL/mL. Tamarind seed were not toxic to Artemia salina since the LC50 of the extracts was higher than 1000 μL/mL.
Fulltext Improving the clinical recognition, prognosis, and treatment of melioidosis through epidemiology and clinical findings: The Sabah perspective

Hussin A, Nor Rahim MY, Dalusim F, Shahidan MA, Nathan S, Ibrahim N

PLoS Negl Trop Dis, 2023 Oct;17(10):e0011696.
PMID: 37844130 DOI: 10.1371/journal.pntd.0011696

INTRODUCTION: Melioidosis is a deadly endemic disease in northern Australia and Southeast Asia, including Sabah, Malaysia, which is caused by the bacterium Burkholderia pseudomallei. It contributes to high fatality rates, mainly due to misdiagnosis leading to the wrong treatment being administered to the patients. Local epidemiology and data on clinical features could assist clinicians during diagnosis and treatment. However, these details are still scarce, particularly in Sabah.
METHODS: A retrospective study of 246 culture-confirmed melioidosis cases in Queen Elizabeth Hospital, Sabah, Malaysia was performed between 2016 and 2018. The epidemiological data and clinical and laboratory findings were extracted and analysed.
RESULTS: The annual incidence of culture-confirmed melioidosis cases was estimated to be 4.97 per 100,000 people. The mean age of the patients was 50±15 years. Males and members of the Kadazan-Dusun ethnic group accounted for the majority of the melioidosis cases. The odds ratio analysis indicated that bacteraemic melioidosis in this region was significantly associated with fever (76%), and patients having at least one underlying illness (43%), including diabetes mellitus (32%). Sixty-eight patients (28%) succumbed to melioidosis. Contrary to what is known regarding factors that promote bacteraemic melioidosis, neither patients with fever nor patients with at least one comorbid disease, including diabetes mellitus, were significantly associated with death from melioidosis. There was no statistically significant difference between patients without comorbidities (24, 27%) and those with at least one comorbid disease (26, 25%), including diabetes mellitus (18, 23%). The odds ratios indicate that melioidosis mortality in this region is related to patients showing respiratory organ-associated symptoms (29%), bacteraemia (30%), and septic shock (47%). Burkholderia pseudomallei isolates in this study were highly susceptible to ceftazidime (100%), imipenem (100%), and trimethoprim-sulfamethoxazole (98%).
CONCLUSIONS: Information obtained from this study can be used by clinicians to recognise individuals with the highest risk of acquiring melioidosis, estimate an accurate prognosis, and provide effective treatment for melioidosis patients to reduce death from melioidosis.
Identification and mechanism determination of the efflux pump subunit amrB gene mutations linked to gentamicin susceptibility in clinical Burkholderia pseudomallei from Malaysian Borneo

Hussin A, Nathan S, Shahidan MA, Nor Rahim MY, Zainun MY, Khairuddin NAN, et al.

Mol Genet Genomics, 2024 Feb 21;299(1):12.
PMID: 38381232 DOI: 10.1007/s00438-024-02105-w

The bacterium Burkholderia pseudomallei is typically resistant to gentamicin but rare susceptible strains have been isolated in certain regions, such as Thailand and Sarawak, Malaysia. Recently, several amino acid substitutions have been reported in the amrB gene (a subunit of the amrAB-oprA efflux pump gene) that confer gentamicin susceptibility. However, information regarding the mechanism of the substitutions conferring the susceptibility is lacking. To understand the mechanism of amino acid substitution that confers susceptibility, this study identifies the corresponding mutations in clinical gentamicin-susceptible B. pseudomallei isolates from the Malaysian Borneo (n = 46; Sarawak: 5; Sabah: 41). Three phenotypically confirmed gentamicin-susceptible (GENs) strains from Sarawak, Malaysia, were screened for mutations in the amrB gene using gene sequences of gentamicin-resistant (GENr) strains (QEH 56, QEH 57, QEH20, and QEH26) and publicly available sequences (AF072887.1 and BX571965.1) as the comparator. The effect of missense mutations on the stability of the AmrB protein was determined by calculating the average energy change value (ΔΔG). Mutagenesis analysis identified a polymorphism-associated mutation, g.1056 T > G, a possible susceptible-associated in-frame deletion, Delta V412, and a previously confirmed susceptible-associated amino acid substitution, T368R, in each of the three GENs isolates. The contribution of Delta V412 needs further confirmation by experimental mutagenesis analysis. The mechanism by which T368R confers susceptibility, as elucidated by in silico mutagenesis analysis using AmrB-modeled protein structures, is proposed to be due to the location of T368R in a highly conserved region, rather than destabilization of the AmrB protein structure.
Enzyme activity profiles and ELISA analysis of biomarkers from human saliva and gingival crevicular fluid during orthodontic tooth movement using self-ligating brackets

Abdul Wahab RM, Abu Kasim N, Senafi S, Jemain AA, Zainol Abidin IZ, Shahidan MA, et al.

Oral Health Dent Manag, 2014 Jun;13(2):194-9.
PMID: 24984622

Profiles of orthodontic tooth movement biomarkers, i.e., Lactate Dehydrogenase (LDH), Aspartate Aminotransferase (AST), Tartrate-resistant Acid Phosphatase (TRAP) and Alkaline Phosphatase (ALP), using Self-ligating Brackets (SLBs) and possible relationships among their activities and total enzymes produced were determined.
Fulltext Evaluation of in vitro osteoblast and osteoclast differentiation from stem cell: a systematic review of morphological assays and staining techniques

Zainal Ariffin SH, Megat Abdul Wahab R, Abdul Razak M, Yazid MD, Shahidan MA, Miskon A, et al.

PeerJ, 2024;12:e17790.
PMID: 39071131 DOI: 10.7717/peerj.17790

BACKGROUND: Understanding human stem cell differentiation into osteoblasts and osteoclasts is crucial for bone regeneration and disease modeling. Numerous morphological techniques have been employed to assess this differentiation, but a comprehensive review of their application and effectiveness is lacking.
METHODS: Guided by the PRISMA framework, we conducted a rigorous search through the PubMed, Web of Science and Scopus databases, analyzing 254 articles. Each article was scrutinized against pre-defined inclusion criteria, yielding a refined selection of 14 studies worthy of in-depth analysis.
RESULTS: The trends in using morphological approaches were identified for analyzing osteoblast and osteoclast differentiation. The three most used techniques for osteoblasts were Alizarin Red S (mineralization; six articles), von Kossa (mineralization; three articles) and alkaline phosphatase (ALP; two articles) followed by one article on Giemsa staining (cell morphology) and finally immunochemistry (three articles involved Vinculin, F-actin and Col1 biomarkers). For osteoclasts, tartrate-resistant acid phosphatase (TRAP staining) has the highest number of articles (six articles), followed by two articles on DAPI staining (cell morphology), and immunochemistry (two articles with VNR, Cathepsin K and TROP2. The study involved four stem cell types: peripheral blood monocyte, mesenchymal, dental pulp, and periodontal ligament.
CONCLUSION: This review offers a valuable resource for researchers, with Alizarin Red S and TRAP staining being the most utilized morphological procedures for osteoblasts and osteoclasts, respectively. This understanding provides a foundation for future research in this rapidly changing field.
Fulltext Gene expression profiles for in vitro human stem cell differentiation into osteoblasts and osteoclasts: a systematic review

Zainal Ariffin SH, Lim KW, Megat Abdul Wahab R, Zainal Ariffin Z, Rus Din RD, Shahidan MA, et al.

PeerJ, 2022;10:e14174.
PMID: 36275474 DOI: 10.7717/peerj.14174

BACKGROUND: There have been promising results published regarding the potential of stem cells in regenerative medicine. However, the vast variety of choices of techniques and the lack of a standard approach to analyse human osteoblast and osteoclast differentiation may reduce the utility of stem cells as a tool in medical applications. Therefore, this review aims to systematically evaluate the findings based on stem cell differentiation to define a standard gene expression profile approach.
METHODS: This review was performed following the PRISMA guidelines. A systematic search of the study was conducted by retrieving articles from the electronic databases PubMed and Web of Science to identify articles focussed on gene expression and approaches for osteoblast and osteoclast differentiation.
RESULTS: Six articles were included in this review; there were original articles of in vitro human stem cell differentiation into osteoblasts and osteoclasts that involved gene expression profiling. Quantitative polymerase chain reaction (qPCR) was the most used technique for gene expression to detect differentiated human osteoblasts and osteoclasts. A total of 16 genes were found to be related to differentiating osteoblast and osteoclast differentiation.
CONCLUSION: Qualitative information of gene expression provided by qPCR could become a standard technique to analyse the differentiation of human stem cells into osteoblasts and osteoclasts rather than evaluating relative gene expression. RUNX2 and CTSK could be applied to detect osteoblasts and osteoclasts, respectively, while RANKL could be applied to detect both osteoblasts and osteoclasts. This review provides future researchers with a central source of relevant information on the vast variety of gene expression approaches in analysing the differentiation of human osteoblast and osteoclast cells. In addition, these findings should enable researchers to conduct accurately and efficiently studies involving isolated human stem cell differentiation into osteoblasts and osteoclasts.