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This study sought to determine the potential role of HBB haplotypes to predict beta-thalassemia in the Malaysian population. A total of 543 archived samples were selected for this study. Five tagging SNPs in the beta-globin gene (HBB; NG_000007.3) were analyzed for SNP-based and haplotype association using SHEsis online software. Single-SNP-based association analysis showed three SNPs have a statistically significant association with beta-thalassemia. When Bonferroni correction was applied, four SNPs were found statistically significant with beta-thalassemia; IVS2-74T>G (padj = 0.047), IVS2-16G>C (padj = 0.017), IVS2-666C>T (padj = 0.017) and 3’UTR + 314G>A (padj = 0.002). However, 3'UTR + 233G>C did not yield a significant association with padj value = 0.076. Further investigation using combined five SNPs for haplotype association analysis revealed three susceptible haplotypes with significant p values of which, haplotypes 1-2-2-1-1 (p = 6.49 × 10−7, OR = 10.371 [3.345–32.148]), 1-2-1-1-1 (p = 0.009, OR = 1.423 [1.095–1.850] and 1-1-1-1-1 (p = 1.39 × 10−4, OR = 10.221 [2.345–44.555]). Three haplotypes showed protective effect with significant p value of which, 2-2-1-1-1 (p = 0.006, OR = 0.668 [0.500–0.893]), 1-1-2-2-1 (p = 0.013, OR = 0.357 [0.153–0.830]) and 1-1-2-1-1 (p = 0.033, OR = 0.745 [0.567–0.977]). This study has identified the potential use of intragenic polymorphic markers in the HBB gene, which were significantly associated with beta-thalassemia. Combining these five SNPs defined a new haplotype model for beta-thalassemia and further evaluation for predicting severity in beta-thalassemia.

Kelulut and Yemeni Sidr honey has been documented to have various therapeutic properties. Investigations associated with the medicinal properties and physicochemical characteristics of Kelulut and Yemeni Sidr honey are growing broadly and receiving raised awareness. This study incorporated and analysed the findings on the biological and physicochemical properties of Kelulut and Yemeni Sidr honey. Kelulut and Yemeni Sidr honey was found to have a wide variety of biological effects attributed to their physicochemical characteristics. Findings showed that Kelulut and Yemeni Sidr honey have anti-bacterial, antibiofilm, anti-virulence, anti-oxidative, anti-cancer, anti-inflammatory, anti-diabetic, antiobesity and wound-healing properties. The physicochemical properties of Kelulut and Yemeni Sidr honey were compared and discussed and results revealed that they have high-quality contents and excellent antioxidant sources.

Introduction Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients. Methods Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing. Results CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2. Conclusion CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

Iron homeostasis is regulated by hepcidin (HEPC) that controls the dietary iron absorption and iron recycling. HEPC deficiency contributes to iron overload in β-thalassemia patients. The present study aimed to investigate the correlation between HEPC concentration and serum iron status among hemoglobin E (HbE)/β-thalassemia patients and their parents (HbE trait and β-thalassemia trait) compared with healthy controls. This study is a comparative cross-sectional study in which iron profile and HEPC level were examined in 65 HbE/β-thalassemia patients (pretransfusion) and 65 parents at the Hospital Sultanah Nur Zahirah and in 130 students as healthy controls from Univesiti Sultan Zainal Abidin, Terengganu, Malaysia. Furthermore, six samples from each group (HbE/β-thalassemia patients, parents and healthy controls) were randomly selected for gene expression analysis of HEPC and ferroportin1 (FPN1) using reverse transcription quantitative PCR. The results demonstrated that serum HEPC level were significantly decreased in HbE/β-thalassemia patients and their parents (P

Background: The study aims to evaluate the antibacterial activity of Trigona honey against S. pneumonia. Methods: The effect of Trigona honey on S. pneumonia investigated using agar well diffusion, MIC, MBC, biofilm formation and RT-qPCR. Results: Trigona honey samples showed the larger zones of inhibition against S. pneumonia, 22.2±0.4 at 100% concentration. Trigona honey possessed the lowest MIC, MBC, MIC50 and MIC90 against S. pneumoniae, 25%, 30%, 12.5% and 25% (w/v) respectively. Trigona honey permeated established biofilms of S. pneumonia, resulting in significant decreased the cells from the biofilm. RT-qPCR revealed that the expression of genes amiF, ftsY, mvaS, pnpA, argG, mvd1, purN, miaA and pbp2a were upregulated, glcK, marR, prmA and ccpA­­­ were downregulated after exposure to honey. Conclusion: Trigona honey demonstrated the highest antibacterial activity against S. pneumoniae. By limiting study in vitro on Trigona honey, we infer that Trigona honey impacts on S. pneumoniae.

Pseudomonas aeruginosa and Streptococcus pyogenes are the most common pathogens to humans and are able to form a biofilm following ineffective precautionary approach. Biofilm is defined as a surface-attached community of bacterium embedded in an extracellular matrix which leads to tremendous problems in the environment, among humans and animals. This study aims to investigate the ability of P. aeruginosa and S. pyogenes to form biofilms in 96-well plate before further study in antibiofilm will be done. Initially, the 96-well plate was added with 100 μl of overnight P. aeruginosa culture with optical density (OD) 0.1 and S. pyogenes culture with OD 0.05. The cultures were incubated for 7 days at 37 °C to justify the formation of biofilm. Subsequently, stained blue biofilm was detached from the plate by using 95% ethanol. Biofilms were finally measured using a micro plate reader at 570 nm and were classified based on the adherence strength formula. P. aeruginosa and S. pyogenes biofilms strongly adhered to the plates on days three, four, five and six. Interestingly on day three, biofilms showed the highest formation. However, moderate biofilm formation onto the plates by both P. aeruginosa and S. pyogenes were observed on day two, but non-adherence was observed on days one and seven. Day three is the optimum cultivation period for P. aeruginosa and S. pyogenes to switch into a strong biofilm in microtiter plate and could be beneficial for antibiofilm experiments.

Background: The purpose of this study was to investigate antibacterial activity of three varieties of Malaysian honey; Tualang honey (TH), Gelam honey (GH), and Acacia honey (AH) against Escherichia coli. Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the honey samples against E. coli were determined by the broth microdilution assay in the presence and absence of catalase enzyme. The mode of inhibition of honey samples against E. coli was investigated by the effect of time on viability. Impacts of the honeys on the expression profiles of the selected genes of E. coli were examined using RT-qPCR analysis. Results: The results showed that TH and GH honey possessed lowest MIC and MBC values against E. coli with 20% and 25% (w/v) respectively. Highest MIC and MBC values were observed by AH honey against E. coli with 25% (w/v) and 50% (w/v) values respectively. Among the tested honeys, TH and GH exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. Time–kill curve demonstrated a bactericidal rather than a bacteriostatic effect; with a 2-log reduction estimated within 540 min. Viable cells were not recovered after 9 hours exposure to MIC of all honey-treated. The RT-qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Conclusion: This study demonstrates that Malaysian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets.

The natural compound, thymoquinone (TQ) has demonstrated potential anticancer properties in inhibiting cell proliferation and promoting apoptosis in myeloid leukemia cells, breast cancer cells, and others. However, the effect mechanism of TQ on AML cells still not fully understood. In this study, the authors examined the effects of TQ on the expression of JAK/STAT-negative regulator genes SOCS-1, SOCS-3, and SHP-1, and their consequences on cell proliferation and apoptosis in HL60 leukemia cells. MTT and trypan blue exclusion tests were conducted to determine the 50% inhibitory concentration (IC50) and cell proliferation. FITC Annexin and Guava® reagent were used to study the cell apoptosis and examine the cell cycle phases, respectively. The expression of JAK/STAT-negative regulator genes, SOCS-1, SOCS-3, and SHP-1, was investigated using reverse transcriptase- quantitative PCR (RT-qPCR). TQ demonstrated a potential inhibition of HL60 cell proliferation and a significant increase in apoptotic cells in dose and time-dependent manner. TQ significantly induced cycle arrest at G0-G1 phase (P < 0.001) and enhanced the re-expression of JAK/STAT-negative regulator genes. TQ potentially inhibited HL60 cell proliferation and significantly increased apoptosis with re-expression of JAK/STAT-negative regulator genes suggesting that TQ could be a new therapeutic candidate for leukemia therapy.. This work was funded by the Fundamental Research Grant Scheme of the Ministry of Education, Malaysia [FRGS/1/2019/SKK08/UNISZA/02/3 (RR330)].

Background: Acute myeloid leukemia (AML) is the most common form of acute leukemias in adults which is clinically and molecularly heterogeneous. Several risk and genetic factors have been widely investigated to characterize AML. However, the concomitant epigenetic factors in controlling the gene expression lead to AML transformation was not fully understood. This study was aimed to identify epigenetically regulated genes in AML cell lines induced by epigenetic modulating agents, Trichostatin A (TSA) and 5-Azacytidine (5-Aza). Materials and Methods: MV4-11 and Kasumi 1 were treated with TSA and/or 5-Aza at IC50 concentration. Gene expression profiling by microarray was utilized using SurePrint G3 Human Gene Expression v3. Gene ontology and KEGG pathway annotations were analyzed by DAVID bioinformatics software using EASE enrichment score. mRNA expression of the differentially expressed genes were verified by quantitative real time PCR. Results: Gene expression analysis revealed a significant changes in the expression of 24,822, 15,720, 15,654 genes in MV4-11 and 12,598, 8828, 18,026 genes in Kasumi 1, in response to TSA, 5-Aza and combination treatments, respectively, compared to non-treated (p

Background MicroRNAs (miRNAs) are known for their pivotal roles in the regulation of gene expression through degradation or translational repression of their target messenger RNAs (mRNAs). Mounting evidence implicates miRNAs as the main actor in leukemia pathogenesis, thus miRNAs emerged as attractive targets for therapeutics. Aim Based on the available data from previous studies, this review focuses on the expressions and effects of particular miRNAs on the clinical outcomes in acute myeloid leukemia (AML) patients and strategies adopted for the treatment of AML using miRNA-based technology. Conclusions Deregulated expressions of miRNAs pose significant impact on the clinical outcomes in AML patients. MicroRNA therapeutics indeed has a tremendous potential in the treatment of AML.

Background: Epigenetic silencing of tumor suppressor genes (TSG) is involved in development and progression of cancers. Re-expression of TSG is inversely proportionate with STAT3 signaling pathways. Demethylation of DNA by 5-Azacytidine (5-Aza) results in re-expression of silenced TSG. Forced expression of PRG2 by 5-Aza induced apoptosis in cancer cells. Imatinib is a tyrosine kinase inhibitor that potently inhibits BCR/ ABL tyrosine kinase resulting in hematological remission in CML patients. However, majority of CML patients treated with imatinib would develop resistance under prolonged therapy. Methods: CML cells resistant to imatinib were treated with 5-Aza and cytotoxicity of imatinib and apoptosis were determined by MTS and annexin-V, respectively. Gene expression analysis was detected by real time-PCR, STATs activity examined using Western blot and methylation status of PRG2 was determined by pyrosequencing analysis. Result: Expression of PRG2 was significantly higher in K562-R+5-Aza cells compared to K562 and K562-R (p=0.001). Methylation of PRG2 gene was significantly decreased in K562-R+5-Aza cells compared to other cells (p=0.021). STAT3 was inactivated in K562-R+5-Aza cells which showed higher sensitivity to imatinib. Conclusion: PRG2 gene is a TSG and its overexpression might induce sensitivity to imatinib. However, further studies are required to evaluate the negative regulations of PRG2 on STAT3 signaling.

Background: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials and Methods: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and IC 50 values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting. Results: The IC 50 for imatinib on K562 was 362nM compared to 3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p

Background: Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia. Materials and Methods: Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC 50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting. Results: P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells. Conclusions: These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.

Background Tumor-suppressor genes are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Src homology-2 (SH2)-containing protein-tyrosine phosphatase 1 (SHP-1) is a negative regulator of the JAK/STAT pathway. Transcriptional silencing of SHP-1 plays a critical role in the development and progression of cancers through STAT3 activation. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA demethylation resulting in re-expression of silenced SHP-1. Lestaurtinib (CEP-701) is a multi-targeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in AML patients harboring the internal tandem duplication of the FLT3 gene (FLT3-ITD). However, the majority of patients in clinical trials developed resistance to CEP-701. Therefore, the aim of this study, was to assess the effect of re-expression of SHP-1 on sensitivity to CEP-701 in resistant AML cells. Methods Resistant cells harboring the FLT3-ITD were developed by overexposure of MV4-11 to CEP-701, and the effects of 5-Aza treatment were investigated. Apoptosis and cytotoxicity of CEP-701 were determined using Annexin V and MTS assays, respectively. Gene expression was performed by quantitative real-time PCR. STATs activity was examined by western blotting and the methylation profile of SHP-1 was studied using MS-PCR and pyrosequencing analysis. Repeated-measures ANOVA and Kruskal–Wallis tests were used for statistical analysis. Results The cytotoxic dose of CEP-701 on resistant cells was significantly higher in comparison with parental and MV4-11R-cep + 5-Aza cells (p = 0.004). The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p

Objective: So far no studies have been performed in Malaysia to look at association of diabetes mellitus (DM) with blood groups. We studied the association of ABO blood groups with DM type 2. Patients and methodology: It was a case control study conducted at Kepala Batas Hospital Batas, Penang, Malaysia in the year 2009, involving 70 patients with DM type 2 and 140 healthy controls. Ethical approval was obtained from Universiti Sains Malaysia. Blood samples were collected from the patients after consent. Samples were tested for ABO blood groups using ID-Card gel method. Results: Chi-square test results showed that there was an association between the ABO blood groups and DM type 2. It was found that A and O blood groups were negatively associated with DM type 2 (P

Background Mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor gene may promote proliferation via activation of multiple signaling pathways. FLT3-internal tandem duplication (FLT3-ITD) is the most common gene alteration found in patients diagnosed with acute myeloid leukaemia (AML) and has been associated with poor prognosis. Materials and methods We performed mutational analysis of exons 14-15 and 20 of the FLT3 gene in 54 AML patients using PCR-CSGE (conformational sensitive gel electrophoresis) followed by sequencing analysis to characterise FLT3 mutations in adult patients diagnosed with AML at Hospital USM, Kelantan, Northeast Peninsular Malaysia. Results FLT3 exon 14-15 mutations were identified in 7 of 54 patients (13%) whereas no mutation was found in FLT3 exon 20. Six ITDs and one non-ITD mutation were found in exon 14 of the juxtamembrane (JM) domain of FLT3. FLT3-ITD mutations were associated with a significantly higher blast percentage (p-value=0.008) and white blood cell count (p-value=0.023) but there was no significant difference in median overall survival time for FLT3-ITD+/FLT3-ITD- within 2 years (p-value=0.374). Conclusions The incidence of FLT3-ITD in AML patients in this particular region of Malaysia is low compared to the Western world and has a significant association with WBC and blast percentage.

Background: Tumor-suppressor genes Tumour suppressor genes (TSG) are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Transcriptional silencing of PRG2 gene could be involved in development and progression of cancers. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA de-methylation resulting in re-expression of silenced TSG. Midostaurin (PKC412) is a multitargeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in patients with AML. However, majority of AML patients in clinical trials developed resistance to PKC412. Methods: Resistant cells were developed by overexposure of MV4-11 cells to PKC-412 and treated with 5-Aza. Apoptosis and cytotoxicity of PKC-412 were determined using Annexin V and MTS assays, respectively. Gene expression profiling was performed using microarray followed by quantitative real-time PCR. STATs activity was examined using Western bloting and methylation of PRG2 was studied using pyrosequencing analysis. Results: The cytotoxic dose of PKC-412 on resistant cells was significantly higher than parental and MV4-11Rpkc+5-Aza cells (p=0.003). The resistant cells showed significant higher viability and lower apoptotic cells compared to other cells (p