Pre-mineralisation effect of nanobiocomposite bone scaffold towards bone marrow-derived stem cells growth and differentiation

Apatite layers formed by simulated body fluid (SBF) on the surface of calcium-based scaffolds have been proven to enhance the osteoblastic activity of pre-osteoblasts and osteogenic activity of bone marrow-derived stem cell (BM-SCs). Previously developed Alginate/Cockle shell powder nanobiocomposite bone scaffold (Alg/nCP) has been shown to possess excellent osteoconductive properties. The effect of pre-mineralization of the scaffold surface towards the growth and differentiation of BM-SCs’ were evaluated using microscopic and biochemical methods in scaffolds divided into SBF pre-treated and control groups at two time points. MTT proliferation assay showed statistically significant decrease in cell proliferation in SBF group for both culture periods. SEM observation revealed growth of BM-SCs and scaffold surface mineralisation and calcium deposition in both groups with higher intensity observable in the control group. Supporting biochemical studies showed a significant decrease in alkaline phosphatase (ALP) level indicating a lesser osteogenic differentiation in the SBF group as compared to control. Pre-mineralisation of scaffolds in SBF produced a contradicting result in which it did not provide a better environment for growth and proliferation of BM-SCs. However, the Alg/nCP scaffold did show potentials in supporting the osteogenic differentiation of the stem cells

Establishment of stable and secretable Tatκ-GFP recombinant protein: a preliminary report of promoter methylation in 293t cell line

Induced pluripotent stem cells (iPSC) is a novel technology useful for therapeutic and research applications. To date, iPSCs is produced through genetic modification that can promote mutation; making it harmful for therapeutic use. Therefore, application of non-genetic modification through direct delivery of recombinant proteins aided by protein transduction domain (PTD) enable a safer production of iPSC. This study is aimed to establish a stable production of secretable recombinant protein via recombination of green fluorescence protein (GFP) and a novel PTD peptide, namely TATκ-GFP. 293Tcell line was transfected with 20 μg/ml of TATκ-GFP plasmid and the stably transfected 293T cells were then cultured for 54 days to determine the stability of expression and secretion of TATκ-GFP recombinant protein in prolonged culture. Methylation at the CMV promoter of the TATκ-GFP plasmid was investigated following treatment of transfected cells with 3 μM/mL of demethylation agent, namely 5-Azacytidine for 72 h in three cycles. Flow cytometry analysis demonstrated a transfection efficiency of 9.33% and successful secretion of TATκ-GFP proteins into the culture medium as analysed by Western blot at 72 h post-transfection. However, the transfected cells exhibited a decreasing level of GFP expression and secretion following prolonged culture with notable stability that only sustained for two weeks. 5-Azacytidine-treated cells showed a slight increase of GFP expression compared to non-treated control, suggesting possible promoter methylation which could cause instability of TATκ-GFP expression. Conclusively, promoter methylation should be considered for future establishment of iPSCs as it could inhibit stable expression and secretion of recombinant proteins

Pemodelan semula jantung dalam kardiomiopati diabetes: peranan inflamasi, tekanan oksidatif dan apoptosis yang mendasari pembentukan dan perkembangannya

Kardiomiopati diabetes (DCM) merupakan komplikasi kronik diabetes melitus akibat daripada perubahan pada fungsi dan struktur jantung yang diaruh oleh keadaan aras gula darah yang tinggi (hiperglisemia) secara berpanjangan. Walaupun pengawalan hiperglisemia dilakukan dengan komprehensif serta perubahan gaya hidup yang lebih sihat, komplikasi kardiovaskular termasuklah DCM terus menjadi antara punca kematian utama pesakit diabetes. Pembentukan dan perkembangan DCM adalah akibat proses kompensasi melalui pemodelan semula jantung yang melibatkan kematian kardiomiosit hasil daripada kerosakan oksidatif, apoptosis dan inflamasi susulan aruhan hiperglisemia yang tidak terkawal. Walaupun permodelan semula jantung merupakan proses yang penting dalam memulihara struktur dan fungsi jantung, namun dalam keadaan diabetes, rangsangan pemodelan semula jantung yang berpanjangan boleh membawa kepada kemorosotan fungsi yang kekal dan akhirnya menyebabkan kegagalan jantung. Memahami mekanisme yang terlibat dalam pembentukan dan perkembangan DCM adalah sangat penting bagi merangka strategi untuk mengurangkan komplikasi akibat penyakit ini. Oleh itu, dalam kertas ulasan ini, hasil kajian terkini mengenai proses pemodelan semula jantung dalam perkembangan DCM dan mekanisme utama yang mendasari pembentukan dan perkembangannya akan diperjelaskan

Future stem cell analysis: progress and challenges towards state-of-the art approaches in automated cells analysis

Background and Aims A microscopic image has been used in cell analysis for cell type identification and classification, cell counting and cell size measurement. Most previous research works are tedious, including detailed understanding and time-consuming. The scientists and researchers are seeking modern and automatic cell analysis approaches in line with the current in-demand technology. Objectives This article provides a brief overview of a general cell and specific stem cell analysis approaches from the history of cell discovery up to the state-of-the-art approaches. Methodology A content description of the literature study has been surveyed from specific manuscript databases using three review methods: manuscript identification, screening, and inclusion. This review methodology is based on Prism guidelines in searching for originality and novelty in studies concerning cell analysis. Results By analysing generic cell and specific stem cell analysis approaches, current technology offers tremendous potential in assisting medical experts in performing cell analysis using a method that is less laborious, cost-effective, and reduces error rates. Conclusion This review uncovers potential research gaps concerning generic cell and specific stem cell analysis. Thus, it could be a reference for developing automated cells analysis approaches using current technology such as artificial intelligence and deep learning

Evaluation of the cytotoxic and genotoxic effects of goniothalamin in leukemic cell lines

The cytotoxic and genotoxic effects of goniothalamin, a plant styryllactone, were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the Alkaline Comet assay respectively in human leukemic cell lines. Following 72 h of treatment, the IC50 values of goniothalamin in human HL-60 promyelocytic leukemia cells and CEM-SS T-lymphoblastic cells were 4.5 μg/mL and 2.4 μg/mL respectively. The genotoxicity of goniothalamin in both HL-60 and CEM-SS cells was detected as early as 2 h following treatment at IC10 and IC25 concentrations. However, pretreatment with the antioxidant N-acetyl-cysteine (NAC) at 1 mM for 30 minutes did not abrogate genotoxicity of this compound. This result suggests that primary induction of DNA damage by goniothalamin may not involve oxidative damage. In conclusion, our results demonstrate genotoxic damage induced by goniothalamin in leukemic cells. Further studies are needed to ascertain the mode of action of goniothalamin in inducing DNA damage

Study of Heavy Metal Levels among Farmers of Muda Agricultural Development Authority, Malaysia

Heavy metals, particularly cadmium, lead, and arsenic, constitute a significant potential threat to human health. This study was conducted to determine the levels of cadmium, lead, and arsenic in nail samples from farmers at Muda Agricultural Development Authority (MADA), Kedah, Malaysia, and evaluate factors that can contribute to their accumulations. A total of 116 farmers participated in this study. Inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze concentration of heavy metals in the nail samples and questionnaires were given to participants to get demographic, health status, and their agricultural activities data. In this paper, the level of heavy metals was within the normal range and varies according to demographic factors. We found that there were significant correlations between working period with level of lead and arsenic (r = 0.315 and r = 0.242, resp., P < 0.01) and age with lead level (r = 0.175, P < 0.05). Our findings suggested that agricultural activities could contribute to the accumulation of heavy metals in farmers. Hence, the control of environmental levels of and human exposure to these metals to prevent adverse health effects is still an important public health issue

Analisa sitogenetik sel bukal petani di Tanjung Karang dan Kelantan yang terdedah kepada pestisid

Pestisid dan baja kimia digunakan secara meluas dalam sektor pertanian bagi meningkatkan hasil pertanian dalam kalangan petani. Namun, pendedahan kepada pestisid akan memberi potensi risiko kepada kesihatan manusia. Kajian ini bertujuan menganalisa kekerapan pembentukan mikronukleus (MN) dan binukleus (BNu) pada mukosa sel bukal petani yang terdedah kepada pestisid dengan menggunakan asai MN. Perbandingan kekerapan MN dan Bnu dilakukan di dua kawasan iaitu Tanjung Karang, Selangor dan Kelantan kerana aktiviti pertanian dan jenis pestisid yang digunakan adalah berbeza. Pengambilan sel bukal dilakukan pada petani di Tanjung Karang (n = 32) dan petani di Kelantan (n = 43) dengan mnggunakan kayu penyendal lidah. Borang soal selidik juga digunakan untuk mendapatkan data demografik para petani. Analisa sitogenetik dilakukan dengan menggunakan pewarnaan Akridin Jingga (AO) 0.0025% (w/v). Kekerapan MN dan BNu yang terbentuk melalui analisa dibawah mikroskop fluoresen dijadikan sebagai petunjuk kerosakan sitogenetik. Keputusan kajian menunjukkan kekerapan MN dan BNu petani di Tanjung Karang adalah lebih tinggi secara signifikan (p 0.05) dan amalan pemakaian PPE (Personal Protective Equipment) (p > 0.05). Selain itu, ujian korelasi yang dijalankan menunjukkan terdapat korelasi positif antara kekerapan MN dengan tempoh pendedahan pestisid petani di Tanjung Karang (p > 0.05, r = 0.015) dan Kelantan (p > 0.05, r = 0.0158). Manakala, kekerapan BNu juga mempunyai korelasi positif dengan pendedahan pestisid petani di Tanjung Karang (p > 0.05, r = 0.036) dan petani di Kelantan (p > 0.05, r = 0.013). Justeru, kajian ini membuktikan bahawa pendedahan pestisid boleh meningkatkan pembentukan MN dan BNu dalam kalangan petani dan ini menjelaskan bahawa penggunaan pestisid dalam jangka masa panjang boleh mengaruh genotoksisiti dan kerosakan DNA kepada manusia

Penetration and Silencing Activity of VEGF Dicer Substrate siRNA Vectorized by Chitosan Nanoparticles in Monolayer Culture and a Solid Tumor Model In Vitro for Potential Application in Tumor Therapy

Penetration and distribution of drug through the avascular regions of human solid tumors after extravasation are crucial concerns for antitumor efficacy. To address this issue, an in vitro solid tumor model of multicellular layers (MCLs) of human colorectal cancer cells (DLD-1) was established. In an attempt to deliver Dicer substrate small interfering RNA (DsiRNA), chitosan (CS) nanoparticles have been developed for targeting vascular endothelial growth factor (VEGF) gene for tumor growth inhibition. The DsiRNA-CS nanoparticles prepared by ionic gelation method had provided maximal protection of DsiRNA in full human serum up to 48 h incubation. RT-PCR studies revealed significant concentration- and time-dependent knock-down of VEGF mRNA and its product due to uniform penetration of DsiRNA-CS nanoparticles throughout MCLs. Taken together, this study also demonstrated that DsiRNA-CS nanoparticles could effectively knock down VEGF gene as therapeutic target in monolayer culture or in solid tumor model for potential treatment of human colorectal carcinoma

Physicochemical Properties and In Vitro Cytotoxicity Studies of Chitosan as a Potential Carrier for Dicer-Substrate siRNA

Recently, Dicer-substrate small interfering RNA (DsiRNA) has gained attention owing to its greater potency over small interfering RNA (siRNA). However, the use of DsiRNA is restricted by its rapid degradation in vitro. To address this issue, chitosan nanoparticulate deliver yplatform for the Dicer-substrate siRNA (DsiRNA) was developed and characterized. Nanoparticles were prepared by simple complexation and ionic gelation methods. The mean particle size of DsiRNA-adsorbed chitosan nanospheres (DsiRNA-CS NPs) prepared by the ionic gelation method ranged from 225 to 335 nm, while simple complexation yielded DsiRNA-chitosan complexes (DsiRNA-CS complexes) ranging from 270 to 730 nm. The zeta potential of both types of nanoparticles ranged from +40 to +65 mV. TEM and AFM micrographs revealed spherical and irregular morphology of DsiRNA-CS NPs and DsiRNA-CS complexes. ATR-FTIR spectroscopy confirmed the presence of DsiRNA in the CS NPs/complexes. Both types of nanoparticles exhibited sustained release and high binding and encapsulation (100%) efficiency of DsiRNA. DsiRNA-CS NPs/complexes showed low, concentration-dependent cytotoxicity in vitro. DsiRNA-CS NPs showed better stability than the complexes when stored at 4 and 25°C. Thus, it is anticipated that CS NPs are promising vectors for DsiRNA delivery due to their stability, safety, and cost-effectiveness

Linking Benzene, in Utero Carcinogenicity and Fetal Hematopoietic Stem Cell Niches: A Mechanistic Review

Previous research reported that prolonged benzene exposure during in utero fetal development causes greater fetal abnormalities than in adult-stage exposure. This phenomenon increases the risk for disease development at the fetal stage, particularly carcinogenesis, which is mainly associated with hematological malignancies. Benzene has been reported to potentially act via multiple modes of action that target the hematopoietic stem cell (HSCs) niche, a complex microenvironment in which HSCs and multilineage hematopoietic stem and progenitor cells (HSPCs) reside. Oxidative stress, chromosomal aberration and epigenetic modification are among the known mechanisms mediating benzene-induced genetic and epigenetic modification in fetal stem cells leading to in utero carcinogenesis. Hence, it is crucial to monitor exposure to carcinogenic benzene via environmental, occupational or lifestyle factors among pregnant women. Benzene is a well-known cause of adult leukemia. However, proof of benzene involvement with childhood leukemia remains scarce despite previously reported research linking incidences of hematological disorders and maternal benzene exposure. Furthermore, accumulating evidence has shown that maternal benzene exposure is able to alter the developmental and functional properties of HSPCs, leading to hematological disorders in fetus and children. Since HSPCs are parental blood cells that regulate hematopoiesis during the fetal and adult stages, benzene exposure that targets HSPCs may induce damage to the population and trigger the development of hematological diseases. Therefore, the mechanism of in utero carcinogenicity by benzene in targeting fetal HSPCs is the primary focus of this review