GC-MS Analysis of Strobilanthes crispus Plants and Callus

Strobilanthes crispus or locally known as “bayam karang”, “pecah kaca”, “jin batu” and “pecah beling” in Malaysia, has been traditionally used to increase immune system, treating kidney stones, treatment of diabetes mellitus, treatment of high blood pressure and treatment of wound. Studies examining the phytochemical constituents reported that the leaves of this plant contain ester glycosidic compound of caffeic acid, -voumaric acid, , vanilic acid, ferulic acid, syringic acids, sitosterol, campesterol, hexadecanoic acid, methylester, lupeol, phytol, stigmasterol, flavonoid compounds such as (+)-catechin, (-)-epicatechin, rutin, and etc. While most of the literatures focused on the chemical compounds present in the leaves of S. crispus, none have been reported for the phytochemical constituents of the whole S. crispus plant including the leaf, stem, root or flower part. Besides, there is also lacking report on the tissue culture generated from this plant too. Thus, this study was carried out to profile the leaves, stems and roots and callus cultures of S. crispus using gas chromatography mass spectrometry (GC-MS) approach. Results revealed that this plant is rich with squalene, phytosterols such as stigmasterols and derivatives, sito-sterol, campesterols, as well as triterpenoids such as lupeol, amyrin and betulin

Effectiveness of DNase and washing steps in removing dead cells' DNA for PCR detection of viable Escherichia coli

This study investigates the use of DNase and washing steps in removing dead cells’ DNA during sample preparation and their effect on the detection of viable cells using polymerase chain reaction (PCR). The results indicated that the DNA from heat-killed cells could be completely removed by DNase; thus, would not be detected by PCR. Inclusion of washing steps in centrifugation during sample preparation fails to remove DNA from heat-killed cells, but it reduces the amount of DNA from dead cells as well as viable cells. DNase could selectively remove DNA of heat-killed cells in the water sample without influencing the PCR amplification of viable cells’ DNA. The inclusion of washing steps in the centrifugation procedure was ineffective, because viable cells might be lost during washing steps. This method allows the detection of viable bacteria and subsequently contributes to research concerning environmental samples

Ruellia tuberosa Ethyl Acetate Leaf Extract Induces Apoptosis and Cell Cycle Arrest in Human Breast Cancer Cell Line, MCF-7

Ruellia tuberosa L. has been previously shown to possess antioxidant and antiproliferative activities on cancer cells but its underlying mechanisms are largely unknown. This study aimed to elucidate the mode of action underlying this inhibitory effect on MCF-7 using ethyl acetate extract obtained after liquid-liquid partition of methanol crude extract. Antiproliferative effect of R. tuberosa ethyl acetate leaf extract (RTEAL) was evaluated using MTT assay. Its ability to induce apoptosis was assessed by DNA ladder formation, JC-1, Annexin V, and methylene blue staining assays. Perturbation of cell cycle progression was determined using flow cytometry. RTEAL was found to selectively inhibit the proliferation of MCF-7 cells with the IC₅₀ value of 28 µg/mL. Morphological changes such as nuclear fragmentation and chromatin condensation were observed although DNA laddering was undetected in agarose gel. RTEAL-induced apoptotic pathways by inhibiting the expression of anti-apoptotic BCL-2 while upregulating pro-apoptotic BAX, caspase 7 and caspase 8. RTEAL also caused cell cycle arrests at the S and G2/M phase and dysregulation of cell cycle regulators. These findings collectively demonstrate that RTEAL extract inhibited cell growth by inducing apoptosis and cell cycle arrest, suggesting its therapeutic potential against breast cancer

Detection of viable bacteria in environmental water samples using DNase I and PCR method. International Journal of Environmental Studies

In this study, we tested the potential application of a previously developed method in detecting Escherichia coli in environmental water samples. To increase the sensitivity of the method, and the recovery of microbial cells, water samples were filtered before being subjected to DNase treatment and polymerase chain reaction amplification. Results showed that DNase I treatment and PCR reaction were not affected by inhibitors as the expected amplicon was successfully amplified in autoclaved environmental waters spiked with E. coli. Then, we applied this method to naturally contaminated environmental water samples. We firstly confirmed the presence of coliforms and E. coli in these water samples by plating in eosin methylene blue agar. Simultaneous PCR amplification targeting Lac Z and uidR gene of total coliforms and E. coli respectively demonstrated that this developed method is potentially applicable for routine microbial assessment of health risks related to viable microorganisms in environmental or drinking waters

First Report on The Occurrence of Cochlodinium Blooms in Sabah, Malaysia

Harmful algal blooms (HABs) resulting in red discoloration of coastal waters in Sepanggar Bay, off Kota Kinabalu, Sabah, East Malaysia, were first observed in January 2005. The species responsible for the bloom, which was identified as Cochlodinium polykrikoides, coincided with fish mortalities in cage-cultures. Determinations of cell density between January 2005 and June 2006 showed two peaks that occurred in March–June 2005 and June 2006. Cell abundance reached a maximum value of 6 × 106 cells L−1 at the fish cage sampling station where the water quality was characterized by high NO3–N and PO4–P concentrations. These blooms persisted into August 2005, were not detected during the north–east monsoon season and occurred again in May 2006. Favorable temperature, salinity and nutrient concentrations, which were similar to those associated with other C. polykrikoides blooms in the Asia Pacific region, likely promoted the growth of this species. Identification of C. polykrikoides as the causative organism was based on light and scanning microscopy, and confirmed by partial 18S ribosomal DNA sequences of two strains isolated during the bloom event (GenBank accession numbers DQ915169 and DQ915170)

Chemical composition and cytotoxic properties of Clinacanthus nutans root extracts

Context: Clinacanthus nutans Lindau (Acanthaceae) is a medicinal plant that has been reported to have anti-inflammatory, antiviral, antimicrobial and antivenom activities. In Malaysia, it has been widely claimed to be effective in various cancer treatments but scientific evidence is lacking. Objective: This study investigates the chemical constituents, anti-proliferative, and apoptotic properties of C. nutans root extracts. Materials and methods: The roots were subjected to solvent extraction using methanol and ethyl acetate. The anti-proliferative effects of root extracts were tested at the concentrations of 10 to 50 μg/mL on MCF-7 and HeLa by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for 72 h. Morphological changes were observed under light microscope. Pro-apoptotic effects of root extracts were examined using flow cytometric analysis and RT-PCR. The chemical compositions of root extracts were detected using GC-MS. Results: The proliferation of MCF-7 cells was inhibited with the IC50 values of 35 and 30 μg/mL, respectively, for methanol and ethyl acetate root extracts. The average inhibition of HeLa cells was ∼25%. Induction of apoptosis in MCF-7 was supported by chromatin condensation, down-regulation of BCL2 and unaltered expression of BAX. However, only ethyl acetate extract caused the loss of mitochondrial membrane potential. GC-MS analysis revealed the roots extracts were rich with terpenoids and phytosterols. Discussion and conclusions: The results demonstrated that root extracts promote apoptosis by suppressing BCL2 via mitochondria-dependent or independent manner. The identified compounds might work solely or cooperatively in regulating apoptosis. However, further studies are required to address this

The Advancement of Biomaterials in Regulating Stem Cell Fate.

Stem cells are well-known to have prominent roles in tissue engineering applications. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can differentiate into every cell type in the body while adult stem cells such as mesenchymal stem cells (MSCs) can be isolated from various sources. Nevertheless, an utmost limitation in harnessing stem cells for tissue engineering is the supply of cells. The advances in biomaterial technology allows the establishment of ex vivo expansion systems to overcome this bottleneck. The progress of various scaffold fabrication could direct stem cell fate decisions including cell proliferation and differentiation into specific lineages in vitro. Stem cell biology and biomaterial technology promote synergistic effect on stem cell-based regenerative therapies. Therefore, understanding the interaction of stem cell and biomaterials would allow the designation of new biomaterials for future clinical therapeutic applications for tissue regeneration. This review focuses mainly on the advances of natural and synthetic biomaterials in regulating stem cell fate decisions. We have also briefly discussed how biological and biophysical properties of biomaterials including wettability, chemical functionality, biodegradability and stiffness play their roles

Collagen type I promotes osteogenic differentiation of amniotic membrane-derived mesenchymal stromal cells in basal and induction media

Collagen has been widely shown to promote osteogenesis of bone marrow mesenchymal stromal cells (BM-MSCs). Due to the invasive procedure of obtaining BM-MSCs, MSCs from other tissues have emerged as a promising alternative for regenerative therapy. MSCs originated from different sources, exhibiting different differentiation potentials. Therefore, the applicability of collagen type I (COL), combining with amniotic membrane (AM)-MSCs was examined through proliferation and differentiation assays together with the expression of surface markers and genes associated with stemness and differentiation under basal or induction conditions. No increase in cell growth was observed because AM-MSCs might be directed toward spontaneous osteogenesis. This was evidenced by the calcium deposition and elevated expression of osteogenic genes when AM-MSCs were cultured in collagen plate with basal media. Under the osteogenic condition, reciprocal expression of OCN and CEBPA suggested a shift toward adipogenesis. Surprisingly, adipogenic genes were not elevated upon adipogenic induction, although oil droplets deposition was observed. In conclusion, our findings demonstrated that collagen causes spontaneous osteogenesis in AM-MSCs. However, the presence of exogenous inductors could shift the direction of adipo-osteogenic gene regulatory network modulated by collagen. © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)