Anti-Leukemic Effects of Typhonium Flagelliforme on Human Lymphoblastoid Cells (CEMss) and Murine Leukemic (WEHI-3) Model

To date, there has been no literature reported on the mechanism of Typhonium flagelliforme and its effects on leukemia. Hence, the anti-leukemia effect of Typhonium flagelliforme was investigated in vitro and in vivo leukemic model. Extraction and fractionation using organic solvents were applied to obtain fractions from T. flagelliforme and subsequently, chemical analysis was done using GC-MS. In vitro cytotoxic effects of extracts and fractions were tested in several human cancer cell lines including leukemia (CEMss cells) using MTT assay. Various microscopy techniques were used to study morphological changes occurring during treatment. The Annexin V assay, TUNEL assay, cell cycle analysis and DNA laddering were employed to detect apoptosis. Colourimetric assays for caspase-3 and 9, immunoblot analysis for cytochrome c, BcL-2, PARP, FasL and β-actin were analysed. The in vivo model of leukemia was induced in male BALB/c mice using WEHI-3 cells. The DCM extract of the plant tuber was used for treatment at various doses. Amongst 8 plant extracts investigated, the dichloromethane (DCM) extracts of T. flagelliforme tuber demonstrated low and significant anti proliferative effect against both CEMss (6.5±0.4 μg/ml) and WEHI-3 cells (24.0±5.2 μg/ml) (p<0.05). Further fractionation of the DCM tuber extract resulted into 12 fractions. Seven of these 12 fractions showed significant cytotoxicity against CEMss, in which the DCM/F7, DCM/F11 and DCM/F12 fractions showed highest anti-cancer activities of 3.0, 5.0 and 6.2 μg/ml respectively. Further studies of these fractions towards non cancerous Peripheral Blood Lymphocytes (PBL) exhibited significant selectivity of DCM/F7 compared to other fractions. Phytochemical analysis using GC-MS revealed that the DCM/F7 fraction contains linoleic acid (51.20%), n-hexadecanoic acid (17.89%), 9-hexadecanoic acid (6.99%) and Stigmasta-5,22-dien-3-ol (6.06%). Cytological observations exhibited chromatin condensation, cell shrinkage, abnormalities of cristae, membrane blebbing, cytoplasmic extrusions and formation of apoptotic bodies, further confirmed using AO/PI, SEM and TEM analysis. The Annexin V and TUNEL assay revealed apoptotic induction in CEMss cells exposed to the DCM/F7 in a time-dependent manner, whilst DNA fragmentation of CEMss cells were detected using 1.0% agarose gel electrophoresis. The DCM/F7 fraction significantly (p<0.05) stimulated both caspases 3 and 9 activities. The immunoblot results revealed that DCM/F7 caused the release of mitochondrial cytochrome c and cleaved 116 kDa PARP into 85 kDa fragments. The Bcl-2 protein was found to decrease during treatment. Meanwhile, FasL did not exhibit up or down regulation on treatment. Cell cycle analysis revealed that there is significant (p<0.05) G1 phase arrest in a time-depended manner. The DCM extract of T. flagelliforme tuber in vivo markedly inhibited the proliferation of WEHI-3 in male BALB/c mice as evidenced by reduction in the percentage of immature monocytes as well as granulocytes, liver weight, spleen weight and histopathological profiles of H&E stained spleen tissue. The DCM tuber extract of T. flagelliforme significantly decreased the spleen tumor size, which had dose-dependent effects. Sections of spleen tissue of the BALB/c mice treated with the extract. Treatment at 800 mg/kg dose showed evidence of apoptosis in comparison to the control groups. Collectively, results presented in this study demonstrate that T. flagelliforme, a local herbal medicinal plant in Malaysia inhibited the proliferation of leukemia in vitro selectively, leading to the programmed cell death, which was later confirmed to lead through mitochondrial pathways. Moreover, in vivo study on an orthotopic BALB/c mice model clearly shows that, T. flagelliforme tuber extract has inhibited the proliferation of leukemia via the induction of apoptosis

β-mangostin suppresses LA-7 cells proliferation in vitro and in vivo: involvement of antioxidant enzyme modulation; suppression of matrix metalloproteinase and α6β4 integrin signalling pathways

β-mangostin (βM) was isolated from Cratoxylum arborescens to investigate anti-breast cancer effect in vitro and in vivo. βM exhibited an inhibitory effect on the growth of LA-7 cells in vitro with apoptosis formation. In the animal model, βM treatment was found to be effective in improving the tissue antioxidant enzymes such as superoxide dismutase and catalase activity (P < 0.05). βM treatment clearly exhibited apoptosis in mammary tumour tissues, and it was associated with regulation of PCNA and p53. The cDNA microarray gene expression followed by qRT-PCR based validation demonstrated that βM could mediate tumour reduction and prevent metastasis by reduction of MMP-9, MMP-13, and MMP-27. Moreover, the reduction of both 14-3-3β and ITGB4 genes indicated the involvement of α6β4 integrin signalling pathway. These findings showed that β-mangostin is a promising compound candidate as an anti-tumour agent against breast cancer

Design of an Automatic Traffic Control System with Real Time Vehicle Density Analysis

Growth in developing countries forcefully leads their roads becoming increasingly congested thus bringing traffic to a standstill. There are two major measures that can address this issue viz. manual and automatic approaches. Because of its inefficiency, manual approach is not considered under the preview of this study and the automatic approach, which is the focus of this study, can further be classified into two viz. timer based and density based approaches. Timer based approaches are primarily static in nature and hence cannot consider real time requirements. Incorporating Density analysis on to the problem under preview adds dynamism to the solution and in the proposed study, traffic signals are controlled by considering real time density of vehicles on each roads. After capturing real time images and performing appropriate preprocessing, density of vehicles are measured using Canny Edge detection approach; which is used as a parameter for later decision making. Considering density parameters alone may lead to starvation to certain roads and here in the proposed study, authors consider a weight factor to propose a better solution. A prototype was developed using Arduino mega 2560 board, Webcams and Airplane 9g Mini Servo - SG-90 and led lights and it was observed that the efficacy of the proposed system is higher while comparing it to other prevailing methods

Apoptosis effect of girinimbine isolated from Murraya koenigii on lung cancer cells in vitro

Murraya koenigii Spreng has been traditionally claimed as a remedy for cancer. The current study investigated the anticancer effects of girinimbine, a carbazole alkaloid isolated from Murraya koenigii Spreng, on A549 lung cancer cells in relation to apoptotic mechanistic pathway. Girinimbine was isolated from Murraya koenigii Spreng. The antiproliferative activity was assayed using MTT and the apoptosis detection was done by annexin V and lysosomal stability assays. Multiparameter cytotoxicity assays were performed to investigate the change in mitochondrial membrane potential and cytochrome c translocation. ROS, caspase, and human apoptosis proteome profiler assays were done to investigate the apoptotic mechanism of cell death. The MTT assay revealed that the girinimbine induces cell death with an IC50 of 19.01 μM. A significant induction of early phase of apoptosis was shown by annexin V and lysosomal stability assays. After 24 h treatment with 19.01 μM of girinimbine, decrease in the nuclear area and increase in mitochondrial membrane potential and plasma membrane permeability were readily visible. Moreover the translocation of cytochrome c also was observed. Girinimbine mediates its antiproliferative and apoptotic effects through up- and downregulation of apoptotic and antiapoptotic proteins. There was a significant involvement of both intrinsic and extrinsic pathways. Moreover, the upregulation of p53 as well as the cell proliferation repressor proteins, p27 and p21, and the significant role of insulin/IGF-1 signaling were also identified. Moreover the caspases 3 and 8 were found to be significantly activated. Our results taken together indicated that girinimbine may be a potential agent for anticancer drug development

Primary total knee replacement using dished polyethylene with resected posterior cruciate ligament

Background: The choice between preserving, sacrificing or substituting the posterior cruciate ligament (PCL) is always a controversial topic in total knee replacement (TKR). Dished polyethylene insert with PCL resection enables correction of the commonly present fixed flexion and varus deformities. Additionally, the risk of premature wear of polyethylene is less because of the confirming articular geometry between the femoral and tibial component.Methods: This is a retrospective study in which we studied 120 knees in 95 consecutive patients undergoing primary TKR by the senior author at our institute. We used TKR system with dished metal backed polyethylene tibial component. PCL resection was performed in all cases. Pre-operative and post-operative functional assessment were done using knee society clinical scores and Western Ontario and McMaster universities osteoarthritis index (WOMAC). All radiographs were assessed using the knee society Roentgenographic scoring system (KSRES). Statistical analysis was performed using paired student t tests. Survivorship was determined using Kaplan-Meier survivorship curves. Results: Mean follow-up was 8 years. Range of motion increased from 75 degrees to 110 degrees.  The knee society pain score increased from 30 to 94. The knee society function score increased from 35 to 75. WOMAC score increased in terms of pain, stiffness and physical function.Conclusions: We conclude that deep dish bearing is a viable option in presence of deficient PCL and provides adequate stability and functional outcome. We need a larger sample size, multicentre trial and longer follow-up to see for complication rate, revision rate and survival

Riboflavin-Vancomycin Conjugate Enables Simultaneous Antibiotic Photo-Release and Photodynamic Killing against Resistant Gram-Positive Pathogens

Decades of antibiotic misuse have led to alarming levels of antimicrobial resistance, and the development of alternative diagnostic and therapeutic strategies to delineate and treat infections is a global priority. In particular, the nosocomial, multidrug-resistant "ESKAPE" pathogens such as Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus spp (VRE) urgently require alternative treatments. Here, we developed light-activated molecules based on the conjugation of the FDA-approved photosensitizer riboflavin to the Gram-positive specific ligand vancomycin to enable targeted antimicrobial photodynamic therapy. The riboflavin-vancomycin conjugate proved to be a potent and versatile antibacterial agent, enabling the rapid, light-mediated, killing of MRSA and VRE with no significant off-target effects. The attachment of riboflavin on vancomycin also led to an increase in antibiotic activity against S. aureus and VRE. Simultaneously, we evidenced for the first time that the flavin subunit undergoes an efficient photoinduced bond cleavage reaction to release vancomycin, thereby acting as a photoremovable protecting group with potential applications in drug delivery

β-Mangostin induces p53-dependent G2/M cell cycle arrest and apoptosis through ROS mediated mitochondrial pathway and NfkB suppression in MCF-7 cells

β-Mangostin (βM) was isolated from Cratoxylum arborescens to investigate its anti-cancer effect in MCF-7 cells. βM induced apoptosis by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol. The release of caspase-9 and -7 and consequently cleaved PARP leading to apoptotic was observed upon treatment. Reduction of both bid and caspase 8 and the up regulation of Fas showed the involvement of the extrinsic pathway. Significantly up regulated GADD45A and HRK genes were observed upon treatment, with concomitant inhibition of NF-kB to nucleus. The protein array had demonstrated the expression of HSP 70, HSP 60, XIAP, Survivin, p53 and Bax. Moreover, βM had showed p53-dependent G2/M cell cycle arrest by down regulation of cdc2 and PCNA. Together, the results demonstrated that the βM induced anti-proliferative effect, leading to G2/M phase cell cycle arrest and apoptosis through both the extrinsic and mitochondrial pathways with the involvement of the multiple pro and anti-apoptosis and NF-kB signalling pathways

Attenuation of cisplatin-induced hepatotoxicity in rats using zerumbone.

Zerumbone is a natural compound isolated from the fresh rhizomes of Zingiber zerumbet. This bioactive compound has shown a chemo-preventive, anti-inflammatory and free radical scavenging activities. This study examines, the effect of zerumbone on the extent of tissue damage in Cisplatin-induced hepatotoxicity in rats. The rats received a single dose injection of 45 mg kg-1 Cisplatin. Other groups of rats received zerumbone (100 and 200 mg kg-1), corn oil or the vehicle (DMSO) intraperitoneally for 4 days prior to Cisplatin injections. All animals were decapitated 16 h after Cisplatin injection. Trunk blood was collected and analyzed for alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase and gama-glutamyl transferase. Liver tissue was kept for the quantification of malondialdehyde and glutathione levels. Histopathological investigations were carried out and severity of lesions was scored to obtain quantitative data. This study revealed that zerumbone reduced the extent of liver damage and preserved liver functions as proved by microscopic observations and lesion scoring. Increase in liver MDA levels with a simultaneous reduction in GSH in the Cisplatin 45 mg kg-1 group was attenuated by zerumbone treatment (p<0.05). Zerumbone is beneficial in Cisplatin-induced liver dysfunction and organ damage in rats via prevention of lipid peroxidation and preservation of antioxidant glutathione

In Vitro Ultramorphological Assessment of Apoptosis Induced by Zerumbone on (HeLa)

Zerumbone (ZER), a potential anticancer compound, isolated from the fresh rhizomes of Zingiber zerumbet. In this investigation, the cytotoxic properties of ZER were evaluated, on cancer cells of human cervix (HeLa), breast and ovary, and normal cells of Chinese Hamster ovary, using MTT assay. Apoptogenic effects of ZER on HeLa were studied using fluorescence microscopy (AO/PI double staining), scanning and transmission electron microscopy (SEM and TEM), and colorimetric assay of the apoptosis promoter enzyme, caspase-3. The results of MTT assay showed that ZER has less effect on normal cells compared to cancer cells. The lowest IC50 of ZER was observed on HeLa cells. Cytological observations showed nuclear and chromatin condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double staining of AO/PI, SEM and TEM. Statistical analysis (two-tailed t-test) of differential counting of 200 cells under fluorescence microscope revealed significant difference in apoptotic cells populations between treated and untreated HeLa cells. In addition, ZER has increased the cellular level of caspase-3 on the treated HeLa cells. It could be concluded that ZER was able to produce distinctive morphological features of cell death that corresponds to apoptosis

β Mangostin suppress LPS-induced inflammatory response in RAW 264.7 macrophages in vitro and carrageenan-induced peritonitis in vivo

Ethnopharmacological relevance: The fruit hull of Garcinia mangostana Linn. has been used in traditional medicine for treatment of various inflammatory diseases. Hence, this study aims to investigate the in vitro and in vivo anti-inflammatory effect of β mangostin (βM), a major compound present in Garcinia mangostana. Materials and methods: The in silico analysis of inflammatory mediators such as cyclooxygenase (COX) and nuclear factor-kappa B (NF-kB) were performed via molecular docking. Further evaluation of anti-inflammatory effect was conducted in lipopolysaccharide (LPS) induced RAW 264.7 macrophages. Suppression of activated NF-kB was analyzed by high content screening. βM triggered inhibition of COX-1 and COX-2 in vitro were studied using biochemical kit. The in vivo model used in this study was carrageenan-induced peritonitis model, where reduction in carrageenan-induced peritonitis is measured by leukocyte migration and vascular permeability. In addition, the evaluation of βM׳s effect on carrageenan induced TNF-α and IL-1β release on peritoneal fluid was also carried out. Results: Treatment with βM could inhibit the LPS-induced NO production but not the viability of RAW 264.7. Similarly, βM inhibited PGE2 production and the cytokines: TNF-α and IL-6. The COX catalyzed prostaglandin biosynthesis assay had showed selective COX-2 inhibition with a 53.0±6.01% inhibition at 20 µg/ml. Apart from this, βM was capable in repressing translocation of NF-kB into the nucleus. These results were concurrent with molecular docking which revealed COX-2 selectivity and NF-kB inhibition. The in vivo analysis showed that after four hours of peritonitis, βM was unable to reduce vascular permeability, yet could decrease the total leukocyte migration; particularly, neutrophils. Meanwhile, dexamethasone 0.5 mg/kg, successfully reduced vascular permeability. The levels of TNF-α and IL-1β in peritoneal fluid was reduced significantly by βM treatment. Conclusion: The current study supports the traditional use of Garcinia mangostana fruit hull for treatment of inflammatory conditions. In addition, it is clear that the anti-inflammatory efficacy of this plant is not limited to the presence of α and γ, but β also with significant activity