Oxidative stress is thought to be one of the factors that cause neurodegeneration and that this can be inhibited by antioxidants. Since astrocytes support the survival of central nervous system (CNS) neurons, we compared the effect of alpha-tocopherol and gamma-tocotrienol in minimizing the cytotoxic damage induced by H(2)O(2), a pro-oxidant. Primary astrocyte cultures were pretreated with either alpha-tocopherol or gamma-tocotrienol for 1 h before incubation with 100 microM H(2)O(2) for 24 h. Cell viability was then assessed using the MTS assay while apoptosis was determined using a commercial ELISA kit as well as by fluorescent staining of live and apoptotic cells. The uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes were also determined using HPLC. Results showed that gamma-tocotrienol is toxic at concentrations >200 microM but protects against H(2)O(2) induced cell loss and apoptosis in a dose dependent manner up to 100 microM. alpha-Tocopherol was not cytotoxic in the concentration range tested (up to 750 microM), reduced apoptosis to the same degree as that of gamma-tocotrienol but was less effective in maintaining the viable cell number. Since the uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes is similar, this may reflect the roles of these 2 vitamin E subfamilies in inhibiting apoptosis and stimulating proliferation in astrocytes.
Exposure to silver nanoparticles (AgNP) used in consumer products carries potential health risks including increased susceptibility to infectious pathogens. Systematic assessments of antimicrobial macrophage immune responses in the context of AgNP exposure are important because uptake of AgNP by macrophages may lead to alterations of innate immune cell functions. In this study we examined the effects of exposure to AgNP with different particle sizes (20 and 110 nm diameters) and surface chemistry (citrate or polyvinlypyrrolidone capping) on cellular toxicity and innate immune responses against Mycobacterium tuberculosis (M.tb) by human monocyte-derived macrophages (MDM). Exposures of MDM to AgNP significantly reduced cellular viability, increased IL8 and decreased IL10 mRNA expression. Exposure of M.tb-infected MDM to AgNP suppressed M.tb-induced expression of IL1B, IL10, and TNFA mRNA. Furthermore, M.tb-induced IL-1β, a cytokine critical for host resistance to M.tb, was inhibited by AgNP but not by carbon black particles indicating that the observed immunosuppressive effects of AgNP are particle specific. Suppressive effects of AgNP on the M.tb-induced host immune responses were in part due to AgNP-mediated interferences with the TLR signaling pathways that culminate in the activation of the transcription factor NF-κB. AgNP exposure suppressed M.tb-induced expression of a subset of NF-κB mediated genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). In addition, AgNP exposure increased the expression of HSPA1A mRNA and the corresponding stress-induced Hsp72 protein. Up-regulation of Hsp72 by AgNP can suppress M.tb-induced NF-κB activation and host immune responses. The observed ability of AgNP to modulate infectious pathogen-induced immune responses has important public health implications.
Artonin E is a prenylated flavonoid isolated from the stem bark of Artocarpus elasticus Reinw.(Moraceae). This study aimed to investigate the apoptotic mechanisms induced by artonin E in a metastatic human ovarian cancer cell line SKOV-3 in vitro. MTT assay, clonogenic assay, acridine orange and propidium iodide double staining, cell cycle and annexin V analyses were performed to explore the mode of artonin E-induced cell death at different time points. DNA laddering, activation of caspases-3, -8, and -9, multi-parametric cytotoxicity-3 analysis by high-content screening, measurement of reactive oxygen species generation, and Western blot were employed to study the pathways involved in the apoptosis. MTT results showed that artonin E inhibited the growth of SKOV-3 cells, with IC50 values of 6.5±0.5 μg/mL after 72 h treatment, and showed less toxicity toward a normal human ovarian cell line T1074, with IC50 value of 32.5±0.5 μg/mL. Results showed that artonin E induced apoptosis and cell cycle arrest at the S phase. This compound also promoted the activation of caspases-3, -8, and -9. Further investigation into the depletion of mitochondrial membrane potential and release of cytochrome c revealed that artonin E treatment induced apoptosis via regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The expression levels of survivin and HSP70 proteins were also down regulated in SKOV-3 cells treated with artonin E. We propose that artonin E induced an antiproliferative effect that led to S phase cell cycle arrest and apoptosis through dysregulation of mitochondrial pathways, particularly the pro- and anti-apoptosis signaling pathways.
To measure plasma pro-apoptotic and pro-necrotic activity in severe acute kidney injury (AKI) patients within a randomized controlled trial of continuous veno-venous hemofiltration with high cut-off filters (CVVH-HCO) versus standard filters (CVVH-Std).
The growing number of people with diabetes worldwide suggests that diabetic retinopathy (DR) and diabetic macular edema (DME) will continue to be sight threatening factors. The pathogenesis of diabetic retinopathy is a widespread cause of visual impairment in the world and a range of hyperglycemia-linked pathways have been implicated in the initiation and progression of this condition. Despite understanding the polyol pathway flux, activation of protein kinase C (KPC) isoforms, increased hexosamine pathway flux, and increased advanced glycation end-product (AGE) formation, pathogenic mechanisms underlying diabetes induced vision loss are not fully understood. The purpose of this paper is to review molecular mechanisms that regulate cell survival and apoptosis of retinal cells and discuss new and exciting therapeutic targets with comparison to the old and inefficient preventive strategies. This review highlights the recent advancements in understanding hyperglycemia-induced biochemical and molecular alterations, systemic metabolic factors, and aberrant activation of signaling cascades that ultimately lead to activation of a number of transcription factors causing functional and structural damage to retinal cells. It also reviews the established interventions and emerging molecular targets to avert diabetic retinopathy and its associated risk factors.
Acrylated abietic acid (acrylated AbA) and acrylated abietic acid-grafted bacterial cellulose pH sensitive hydrogel (acrylated AbA-g-BC) were prepared by a one-pot synthesis. The successful dimerization of acrylic acid (AA) and abietic acid (AbA) and grafting of the dimer onto bacterial cellulose (BC) was confirmed by 13C solid state NMR as well as FT-IR. X-ray diffraction analysis showed characteristic peaks for AbA and BC; further, there was no effect of increasing amorphous AA content on the overall crystallinity of the hydrogel. Differential scanning calorimetry revealed a glass transition temperature of 80°C. Gel fraction and swelling studies gave insight into the features of the hydrogel, suggesting that it was suitable for future applications such as drug delivery. Scanning electron microscopy observations showed an interesting interpenetrating network within the walls of hydrogel samples with the lowest levels of AA and gamma radiation doses. Cell viability test revealed that the synthesized hydrogel is safe for future use in biomedical applications.
The hybridoma 192 was used to produce a monoclonal antibody (MAb) against 17-hydroxyprogesterone (17-OHP), for possible use in screening for congenital adrenal hyperplasia (CAH). The factors influencing the MAb production were screened and optimized in a 2 L stirred bioreactor. The production was then scaled up to a 20 L bioreactor. All of the screened factors (aeration rate, stirring speed, dissolved oxygen concentration, pH, and temperature) were found to significantly affect production. Optimization using the response surface methodology identified the following optimal production conditions: 36.8°C, pH 7.4, stirring speed of 100 rpm, 30% dissolved oxygen concentration, and an aeration rate of 0.09 vvm. Under these conditions, the maximum viable cell density achieved was 1.34 ± 0.21 × 10(6) cells mL(-1) and the specific growth rate was 0.036 ± 0.004 h(-1) . The maximum MAb titer was 11.94 ± 4.81 μg mL(-1) with an average specific MAb production rate of 0.273 ± 0.135 pg cell(-1) h(-1) . A constant impeller tip speed criterion was used for the scale-up. The specific growth rate (0.040 h(-1) ) and the maximum viable cell density (1.89 × 10(6) cells mL(-1) ) at the larger scale were better than the values achieved at the small scale, but the MAb titer in the 20 L bioreactor was 18% lower than in the smaller bioreactor. A change in the culture environment from the static conditions of a T-flask to the stirred bioreactor culture did not affect the specificity of the MAb toward its antigen (17-OHP) and did not compromise the structural integrity of the MAb.
Trichomonas vaginalis, a flagellated protozoan parasite causes a variety of adverse health consequences in both men and women. The parasite exists in the trophozoite and the pseudocystic stage. The study reports for the first time that pseudocyst forms of T. vaginalis isolated from cervical neoplasia (CN) patients demonstrated distinct, different and significant in vitro growth profiles when grown in vitro cultures from day 1 up to day 5 (p<0.05, Mann-Whitney test) when compared with the same life cycle stages isolated from non-cervical neoplasia but symptomatic patients (NCN). Pseudocysts from CN and NCN isolates remained viable in distilled water until 3 h 10 min and 2 h 10 min, respectively. The nucleus of pseudocysts in CN isolates using acridine orange and DAPI showed more intense staining revealing higher nuclear content. The FITC-labeled Concanavalin A stained stronger green fluorescence with surface of pseudocysts in CN isolates showing more rough and creased surface with higher numbers of deep micropores with larger numbers of chromatin masses, vacuoles, and hydrogenosomes. The study confirms that pseudocystic stage from CN, despite the uniformity in appearance of being rounded and showing no motility without a true cyst wall under light microscopy, demonstrated different biochemical, surface, and ultrastructural properties. The study provides evidence that phenotypic variant forms of pseudocysts does exist and possibly does play a role in exacerbating cervical cancer.
Tracking of transplanted cells has become an important procedure in cell therapy. We studied the in vitro dye retention, survival and in vivo tracking of stem cells with PKH26 dye. Sheep BMSCs and ADSCs were labeled with 2, 4 and 8 μmol of PKH26 and monitored for six passages. Labeled BMSCs and ADSCs acquired mean cumulative population doubling of 12.7±0.4 and 14.6±0.5; unlabeled samples had 13.8±0.5 and 15.4±0.6 respectively. Upon staining with 2, 4 and 8 μmol PKH26, BMSCs had retentions of 40.0±5.8, 60.0±2.9 and 95.0±2.9%, while ADSCs had 92.0±1.2, 95.0±1.2 and 98.0±1.2%. ADSCs retentions were significantly higher at 2 and 4 μmol. On dye retention comparison at 8 μmol and 4 μmol for BMSCs and ADSCs; ADSCs were significantly higher at passages 2 and 3. The viability of BMSCs reduced from 94.0±1.2% to 90.0±0.6% and ADSCs from 94.0±1.2% to 52.0±1.2% (p<0.05) after 24h. BMSCs had significant up regulation of the cartilage genes for both the labeled and the unlabeled samples compared to ADSCs (p<0.05). PKH26 fluorescence was detected on the resected portions of the regenerated neo-cartilage. The recommended concentration of PKH26 for ADSCs is 2 μmol and BMSCs is 8 μmol, and they can be tracked up to 49 days.
Systemic infections of Candida albicans, the most prevalent fungal pathogen in humans, are on the rise in recent years. However, the exact mode of pathogenesis of this fungus is still not well elucidated. Previous studies using C. albicans mutants locked into the yeast form via gene deletion found that this form was avirulent and did not induce significant differential expression of host genes in vitro. In this study, a high density of C. albicans was used to infect human umbilical vein endothelial cells (HUVEC), resulting in yeast-form infections, whilst a low density of C. albicans resulted in hyphae infections. Transcriptional profiling of HUVEC response to these infections showed that high densities of C. albicans induced a stronger, broader transcriptional response from HUVEC than low densities of C. albicans infection. Many of the genes that were significantly differentially expressed were involved in apoptosis and cell death. In addition, conditioned media from the high-density infections caused a significant reduction in HUVEC viability, suggesting that certain molecules released during C. albicans and HUVEC interactions were capable of causing cell death. This study has shown that C. albicans yeast-forms, at high densities, cannot be dismissed as avirulent, but instead could possibly contribute to C. albicans pathogenesis.
Photodynamic therapy (PDT) is a promising cancer treatment which involves activation of a photosensitizing drug with light to produce reactive oxygen species that kill tumors without causing damage to unirradiated normal tissues. To date, only Photofrin, Foscan and Levulan have been approved for clinical treatment of cancer. Tropical habitats such as those found in Malaysia are attractive sources of new therapeutic compounds as tremendous chemical diversity is found in a large number of plants, animals, marine- and micro-organisms. In our screening program for novel photosensitizers from nature, colorful strains of fungi (from Aspergillus and Penicillium genus) and bacteria (including actinomycetes and photosynthetic bacteria) were collected from various habitats in Peninsular Malaysia, such as coastal soil, peat soil, marine sponges and wastewater ponds. Methanolic extracts from a total of 85 different species were evaluated with a short-term cell viability assay for photo-cytotoxicity, where a promyelocytic leukemia cell-line, HL60 incubated with 20 microg/ml of extracts was irradiated with 9.6 J/cm(2) of a broad spectrum light. Two of these extracts, one from Rhodobacter sphaeroides (PBUM003) and one from Rhodopseudomonas palustris (PBUM001) showed moderate to strong photo-cytotoxicity. Subsequent bioassay guided isolation of the PBUM001 extract yielded known photosensitisers that are based on bacteriochlorophyll-a by comparing their molecular weight data, HPLC profiles and UV-vis absorption spectra with literature values, thereby demonstrating the validity of our screening approach.
Microgravity, hypergravity, vibration, ionizing radiation and temperature fluctuations are major factors of outer space flight affecting human organs and tissues. There are several reports on the effect of space flight on different human cell types of mesenchymal origin while information regarding changes to vascular endothelial cells is scarce. Ultrastructural and cytophysiological features of macrovascular endothelial cells in outer space flight and their persistence during subsequent culturing were demonstrated in the present investigation. At the end of the space flight, endothelial cells displayed profound changes indicating cytoskeletal lesions and increased cell membrane permeability. Readapted cells of subsequent passages exhibited persisting cytoskeletal changes, decreased metabolism and cell growth indicating cellular senescence.
Cell-based therapy has great potential to treat patients with lung diseases. The administration of cells into an injured lung is one method of repairing and replacing lost lung tissue. However, different types of delivery have been studied and compared, and none of the techniques resulted in engraftment of a high number of cells into the targeted organ. In this in vitro study, a novel method of cell delivery was introduced to investigate the possibility of delivering aerosolized skin-derived fibroblasts.
Nigella sativa Linn. (N. sativa) and its bioactive constituent Thymoquinone (TQ) have demonstrated numerous pharmacological attributes. In the present study, the neuroprotective properties of Thymoquinone-rich fraction (TQRF) and TQ against hydrogen peroxide- (H2O2-) induced neurotoxicity in differentiated human SH-SY5Y cells were investigated. TQRF was extracted using supercritical fluid extraction while TQ was acquired commercially, and their effects on H2O2 were evaluated using cell viability assay, reactive oxygen species (ROS) assay, morphological observation, and multiplex gene expression. Both TQRF and TQ protected the cells against H2O2 by preserving the mitochondrial metabolic enzymes, reducing intracellular ROS levels, preserving morphological architecture, and modulating the expression of genes related to antioxidants (SOD1, SOD2, and catalase) and signaling genes (p53, AKT1, ERK1/2, p38 MAPK, JNK, and NF-κβ). In conclusion, the enhanced efficacy of TQRF over TQ was likely due to the synergism of multiple constituents in TQRF. The efficacy of TQRF was better than that of TQ alone when equal concentrations of TQ in TQRF were compared. In addition, TQRF also showed comparable effects to TQ when the same concentrations were tested. These findings provide further support for the use of TQRF as an alternative to combat oxidative stress insults in neurodegenerative diseases.
Hydroxyapatite (Ca10(PO4)6(OH)2) is widely investigated as an implantable material for hard tissue restoration due to its osteoconductive properties. However, hydroxyapatite in bulk form is limited as its mechanical properties are insufficient for load-bearing orthopedic applications. Attempts have been made to improve the mechanical properties of hydroxyapatite, by incorporating ceramic fillers, but the resultant composite materials require high sintering temperatures to facilitate densification, leading to the decomposition of hydroxyapatite into tricalcium phosphate, tetra-calcium phosphate and CaO phases. One method of improving the properties of hydroxyapatite is to incorporate bioactive glass particles as a second phase. These typically have lower softening points which could possibly facilitate sintering at lower temperatures. In this work, a bioactive glass (SiO2-CaO-ZnO-Na2O-TiO2) is incorporated (10, 20 and 30 wt%) into hydroxyapatite as a reinforcing phase. X-ray diffraction confirmed that no additional phases (other than hydroxyapatite) were formed at a sintering temperature of 560 ℃ with up to 30 wt% glass addition. The addition of the glass phase increased the % crystallinity and the relative density of the composites. The biaxial flexural strength increased to 36 MPa with glass addition, and there was no significant change in hardness as a function of maturation. The pH of the incubation media increased to pH 10 or 11 through glass addition, and ion release profiles determined that Si, Na and P were released from the composites. Calcium phosphate precipitation was encouraged in simulated body fluid with the incorporation of the bioactive glass phase, and cell culture testing in MC-3T3 osteoblasts determined that the composite materials did not significantly reduce cell viability.
Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.
Myocardial atrophy, characterized by the decreases in size and contractility of cardiomyocytes, is caused by severe malnutrition and/or mechanical unloading. Extracellular adenosine 5'-triphosphate (ATP), known as a danger signal, is recognized to negatively regulate cell volume. However, it is obscure whether extracellular ATP contributes to cardiomyocyte atrophy. Here, we report that ATP induces atrophy of neonatal rat cardiomyocytes (NRCMs) without cell death through P2Y2 receptors. ATP led to overproduction of reactive oxygen species (ROS) through increased amount of NADPH oxidase (Nox) 2 proteins, due to increased physical interaction between Nox2 and canonical transient receptor potential 3 (TRPC3). This ATP-mediated formation of TRPC3-Nox2 complex was also pathophysiologically involved in nutritional deficiency-induced NRCM atrophy. Strikingly, knockdown of either TRPC3 or Nox2 suppressed nutritional deficiency-induced ATP release, as well as ROS production and NRCM atrophy. Taken together, we propose that TRPC3-Nox2 axis, activated by extracellular ATP, is the key component that mediates nutritional deficiency-induced cardiomyocyte atrophy.
Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.
Arabinoxylan (AX) is a natural biological macromolecule with several potential biomedical applications. In this research, AX, nano-hydroxyapatite (n-HAp) and titanium dioxide (TiO2) based polymeric nanocomposite scaffolds were fabricated by the freeze-drying method. The physicochemical characterizations of these polymeric nanocomposite scaffolds were performed for surface morphology, porosity, swelling, biodegradability, mechanical, and biological properties. The scaffolds exhibited good porosity and rough surface morphology, which were efficiently controlled by TiO2 concentrations. MC3T3-E1 cells were employed to conduct the biocompatibility of these scaffolds. Scaffolds showed unique biocompatibility in vitro and was favorable for cell attachment and growth. PNS3 proved more biocompatible, showed interconnected porosity and substantial mechanical strength compared to PNS1, PNS2 and PNS4. Furthermore, it has also showed more affinity to cells and cell growth. The results illustrated that the bioactive nanocomposite scaffold has the potential to find applications in the tissue engineering field.
Skin grafts are indicated when there is a major loss of skin. Full-thickness skin graft is an ideal choice to reconstruct defect of irregular surface that is difficult to immobilize. Full-thickness mesh grafts can be applied to patch large skin defect when there is less donor site in extensively traumatized and burned surgical patients. The concept of using natural biomaterials such as keratin, basic fibroblast growth factor is slowly gaining popularity in the field of medical research to achieve early healing. The main objective of this study is to evaluate the efficacy of gelatin conjoined with keratin processed from the poultry feather and commercially available basic fibroblast growth factor (bFGF) as a sandwich layer in promoting the viability of full-thickness skin mesh grafts. The efficacy was assessed from the observation of clinical, bacteriological, and histopathological findings in three groups of experimental dogs. The clinical observations such as color, appearance and discharge, and hair growth were selected as criteria which indicated good and early acceptance of graft in keratin-gelatin (group II). On bacteriological examination, Staphylococcus aureus and Proteus was identified in few animals. Histopathological study of the patched graft revealed early presences of hair follicles; sebaceous gland, and normal thickness of the epidermis in keratin-gelatin in group II treated animals compared with other group (group I-control, group III-bFGF-gelatin).