Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Candida glabrata, Candida parapsilosis and Candida rugosa from two major hospitals in Kuala Lumpur, Malaysia.

The purpose of this study is to characterize 3 non-albicans Candida spp. that were collected from two major hospitals in a densely populated area of Kuala Lumpur for their susceptibilities to azole and genetic background. Fifteen non-albicans Candida clinical isolates in two major hospitals in Kuala Lumpur area of Malaysia were collected by convenience sampling during 2007 and 2010. The genetic diversity of 15 non-albicans Candida species comprising C. glabrata (n = 5), C. parapsilosis (n = 5) and C. rugosa (n = 5) were assessed by RAPD-PCR typing. Strains were initially identified using biochemical tests and CHROMagar Candida medium. Fluconazole and voriconazole susceptibilities were determined by E-test method. Commercial kits were used for DNA extraction and amplification with RAPD primers (OPA02, OPA03 and OPA08). PCR conditions were optimized and simultaneous identification was possible by agarose gel electrophoresis of PCR products and the bands obtained were analyzed using BioNumerics Applied Maths v.6.6 software. The RAPD primers used in this study generated 100% polymorphic profile. Cluster analysis using the RAPD-PCR profile showed 12.5-25% similarity among the strains. The genetic diversity was based on the strain susceptibility towards both the azoles, site of isolation and place according to their unique banding patterns. In contrast, strains susceptible to azoles were found to be genetically similar with clonal dissimilarity. The use of OPA02, OPA03 and OPA08 primers in differentiating non-albicans Candida spp. underscores the higher resolution of RAPD-PCR as a reliable tool for strain/species level differentiation.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Candida glabrata, Candida parapsilosis and Candida rugosa from two major hospitals in Kuala Lumpur, Malaysia

@#The purpose of this study is to characterize 3 non-albicans Candida spp. that were collected from two major hospitals in a densely populated area of Kuala Lumpur for their susceptibilities to azole and genetic background. Fifteen non-albicans Candida clinical isolates in two major hospitals in Kuala Lumpur area of Malaysia were collected by convenience sampling during 2007 and 2010. The genetic diversity of 15 non-albicans Candida species comprising C. glabrata (n = 5), C. parapsilosis (n = 5) and C. rugosa (n = 5) were assessed by RAPD-PCR typing. Strains were initially identified using biochemical tests and CHROMagar Candida medium. Fluconazole and voriconazole susceptibilities were determined by E-test method. Commercial kits were used for DNA extraction and amplification with RAPD primers (OPA02, OPA03 and OPA08). PCR conditions were optimized and simultaneous identification was possible by agarose gel electrophoresis of PCR products and the bands obtained were analyzed using BioNumerics Applied Maths v.6.6 software. The RAPD primers used in this study generated 100% polymorphic profile. Cluster analysis using the RAPD-PCR profile showed 12.5-25% similarity among the strains. The genetic diversity was based on the strain susceptibility towards both the azoles, site of isolation and place according to their unique banding patterns. In contrast, strains susceptible to azoles were found to be genetically similar with clonal dissimilarity. The use of OPA02, OPA03 and OPA08 primers in differentiating non-albicans Candida spp. underscores the higher resolution of RAPD-PCR as a reliable tool for strain/species level differentiation.

Differential osteogenic activity of osteoprogenitor cells on HA and TCP/HA scaffold of tissue engineered bone.

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering.

Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold.

Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.

Effects of Strobilanthes crispus tea aqueous extracts on glucose and lipid profile in normal and streptozotocin-induced hyperglycemic rats.

Strobilanthes crispus (Acanthaceae) has been used traditionally as antidiabetic, diuretic, antilytic, and laxative and has been proven scientifically to possess high antioxidant activity, anti-AIDS, and anticancer properties. It is commonly consumed in the form of herbal tea. The ethnopharmacological value of this plant, such as the development of nutraceutical S. crispus herbal tea (fermented and unfermented) and assessment of their antihyperglycemic properties were investigated. The antidiabetic properties of S. crispus fermented and unfermented tea was carried out in normal and streptozotocin-induced hyperglycaemic rats for 21 days. Glucose and lipid profile (total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol) were determined at day 0 (baseline), day 7, and day 21. The results showed that the hot water extract of both fermented and unfermented S. crispus tea reduced blood glucose in hyperglycaemic rats. S. crispus unfermented tea also reduced glucose level in normal rat. Both fermented and unfermented S. crispus tea also showed to improve lipid profile. Antioxidant and polyphenol content that present in the extracts might contribute to the antihyperglycemic and antilipidemic properties. Further study is needed to be carried out in pre-clinical and clinical environment to prove its efficacy in human.

Quality evaluation analysis of bioengineered human skin.

Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.

Strategy for generating tissue-engineered human bone construct.

The strategy used to generate tissue-engineered bone construct, in view of future clinical application is presented here. Osteoprogenitor cells from periosteum of consenting scoliosis patients were isolated. Growth factors viz TGF-B2, bFGF and IGF-1 were used in concert to increase cell proliferation during in vitro cell expansion. Porous tricalcium phosphate (TCP)-hydroxyapatite (HA) scaffold was used as the scaffold to form 3D bone construct. We found that the addition of growth factors, greatly increased cell growth by 2 to 7 fold. TCP/HA proved to be the ideal scaffold for cell attachment and proliferation. Hence, this model will be further carried out on animal trial.

Collagen fibers an important entity in skin tissues remodeling.

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) evaluation were carried out in the in vivo skin construct using fibrin as biomaterial. To investigate its progressive remodeling, nude mice were grafted and the Extracellular Matrix (ECM) components were studied at four and eight weeks post-grafting. It was discovered that by 4 weeks of remodeling the skin construct acquired its native structure.

Comparison of chitosan scaffold and chitosan-collagen scaffold: a preliminary study.

Chitosan has similar structure to glycosaminoglycans in the tissue, thus may be a good candidates as tissue engineering scaffold. However, to improve their cell attachment ability, we try to incorporate this natural polymer with collagen by combining it via cross-linking process. In this preliminary study we evaluate the cell attachment ability of chitosan-collagen scaffold versus chitosan scaffold alone. Chitosan and collagen were dissolved in 1% acetic acid and then were frozen for 24 hours before the lyophilizing process. Human skin fibroblasts were seeded into both scaffold and were cultured in F12: DMEM (1:1). Metabolic activity assay were used to evaluate cell attachment ability of scaffold for a period of 1, 3, 7 and 14 days. Scanning electron micrographs shows good cell morphology on chitosan-collagen hybrid scaffold. In conclusion, the incorporation of collagen to chitosan will enhance its cell attachment ability and will be a potential scaffold in tissue engineering.

Evaluation of suitable biodegradable scaffolds for engineered bone tissue.

Tricalcium phosphate/hydroxyapatite (TCP/HA), hydroxyapatite (HA), chitosan and calcium sulphate (CaSO4) were studied and evaluated for possible bone tissue engineered construct acting as good support for osteogenic cells to proliferate, differentiate, and eventually spread and integrate into the scaffold. Surface morphology visualized by SEM showed that scaffold materials with additional fibrin had more cell densities attached than those without, depicting that the presence of fibrin and collagen fibers were truly a favourite choice of cells to attach. In comparison of various biomaterials used incorporated with fibrin, TCP/HA had the most cluster of cells attached.

The effect of neem (Azadirachta indica) extract and dietary selenium on distribution of selenium in hepatocarcinogenesis induced rat.

Neem, Azadirachta indica, is a plant from the family Meliaceae, known as "Pokok Semambu" in Malay community. It has been extensively used in India as traditional Ayurvedic and folklore minedicine for the treatment of various diseases. This study aimed to determine the distribution of selenium in the liver of rats during hepatocarcinogenesis when neem aqueous extract and dietary selenium was supplemented.

Comparison of chitosan scaffold and chitosan-collagen scaffold: a preliminary study

Chitosan has similar structure to glycosaminoglycans in the tissue, thus may be a good candidates as tissue engineering scaffold. However, to improve their cell attachment ability, we try to incorporate this natural polymer with collagen by combining it via cross-linking process. In this preliminary study we evaluate the cell attachment ability of chitosan-collagen scaffold versus chitosan scaffold alone. Chitosan and collagen were dissolved in 1% acetic acid and then were frozen for 24 hours before the lyophilizing process. Human skin fibroblasts were seeded into both scaffold and were cultured in F12: DMEM (1:1). Metabolic activity assay were used to evaluate cell attachment ability of scaffold for a period of 1, 3, 7 and 14 days. Scanning electron micrographs shows good cell morphology on chitosan-collagen hybrid scaffold. In conclusion, the incorporation of collagen to chitosan will enhance its cell attachment ability and will be a potential scaffold in tissue engineering.

Collagen fibers an important entity in skin tissues remodeling

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) evaluation were carried out in the in vivo skin construct using fibrin as biomaterial. To investigate its progressive remodeling, nude mice were grafted and the Extracellular Matrix (ECM) components were studied at four and eight weeks post-grafting. It was discovered that by 4 weeks of remodeling the skin construct acquired its native structure.

Strategy for generating tissue-engineered human bone construct

The strategy used to generate tissue-engineered bone construct, in view of future clinical application is presented here. Osteoprogenitor cells from periosteum of consenting scoliosis patients were isolated. Growth factors viz TGF-B2, bFGF and IGF-1 were used in concert to increase cell proliferation during in vitro cell expansion. Porous tricalcium phosphate (TCP)-hydroxyapatite (HA) scaffold was used as the scaffold to form 3D bone construct. We found that the addition of growth factors, greatly increased cell growth by 2 to 7 fold. TCP/HA proved to be the ideal scaffold for cell attachment and proliferation. Hence, this model will be further carried out on animal trial.

Quality evaluation analysis of bioengineered human skin

Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.

Respiratory epithelium, production performance and behaviour of formaldehyde-exposed broiler chicks.

Two experiments were conducted to evaluate the effect of formaldehyde vaporization of a hatcher on the tracheal epithelium of chick embryos, and on the production performance and behaviour of commercial broiler chicks. In experiment 1, chick embryos were exposed to 23.5 ppm of formaldehyde vapour during the last 3 days of incubation. Tracheal samples were taken at 0, 6, 30 and 54 h after exposure to formaldehyde and examined by scanning electron microscopy for pathological changes. Observable lesions included excessive accumulation of mucus, matted cilia, loss of cilia and sloughing of the epithelium. The lesions were more severe in chicks exposed for 54 h as compared to those exposed for 6 or 30 h. In experiment 2, 60 chicks that had been exposed to formaldehyde vapour as above and 60 control chicks were used to investigate the effect of formaldehyde fumigation on production performance and behaviour. Formaldehyde vaporization resulted in higher weekly (days 0-6 and 21-27) and total (days 0-41) feed intake and poorer weekly (days 0-6, 7-13, 21-27 and 28-34) and overall (days 0-41) feed conversion ratios. Body weight, mortality and behaviour (eating, drinking, sitting and standing activities) were not affected by formaldehyde fumigation.

Scanning electron microscopy of the respiratory epithelium of chicks fumigated with formaldehyde vapour.

1. Hatching chicks were exposed to 10.9 ppm of formaldehyde vapour during the last 3 d of incubation in a commercial situation. 2. Samples from pre-selected regions of the entire respiratory tract, taken at 0, 6, 30 and 54 h post-vaporisation of formaldehyde, were examined by scanning electron microscopy for surface morphological features and associated pathological changes. 3. Clumping of cilia, blebs on the cilial wall, deciliation and exfoliation of the epithelium were all observed under the scanning electron microscope. 4. Lesions were more severe in chicks exposed for 54 h as compared to those exposed for 6 and 30 h. There were no regional differences in the lesions throughout the respiratory tract of all the chicks. 5. Limited observations suggest that the passage of formaldehyde vapour across the egg shell wall may influence the morphology of the respiratory lining of the developing embryo.