Minocycline Loaded Hybrid Composites Nanoparticles for Mesenchymal Stem Cells Differentiation into Osteogenesis.

Bone transplants are used to treat fractures and increase new tissue development in bone tissue engineering. Grafting of massive implantations showing slow curing rate and results in cell death for poor vascularization. The potentials of biocomposite scaffolds to mimic extracellular matrix (ECM) and including new biomaterials could produce a better substitute for new bone tissue formation. A purpose of this study is to analyze polycaprolactone/silk fibroin/hyaluronic acid/minocycline hydrochloride (PCL/SF/HA/MH) nanoparticles initiate human mesenchymal stem cells (MSCs) proliferation and differentiation into osteogenesis. Electrospraying technique was used to develop PCL, PCL/SF, PCL/SF/HA and PCL/SF/HA/MH hybrid biocomposite nanoparticles and characterization was analyzed by field emission scanning electron microscope (FESEM), contact angle and Fourier transform infrared spectroscopy (FT-IR). The obtained results proved that the particle diameter and water contact angle obtained around 0.54 ± 0.12 to 3.2 ± 0.18 µm and 43.93 ± 10.8° to 133.1 ± 12.4° respectively. The cell proliferation and cell-nanoparticle interactions analyzed using (3-(4,5-dimethyl thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) MTS assay (Promega, Madison, WI, USA), FESEM for cell morphology and 5-Chloromethylfluorescein diacetate (CMFDA) dye for imaging live cells. Osteogenic differentiation was proved by expression of osteocalcin, alkaline phosphatase activity (ALP) and mineralization was confirmed by using alizarin red (ARS). The quantity of cells was considerably increased in PCL/SF/HA/MH nanoparticles when compare to all other biocomposite nanoparticles and the cell interaction was observed more on PCL/SF/HA/MH nanoparticles. The electrosprayed PCL/SF/HA/MH biocomposite nanoparticle significantly initiated increased cell proliferation, osteogenic differentiation and mineralization, which provide huge potential for bone tissue engineering.

Breathable Medicine: Pulmonary Mode of Drug Delivery.

Pharmaceutically active compounds require different modes of drug delivery systems to accomplish therapeutic activity without loss of its activity and lead to exhibit no adverse effects. Originating from ancient days, pulmonary mode of drug delivery is gaining much importance compared to other modes of drug delivery systems with respect to specific diseases. Pulmonary drug delivery is a non-invasive route for local and systemic therapies together with more patient convenience, compliance and is a needleless system. In this review, we addressed the vaccine delivery via non- or minimally invasive routes. Polymeric nanoparticles are preferred for use in the pulmonary delivery devices owing to a prolonged retention in lungs. Small site for absorption, mucociliary clearance, short residence time and low bioavailability are some of the limitations in pulmonary drug delivery have been resolved by generating micro- and nano-sized aerosol particles. We have classified the breathable medicine on the basis of available devices for inhalation and also prominent diseases treated through pulmonary mode of drug delivery. Owing to increasing toxicity of pharmacological drugs, the use of natural medicines has been rapidly gaining importance recently. The review article describes breathability of medicines or the pulmonary mode of drug delivery system and their drug release profile, absorption, distribution and efficacy to cure asthma and diabetes.

Biomimetic hybrid nanofibrous substrates for mesenchymal stem cells differentiation into osteogenic cells.

Mimicking native extracellular matrix with electrospun porous bio-composite nanofibrous scaffolds has huge potential in bone tissue regeneration. The aim of this study is to fabricate porous poly(l-lactic acid)-co-poly-(ε-caprolactone)/silk fibroin/ascorbic acid/tetracycline hydrochloride (PLACL/SF/AA/TC) and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). Fabricated nanofibrous scaffolds were characterized for fiber morphology, hydrophilicity, porosity, mechanical test and chemical properties by FT-IR and EDX analysis. The results showed that the fiber diameter and pore size of scaffolds observed around 228±62-320±22nm and 1.5-6.9µm respectively. Resulting nanofibrous scaffolds are highly porous (87-94%) with ultimate tensile strength observed in the range of 1.51-4.86MPa and also showed better hydrophilic properties after addition of AA, TC and n-HA. Human mesenchymal stem cells (MSCs) cultured on these bio-composite nanofibrous scaffolds and stimulated to osteogenic differentiation in the presence of AA/TC/n-HA for BTE. The cell proliferation and biomaterial interactions were studied using MTS assay, SEM and CMFDA dye exclusion methods. Osteogenic differentiation of MSCs was proven by using alkaline phosphatase activity, mineralization and double immunofluorescence staining of both CD90 and osteocalcin. The observed results suggested that the fabricated PLACL/SF/AA/TC/n-HA biocomposite hybrid nanofibrous scaffolds have good potential for the differentiation of MSCs into osteogenesis for bone tissue engineering.

Biocomposite nanofibrous strategies for the controlled release of biomolecules for skin tissue regeneration.

Nanotechnology and tissue engineering have enabled engineering of nanostructured strategies to meet the current challenges in skin tissue regeneration. Electrospinning technology creates porous nanofibrous scaffolds to mimic extracellular matrix of the native tissues. The present study was performed to gain some insights into the applications of poly(l-lactic acid)-co-poly-(ε-caprolactone) (PLACL)/silk fibroin (SF)/vitamin E (VE)/curcumin (Cur) nanofibrous scaffolds and to assess their potential for being used as substrates for the culture of human dermal fibroblasts for skin tissue engineering. PLACL/SF/VE/Cur nanofibrous scaffolds were fabricated by electrospinning and characterized by fiber morphology, membrane porosity, wettability, mechanical strength, and chemical properties by Fourier transform infrared (FTIR) analysis. Human dermal fibroblasts were cultured on these scaffolds, and the cell scaffold interactions were analyzed by cell proliferation, cell morphology, secretion of collagen, expression of F-actin, and 5-chloromethylfluorescein diacetate (CMFDA) dye. The electrospun nanofiber diameter was obtained between 198±4 nm and 332±13 nm for PLACL, PLACL/SF, PLACL/SF/VE, and PLACL/SF/VE/Cur nanofibrous scaffolds. FTIR analysis showed the presence of the amide groups I, II, and III, and a porosity of up to 92% obtained on these nanofibrous scaffolds. The results showed that the fibroblast proliferation, cell morphology, F-actin, CMFDA dye expression, and secretion of collagen were significantly increased in PLACL/SF/VE/Cur when compared to PLACL nanofibrous scaffolds. The accessibility of human dermal fibroblasts cultured on PLACL/SF/VE/Cur nanofibrous scaffolds proved to be a potential scaffold for skin tissue regeneration.

Effect of feeding a high fat diet on hydrogen sulfide (H2S) metabolism in the mouse.

Hydrogen sulfide (H2S) has complex effects in inflammation with both pro- and anti-inflammatory actions of this gas reported. Recent work suggests that a deficiency of H2S occurs in, and may contribute to, the chronic inflammation which underpins ongoing atherosclerotic disease. However, whether a high fat diet, predisposing to atherosclerosis, affects H2S metabolism is not known. In this study we assessed H2S metabolism in different tissues of mice fed a high fat diet for up to 16 weeks. Ex vivo biosynthesis of H2S was reduced in liver, kidney and lung of high fat fed mice. Western blotting revealed deficiency of cystathionine γ lyase (CSE) in liver and lung with increased expression of cystathionine ß synthetase (CBS) in liver and kidney. Expression of 3-mercaptopyruvate sulfurtransferase (3-MST) was reduced in liver but not other tissues. Aortic endothelial cell CSE was also reduced in high fat fed animals as determined immunohistochemically. Plasma H2S concentration was not changed in these animals. No evidence of lipid deposition was apparent in aortae from high fat fed animals and plasma serum amyloid A (SAA) and C-reactive protein (CRP) were also unchanged suggesting lack of frank atherosclerotic disease. Plasma IL-6, IL12p40 and G-CSF levels were increased by high fat feeding whilst other cytokines including IL-1α, IL-1b and TNF-α were not altered. These results suggest that deficiency of tissue CSE and H2S occurs in mice fed a high fat diet and that this change takes place prior to development of frank atherosclerotic disease.

Hydrogen sulfide is an endogenous regulator of aging in Caenorhabditis elegans.

AIMS: To investigate the role of endogenous hydrogen sulfide (H2S) in the control of aging and healthspan of Caenorhabditis elegans. RESULTS: We show that the model organism, C. elegans, synthesizes H2S. Three H2S-synthesizing enzymes are present in C. elegans, namely cystathionine γ lyase (CSE), cystathionine ß synthetase, and 3-mercaptopyruvate transferase (MPST or 3-MST). Genetic deficiency of mpst-1 (3-MST orthologue 1), but not cth-2 (CSE orthologue), reduced the lifespan of C. elegans. This effect was reversed by a pharmacological H2S donor (GYY4137). GYY4137 also reduced detrimental age-dependent changes in a range of physiological indices, including pharyngeal contraction and defecation. Treatment of C. elegans with GYY4137 increased the expression of several age-related, stress response, and antioxidant genes, whereas MitoSOX Red fluorescence, indicative of reactive oxygen species generation, was increased in mpst-1 knockouts and decreased by GYY4137 treatment. GYY4137 additionally increased the lifespan in short-lived mev-1 mutants with elevated oxidative stress and protected wild-type C. elegans against paraquat poisoning. The lifespan-prolonging and health-promoting effects of H2S in C. elegans are likely due to the antioxidant action of this highly cell-permeable gas. INNOVATION: The possibility that novel pharmacological agents based on the principle of H2S donation may be able to retard the onset of age-related disease by slowing the aging process warrants further study. CONCLUSION: Our results show that H2S is an endogenous regulator of oxidative damage, metabolism, and aging in C. elegans and provide new insight into the mechanisms, which control aging in this model organism.

Differential gene expression profiles during embryonic heart development in diabetic mice pregnancy.

Congenital heart defects (CHD) are one of the most common defects in offspring of diabetic mothers. There is a clear association between maternal diabetes and CHD; however the underlying molecular mechanism remains unknown. We hypothesized that maternal diabetes affects with the expression of early developmental genes that regulate the essential developmental processes of the heart, thereby resulting in the pathogenesis of CHD. We analyzed genome-wide expression profiling in the developing heart of embryos from diabetic and control mice by using the oligonucleotide microarray. Microarray analysis revealed that a total of 878 genes exhibited more than 1.5 fold changes in expression level in the hearts of experimental embryos in either E13.5 or E15.5 compared with their respective controls. Expression pattern of genes that is differentially expressed in the developing heart was further examined by the real-time reverse transcriptase-polymerase chain reaction. Several genes involved in a number of molecular signaling pathways such as apoptosis, proliferation, migration and differentiation in the developing heart were differentially expressed in embryos of diabetic pregnancy. It is concluded that altered expression of several genes involved in heart development may contribute to CHD in offspring of diabetic mothers.

Zinc supplementation prevents cardiomyocyte apoptosis and congenital heart defects in embryos of diabetic mice.

Oxidative stress induced by maternal diabetes plays an important role in the development of cardiac malformations. Zinc (Zn) supplementation of animals and humans has been shown to ameliorate oxidative stress induced by diabetic cardiomyopathy. However, the role of Zn in the prevention of oxidative stress induced by diabetic cardiac embryopathy remains unknown. We analyzed the preventive role of Zn in diabetic cardiac embryopathy by both in vivo and in vitro studies. In vivo study revealed a significant decrease in lipid peroxidation, superoxide ions, and oxidized glutathione and an increase in reduced glutathione, nitric oxide, and superoxide dismutase in the developing heart at embryonic days (E) 13.5 and 15.5 in the Zn-supplemented diabetic group when compared to the diabetic group. In addition, significantly down-regulated protein and mRNA expression of metallothionein (MT) in the developing heart of embryos from diabetic group was rescued by Zn supplement. Further, the nuclear microscopy results showed that trace elements such as phosphorus, calcium, and Zn levels were significantly increased (P<0.001), whereas the iron level was significantly decreased (P<0.05) in the developing heart of embryos from the Zn-supplemented diabetic group. In vitro study showed a significant increase in cellular apoptosis and the generation of reactive oxygen species (ROS) in H9c2 (rat embryonic cardiomyoblast) cells exposed to high glucose concentrations. Supplementation with Zn significantly decreased apoptosis and reduced the levels of ROS. In summary, oxidative stress induced by maternal diabetes could play a role in the development and progression of cardiac embryopathy, and Zn supplementation could be a potential therapy for diabetic cardiac embryopathy.

Effect of blast exposure on the brain structure and cognition in Macaca fascicularis.

Blast injury to the brain is one of the major causes of death and can also significantly affect cognition and physical and psychological skills in survivors of blast. The complex mechanisms via which blast injury causes impairment of cognition and other symptoms are poorly understood. In this study, we investigated the effects of varying degrees of primary blast overpressure (BOP; 80 and 200 kPa) on the pathophysiological and magnetic resonance imaging (MRI) changes and neurocognitive performance as assessed by the monkey Cambridge Neuropsychological Test Automated Battery (mCANTAB) in non-human primates (NHP). The study aimed to examine the effects of neurobehavioral and histopathological changes in NHP. MRI and histopathology revealed ultrastructural changes in the brain, notably in the Purkinje neurons in the cerebellum and pyramidal neurons in the hippocampus, which were most vulnerable to the blast. The results correlated well with the behavioral changes and changes in motor coordination and working memory of the affected monkeys. In addition, there was white matter damage affecting myelinated axons, astrocytic hypertrophy, and increased aquaporin-4 (AQP-4) expression in astrocytes, suggesting cerebral edema. Increased apoptosis appeared to involve astrocytes and oligodendrocytes in the animals following blast exposure. The small sample size could have contributed to the non-significant outcome in cognitive performance post-blast and limited quantitative analyses. Nevertheless, the study has provided initial descriptive changes for establishing a primary BOP threshold for brain injury to serve as a useful platform for future investigations that aim to estimate brain injury potential and set safe limits of exposure.

Deciphering the key molecular and cellular events in neutrophil transmigration during acute inflammation.

Recruitment of leukocytes circulating in our blood to the sites of infection or tissue damage is the key phenomenon in the acute inflammatory response(s). Among the leukocytes, neutrophils are primarily recruited into the areas of acute inflammation. When neutrophils interact with activated endothelium of the blood vessels, they become migratory and cross the endothelial layer of the blood vessel wall in a process called as leukocyte extravasation. Identifying and understanding the gene regulation of this extravasation phenomenon is one of the key objective of biomedical research aimed at ameliorating or alleviating the symptoms of various diseases, such as rheumatoid arthritis, asthma, anaphylaxis, atherosclerosis, ulcerative colitis etc., that are exacerbated by inappropriate inflammatory stimuli. Here, we decipher and discuss the key genes implicated in the leukocyte transmigration using the acute inflammation model called as the Dextran Sulphate Sodium (DSS) induced Colitis in mice as a classic paradigm.

Function of sirtuins in biological tissues.

Sirtuins are protein deacetylases, which are dependent on nicotine adenine dinucleotide. They are phylogenetically conserved from bacteria to humans. Seven sirtuin proteins localized in a wide variety of subcellular locations have been identified in the human genome. The most important known function of sirtuins is their regulation of transcriptional repression, mediated through binding of a complex containing sirtuins and other proteins. Studies have shown that sirtuins have pathophysiological relevance to neurodegeneration, muscle differentiation, inflammation, obesity, and cancer. In addition, sirtuin activity extends the lifespan of several organisms. In this review, we discuss the mode(s) of action of sirtuins, and their biological role(s) in health and disease.

Y-Box-binding protein-1 is a promising predictive marker of radioresistance and chemoradioresistance in nasopharyngeal cancer.

The Y-Box-binding protein-1, a member of the cold-shock domain DNA- and RNA-binding protein superfamily, is known to mediate chemoresistance. The aim of this study was to determine the expression of Y-Box-binding protein-1 in nasopharyngeal cancer in vitro and in tumor tissue samples as well as analyze the clinicopathological significance of Y-Box-binding protein-1 expression in nasopharyngeal cancer, in particular as a predictor of outcome after treatment. The Y-Box-binding protein-1 expression profile was evaluated at the mRNA and protein levels in poorly differentiated CNE-2 nasopharyngeal cancer cells by real-time RT-PCR, western blot analysis and immunohistochemistry. Y-Box-binding protein-1 expression in 143 nasopharyngeal cancer specimens was examined by immunohistochemistry and correlated with clinicopathologic parameters. Y-Box-binding protein-1 mRNA and protein were found to be expressed in CNE-2 nasopharyngeal cancer cells in vitro. Of 143 patient tissue sections, 137 (96%) were stained positive for the Y-Box-binding protein-1 protein. Y-Box-binding protein-1 immunostaining was observed to be predominantly cytoplasmic. A higher recurrence of nasopharyngeal cancer was found in patients whose tissues had increased Y-Box-binding protein-1 expression (P<0.001). The Cox proportionate hazard regression model also established that high Y-Box-binding protein-1 immunoreactivity was significantly correlated with increased risk (2.13 times) of recurrence as compared to low Y-Box-binding protein-1 immunoreactivity (P=0.01). Within groups of patients treated by radiotherapy or chemoradiotherapy, recurrent cases had significantly higher Y-Box-binding protein-1 expression than nonrecurrent cases (P<0.001 and P=0.0035, respectively). These data suggest that Y-Box-binding protein-1 expression has clinicopathological significance with potential as a predictive marker of recurrence in nasopharyngeal cancer patients who undergo radiotherapy or chemoradiotherapy.

Cardiac malformations are associated with altered expression of vascular endothelial growth factor and endothelial nitric oxide synthase genes in embryos of diabetic mice.

The aim of this study was to investigate the role of nitric oxide (NO), and the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) genes in developing hearts at embryonic day 13.5 of embryos from diabetic mice. The protein and mRNA expression levels of eNOS and VEGF were significantly altered in the developing hearts of embryos from diabetic mice. The NO level was significantly decreased, whereas the VEGF concentration was significantly increased in the developing hearts of the embryos from diabetic mice. In vitro study showed a significant reduction in eNOS expression and cell proliferation in cardiac myoblast cells exposed to high glucose concentrations. Further, high glucose induced apoptosis in myoblast cells. Ultrastructural changes characteristics of apoptosis, including cell blebbing, aggregation of ribosomes and vacuoles in the cytoplasm were also evident in myoblast cells exposed to high glucose. It is suggested that hyperglycemia alters the expression of eNOS and VEGF genes that are involved in the regulation of cell growth and vasculogenesis, thereby contributing to the cardiac malformations seen in embryos from diabetic mice.

Oncomirs: the potential role of non-coding microRNAs in understanding cancer.

MicroRNAs (miRNAs) are members of a family of non-coding RNAs of 8-24 nucleotide RNA molecules that regulate target mRNAs. The first miRNAs, lin-4 and let-7, were first discovered in the year 1993 by Ambros, Ruvkun, and co-workers while studying development in Caenorhabditis elegans. miRNAs can play vital functions form C. elegans to higher vertebrates by typical Watson-Crick base pairing to specific mRNAs to regulate the expression of a specific gene. It has been well established that multicellular eukaryotes utilize miRNAs to regulate many biological processes such as embryonic development, proliferation, differentiation, and cell death. Recent studies have shown that miRNAs may provide new insight in cancer research. A recent study demonstrated that more than 50% of miRNA genes are located in fragile sites and cancer-associated genomic regions, suggesting that miRNAs may play a more important role in the pathogenesis of human cancers. Exploiting the emerging knowledge of miRNAs for the development of new human therapeutic applications will be important. Recent studies suggest that miRNA expression profiling can be correlated with disease pathogenesis and prognosis, and may ultimately be useful in the management of human cancer. In this review, we focus on how miRNAs regulate tumorigenesis by acting as oncogenes and anti-oncogenes in higher eukaryotes.

RNAi and RNAa--the yin and yang of RNAome.

RNA interference (RNAi) is a powerful technology with huge applications for functional genomics, target identification in drug discovery and elucidation of molecular signaling pathways. Current RNAi studies have demonstrated the clinical potential of small interfering RNAs (siRNAs) in metabolic diseases, cancer, AIDS, malaria, neurodegenerative disorders, dental diseases and other illnesses. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs could effectively activate a particular gene in a sequence specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed as RNA activation (RNAa). The paradoxical concept of Yin and Yang, which describe two primal opposing but complementary principles, can potentially be applied to elucidate the complex phenomenon of RNAa/RNAi in the RNAome. This warrants a proper understanding of the RNAi/RNAa molecular pathways in living organisms before any of the small dsRNAs can potentially be exploited for therapeutics in human beings.

Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development.

BACKGROUND: Congenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice. METHODS AND RESULTS: We analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos. CONCLUSION: It is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.