Biological role of MicroRNA-103 based on expression profile and target genes analysis in pigs

MicroRNAs (miRNAs) are endogenously expressed RNAs consisting of 20–24 nucleotides. These molecules are thought to repress protein translation by binding to target mRNAs. However, biological functions have not been assigned to most of the 175 porcine miRNAs registered in miRBase (release 15.0). In an effort to uncover miR-103 important in pigs, we examined the integrative tissue expression profile and gene ontology (GO) term enrichment of predicted target genes to determine the global biological functions of miR-103. Our results demonstrated that miR-103 is involved in various biological processes including brain development, lipid metabolism, adipocyte differentiation, hematopoiesis, and immunity. Moreover, we also experimentally verified effects of miR-103 in porcine preadipocytes. miR-103 levels increased in differentiating adipocytes, and inhibition of miR-103 effectively inhibited preadipocyte differentiation. In addition, mRNA levels of the putative miR-103 target RAI14 were higher in miR-103 inhibitor-treated adipocytes. These results demonstrate that miR-103 is involved in porcine preadipocyte differentiation and may act through the putative target gene RAI14. In a word, our data provide new insights into the global biological role of miR-103.     

Multiple strategies to improve sensitivity,speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection

Background  

In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).     

Premalignant Variations in Extracellular Matrix Composition in Chemically Induced Hepatocellular Carcinoma in Rats

Chemical composition of extracellular matrix (ECM) plays a pivotal role in cellular and tissue development, regeneration, and differentiation. It also plays a key role in pathogenesis of hepatocellular carcinoma (HCC). This study explored premalignant changes in the liver tissue content of collagen (as hydroxyproline, HP), total glycosaminoglycans (TGAGs), free glucosamine (FGA), total sialic acid (TSA), lysosomal membrane integrity variations (calculated as total and free cathepsin D activities), and liver histology. Serum alfa-fetoprotein (AFP) level was used as an early marker for HCC in two groups of Wistar rats. One group of rats served as control and was provided normal saline orally. The other group was provided trichloroacetic acid (TCA) as 0.5 g/kg/day for five consecutive days by oral gavage. Animals were killed before tumor development. The treatment revealed dysplastic changes in addition to microsteatosis (fatty changes). Both sinusoids and the portal vein among dysplastic cells were dilated and congested. These dysplastic foci are believed to be premalignant and may be precancerous lesions. The following things were observed: a highly significant increase in serum AFP (as a key marker for HCC), a significant decrease in HP and TSA, a significant increase in FGA, nonsignificant decrease in TGAGs, significant up-regulation of free cathepsin D, nonsignificant decrease in total cathepsin D activities, and destabilization of lysosomal membrane integrity. Down-regulation of HP, TSA, and TGAGs seems to be a prerequisite for cancer development. This might be stimulated by up-regulation of free cathepsin D activity. Perhaps tissue fibrosis is not a condition for developing HCC because collagen was significantly depressed. Up-regulated FGA could be assumed to be a defense mechanism against TCA-induced proteolysis of membrane proteins because it is frequently reported to be of value in cancer chemotherapy. Studied ECM perturbations can be assumed as preliminary changes during chemical hepatocarcinogenesis at the tissue level. Prospective studies on blood levels of cathepsins, TGAGs, and individual ECM variables such as TSA, FGA, and Hp in patients at risk for HCC, performed in parallel with assessments of AFP, may provide a cost-effective way to find new links between tissue changes and circulation that would permit early prediction of disease. It may also provide a way to monitor HCC and compensate for the missed peak AFP values.     

Possible role of ionic gradients in the apical growth of <Emphasis Type="Italic">Neurospora crassa</Emphasis>

The effects of the Ca²⁺/H⁺ exchanger A23187 and the K⁺/H⁺ exchanger nigericin on the growth of Neurospora crassa were analyzed. Both ionophores had the same effects on the fungus. They both inhibited growth in liquid media, apical extension being more affected than protein synthesis. A sudden challenge to either ionophore on solid media rapidly stopped hyphal extension. Additionally, both ionophores induced profuse mycelium branching and upward hyphal growth. Hyphae growing on nigericin-containing media also burst at the apex. Both ionophores caused a rapid inhibition in the apically-occurring synthesis of structural wall polysaccharides, but they did not affect mitochondrial energy conservation. With the use of DiBAC, a membrane-potential sensitive fluorophore, it was excluded that their effects were due to depletion of the plasma membrane potential. Considering that both ionophores exchange H⁺ for different metallic ions, we concluded that their effect was due to dissipation of a proton gradient, which is directly or indirectly involved in the apical growth of the fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA indels in coding regions reveal selective constraints on protein evolution in the human lineage

Background  

Insertions and deletions of DNA segments (indels) are together with substitutions the major mutational processes that generate genetic variation. Here we focus on recent DNA insertions and deletions in protein coding regions of the human genome to investigate selective constraints on indels in protein evolution.     

Preparation of cross-linked carboxymethyl chitosan for repairing sciatic nerve injury in rats

A successful nerve regeneration process was achieved with nerve repair tubes made up of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) cross-linked carboxymethyl chitosan (CM-chitosan) with improved biodegradability. Chitosan has a very slow degradation rate, while the EDC cross-linked CM-chitosan tubes degraded to 30% of original weight during 8 weeks of incubation in lysozyme solution. In vitro cell culture indicated that the CM-chitosan films presented no cytotoxicity to Schwann cells. From in vivo studies using a 10 mm rat sciatic nerve defect model investigated by histomorphometry analysis, the average diameter of the fibers and the average thickness of myelin sheath in the CM-chitosan tubes were 3.7 ± 0.33 and 0.33 ± 0.04 μm, respectively, which demonstrated equivalence to nerve autografts (the current “gold” standard); furthermore, the average fiber density in the CM-chitosan tubes was 20.5 × 10³/mm², which was similar to that of autografts (21 × 10³/mm²) and significantly higher than that of common chitosan tubes (15.3 × 10³/mm²).     

Anti-Osteoporotic Drug Release from Ordered Mesoporous Bioceramics: Experiments and Modeling

The release of a potent bone-resorption inhibitor such as zoledronate from a versatile drug delivery system such as SBA 15 has been modeled. The initial and boundary conditions have been defined, together with the system parameters, including the determination of equilibrium and transport parameters. Additionally, the experimental model of the same system has been observed to validate the prediction here developed. This approach represents a powerful tool for the designing of mesoporous implantable drug delivery systems because their release kinetics can be predicted in advance, and this leads to a considerable time and resources saving.     

Oxidative stress and neurodegeneration: the involvement of iron

Many evidences indicate that oxidative stress plays a significant role in a variety of human disease states, including neurodegenerative diseases. Iron is an essential metal for almost all living organisms due to its involvement in a large number of iron-containing proteins and enzymes, though it could be also toxic. Actually, free iron excess generates oxidative stress, particularly in brain, where anti-oxidative defences are relatively low. Its accumulation in specific regions is associated with pathogenesis in a variety of neurodegenerative diseases (i.e., Parkinson’s disease, Alzheimer’s disease, Huntington’s chorea, Amyotrophic Lateral Sclerosis and Neurodegeneration with Brain Iron Accumulation). Anyway, the extent of toxicity is dictated, in part, by the localization of the iron complex within the cell (cytosolic, lysosomal and mitochondrial), its biochemical form, i.e., ferritin or hemosiderin, as well as the ability of the cell to prevent the generation and propagation of free radical by the wide range of antioxidants and cytoprotective enzymes in the cell. Particularly, ferrous iron can act as a catalyst in the Fenton reaction that potentiates oxygen toxicity by generating a wide range of free radical species, including hydroxyl radicals (·OH). The observation that patients with neurodegenerative diseases show a dramatic increase in their brain iron content, correlated with the production of reactive oxigen species in these areas of the brain, conceivably suggests that disturbances in brain iron homeostasis may contribute to the pathogenesis of these disorders. The aim of this review is to describe the chemical features of iron in human beings and iron induced toxicity in neurodegenerative diseases. Furthermore, the attention is focused on metal chelating drugs therapeutic strategies.     

MPEG-PCL Copolymeric Micelles for Encapsulation of Azithromycin

Macrolide antibiotics are lipophilic drugs with some limitations including low solubility, limited cellular permeation, patients discomfort, etc. With amphiphilic methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) (MPEG-PCL) copolymer and azithromycin (AZT) as drug carrier and model drug, AZT-loaded micelles were prepared via thin-membrane hydration method in order to overcome these limitations. Encapsulation efficiency of AZT-loaded micelles was 94.40% with good storage stability for 28 days, and AZT’s water solubility was enhanced to 944 μg/mL. Fourier transform infrared spectrum and x-ray diffraction analysis indicated that AZT was enveloped into the micelles in amorphous form due to its interaction with the copolymer. AZT’s in vitro release from the AZT-loaded micelles demonstrated a slow and continuous behavior when compared with raw AZT. The release dynamics was accorded with Weibull equation, meaning that release amount of AZT lowered with time and was proportional to remaining amount of drug in the AZT-loaded micelles. Korsmeyer-Peppas fitting result suggested that drug release process was a classical Fickian diffusion-controlled manner. With Staphylococcus aureus as bacterial strain, antibacterial activity of the AZT-loaded micelles displayed was comparable with raw AZT. In conclusion, MPEG-PCL should be a promising carrier for macrolide antibiotic delivery in treatment of bacterial infections.     

Selenite and ebselen supplementation attenuates <Emphasis Type="SmallCaps">d</Emphasis>-galactose-induced oxidative stress and increases expression of SELR and SEP15 in rat lens

Selenite and ebselen supplementation has been shown to possess anti-cataract potential in some experimental animal models of cataract, however, the underlying mechanisms remain unclear. The present study was designed to evaluate the anti-cataract effects and the underlying mechanisms of selenite and ebselen supplementation on galactose induced cataract in rats, a common animal model of sugar cataract. Transmission electron microscopy images of lens fiber cells (LFC) and lens epithelial cells (LEC) were observed in d-galactose-induced experimental cataractous rats treated with or without selenite and ebselen, also redox homeostasis and expression of proteins such as selenoprotein R (SELR), 15kD selenoprotein (SEP15), superoxide dismutase 1 (SOD1), catalase (CAT), β-crystallin protein, aldose reductase (AR) and glucose-regulated protein 78 (GRP78) were estimated in the lenses. The results showed that d-galactose injection injured rat lens and resulted in cataract formation; however, selenite and ebselen supplementation markedly alleviated ultrastructural injury of LFC and LEC. Moreover, selenite and ebselen supplementation could mitigate the oxidative damage in rat lens and increase the protein expressions of SELR, SEP15, SOD1, CAT and β-crystallin, as well as decrease the protein expressions of AR and GRP78. Taken together, these findings for the first time reveal the anti-cataract potential of selenite and ebselen in galactosemic cataract, and provide important new insights into the anti-cataract mechanisms of selenite and ebselen in sugar cataract.