Preparation of Chitosan Cryostructurates with Controlled Porous Morphology and Their Use as 3D-Scaffolds for the Cultivation of Animal Cells

The influence of the conditions of the formation of genipin cross-linked chitosan cryostructurates on the porous morphology and physicochemical properties of these cryostructurates and on the possibility of their use as biopolymer 3D scaffolds for tissue engineering was studied. The chitosan cryostructurates were obtained by freeze-drying a chitosan acetate solution, treating the resulting sponge with an alcohol solution of ammonia to transform the polyaminosaccharide from a salt into a chitosan-base, and then cross-linking the polymer with genipin (the molar ratios of genipin to the number of chitosan amino groups were 0.05, 0.033, and 0.02, respectively). The pore sizes, water-holding capacity, and in vitro biodegradation rate of the cryostructurates were shown to depend on the aforementioned ratio. The properties of the prepared chitosan cryostructurates, the hydrogels formed by chitosan cross-linking with genipin at positive temperatures, and the films cast from genipin-containing chitosan solutions after solvent evaporation were studied and compared. The biocompatibility of the obtained macroporous sponge materials was demonstrated using L929 mouse fibroblasts. Confocal laser microscopy showed that the cells in all of the 3D scaffolds obtained were evenly distributed; they grew and proliferated when cultured in vitro for seven days.     

Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis

Antibodies to citrullinated proteins (anti-cyclic-citrullinated peptide [anti-CCP] antibodies) are highly specific for rheumatoid arthritis (RA) and precede the onset of disease symptoms, indicating a pathogenetic role for these antibodies in RA. We recently showed that distinct genetic risk factors are associated with either anti-CCP-positive disease or anti-CCP-negative disease. These data are important as they indicate that distinct pathogenic mechanisms are underlying anti-CCP-positive disease or anti-CCP-negative disease. Likewise, these observations raise the question of whether anti-CCP-positive RA and anti-CCP-negative RA are clinically different disease entities. We therefore investigated whether RA patients with anti-CCP antibodies have a different clinical presentation and disease course compared with patients without these autoantibodies. In a cohort of 454 incident patients with RA, 228 patients were anti-CCP-positive and 226 patients were anti-CCP-negative. The early symptoms, tender and swollen joint count, and C-reactive protein level at inclusion, as well as the swollen joint count and radiological destruction during 4 years of follow-up, were compared for the two groups. There were no differences in morning stiffness, type, location and distribution of early symptoms, patients' rated disease activity and C-reactive protein at inclusion between RA patients with and without anti-CCP antibodies. The mean tender and swollen joint count for the different joints at inclusion was similar. At follow-up, patients with anti-CCP antibodies had more swollen joints and more severe radiological destruction. Nevertheless, the distribution of affected joints, for swelling, bone erosions and joint space narrowing, was similar. In conclusion, the phenotype of RA patients with or without anti-CCP antibodies is similar with respect to clinical presentation but differs with respect to disease course.     

Immobilization of Champagne Yeasts by Inclusion into Cryogels of Polyvinyl Alcohol: Means of Preventing Cell Release from the Carrier Matrix

Wine champagnizing, a process involving the use of champagne yeasts immobilized by inclusion into cryogels of polyvinyl alcohol, has been studied. Treatment of yeast cells with the autoregulatory factor d ₁ was proposed as a means of preventing the cell release from the carrier matrix. Such a treatment inhibited growth and proliferation processes in yeast cells, without affecting the activity of fermentation; the resulting champagne had the same organoleptic and chemical characteristics as its counterparts obtained using conventional techniques.     

Beneficial effect of dinitrosyl iron complexes with thiol ligands on the rat penile cavernous bodies

Dinitrosyl iron complexes (DNIC) with thiol ligands were found to beneficially affect the state of the penile cavernous tissue upon its experimental denervation in rats. Histological and histochemical analysis showed that intracavernous administration of DNIC (twice weekly over six months) almost completely abolished the proliferation of endothelial cells typical of denervated cavernous tissue. On the other hand, this treatment sustained the mitotic activity of smooth myocytes and prevented the appearance of collagenase, a marker of their fibrotic transformation. The DNIC treatment had a pronounced effect on penile erection in neurotomized as well as in intact animals. Introduction of low-molecular DNIC into cavernous tissue was found to cause formation of protein-bound complexes observed by EPR and probably acting as depots of nitric oxide, ensuring steady erection.     

A comparative study on efficiency of adult fibroblasts and amniotic fluid-derived stem cells as donor cells for production of hand-made cloned buffalo (Bubalus bubalis) embryos

The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical “S” shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10–15 and 8–12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.     

Replicated high-density genetic maps of two great tit populations reveal fine-scale genomic departures from sex-equal recombination rates

Linking variation in quantitative traits to variation in the genome is an important, butchallenging task in the study of life-history evolution. Linkage maps provide a valuabletool for the unravelling of such trait−gene associations. Moreover, they giveinsight into recombination landscapes and between-species karyotype evolution. Here weused genotype data, generated from a 10k single-nucleotide polymorphism (SNP) chip, ofover 2000 individuals to produce high-density linkage maps of the great tit (Parusmajor), a passerine bird that serves as a model species for ecological andevolutionary questions. We created independent maps from two distinct populations: acaptive F2-cross from The Netherlands (NL) and a wild population from the United Kingdom(UK). The two maps contained 6554 SNPs in 32 linkage groups, spanning 2010 cM and1917 cM for the NL and UK populations, respectively, and were similar in size andmarker order. Subtle levels of heterochiasmy within and between chromosomes wereremarkably consistent between the populations, suggesting that the local departures fromsex-equal recombination rates have evolved. This key and surprising result would have beenimpossible to detect if only one population was mapped. A comparison with zebra finchTaeniopygia guttata, chicken Gallus gallus and the green anole lizardAnolis carolinensis genomes provided further insight into the evolution ofavian karyotypes.     

A histological and immunohistochemical study of the effects of N-acetyl cysteine on retinopathy of prematurity by modifying insulin-like growth factor-1

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that occurs in premature infants and may lead to permanent visual impairment. We investigated both the possible protective role of N-acetyl cysteine (NAC) for preventing ROP and the role of IGF-1 in the disorder. Forty-five newborn rats were divided into three groups. Group 1 was raised in room air as controls. Group 2 was exposed to 60% oxygen for 14 days after birth, then transferred to room air. Group 3 was exposed to the same conditions as group 2, but received intraperitoneal injections of NAC on postnatal days 7–17. After 35 days, both eyes of all rats were processed for histology. Some sections were stained with hematoxylin and eosin to assess structural changes and other sections were immunostained to determine the location of IGF-1. Frozen sections also were prepared and stained for adenosine triphosphatase to detect retinal blood vessels. Compared to the controls, more blood vessels, many of which were abnormal, and increased IGF-1 expression were observed in group 2. In group 3, abnormal blood vessels and IGF-1 expression were less evident. NAC appeared to be an effective vascular-protective agent for ROP by decreasing IGF-1 expression.     

Gap Junction Intercellular Communication Mediated by Connexin43 in Astrocytes Is Essential for Their Resistance to Oxidative Stress

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H₂O₂) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H₂O₂ transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H₂O₂-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H₂O₂ insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H₂O₂ treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.