The association between the macrolide efflux gene mef(E) and the tet(M) gene was studied in two clinical strains of Streptococcus pneumoniae that belonged to serotypes 19F and 6A, respectively, and that were resistant to both tetracycline and erythromycin. The mef(E)-carrying element mega (macrolide efflux genetic assembly; 5,511 bp) was found to be inserted into a Tn916-like genetic element present in the chromosomes of the two pneumococcal strains. In both strains, mega was integrated at the same site, an open reading frame identical to orf6 of Tn916. The new composite element, Tn2009, was about 23.5 kb and, with the exception of the tet(M)-coding sequence, appeared to be identical in both strains. By sequencing of the junction fragments of Tn2009 at the site of insertion into the chromosome, it was possible to show that (i) the insertion site was identical in the two clinical strains and (ii) the integration of Tn2009 caused a 9.5 kb-deletion in the pneumococcal chromosome. It was not possible to detect the conjugal transfer of Tn2009 to a recipient pneumococcal strain; however, transfer of the whole element by transformation was shown to occur. It is possible to hypothesize that Tn2009 relies on transformation for its spread among clinical strains of S. pneumoniae.

This report unravels two novel mutations of the LH gene critical for synthesis and activity of the LH molecule. The insight gained from the study is that normal pubertal maturation in women can occur in a state of LH deficiency, whereas LH is essential for maturation of Leydig cells and thus steroidogenesis, puberty, and spermatogenesis in man. These mutations should be considered in girls and boys with selective deficiency of LH.

Scaffolds are three-dimensional porous structures that must have specific requirements to be applied in tissue engineering. Therefore, the study of factors affecting scaffold performance is of great importance. In this work, the optimal conditions for cross-linking preformed chitosan (CS) scaffolds by the tripolyphosphate polyanion (TPP) were investigated. The effect on scaffold physico-chemical properties of different concentrations of chitosan (1 and 2% w/v) and tripolyphosphate (1 and 2% w/v) as well as of cross-linking reaction times (2, 4, or 8 h) were studied. It was evidenced that a low CS concentration favored the formation of three-dimensional porous structures with a good pore interconnection while the use of more severe conditions in the cross-linking reaction (high TPP concentration and crosslinking reaction time) led to scaffolds with a suitable pore homogeneity, thermal stability, swelling behavior, and mechanical properties, but having a low pore interconnectivity. Preliminary biocompatibility tests showed a good osteoblasts’ viability when cultured on the scaffold obtained by CS 1%, TPP 1%, and an 8-h crosslinking time. These findings suggest how modulation of scaffold cross-linking conditions may permit to obtain chitosan scaffold with properly tuned morphological, mechanical and biological properties for application in the tissue regeneration field.

IntroductionNuclear factor-κB (NF-κB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor κB kinase (IKK)α and IKKβ are kinases involved in the activation of the NF-κB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-κB activation in human chondrocytes.MethodsThe human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)α after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor κB protein (IκB)α phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKα nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay.ResultsAfter TNFα stimulation, the mRNA expression level of some of the genes under NF-κB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKα kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKα nuclear re-localization.ConclusionsOur results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-κB signaling pathway by inhibiting IKKα activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKα kinase activity and IKKα nuclear re-localization, GlcN only acts on IKKα nuclear re-localization.

Harpagophytum procumbens (Burch.) DC. ex Meisn. is a traditional remedy for osteoarticular diseases, including osteoarthritis (OA), although the bioactive constituents and mechanisms involved are yet to be clarified. In the present study, an aqueous H. procumbens root extract (HPE; containing 1.2% harpagoside) was characterized for its effects on synoviocytes from OA patients and phytochemical composition in polyphenols, and volatile compounds were detected. HPE powder was dissolved in different solvents, including deionized water (HPEH2O), DMSO (HPEDMSO), 100% v/v ethanol (HPEEtOH100), and 50% v/v ethanol (HPEEtOH50). The highest polyphenol levels were found in HPEDMSO and HPEEtOH50, whereas different volatile compounds, mainly β-caryophyllene and eugenol, were detected in all the extracts except for HPEH2O. HPEH2O and HPEDMSO were able to enhance CB2 receptor expression and to downregulate PI-PLC β2 in synovial membranes; moreover, all the extracts inhibited FAAH activity. The present results highlight for the first time a multitarget modulation of the endocannabinoid system by HPE, likely ascribable to its hydrosoluble compounds, along with the presence of volatile compounds in H. procumbens root. Although hydrosoluble compounds seem to be mainly responsible for endocannabinoid modulation by HPE, a possible contribution of volatile compounds can be suggested, strengthening the hypothesis that the entire phytocomplex can contribute to the H. procumbens healing properties.

NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo.

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The aim of this study was to determine the effects of glucosamine on matrix metalloprotease (MMP) production, on mitogen-activated protein kinase (MAPK) phosphorylation, and on activator protein (AP)-1 transcription factor activation in human chondrocytes. The human immortalized cell line lbpva55 and healthy human chondrocytes (obtained from healthy donors) were subjected to challenge with 10 ng/ml IL-1β after pretreatment with 2.5 or 10 mmol/l glucosamine. MMP mRNA expression levels were evaluated using quantitative real-time PCR, and MMP protein production levels were evaluated in the culture supernatant using ELISA. MAPK phosphorylation was evaluated using Western blotting. AP-1 transcription factor activation was evaluated by measuring AP-1 DNA-binding activity. After IL-1β stimulation, levels of MMP-1, MMP-3 and MMP-13 production were markedly increased. Treatment with 2.5 and 10 mmol/l glucosamine reduced expression of these metalloproteases. MMP expression is regulated by transcription factors such as the AP-1 complex, which is activated by phosphorylated MAPKs. IL-1β stimulated phosphorylation of cjun amino-terminal kinase, p38 MAPK and extracellular signalregulated kinase-1/2. Glucosamine inhibited c-jun aminoterminal kinase and p38 phosphorylation, and consequently cjun binding activity. These findings demonstrate, for the first time, that glucosamine inhibits IL-1β-stimulated MMP production in human chondrocytes by affecting MAPK phosphorylation.