Arachidonic acid-induced IL-6 expression is mediated by PKC alpha activation in osteoblastic cells

There are several pieces of evidence supporting the important role that essential fatty acids (EFAs) and their metabolites play in regulating calcium and bone metabolism, and their relevance to the pathobiology of bone disease, with particular reference to modulating effects on cytokines. We found that arachidonic acid (AA) triggers a cell signal in osteoblasts and leads to the expression of IL-6. To explore the biochemical pathways involved in AA induction of cytokine gene expression, we evaluated the potential protein kinase C (PKC) dependent mechanism accounting for the AA effect on IL-6 gene expression. The osteoblast-like cell line MG-63 was pretreated with calphostin C, a PKC inhibitor, or phorbol 12-myristate 13-acetate (PMA) for an extended period, a condition which causes PKC downregulation, and subsequently with AA. After these treatments, IL-6 gene expression was no longer evident. We also showed that PKC and, in particular, PKC alpha, which are both recruited to the particulate fraction, undergo proteolysis and autophosphorylation; all of these steps are required for PKC activation and, subsequently, for AA-induced signaling. It is interesting that other unsaturated fatty acids, such as oleic acid (OA) or eicosapentaenoic acid (EPA), are unable to induce either PKC activation or IL-6 gene expression.     

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.     

Human platelet lysate‐derived extracellular vesicles enhance angiogenesis through miR‐126

ObjectivesExtracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL.MethodsWe tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL‐derived EVs.ResultsA subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly‐like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR‐126, the most abundant angiogenic miRNA in platelets. The transfer of miR‐126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system.ConclusionPL is a biological source of available EVs with angiogenic effects involving a miRNAs‐based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.

A high amount of small‐size extracellular vesicles (EVs) can be isolated from platelet lysate (PL)‐based preparations. When endothelial cells (HUVEC) are cultured in presence of EVs of platelet origin, they are able to significantly enhance the formation of angiogenic tubularly‐like structures in 3D endothelial bioprinted constructs (2A,B). PL‐derived EVs reflect a similar angiogenic microRNA profile (3A). Accordingly, EVs are mainly enriched with miR‐126, also known as angio‐miRNA, the most expressed miRNA by platelets in the blood. Hence, the silencing of the endogenous levels of miR‐126 in HUVEC and the retransferring of the same through PL‐derived EVs, restore angiogenesis in endothelial cells (3B). Images were created with the Biorender software.     

Immunogenicity of an Electron Beam Inactivated Rhodococcus equi Vaccine in Neonatal Foals

Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals.     

Studies on the photosensitizing properties of angelicin, an angular furocoumarin forming only monofunctional adducts with the pyrimidine bases of DNA.

The bioligical photosensitizing properties of furocoumarins are due to the formation of adducts with the pyrimidine bases of DNA under irradiation with long wavelength ultraviolet light. The greatest importance is attributed to the difunctional adducts, which form cross-linkings between the 2 strands of DNA. As angelicin, photoreacting with DNA, forms only monofunctional adducts, and therefore no cross-linkings, its photosensitizing properties have been studied in order to evaluate the ability of monofunctional adducts to produce biological effects. The results obtained studying the inhibition of DNA, RNA and protein synthesis in Ehrlich ascite tumor cells after irradiation in the presence of angelicin and psoralen (for a comparison), and the inhibition of the ability of identically treated cells to transmit the tumor showed a remarkable ability of monofunctional adducts to produce biological effects.     

Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8-methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4',8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 microM in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.     

The thioredoxin reductase-thioredoxin system is involved in the entry of tetanus and botulinum neurotoxins in the cytosol of nerve terminals

Tetanus and botulinum neurotoxins cause paralysis by cleaving SNARE proteins within the cytosol of nerve terminals. They are endocytosed inside acidic vesicles and the pH gradient across the membrane drives the translocation of their metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-thioredoxin reductase-thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins.     

Review: Epidemiology of Helicobacter pylori

This review summarizes the recent knowledge on the epidemiology of Helicobacter pylori and the potential modes of transmission. In addition to English language publications, the authors have included original full‐text publications from Russia and Latin America published in the original languages. High H pylori prevalence has been reported in Russia, Jordan, Iran, China, and Latin American countries as well as in Arctic populations in Canada. Indigenous inhabitants in the Arctic were found to be infected substantially more frequently than non‐indigenous inhabitants. In Amsterdam, the Netherlands, the ethnic minority groups were at a significantly higher risk of being H pylori seropositive compared to the Dutch population. For the first time, data on the prevalence from Armenia have been published indicating 41.5% H pylori prevalence. Convincing evidence on the decline of H pylori prevalence in Southeast Hungary and Taiwan was published. A study from Chile suggested high infection rates in newborns during the first month after birth. Two meta‐analyses covered the potential correlation between H pylori and periodontal diseases, therefore addressing the potential oro‐oral transmission rates. Periodontal disease was found to be more prevalent in H pylori‐infected subjects. Other studies addressed the potential role of drinking water and food products as well as socioeconomic factors in transmitting the infection. Several studies in Asia addressed annual reinfection rates of H pylori, ranging from 1.5% in China to 3.1% in Korea. Finally, a review was published on the current evidence and future perspective of analysing H pylori in ancient human remains by a metagenomic approach.     

DIDS-effect on Ser/Thr- and Tyr-phosphorylation of membrane proteins in human erythrocytes.

Band 3, the major transmembrane multifunctional protein of human erythrocytes, has been found to be phosphorylated-dephosphorylated on both Ser/Thr- and Tyr-residues by specific protein kinases and protein phosphatases. The results reported here would indicate that the ghosts prepared from human erythrocytes pretreated with DIDS, well known inhibitor of band 3-mediated anion transport, exhibit a markedly reduced Ser/Thr-phosphorylation of spectrin and band 3, when incubated with [gamma-32P]ATP in the presence of Mg2+. On the other hand, Tyr-phosphorylation of this latter protein is practically unchanged or even slightly enhanced. This suggests that Ser/Thr- and Tyr-phosphorylation of band 3 display a different functional role.