Determination of total aluminum, chromium, copper, iron, manganese, and nickel and their fractions leached to the infusions of black tea, green tea, Hibiscus sabdariffa, and Ilex paraguariensis (mate) by ETA-AAS

Total aluminum, chromium, copper, iron, manganese, and nickel were determined in black tea, green tea, Hibiscus sabdariffa, and Ilex paraguariensis (mate) by electrothermal atomic absorption spectrometry after nitric/perchloric acid digestion. In each case, one ground sample of commercially available leafy material was prepared and three 0.5-g subsamples were run in parallel. The infusions were also analyzed and the percentage of each element leached into the liquor was evaluated. The obtained results indicated that hibiscus and mate contained lower levels of aluminum (272±19 μg/g and 369±22 μg/g, respectively) as referred to black tea (759±31 μg/g) or green tea (919±29 μg/g) and suggested that mate drinking could be a good dietary source of essential micronutrient manganese (total content 2223±110 μg/g, 48.1% leached to the infusion). It was also found that the infusion of hibiscus could supply greater amounts of iron (111±5 μg/g total, 40.5% leached) and copper (5.9±0.3 μg/g total, 93.4% leached) as compared to other infusions. Moreover, it was found that the percentage of element leached to the infusion was strongly related to the tannins content in the beverage (correlation coefficients >0.82 with the exception for nickel); for lower tannins level, better leaching was observed.     

Haemagglutinin substitutions N125D,D127E,D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding

During 2013–14 and 2015–16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.     

N‐terminal guanidinylation of the cyclic 1,4‐ureido‐deltorphin analogues: the synthesis,receptor binding studies,and resistance to proteolytic digestion

The synthesis of a series of N‐guanidinylated cyclic ureidopeptides, analogues of 1,4‐ureido‐deltorphin/dermorphine tetrapeptide is described. The δ‐ and μ‐opioid receptor affinity of new guanidinylated analogues and their non‐guanidinylated precursors was determined by the displacement radioligand binding experiments. Our results indicate that the guanidinylation of cyclic 1,4‐ureidodeltorphin peptide analogues does not exhibit a uniform influence on the opioid receptor binding properties, similarly as reported earlier for some linear peptides. All analogues were also tested for their in vitro resistance to proteolysis during incubation with large excess of chymotrypsin, pepsin, and papain by means of mass spectroscopy. Guanidinylated ureidopeptides 1G–4G showed mixed μ agonist/δ agonist properties and high enzymatic stability indicating their potential as therapeutic agents for treatment of pain. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Site-specific Phosphorylation of Kindlin-3 Protein Regulates Its Capacity to Control Cellular Responses Mediated by Integrin αIIbβ3

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin α_IIbβ₃, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), α_IIbβ₃ became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β₃ integrin and kindlin-3 in focal adhesions, and enhanced β₃ integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr⁴⁸² or Ser⁴⁸⁴ was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476–485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin β₃ activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs.     

Vertical Transmission of Babesia microti in BALB/c Mice: Preliminary Report

Babesia spp. (Apicomplexa, Piroplasmida) are obligate parasites of many species of mammals, causing a malaria-like infection- babesiosis. Three routes of Babesia infection have been recognized to date. The main route is by a tick bite, the second is via blood transfusion. The third, vertical route of infection is poorly recognized and understood. Our study focused on vertical transmission of B. microti in a well-established mouse model. We assessed the success of this route of infection in BALB/c mice with acute and chronic infections of B. microti. In experimental groups, females were mated on the 1^st day of Babesia infection (Group G0); on the 28^th day post infection (dpi) in the post- acute phase of the parasite infection (G28); and on the 90^th and 150^th dpi (G90 and G150 group, respectively), in the chronic phase of the parasite infection. Pups were obtained from 58% of females mated in the post-acute phase (G28) and from 33% of females in groups G90 and G150. Mice mated in the pre-acute phase of infection (G0) did not deliver pups. Congenital B. microti infections were detected by PCR amplification of Babesia 18S rDNA in almost all pups (96%) from the experimental groups G28, G90 and G150. Parasitaemia in the F1 generation was low and varied between 0.01–0.001%. Vertical transmission of B. microti was demonstrated for the first time in BALB/c mice.     

Design,Synthesis and In Vitro Activity of Anticancer Styrylquinolines. The p53 Independent Mechanism of Action

A group of styrylquinolines were synthesized and tested for their anti-proliferative activity. Anti-proliferative activity was evaluated against the human colon carcinoma cell lines that had a normal expression of the p53 protein (HCT116 p53^+/+) and mutants with a disabled TP53 gene (HCT116 p53^-/-) and against the GM 07492 normal human fibroblast cell line. A SAR study revealed the importance of Cl and OH as substituents in the styryl moiety. Several of the compounds that were tested were found to have a marked anti-proliferative activity that was similar to or better than doxorubicin and were more active against the p53 null than the wild type cells. The cellular localization tests and caspase activity assays suggest a mechanism of action through the mitochondrial pathway of apoptosis in a p53-independent manner. The activity of the styrylquinoline compounds may be associated with their DNA intercalating ability.     

Host Modulators of H1N1 Cytopathogenicity

Influenza A virus infects 5-20% of the population annually, resulting in ～35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here we employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human bronchial epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to an expanding body of studies directed at decoding vulnerabilities in the command and control networks specified by influenza virulence factors.     

Quantitative determination of creatine kinase release from herring (Clupea harengus) spermatozoa induced by tributyltin

Creatine kinase (CK, ATP creatine phosphotransferase, EC 2.7.3.2) is an enzyme participating in ATP regeneration, which is the primary source of energy in living organisms. We demonstrated that CK from herring spermatozoa has high activity ( approximately 452 micromol/min/g of fresh semen) and has a different electrophoretic mobility from isoenzymes present in skeletal muscle. In our study, we investigated toxic effect of tributyltin (TBT) on herring spermatozoa using a specific sperm viability kit to observe live and dead sperm cells with a confocal microscope. Treatment of herring spermatozoa with TBT caused a time-dependent decrease of viability: 35% nonviable cells with 5 microM TBT and more than 90% nonviable cells with 10 microM TBT after 6 h exposure. We also monitored CK release from damaged spermatozoa into surrounding medium containing different concentrations of TBT. The higher concentration of TBT was used the more CK release from spermatozoa was observed. We suggest that CK could be a good biomarker of sperm cell membranes degradation in the case when lactate dehydrogenase release from permeabilized cells is not possible for rapid determination of the effect of TBT.     

The effect of luminal ghrelin on pancreatic enzyme secretion in the rat

Ghrelin, a 28-amino-acid peptide produced predominantly by oxyntic mucosa has been reported to affect the pancreatic exocrine function but the mechanism of its secretory action is not clear. The effects of intraduodenal (i.d.) infusion of ghrelin on pancreatic amylase outputs under basal conditions and following the stimulation of pancreatic secretion with diversion of pancreato-biliary juice (DPBJ) as well as the role of vagal nerve, sensory fibers and CCK in this process were determined. Ghrelin given into the duodenum of healthy rats at doses of 1.0 or 10.0 microg/kg increased pancreatic amylase outputs under basal conditions or following the stimulation of pancreatic secretion with DPBJ. Bilateral vagotomy as well as capsaicin deactivation of sensory fibers completely abolished all stimulatory effects of luminal ghrelin on pancreatic exocrine function. Pretreatment with lorglumide, a CCK(1) receptor blocker, reversed the stimulation of amylase release produced by intraduodenal application of ghrelin. Intraduodenal ghrelin at doses of 1.0 or 10.0 microg/kg increased plasma concentrations of CCK and ghrelin. In conclusion, ghrelin given into the duodenum stimulates pancreatic enzyme secretion. Activation of vagal reflexes and CCK release as well as central mechanisms could be implicated in the stimulatory effect of luminal ghrelin on the pancreatic exocrine functions.