Novel Phylogenetic Algorithm to Monitor Human Tropism in Egyptian H5N1-HPAIV Reveals Evolution toward Efficient Human-to-Human Transmission

Years of endemic infections with highly pathogenic avian influenza (HPAI) A subtype H5N1 virus in poultry and high numbers of infections in humans provide ample opportunity in Egypt for H5N1-HPAIV to develop pandemic potential. In an effort to better understand the viral determinants that facilitate human infections of the Egyptian H5N1-HPAIVvirus, we developed a new phylogenetic algorithm based on a new distance measure derived from the informational spectrum method (ISM). This new approach, which describes functional aspects of the evolution of the hemagglutinin subunit 1 (HA1), revealed a growing group G2 of H5N1-HPAIV in Egypt after 2009 that acquired new informational spectrum (IS) properties suggestive of an increased human tropism and pandemic potential. While in 2006 all viruses in Egypt belonged to the G1 group, by 2011 these viruses were virtually replaced by G2 viruses. All of the G2 viruses displayed four characteristic mutations (D43N, S120(D,N), (S,L)129Δ and I151T), three of which were previously reported to increase binding to the human receptor. Already in 2006–2008 G2 viruses were significantly (p<0.02) more often found in humans than expected from their overall prevalence and this further increased in 2009–2011 (p<0.007). Our approach also identified viruses that acquired additional mutations that we predict to further enhance their human tropism. The extensive evolution of Egyptian H5N1-HPAIV towards a preferential human tropism underlines an urgent need to closely monitor these viruses with respect to molecular determinants of virulence.     

Febrile temperature leads to significant stiffening of Plasmodium falciparum parasitized erythrocytes

Parasitic infection with Plasmodium falciparum is responsible for the most severe form of human malaria in which patients suffer from periodic fever. It is well established that during intra-erythrocytic maturation of the parasite in the red blood cell (RBC), the RBC becomes significantly more cytoadhesive and less deformable; these and other biochemical factors together with human host factors such as compromised immune status are important contributors to the disease pathology. There is currently substantial interest in understanding the loss of RBC deformability due to P. falciparum infection, but few results are available concerning effects of febrile conditions or parasitization on RBC membrane rheology. Here, for the first time, we report rheology of the single, isolated RBC with and without P. falciparum merozoite invasion, spanning a range from room temperature to febrile conditions (41 degrees C), over all the stages of parasite maturation. As expected, stiffness increased with parasite maturation. Surprisingly, however, stiffness increased acutely with temperature on a scale of minutes, particularly in late trophozoite and schizont stages. This acute stiffening in late falciparum stages may contribute to fever-dependent pathological consequences in the microcirculation.     

Characterization of conserved properties of hemagglutinin of H5N1 and human influenza viruses: possible consequences for therapy and infection control

Background  

Epidemics caused by highly pathogenic avian influenza virus (HPAIV) are a continuing threat to human health and to the world's economy. The development of approaches, which help to understand the significance of structural changes resulting from the alarming mutational propensity for human-to-human transmission of HPAIV, is of particularly interest. Here we compare informational and structural properties of the hemagglutinin (HA) of H5N1 virus and human influenza virus subtypes, which are important for the receptor/virus interaction.     

Etanercept reduces matrix metalloproteinase-9 level in children with polyarticular juvenile idiopathic arthritis and TNF-α-308GG genotype

Genetic contribution of tumor necrosis factor polymorphism (TNF-α-308G/A) in patients with juvenile idiopathic arthritis (JIA) on response to TNF blocking agents, as well as matrix metalloproteinase-9 (MMP-9) production, is not yet well established. We have investigated whether the TNF-α-308G/A polymorphism can influence MMP-9 level and clinical response to etanercept (TNF receptor II-Fc fusion protein) in JIA patients, after 1 year of treatment. A total of 66 patients with polyarticular JIA and 65 healthy children were screened for the polymorphism using the polymerase chain reaction–restriction fragment length polymorphism method. JIA patients donated paired blood samples prior to and 12 months after etanercept therapy. Plasma MMP-9 level was determined using an enzyme-linked immunosorbent assay kit. Clinical assessment was performed according to ACR Pedi 50 improvement criteria. The frequency of the A allele was significantly higher in JIA patients compared to controls (39% vs. 26%, P?=?0.026). Patients with the ?308GG genotype achieved an ACR Pedi 50 response significantly more frequently than those with the ?308AA genotype (P?=?0.035). MMP-9 level in patients with the genotype ?308GG was significantly decreased after 1 year of treatment with etanercept compared to the value from before (P?=?0.036). On the other hand, there was a decrease of MMP-9 levels after treatment, but not statistically significant in patients with the genotypes ?308GA/AA. We conclude that etanercept reduces MMP-9 level in children with polyarticular JIA and TNF-α-308GG genotype. Our results correlate with findings that the ?308A allele is associated with a lower response to etanercept treatment.     

Effects of Cytokinins on In Vitro Seed Germination and Early Seedling Morphogenesis in Lotus corniculatus L.

We determined the effects of zeatin (ZEA), isopentenyl adenine (2iP), kinetin (KIN), benzyladenine (BA), and thidiazuron (TDZ) on seed germination, elongation of seedling shoots and roots, frequency of regeneration, and the number of regenerants per seedling in Lotus corniculatus L. Sterilized seeds were cultured in vitro on Murashige and Skoog (1962) medium containing 3% sucrose, 0.7% agar, and various cytokinins (0, 0.08, 0.22, 0.35, 0.80, 2.20, and 3.50 μM). After 30 days, seedlings were transferred to cytokinin-free medium for another 60 days. All cytokinins stimulated the rate and percentage of seed germination at least twofold in optimum concentrations; TDZ and ZEA were the most active, followed closely by BA, whereas KIN and 2iP stimulated germination in higher concentrations only. Elongation of shoots and roots was strongly inhibited at the lowest TDZ and BA concentrations, whereas ZEA, KIN, and 2iP exerted moderate, dose-dependent inhibition. The frequency of regenerant-producing seeds was highest on ZEA and BA, whereas the greatest number of regenerants per seedling was found on TDZ. It is concluded that the culture of seeds on cytokinin-containing media, followed by transfer to cytokinin-free medium, is a suitable procedure for rapid production of a large number of uniform regenerants. The presumed role of particular cytokinins is discussed.     

Electrospun nano-fibre mats with antibacterial properties from quaternised chitosan and poly(vinyl alcohol)

Nano-fibres containing quaternised chitosan (QCh) have been successfully prepared by electrospinning of QCh solutions mixed with poly(vinyl alcohol) (PVA). The average fibre diameter is in the range of 60-200 nm. UV irradiation of the composite electrospun nano-fibrous mats containing triethylene glycol diacrylate as cross-linking agent has resulted in stabilising of the nano-fibres against disintegration in water or water vapours. Microbiological screening has demonstrated the antibacterial activity of the photo-cross-linked electrospun mats against Staphylococcus aureus and Escherichia coli. The obtained nano-fibrous electrospun mats are promising for wound-healing applications.     

Propionylpiperazines as human melanocortin-4 receptor ligands

A series of alpha-benzylpropionylpiperazines were synthesized and tested as antagonists of the melanocortin-4 receptor. In addition to its high potency and selectivity, R-11a had desirable pharmacokinetic properties including high brain penetration in mice.     

Arylpropionylpiperazines as antagonists of the human melanocortin-4 receptor

A series of 3-arylpropionylpiperazines were synthesized as antagonists of the melanocortin-4 receptor. Their potency was found to be increased by replacing the alpha-methyl substituent of the initial lead 11 with a larger s-Bu or i-Bu group. Further potency enhancement was observed when a glycine or beta-alanine was incorporated onto the benzylamine. Some compounds demonstrated good potency, moderate selectivity, and oral bioavailability.     

The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background

In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen.

Materials and methods

BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 × LD₅₀) of tetanus toxin.

Results

The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFNγ and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p<0.05).

Conclusion

Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively.     

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l^?1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.