Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines

In this study, we describe the synthesis of various lipopeptides based on the sequence of luteinizing hormone-releasing hormone (LHRH) and report on their abilities to induce Abs against this "self" hormone when inoculated into mice in the absence of additional adjuvant. The peptides consisted of a colinear CD4(+) T helper cell epitope from the L chain of influenza virus hemagglutinin and LHRH, which has B cell epitopes but no T cell epitopes present in its sequence. Lipids were attached either at the N terminus or between the T cell epitope and LHRH, in the approximate center of the peptide. The lipopeptide constructs displayed different solubilities and immunological properties that depended not only on the lipid content but also on the position of attachment of the lipids. Some of these constructs were highly immunogenic, inducing high titers of Ab, which were capable of efficiently sterilizing female mice when administered in saline by s.c. or intranasal routes. The most effective vaccines were highly soluble, contained the dipalmitoyl-S-glyceryl cysteine moiety, and had this lipid attached at the center of the molecule. The relative ability of the lipopeptides to induce an Ab response in the absence of external adjuvant was reflected by their ability to up-regulate the surface expression of MHC class II molecules on immature dendritic cells. These results demonstrate that the composition and position within peptide vaccines of self-adjuvanting lipid groups can influence the ability to induce the maturation of dendritic cells and, in turn, the magnitude of the resulting Ab response.     

Biodegradable polymeric nanospheres formed by temperature-induced phase transition in a mixture of poly(lactide-co-glycolide) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer

The mixture of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer(F-127) and PLGA (poly(lactide-co-gycolide)) forms a liquid state above their phase transition temperatures, and the phase-separated state is induced by decreasing the temperature below the phase transition temperature. On the basis of the temperature-induced phase transition behavior in the mixture of F-127 and PLGA, a novel method for the preparation of drug-loaded PLGA nanospheres was designed and characterized by measuring the loading amount, the encapsulation efficiency, and the drug release pattern. Paclitaxel, used as a potent anticancer drug, was selected as a model drug.     

The correlation of protein structure and evolution of a protein-coding gene: phylogenetic inference using cytochrome oxidase III

Discriminating phylogenetic signal from noise in DNA sequence data is a difficult problem in phylogenetic inference at higher systematic levels. For protein-coding genes, noise at synonymous (silent) positions can be filtered by deleting entire codon positions or types of change at a codon position. This method is not appropriate for replacement sites, because changes at each site within a codon may not be independent. This research presents a method using information from protein structure to evaluate variation in replacement sites. Analysis of the correlation of amino acid variation with protein structure identified rapidly evolving codons in the COIII gene. In a series of phylogenetic analyses attempting to recover a known set of vertebrate relationships, downweighting these labile codons produced the most accurate results. Structural correlates of variable and invariant residues identified in this study can be used to increase the accuracy of models used for phylogenetic inference. Viewing amino acid variation within a phylogenetic framework provided insight into residue changes important in the secondary and tertiary structures of the molecule, changes that were correlated between pairs of neighboring residues or between residues in neighboring helices.      

Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells.     

Molecular Determinants of Cardiac Myocyte Performance as Conferred by Isoform-Specific TnI Residues

Troponin I (TnI) is the molecular switch of the sarcomere. Cardiac myocytes express two isoforms of TnI during development. The fetal heart expresses the slow skeletal TnI (ssTnI) isoform and shortly after birth ssTnI is completely and irreversibly replaced by the adult cardiac TnI (cTnI) isoform. These two isoforms have important functional differences; broadly, ssTnI is a positive inotrope, especially under acidic/hypoxic conditions, whereas cTnI facilitates faster relaxation performance. Evolutionary directed changes in cTnI sequence suggest cTnI evolved to favor relaxation performance in the mammalian heart. To investigate the mechanism, we focused on several notable TnI isoform and trans-species-specific residues located in TnI’s helix 4 using structure/function and molecular dynamics analyses. Gene transduction of adult cardiac myocytes by cTnIs with specific helix 4 ssTnI substitutions, Q157R/A164H/E166V/H173N (QAEH), and A164H/H173N (AH), were investigated. cTnI QAEH is similar in these four residues to ssTnI and nonmammalian chordate cTnIs, whereas cTnI AH is similar to fish cTnI in these four residues. In comparison to mammalian cTnI, cTnI QAEH and cTnI AH showed increased contractility and slowed relaxation, which functionally mimicked ssTnI expressing myocytes. cTnI QAEH molecular dynamics simulations demonstrated altered intermolecular interactions between TnI helix 4 and cTnC helix A, specifically revealing a new, to our knowledge, electrostatic interaction between R171of cTnI and E15 of cTnC, which structurally phenocopied the ssTnI conformation. Free energy perturbation calculation of cTnC Ca²⁺ binding for these conformations showed relative increased calcium binding for cTnI QAEH compared to cTnI. Taken together, to our knowledge, these new findings provide evidence that the evolutionary-directed coordinated acquisition of residues Q157, A164, E166, H173 facilitate enhanced relaxation performance in mammalian adult cardiac myocytes.     

The NorM MATE Transporter from N.?gonorrhoeae: Insights into Drug and Ion Binding from Atomistic Molecular Dynamics Simulations

The multidrug and toxic compound extrusion transporters extrude a wide variety of substrates out of both mammalian and bacterial cells via the electrochemical gradient of protons and cations across the membrane. The substrates transported by these proteins include toxic metabolites and antimicrobial drugs. These proteins contribute to multidrug resistance in both mammalian and bacterial cells and are therefore extremely important from a biomedical perspective. Although specific residues of the protein are known to be responsible for the extrusion of solutes, mechanistic details and indeed structures of all the conformational states remain elusive. Here, we report the first, to our knowledge, simulation study of the recently resolved x-ray structure of the multidrug and toxic compound extrusion transporter, NorM from Neisseria gonorrhoeae (NorM_NG). Multiple, atomistic simulations of the unbound and bound forms of NorM in a phospholipid lipid bilayer allow us to identify the nature of the drug-protein/ion-protein interactions, and secondly determine how these interactions contribute to the conformational rearrangements of the protein. In particular, we identify the molecular rearrangements that occur to enable the Na⁺ ion to enter the cation-binding cavity even in the presence of a bound drug molecule. These include side chain flipping of a key residue, GLU-261 from pointing toward the central cavity to pointing toward the cation binding side when bound to a Na⁺ ion. Our simulations also provide support for cation binding in the drug-bound and apo states of NorM_NG.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.