In vitro culture methods in sorghum with shoot tip as the explant material

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.     

Aromatic interactions in model peptide β-hairpins: ring current effects on proton chemical shifts

Crystal structures of eight peptide β-hairpins in the sequence Boc-Leu-Phe-Val-Xxx-Yyy-Leu-Phe-Val-OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spacial proximity, with the centroid-centroid distance (R(cen)) of 4.4-5.4 ? between the two phenyl rings. Proton NMR spectra in chloroform and methanol solution reveal a significant upfield shift of the Phe(7) C(δ,δ') H(2) protons (6.65-7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometrics observed in crystals for all eight peptides. Only one of the C(δ,δ') H proton lies in the shielding zone with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations, which resemble crystal state orientation, may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe side chains provide support for close similarity between the solid state and solution conformation. Temperature dependence of aromatic ring proton chemical shift and line widths for peptide 6 (Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe) and the control peptide Boc-Leu-Val-Val-(D)Pro-Gly-Leu-Phe-Val-OMe establish an enhanced barrier to ring flipping when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustment about C(α)-C(β) (χ(1) ) and C(β)-C(γ) (χ(2) ) bonds of both the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of the specific aromatic ring orientation in organic solvents provides evidence for significant stabilizing interaction.     

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.     

An Alphavirus Vector-Based Tetravalent Dengue Vaccine Induces a Rapid and Protective Immune Response in Macaques That Differs Qualitatively from Immunity Induced by Live Virus Infection

Despite many years of research, a dengue vaccine is not available, and the more advanced live attenuated vaccine candidate in clinical trials requires multiple immunizations with long interdose periods and provides low protective efficacy. Here, we report important contributions to the development of a second-generation dengue vaccine. First, we demonstrate that a nonpropagating vaccine vector based on Venezuelan equine encephalitis virus replicon particles (VRP) expressing two configurations of dengue virus E antigen (subviral particles [prME] and soluble E dimers [E85]) successfully immunized and protected macaques against dengue virus, while antivector antibodies did not interfere with a booster immunization. Second, compared to prME-VRP, E85-VRP induced neutralizing antibodies faster, to higher titers, and with improved protective efficacy. Third, this study is the first to map antigenic domains and specificities targeted by vaccination versus natural infection, revealing that, unlike prME-VRP and live virus, E85-VRP induced only serotype-specific antibodies, which predominantly targeted EDIII, suggesting a protective mechanism different from that induced by live virus and possibly live attenuated vaccines. Fourth, a tetravalent E85-VRP dengue vaccine induced a simultaneous and protective response to all 4 serotypes after 2 doses given 6 weeks apart. Balanced responses and protection in macaques provided further support for exploring the immunogenicity and safety of this vaccine candidate in humans.     

Leukemia Fusion Target AF9 Is an Intrinsically Disordered Transcriptional Regulator that Recruits Multiple Partners via Coupled Folding and Binding

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Highlights? The AF9 AHD is intrinsically disordered ? The AHD recruits AF4, BCoR, Dot1L, and hPC3 by coupled folding and binding ? AF9 binding partners compete for binding to a common site ? Dynamics of the AF4-AF9 complex may facilitate exchange between partners     

Design,synthesis, and biological evaluation of conformationally constrained glycerol 3-phosphate acyltransferase inhibitors

Glycerol 3-phosphate acyltransferase (GPAT) isozymes are central control points for fat synthesis in mammals. Development of inhibitors of these membrane-bound enzymes could lead to an effective treatment for obesity, but is thwarted by an absence of direct structural information. Based on a highly successful study involving conformationally constrained glycerol 3-phosphate analogs functioning as potent glycerol 3-phosphate dehydrogenase inhibitors, several series of cyclic bisubstrate and transition state analogs were designed, synthesized, and tested as GPAT inhibitors. The weaker in vitro inhibitory activity of these compounds compared to a previously described benzoic acid series was then examined in docking experiments with the soluble squash chloroplast GPAT crystal structure. These in silico experiments indicate that cyclopentyl and cyclohexyl scaffolds prepared in this study may be occluded from the enzyme active site by two protein loops that sterically guard the phosphate binding region. In view of these findings, future GPAT inhibitor design will be driven toward compounds based on planar frameworks able to slide between these loops and enter the active site, resulting in improved inhibitory activity.     

Genome-wide association study of coronary heart disease and its risk factors in 8,090 African Americans: the NHLBI CARe Project

Coronary heart disease (CHD) is the leading cause of mortality in African Americans. To identify common genetic polymorphisms associated with CHD and its risk factors (LDL- and HDL-cholesterol (LDL-C and HDL-C), hypertension, smoking, and type-2 diabetes) in individuals of African ancestry, we performed a genome-wide association study (GWAS) in 8,090 African Americans from five population-based cohorts. We replicated 17 loci previously associated with CHD or its risk factors in Caucasians. For five of these regions (CHD: CDKN2A/CDKN2B; HDL-C: FADS1-3, PLTP, LPL, and ABCA1), we could leverage the distinct linkage disequilibrium (LD) patterns in African Americans to identify DNA polymorphisms more strongly associated with the phenotypes than the previously reported index SNPs found in Caucasian populations. We also developed a new approach for association testing in admixed populations that uses allelic and local ancestry variation. Using this method, we discovered several loci that would have been missed using the basic allelic and global ancestry information only. Our conclusions suggest that no major loci uniquely explain the high prevalence of CHD in African Americans. Our project has developed resources and methods that address both admixture- and SNP-association to maximize power for genetic discovery in even larger African-American consortia.