Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)—the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.     

Cryo-electron microscopy reveals the membrane insertion mechanism of V. cholerae hemolysin

Vibrio cholerae hemolysin (HlyA) is a 65?kDa pore-forming toxin which causes lysis of target eukaryotic cells by forming heptameric channels in the plasma membrane. Deletion of the 15?kDa C-terminus β-prism carbohydrate-binding domain generates a 50?kDa truncated variant (HlyA50) with 1000-fold-reduced pore-forming activity. Previously, we showed by cryo-electron microscopy that the two toxin oligomers have central channels, but the 65?kDa toxin oligomer is a seven-fold symmetric structure with bowl-, ring-, and arm-like domains, whereas the 50?kDa oligomer is an asymmetric jar-like heptamer. In the present study, we determined three-dimensional(3D) structures of HlyA and HlyA50 in presence of erythrocyte stroma and observed that interaction of the 65?kDa toxin with the stroma induced a significant decrease in the height of the β-barrel oligomer with a change in conformation of the ring- and arm-like domains of HlyA. These features were absent in interaction of HlyA50 with stroma. We propose that this conformational transition is critical for membrane-insertion of the toxin.     

Highly Air‐Stable Single‐Crystalline β‐CsPbI3 Nanorods: A Platform for Inverted Perovskite Solar Cells

The synthesis of single‐crystalline β‐CsPbI₃ perovskite nanorods (NRs) using a colloidal process is reported, exhibiting their improved photostability under 45–55% humidity. The crystal structure of CsPbI₃ NRs films is investigated using Rietveld refined X‐ray diffraction (XRD) patterns to determine crystallographic parameters and the phase transformation from orthorhombic (γ‐CsPbI₃) to tetragonal (β‐CsPbI₃) on annealing at 150 °C. Atomic resolution transmission electron microscopy images are utilized to determine the probable atomic distribution of Cs, Pb, and I atoms in a single β‐phase CsPbI₃ NR, in agreement with the XRD structure and selected area electron diffraction pattern, indicating the growth of single crystalline β‐CsPbI₃ NR. The calculation of the electronic band structure of tetragonal β‐CsPbI₃ using density functional theory (DFT) reveals a direct transition with a lower band gap and a higher absorption coefficient in the solar spectrum, as compared to its γ‐phase. An air‐stable (45–55% humidity) inverted perovskite solar cell, employing β‐CsPbI₃ NRs without any encapsulation, yields an efficiency of 7.3% with 78% enhancement over the γ‐phase, showing its potential for future low cost photovoltaic devices.     

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD’s localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of I_Na by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.     

Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.     

Synthesis and evaluation of the antimalarial,anticancer, and caspase 3 activities of tetraoxane dimers

The synthesis of a range of mono spiro and dispiro 1,2,4,5-tetraoxane dimers is described. Selected molecules were examined in in vitro assays to determine their antimalarial and anticancer potential. Our studies reveal that several molecules possess potent nanomolar antimalarial and single digit micromolar antiproliferative IC₅₀s versus colon (HT29-AK and leukemia (HL60) cell lines.     

Binding interaction study on human serum albumin with bactericidal gold nanoparticles synthesized from a leaf extract of Musa balbisiana: a multispectroscopic approach

This study describes the eco‐friendly, low‐cost and room‐temperature synthesis of gold nanoparticles from Musa balbisiana leaf extract, which acts as both reducing and stabilizing agent, and characterized by ultraviolet?visible (UV–vis) light spectroscopy, fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FE‐SEM), analytical transmission electron microscopy (TEM), energy‐dispersive X‐ray spectroscopy (EDAX) and dynamic light scattering (DLS) instruments. These nanoparticles showed an average diameter of 33.83 ± 3.39 nm, which was confirmed from the size distribution histogram. The bactericidal activity of these nanoparticles was confirmed using bacteria Escherichia coli and Staphylococcus aureus at 1 and 2 nM minimum inhibitory concentrations, respectively. The interaction between nanoparticles and human serum albumin (HSA) was investigated, as this plays significant roles in biological systems. The nature of interaction, binding parameters and structural variation of HSA in the presence of these nanoparticles have been evaluated using several useful spectroscopic approaches such as UV–vis, FTIR, time‐resolved and steady‐state fluorescence, and circular dichroism in addition to the measurement of zeta potential. This interaction study revealed that static quenching occurs in this process with minimal alteration in the secondary structure, but the native structure of HSA remained unaltered. The binding constant and thermodynamic parameters of this interaction process were also evaluated.     

Dual inhibition of Kif15 by oxindole and quinazolinedione chemical probes

The mitotic spindle is a microtubule-based machine that segregates a replicated set of chromosomes during cell division. Many cancer drugs alter or disrupt the microtubules that form the mitotic spindle. Microtubule-dependent molecular motors that function during mitosis are logical alternative mitotic targets for drug development. Eg5 (Kinesin-5) and Kif15 (Kinesin-12), in particular, are an attractive pair of motor proteins, as they work in concert to drive centrosome separation and promote spindle bipolarity. Furthermore, we hypothesize that the clinical failure of Eg5 inhibitors may be (in part) due to compensation by Kif15. In order to test this idea, we screened a small library of kinase inhibitors and identified GW108X, an oxindole that inhibits Kif15 in vitro. We show that GW108X has a distinct mechanism of action compared with a commercially available Kif15 inhibitor, Kif15-IN-1 and may serve as a lead with which to further develop Kif15 inhibitors as clinically relevant agents.     

An empirical bayes adjustment to increase the sensitivity of detecting differentially expressed genes in microarray experiments

MOTIVATION: Detection of differentially expressed genes is one of the major goals of microarray experiments. Pairwise comparison for each gene is not appropriate without controlling the overall (experimentwise) type 1 error rate. Dudoit et al. have advocated use of permutation-based step-down P-value adjustments to correct the observed significance levels for the individual (i.e. for each gene) two sample t-tests. RESULTS: In this paper, we consider an ANOVA formulation of the gene expression levels corresponding to multiple tissue types. We provide resampling-based step-down adjustments to correct the observed significance levels for the individual ANOVA t-tests for each gene and for each pair of tissue type comparisons. More importantly, we introduce a novel empirical Bayes adjustment to the t-test statistics that can be incorporated into the step-down procedure. Using simulated data, we show that the empirical Bayes adjustment improved the sensitivity of detecting differentially expressed genes up to 16%, while maintaining a high level of specificity. This adjustment also reduces the false non-discovery rate to some degree at the cost of a modest increase in the false discovery rate. We illustrate our approach using a human colon cancer dataset consisting of oligonucleotide arrays of normal, adenoma and carcinoma cells. The number of genes with differential expression level declared statistically significant was about 50 when comparing normal to adenoma cells and about five when comparing adenoma to carcinoma cells. This list includes genes previously known to be associated with colon cancer as well as some novel genes. AVAILABILITY: R code for the empirical Bayes adjustment and step-down P-value calculation via resampling are available from the supplementary web-site. Supplementary information: http://www.mathstat.gsu.edu/~matsnd/EB/supp.htm