Protein structure analysis through Hough Transform and Range Tree

The Generalized Hough Transform (GHT) allows to recognize general patterns once defined a model to be recognized, a reference point (RP) rigid with the model, and a mapping rule. This rule establishes the contributions in the parameters space; this space, generally speaking, is given by the parameters of a rigid motion leading to overlap a model item with an equal item detected on the unknown pattern. In this paper we introduce the GHT applied to motifs, domains and entire proteins retrieval into a protein data base. The spatial attitude of a single protein secondary structure (SS) constitutes the item supporting the contributions. If the unknown pattern contains a block of N SS of the model to be recognized, the N corresponding votes will have a common point, so accumulating N contributions. An analysis of the neighborhoods around the areas with high contributions density is necessary. It is not sufficient and often inaccurate to limit the analysis to the peaks even if the number of contribution is closed to the expected one. Both convenient data structures for effectively operating in the neighborhoods (a range tree data structure) and suitable decision criteria have been introduced. Preliminary results of comparative analysis are given

Tropomyosin-like properties of clathrin light chains allow a rapid, high-yield purification.

The light chains (LCa and LCb) of bovine brain clathrin are resistant to heat denaturation by boiling, a property shared by tropomyosin (Bailey, K., 1948, Biochem. J., 43:271-281). Light chains were partially purified by boiling and centrifugation of a Tris-extract of crude membranes prepared from bovine brains (Keen, J. H., M. C. Willingham, and I. H. Pastan, 1979, Cell., 16:303-312). Contaminant polypeptides were then removed by size-exclusion high-pressure liquid chromatography. The purified light chains were separated from each other by using an immunoaffinity column prepared from a monoclonal antibody CVC.7 specific for LCa and not LCb

Conservation of structure and mechanism in primary and secondary transporters exemplified by SiaP, a sialic acid binding virulence factor from Haemophilus influenzae

Extracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient-dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxyN-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an alpha-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes

Direct Cardiac Reprogramming: Progress and Promise.

The human adult heart lacks a robust endogenous repair mechanism to fully restore cardiac function after insult; thus, the ability to regenerate and repair the injured myocardium remains a top priority in treating heart failure. The ability to efficiently generate a large number of functioning cardiomyocytes capable of functional integration within the injured heart has been difficult. However, the ability to directly convert fibroblasts into cardiomyocyte-like cells both in vitro and in vivo offers great promise in overcoming this problem. In this review, we describe the insights and progress that have been gained from the investigation of direct cardiac reprogramming. We focus on the use of key transcription factors and cardiogenic genes as well as on the use of other biological molecules such as small molecules, cytokines, noncoding RNAs, and epigenetic modifiers to improve the efficiency of cardiac reprogramming. Finally, we discuss the development of safer reprogramming approaches for future clinical application

A morphometric analysis of ultrastructural dynamics in the murine glomerulus following surgically-induced renal hypertension

Chronic kidney disease (CKD) and end stage renal disease (ESRD) are significant causes of adult morbidity and mortality worldwide. Though these conditions are common, the mechanisms of pathogenesis in kidney disease are poorly understood. Genetic predisposition has been established in the African American population; however this does not explain the ubiquity of CKD in the United States and abroad. Diabetes and hypertension are the two most frequently occurring co-morbidities in kidney disease and both have been identified as putative sources of injury to the delicate filtering structures of the kidney. Furthermore, the intrinsic functional relationship between the cardiovascular and renal organ systems adds to the plausibility of a hemodynamic cause. In light of this knowledge, we aim to explore the roles of genetic predisposition and hypertension in the pathogenesis and progression of CKD. The filtering apparatus of the kidney, the glomerulus, is a looping tuft of capillaries specialized to allow the passage of water and certain substances from the blood while restricting others. Glomeruli at the corticomedullary boundary of the kidney experience blood pressures closer to those in systemic arterioles and are subject to similar hemodynamic stresses. To evaluate the role of hypertension in CKD, we employed a well-known model of hypertensive kidney disease in mice involving uninephrectomy (UNX), subcutaneous implantation of a timed-release pellet containing the active aldosterone precursor deoxycorticosterone acetate (DOCA), and a high-salt diet. Given the role of heritability in human CKD pathogenesis, we applied the DOCA-UNX model in two strains of mice with differing susceptibility to kidney damage, the 129S6 and C57BL/6 strains, to evaluate the effects of genetic predisposition. Mice were subjected to varying lengths of hypertension exposure and their kidneys were subsequently examined by transmission electron microscopy (TEM). Ultrastructural lesions of glomeruli were evaluated by a renal pathologist and assigned subjective pathology scores based on the extent and severity of involvement. We hypothesized that certain glomerular lesions, particularly those involving the podocytes of the visceral epithelium, would increase in severity in mice with heritable susceptibility (129S6) as well as those with longer exposure to glomerular hypertension. Our observations demonstrate these hypotheses are partially correct. By TEM histopathology, mouse strain was found to have a significant effect on the severity of certain epithelial lesions while duration of hypertension had a significant effect on the overall morphological pathology of the podocytes, glomerular basement membrane, and glomerulus as a whole. These results provide a promising foundation for further investigation of the pathogenesis of CKD in mice

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs

Noncooperative thermodynamics and kinetic models of ligand binding to polymers: Connecting McGhee-von Hippel model with the Tonks gas model

Ligand binding to polymers modifies the physical and chemical properties of the polymers, leading to physical, chemical, and biological implications. McGhee and von Hippel obtained the equilibrium coverage as a function of the ligand affinity, through the computation of the possible binding sites for the ligand. Here, we complete this theory deriving the kinetic model for the ligand-binding dynamics and the associated equilibrium chemical potential, which turns out to be of the Tonks gas model type. At low coverage, the Tonks chemical potential becomes the Fermi chemical potential and even the ideal gas chemical potential. We also discuss kinetic models associated with these chemical potentials. These results clarify the kinetic models of ligand binding, their relations with the chemical potentials, and their range of validity. Our results highlight the inaccuracy of ideal and simplified kinetic approaches for medium and high coverages

Comparative Genomics, Evolutionary Epidemiology, and RBD-hACE2 Receptor Binding Pattern in B.1.1.7 (Alpha) and B.1.617.2 (Delta) Related to Their Pandemic Response in UK and India

BACKGROUND: The massive increase in COVID-19 infection had generated a second wave in India during May-June 2021 with a critical pandemic situation. The Delta variant (B.1.617.2) was a significant factor during the second wave. Conversely, the UK had passed through the crucial phase of the pandemic from November to December 2020 due to B.1.1.7. The study tried to comprehend the pandemic response in the UK and India to the spread of the B.1.1.7 (Alpha, UK) variant and B.1.617.2 (Delta, India) variant. METHODS: This study was performed in three directions to understand the pandemic response of the two emerging variants. First, we served comparative genomics, such as genome sequence submission patterns, mutational landscapes, and structural landscapes of significant mutations (N501Y, D614G, L452R, E484Q, and P681R). Second, we performed evolutionary epidemiology using molecular phylogenetics, scatter plots of the cluster evaluation, country-wise transmission pattern, and frequency pattern. Third, the receptor binding pattern was analyzed using the Wuhan reference strain and the other two variants. RESULTS: The study analyzed the country-wise and region-wise genome sequences and their submission pattern, molecular phylogenetics, scatter plot of the cluster evaluation, country-wise geographical distribution and transmission pattern, frequency pattern, entropy diversity, and mutational landscape of the two variants. The structural pattern was analyzed in the N501Y, D614G L452R, E484Q, and P681R mutations. The study found increased molecular interactivity between hACE2-RBD binding of B.1.1.7 and B.1.617.2 compared to the Wuhan reference strain. Our receptor binding analysis showed a similar indication pattern for hACE2-RBD of these two variants. However, B.1.617.2 offers slightly better stability in the hACE2-RBD binding pattern through MD simulation than B.1.1.7. CONCLUSION: The increased hACE2-RBD binding pattern of B.1.1.7 and B.1.617.2 might help to increase the infectivity compared to the Wuhan reference strain

Involvement of secondary metabolites in the pathogenesis of the American foulbrood of honey bees caused by Paenibacillus larvae

Covering: 2011 to end of 2014 The Gram-positive, spore-forming bacterium Paenibacillus larvae (P. larvae) is the causative agent of the epizootic American Foulbrood (AFB), a fatal brood disease of the western honey bee (Apis mellifera). AFB is one of the most destructive honey bee diseases since it is not only lethal for infected larvae but also for the diseased colonies. Due to the high impact of honey bees on ecology and economy this epizootic is a severe and pressing problem. Knowledge about virulence mechanisms and the underlying molecular mechanisms remain largely elusive. Recent genome sequencing of P. larvae revealed its potential to produce unknown secondary metabolites, like nonribosomal peptides and peptide-polyketide hybrids. This article highlights recent findings on secondary metabolites synthesized by P. larvae and discusses their role in virulence and pathogenicity towards the bee larvae

Molecular Basis of the Mechanism and Regulation of Receptor-GTP Binding Protein Interactions: A Thesis

The photon receptor, rhodopsin, and the GTP-binding regulatory protein, transducin, belong to a family of G protein-coupled receptors. The activation process through which guanine nucleotide exchange of the G protein is accomplished was investigated utilizing these components of the visual transduction system. Rhodopsin, modelled as an enzyme in its interaction with substrates, transducin and guanine nucleotides, was characterized to catalyze the G protein\u27s activation by a double-displacement mechanism. Remarkable allosteric behavior was observed in these kinetic studies. Equilibrium binding studies were performed to investigate the molecular basis of the positive cooperative behavior between transducin and rhodopsin. These experiments show that the origins of the allosterism must arise from oligomeric assemblies between receptor and G protein. The determined Hill coefficient, nH = 2, suggests that at least two transducin molecules are involved, and the Bmax parameter a1so indicates that multimeric assemblies of rhodopsin may participate in the positive cooperative interactiions. Physical studies of transducin in solution were performed and do not indicate the existence of a dimeric structure, in contrast to the kinetic and binding experiments which analyze interactions at the membrane surface. Since the latter environment represents the native surroundings in vivo, aspects of the allosteric behavior must be considered for a complete understanding of the signal transduction mechanism. The reported findings are interpreted in the context of homologies between other G protein-coupled receptor systems in order to develop a model for the molecular basis of the mechanism and regulation of this mode of signal transduction