Showing papers in "Methods of Molecular Biology in 2017"

Structural Equation Modeling

Abstract: Structural equation modeling (SEM) is a multivariate statistical framework that is used to model complex relationships between directly observed and indirectly observed (latent) variables. SEM is a general framework that involves simultaneously solving systems of linear equations and encompasses other techniques such as regression, factor analysis, path analysis, and latent growth curve modeling. Recently, SEM has gained popularity in the analysis of complex genetic traits because it can be used to better analyze the relationships between correlated variables (traits), to model genes as latent variables as a function of multiple observed genetic variants, and to assess the association between multiple genetic variants and multiple correlated phenotypes of interest. Though the general SEM framework only allows for the analysis of independent observations, recent work has extended SEM for the analysis of data on general pedigrees. Here, we review the theory of SEM for both unrelated and family data, describe the available software for SEM, and provide examples of SEM analysis.

Predicting Secretory Proteins with SignalP

Abstract: SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the secretory pathway, but are not necessarily secreted. After a brief introduction to the biology of signal peptides and the history of signal peptide prediction, this chapter will describe all the options of the current version of SignalP and the details of the output from the program. The chapter includes a case study where the scores of SignalP were used in a novel way to predict the functional effects of amino acid substitutions in signal peptides.

Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive.

Abstract: The Protein Data Bank (PDB)--the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes--was established in 1971, becoming the first open-access digital resource in the biological sciences The PDB archive currently houses ~130,000 entries (May 2017) It is managed by the Worldwide Protein Data Bank organization (wwPDB; wwpdborg), which includes the RCSB Protein Data Bank (RCSB PDB; rcsborg), the Protein Data Bank Japan (PDBj; pdbjorg), the Protein Data Bank in Europe (PDBe; pdbeorg), and BioMagResBank (BMRB; wwwbmrbwiscedu) The four wwPDB partners operate a unified global software system that enforces community-agreed data standards and supports data Deposition, Biocuration, and Validation of ~11,000 new PDB entries annually (depositwwpdborg) The RCSB PDB currently acts as the archive keeper, ensuring disaster recovery of PDB data and coordinating weekly updates wwPDB partners disseminate the same archival data from multiple FTP sites, while operating complementary websites that provide their own views of PDB data with selected value-added information and links to related data resources At present, the PDB archives experimental data, associated metadata, and 3D-atomic level structural models derived from three well-established methods: crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM) wwPDB partners are working closely with experts in related experimental areas (small-angle scattering, chemical cross-linking/mass spectrometry, Forster energy resonance transfer or FRET, etc) to establish a federation of data resources that will support sustainable archiving and validation of 3D structural models and experimental data derived from integrative or hybrid methods

MiRNA Biogenesis and Regulation of Diseases: An Overview.

Abstract: MicroRNAs (miRNAs) are small RNA molecules, with their role in gene silencing and translational repression by binding to target mRNAs. Since it was discovered in 1993, miRNA are found in all eukaryotic cells conserved across the species. In recent years, regulation of miRNAs are extensively studied for their role in biological processes as well as in development and progression of various human diseases including retinal disorder, neurodegenerative diseases, cardiovascular disease and cancer. This chapter summarises miRNA biogenesis and explores their potential roles in a variety of diseases. miRNAs holds huge potential for diagnostic and prognostic biomarkers, and as predictors of drug response.

Data Conversion with ProteoWizard msConvert

Abstract: Recent advances in proteome informatics have led to an explosion in tools to analyze mass spectrometry data. These tools operate across the analysis pipeline doing everything from assessing quality control to matching peptides to spectra to quantification. Unfortunately, the vast majority of these tools are not able to operate directly on the proprietary formats generated by the diverse mass spectrometers. Consequently, the first step in many protocols is the conversion of data from vendor-specific binary files to open-format files. This protocol details the use of ProteoWizard's msConvert and msConvertGUI software for this conversion, taking format features, coding options, and vendor particularities into account. We specifically describe the various options available when doing conversions and the implications of each option.

Thin-Film Hydration Followed by Extrusion Method for Liposome Preparation

Abstract: One of the simplest ways to prepare liposomes in a research laboratory is the thin-film hydration method followed by extrusion. This method involves making a thin lipid film in a round-bottom flask by the removal of organic solvent. Upon the addition and agitation of the dispersion medium, heterogeneous liposomes are formed. Finally, after extrusion through polycarbonate membranes, homogeneous small liposomes are obtained.

Computer-Aided Drug Design Methods.

Abstract: Computational approaches are useful tools to interpret and guide experiments to expedite the antibiotic drug design process. Structure-based drug design (SBDD) and ligand-based drug design (LBDD) are the two general types of computer-aided drug design (CADD) approaches in existence. SBDD methods analyze macromolecular target 3-dimensional structural information, typically of proteins or RNA, to identify key sites and interactions that are important for their respective biological functions. Such information can then be utilized to design antibiotic drugs that can compete with essential interactions involving the target and thus interrupt the biological pathways essential for survival of the microorganism(s). LBDD methods focus on known antibiotic ligands for a target to establish a relationship between their physiochemical properties and antibiotic activities, referred to as a structure-activity relationship (SAR), information that can be used for optimization of known drugs or guide the design of new drugs with improved activity. In this chapter, standard CADD protocols for both SBDD and LBDD will be presented with a special focus on methodologies and targets routinely studied in our laboratory for antibiotic drug discoveries.

Basic Colorimetric Proliferation Assays: MTT, WST, and Resazurin.

Abstract: This chapter describes selected assays for the evaluation of cellular viability and proliferation of cell cultures. The underlying principle of these assays is the measurement of a biochemical marker to evaluate the cell's metabolic activity. The formation of the omnipresent reducing agents NADH and NADPH is used as a marker for metabolic activity in the following assays. Using NADH and NADPH as electron sources, specific dyes are biochemically reduced which results in a color change that can be determined with basic photometrical methods. The assays selected for this chapter include MTT, WST, and resazurin. They are applicable for adherent or suspended cell lines, easy to perform, and comparably economical. Detailed protocols and notes for easier handling and avoiding pitfalls are enclosed to each assay.

ErbB Receptors and Cancer

Abstract: The ErbB receptor family, also known as the EGF receptor family or type I receptor family, includes the epidermal growth factor (EGF) receptor (EGFR) or ErbB1/Her1, ErbB2/Her2, ErbB3/Her3, and ErbB4/Her4. Among all RTKs, EGFR was the first RTK identified and the first one linked to cancer. Thus, EGFR has also been the most intensively studied among all RTKs. ErbB receptors are activated after homodimerization or heterodimerization. The ErbB family is unique among the various groups of receptor tyrosine kinases (RTKs) in that ErbB3 has impaired kinase activity, while ErbB2 does not have a direct ligand. Therefore, heterodimerization is an important mechanism that allows the activation of all ErbB receptors in response to ligand stimulation. The activated ErbB receptors bind to many signaling proteins and stimulate the activation of many signaling pathways. The specificity and potency of intracellular signaling pathways are determined by positive and negative regulators, the specific composition of activating ligand(s), receptor dimer components, and the diverse range of proteins that associate with the tyrosine phosphorylated C-terminal domain of the ErbB receptors. ErbB receptors are overexpressed or mutated in many cancers, especially in breast cancer, ovarian cancer, and non-small cell lung cancer. The overexpression and overactivation of ErbB receptors are correlated with poor prognosis, drug resistance, cancer metastasis, and lower survival rate. ErbB receptors, especially EGFR and ErbB2 have been the primary choices as targets for developing cancer therapies.

The Changing Epidemiology of Invasive Fungal Infections.

Abstract: Invasive fungal infections (IFI) are an emerging problem worldwide with invasive candidiasis and candidemia responsible for the majority of cases. This is predominantly driven by the widespread adoption of aggressive immunosuppressive therapy among certain patient populations (e.g., chemotherapy, transplants) and the increasing use of invasive devices such as central venous catheters (CVCs). The use of new immune modifying drugs has also opened up an entirely new spectrum of patients at risk of IFIs. While the epidemiology of candida infections has changed in the last decade, with a gradual shift from C. albicans to non-albicans candida (NAC) strains which may be less susceptible to azoles, these changes vary between hospitals and regions depending on the type of population risk factors and antifungal use. In certain parts of the world, the incidence of IFI is strongly linked to the prevalence of other disease conditions and the ecological niche for the organism; for instance cryptococcal and pneumocystis infections are particularly common in areas with a high prevalence of HIV disease. Poorly controlled diabetes is a major risk factor for invasive mould infections. Environmental factors and trauma also play a unique role in the epidemiology of mould infections, with well-described hospital outbreaks linked to the use of contaminated instruments and devices. Blastomycosis is associated with occupational exposure (e.g., forest rangers) and recreational activities (e.g., camping and fishing).The true burden of IFI is probably an underestimate because of the absence of reliable diagnostics and lack of universal application. For example, the sensitivity of most blood culture systems for detecting candida is typically 50 %. The advent of new technology including molecular techniques such as 18S ribosomal RNA PCR and genome sequencing is leading to an improved understanding of the epidemiology of the less common mould and dimorphic fungal infections. Molecular techniques are also providing a platform for improved diagnosis and management of IFI.Many factors affect mortality in IFI, not least the underlying medical condition, choice of therapy, and the ability to achieve early source control. For instance, mortality due to pneumocystis pneumonia in HIV-seronegative individuals is now higher than in seropositive patients. Of significant concern is the progressive increase in resistance to azoles and echinocandins among candida isolates, which appears to worsen the already significant mortality associated with invasive candidiasis. Mortality with mould infections approaches 50 % in most studies and varies depending on the site, underlying disease and the use of antifungal agents such as echinocandins and voriconazole. Nevertheless, mortality for most IFIs has generally fallen with advances in medical technology, improved care of CVCs, improved diagnostics, and more effective preemptive therapy and prophylaxis.

UniProt Protein Knowledgebase

Abstract: The Universal Protein Resource (UniProt) is a freely available comprehensive resource for protein sequence and annotation data. UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the Swiss Institute of Bioinformatics (SIB), and the Protein Information Resource (PIR). Across the three institutes more than 100 people are involved through different tasks such as expert curation, software development, and support.This chapter introduces the functionality and data provided by UniProt. It describes example use cases for which you might come to UniProt and the methods to help you achieve your goals.

Semaphorins and their Signaling Mechanisms.

Abstract: Semaphorins are extracellular signaling proteins that are essential for the development and maintenance of many organs and tissues The more than 20-member semaphorin protein family includes secreted, transmembrane and cell surface-attached proteins with diverse structures, each characterized by a single cysteine-rich extracellular sema domain, the defining feature of the family Early studies revealed that semaphorins function as axon guidance molecules, but it is now understood that semaphorins are key regulators of morphology and motility in many different cell types including those that make up the nervous, cardiovascular, immune, endocrine, hepatic, renal, reproductive, respiratory and musculoskeletal systems, as well as in cancer cells Semaphorin signaling occurs predominantly through Plexin receptors and results in changes to the cytoskeletal and adhesive machinery that regulate cellular morphology While much remains to be learned about the mechanisms underlying the effects of semaphorins, exciting work has begun to reveal how semaphorin signaling is fine-tuned through different receptor complexes and other mechanisms to achieve specific outcomes in various cellular contexts and physiological systems These and future studies will lead to a more complete understanding of semaphorin-mediated development and to a greater understanding of how these proteins function in human disease

Antimicrobial Peptides: An Introduction.

Abstract: The "golden era" of antibiotic discovery has long passed, but the need for new antibiotics has never been greater due to the emerging threat of antibiotic resistance. This urgency to develop new antibiotics has motivated researchers to find new methods to combat pathogenic microorganisms resulting in a surge of research focused around antimicrobial peptides (AMPs; also termed host defense peptides) and their potential as therapeutics. During the past few decades, more than 2000 AMPs have been identified from a diverse range of organisms (animals, fungi, plants, and bacteria). While these AMPs share a number of common features and a limited number of structural motifs; their sequences, activities, and targets differ considerably. In addition to their antimicrobial effects, AMPs can also exhibit immunomodulatory, anti-biofilm, and anticancer activities. These diverse functions have spurred tremendous interest in research aimed at understanding the activity of AMPs, and various protocols have been described to assess different aspects of AMP function including screening and evaluating the activities of natural and synthetic AMPs, measuring interactions with membranes, optimizing peptide function, and scaling up peptide production. Here, we provide a general overview of AMPs and introduce some of the methodologies that have been used to advance AMP research.

The Bleomycin Model of Pulmonary Fibrosis

Abstract: Interstitial lung disease (ILD) comprises a large number of chronic lung disease characterized by varying degrees of inflammation and fibrosis. Mostly they are idiopathic including idiopathic pulmonary fibrosis (IPF), which is a specific disorder characterized by progressive fibrosis leading commonly to end-stage lung disease, respiratory failure, and fatal outcome. IPF and many of these fibrotic ILDs lack effective therapy despite recent approval of two drugs to slow progression in certain IPF patients. Because there are no natural models for IPF, the use of animal models that reproduce key known features of the disease is warranted. Thus, different animal models have been developed to investigate key mechanisms underlying pathogenesis of pulmonary fibrosis and identify potential therapeutic targets for IPF. While no animal model can recapitulate all features of human disease, several are available to address select features of IPF and other fibrotic ILDs. Historically, among the first to be developed and used widely is the bleomycin model, which is the best-characterized and currently most extensively used animal model due to its ability to reproduce many aspects of IPF and other fibrotic ILDs, good reproducibility, and ease of induction. Studies using the bleomycin model have identified many of the cellular and molecular mechanisms now recognized as being important in pathogenesis of IPF and other fibrotic ILDs, as well as novel therapies for these diseases, including two recent drugs approved for treatment of IPF. This chapter will describe commonly used techniques for induction of the model by endotracheal administration of bleomycin through surgical and nonsurgical (transoral instillation).

Protein bioinformatics databases and resources.

Abstract: In the past decades, a variety of publicly available data repositories and resources have been developed to support protein related information management, data-driven hypothesis generation and biological knowledge discovery. However, there is also an increasing confusion for the researchers who are trying to quickly find the appropriate resources to help them solve their problems. In this chapter, we present a comprehensive review (with categorization and description) of major protein bioinformatics databases and resources that are relevant to comparative proteomics research. We conclude the chapter by discussing the challenges and opportunities for developing new protein bioinformatics databases.

SPIDER2: A Package to Predict Secondary Structure, Accessible Surface Area, and Main-Chain Torsional Angles by Deep Neural Networks

Abstract: Predicting one-dimensional structure properties has played an important role to improve prediction of protein three-dimensional structures and functions. The most commonly predicted properties are secondary structure and accessible surface area (ASA) representing local and nonlocal structural characteristics, respectively. Secondary structure prediction is further complemented by prediction of continuous main-chain torsional angles. Here we describe a newly developed method SPIDER2 that utilizes three iterations of deep learning neural networks to improve the prediction accuracy of several structural properties simultaneously. For an independent test set of 1199 proteins SPIDER2 achieves 82 % accuracy for secondary structure prediction, 0.76 for the correlation coefficient between predicted and actual solvent accessible surface area, 19° and 30° for mean absolute errors of backbone φ and ψ angles, respectively, and 8° and 32° for mean absolute errors of Cα-based θ and τ angles, respectively. The method provides state-of-the-art, all-in-one accurate prediction of local structure and solvent accessible surface area. The method is implemented, as a webserver along with a standalone package that are available in our website: http://sparks-lab.org .

MicroRNAs: Biomarkers, Diagnostics, and Therapeutics.

Abstract: MicroRNAs (miRNAs) are small noncoding RNAs (21-23 nucleotides in length) that regulate gene expression at translational or posttranslational levels. The major regulatory mechanisms include translational repression or mRNA degradation (Filipowicz et al., Curr Opin Struct Biol 15:331-341, 2005).Aberrant expression of miRNAs has been found to be associated with a variety of human diseases such as cancers/tumors, diabetes, viral infections, cardiovascular diseases, neurodegenerative diseases, and other diseases (Wang et al., J Cell Physiol 23:25-30, 2016; Lawrie, MicroRNAs in medicine, 2013). The expression of miRNAs is tissue specific and can be used to identify tumor type and its origin (Mishra and Merlino, J Clin Invest 119:2119-2123, 2009). Many investigations suggest that the miRNA-expression profiles are novel diagnostic and prognostic biomarkers for multiple human diseases. Manipulating relevant miRNA expression or function may serve as potential therapeutic strategies for different diseases.

Fusarium Species and Their Associated Mycotoxins

Abstract: The genus Fusarium includes numerous toxigenic species that are pathogenic to plants or humans, and are able to colonize a wide range of environments on earth. The genus comprises around 70 well-known species, identified by using a polyphasic approach, and as many as 300 putative species, according to phylogenetic species concepts; many putative species do not yet have formal names. Fusarium is one of the most economically important fungal genera because of yield loss due to plant pathogenic activity; mycotoxin contamination of food and feed products which often render them unaccep for marketing; and health impacts to humans and livestock, due to consumption of mycotoxins. Among the most important mycotoxins produced by species of Fusarium are the trichothecenes and the fumonisins. Fumonisins cause fatal livestock diseases and are considered potentially carcinogenic mycotoxins for humans, while trichothecenes are potent inhibitors of protein synthesis. This chapter summarizes the main aspects of morphology, pathology, and toxigenicity of the main Fusarium species that colonize different agricultural crops and environments worldwide, and cause mycotoxin contamination of food and feed.

Development of the CpG Adjuvant 1018: A Case Study.

Abstract: The development of aluminum salts (alum) as vaccine adjuvants was an empirical process with little understanding of the mechanism of action and, with decades of use, it has become clear that there is a need for alternatives where alum-based adjuvants are suboptimal. Oligonucleotides containing unmethylated CpG sequences represent one alternative as they are potent stimulators of the vertebrate innate immune system through activation of Toll-like receptor-9. This chapter outlines the methods used by Dynavax Technologies to progress a CpG-containing oligonucleotide sequence termed 1018 through preclinical and clinical testing as an adjuvant for immunization against hepatitis B virus (HBV). 1018 is a short (22-mer) oligonucleotide sequence containing CpG motifs active in both rodents and primates. Preclinical testing of hepatitis B surface antigen (HBsAg) + 1018 in comparison to HBsAg + alum demonstrated induction of substantially higher antibody titers and a favorable safety profile for 1018. Most importantly, clinical studies with HBsAg vaccination consistently demonstrate more rapid induction of protective antibody titers with 1018 compared to alum in all populations studied, including groups that are harder to immunize such as the elderly and immunocompromised individuals. These studies represent the basis for use of the CpG-motif-containing oligonucleotide 1018 as an improved adjuvant for HBsAg immunogenicity. HBsAg + 1018 (HEPLISAV-B™) is currently in late-stage clinical testing for prophylactic immunization against HBV.

GeneNetwork: A Toolbox for Systems Genetics

Abstract: The goal of systems genetics is to understand the impact of genetic variation across all levels of biological organization, from mRNAs, proteins, and metabolites, to higher-order physiological and behavioral traits. This approach requires the accumulation and integration of many types of data, and also requires the use of many types of statistical tools to extract relevant patterns of covariation and causal relations as a function of genetics, environment, stage, and treatment. In this protocol we explain how to use the GeneNetwork web service, a powerful and free online resource for systems genetics. We provide workflows and methods to navigate massive multiscalar data sets and we explain how to use an extensive systems genetics toolkit for analysis and synthesis. Finally, we provide two detailed case studies that take advantage of human and mouse cohorts to evaluate linkage between gene variants, addiction, and aging.

The Gene Ontology and the Meaning of Biological Function.

Abstract: The Gene Ontology (GO) provides a framework and set of concepts for describing the functions of gene products from all organisms. It is specifically designed for supporting the computational representation of biological systems. A GO annotation is an association between a specific gene product and a GO concept, together making a statement pertinent to the function of that gene. However, the meaning of the term "function" is not as straightforward as it might seem, and has been discussed at length in both philosophical and biological circles. Here, I first review these discussions. I then present an explicit formulation of the biological model that underlies the GO and annotations, and discuss how this model relates to the broader debates on the meaning of biological function.

Method for Picrosirius Red-Polarization Detection of Collagen Fibers in Tissue Sections.

Abstract: The extracellular matrix is critical in guiding cell behavior in normal and pathologic tissues, particularly in cancers and fibrosis. Highlighting the organization (or disorganization) of a collagen-containing matrix can be very useful for understanding or grading pathophysiological conditions. The picrosirius red stain (also called "Sirius red" stain) is one of the best understood histochemical techniques able to selectively highlight collagen networks. Relatively inexpensive, the technique relies on the birefringent properties of collagen molecules. While the picrosirius red stain alone does not selectively bind collagen network, it becomes more specific than the other common collagen stains when combined with polarized light detection. This is why the selective histochemical procedure for collagen detection should be called the picrosirius-polarization method. In this chapter, we will provide essential explanation and detailed protocols and tips to allow collagen researchers not only to better understand how this staining technique works but also to easily apply this technique to their collagen-related research.

MicroRNAs, Long Noncoding RNAs, and Their Functions in Human Disease.

Abstract: Majority of the human genome is transcribed into RNAs with absent or limited protein-coding potential. microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are two major families of the non-protein-coding transcripts. miRNAs and lncRNAs can regulate fundamental cellular processes via diverse mechanisms. The expression and function of miRNAs and lncRNAs are tightly regulated in development and physiological homeostasis. Dysregulation of miRNAs and lncRNAs is critical to pathogenesis of human disease. Moreover, recent evidence indicates a cross talk between miRNAs and lncRNAs. Herein we review recent advances in the biology of miRNAs and lncRNAs with respect to the above aspects. We focus on their roles in cancer, respiratory disease, and neurodegenerative disease. The complexity, flexibility, and versatility of the structures and functions of miRNAs and lncRNAs demand integration of experimental and bioinformatics tools to acquire sufficient knowledge for applications of these noncoding RNAs in clinical care.

Protein-Protein Interactions: Co-Immunoprecipitation.

Abstract: Proteins often do not function as single substances but rather as team players in a dynamic network. Growing evidence shows that protein-protein interactions are crucial in many biological processes in living cells. Genetic (such as yeast two-hybrid, Y2H) and biochemical (such as co-immunoprecipitation, co-IP) methods are the methods commonly used at the beginning of a study to identify the interacting proteins. Immunoprecipitation (IP), a method using a target protein-specific antibody in conjunction with Protein A/G affinity beads, is a powerful tool to identify molecules that interact with specific proteins. Therefore, co-IP is considered to be one of the standard methods of identifying or confirming the occurrence of protein-protein interaction events in vivo. Co-IP experiments can identify proteins via direct or indirect interactions or in a protein complex. Here, we use Agrobacterium type VI secretion system (T6SS) sheath components TssB-TssC41 interaction as an example to describe the principle, procedure, and experimental problems of co-IP.

Isolation of Extracellular Vesicles by Ultracentrifugation.

Abstract: Extracellular vesicles (EVs) represent a group of heterogeneous vesicles that can be obtained from almost all biofluids. EVs, including microvesicles, exosomes, and apoptotic bodies , can deliver bioactive cargos and signaling molecules. Various physiological roles and pathophysiological roles for EVs in diseases such as cancer, infectious diseases, endocrine diseases, and neurodegenerative disorders have been recognized. These observations highlight EVs as potential novel biomarkers and targets for therapeutic intervention. One of the major limitations in the use of EVs for diagnosis and therapeutic purposes is the lack of standardization of isolation techniques. Here, we describe protocols for ultracentrifugation and sucrose gradient isolation methods, which are the current gold standard, and are the most studied methods for EV isolation.

Methods for Quantifying Fibrillar Collagen Alignment

Abstract: Recent evidence has implicated collagen, particularly fibrillar collagen, in a number of diseases ranging from osteogenesis imperfecta and asthma to breast and ovarian cancer. A key property of collagen that has been correlated with disease has been the alignment of collagen fibers. Collagen can be visualized using a variety of imaging techniques including second-harmonic generation (SHG) microscopy, polarized light microscopy, and staining with dyes or antibodies. However, there exists a great need to easily and robustly quantify images from these modalities for individual fibers in specified regions of interest and with respect to relevant boundaries. Most currently available computational tools rely on calculation of pixel-wise orientation or global window-wise orientation that do not directly calculate or give visible fiber-wise information and do not provide relative orientation against boundaries. We describe and detail how to use a freely available, open-source MATLAB software framework that includes two separate but linked packages "CurveAlign" and "CT-FIRE" that can address this need by either directly extracting individual fibers using an improved fiber tracking algorithm or directly finding optimal representation of fiber edges using the curvelet transform. This curvelet-based framework allows the user to measure fiber alignment on a global, region of interest, and fiber basis. Additionally, users can measure fiber angle relative to manually or automatically segmented boundaries. This tool does not require prior experience of programming or image processing and can handle multiple files, enabling efficient quantification of collagen organization from biological datasets.

Docking and Virtual Screening in Drug Discovery.

Abstract: Stages in a typical drug discovery organization include target selection, hit identification, lead optimization, preclinical and clinical studies. Hit identification and lead optimization are very much intertwined with computational modeling. Structure-based virtual screening (VS) has been a staple for more than a decade now in drug discovery with its underlying computational technique, docking, extensively studied. Depending on the objective, the parameters for VS may change, but the overall protocol is very straightforward. The idea behind VS is that a library of small compounds are docked into the binding pocket of a protein (e.g., receptor, enzyme), a number of solutions per molecule, among the top-ranked, are being returned, and a choice is made on the fraction of compounds to be moved forward for testing toward hit identification. The underlying principle of VS is that it differentiates between active and inactive compounds, thus reducing the number of molecules moving forward and possibly offering a complementary tool to high-throughput screening (HTS). Best practices in library selection, target preparation and refinement, criteria in selecting the most appropriate docking/scoring scheme, and a step-wise approach in performing Glide VS are discussed.

Human Fibrotic Diseases: Current Challenges in Fibrosis Research.

Abstract: Human fibrotic diseases constitute a major health problem worldwide owing to the large number of affected individuals, the incomplete knowledge of the fibrotic process pathogenesis, the marked heterogeneity in their etiology and clinical manifestations, the absence of appropriate and fully validated biomarkers, and, most importantly, the current void of effective disease-modifying therapeutic agents. The fibrotic disorders encompass a wide spectrum of clinical entities including systemic fibrotic diseases such as systemic sclerosis (SSc), sclerodermatous graft vs. host disease, and nephrogenic systemic fibrosis, as well as numerous organ-specific disorders including radiation-induced fibrosis and cardiac, pulmonary, liver, and kidney fibrosis. Although their causative mechanisms are quite diverse and in several instances have remained elusive, these diseases share the common feature of an uncontrolled and progressive accumulation of fibrotic tissue in affected organs causing their dysfunction and ultimate failure. Despite the remarkable heterogeneity in the etiologic mechanisms responsible for the development of fibrotic diseases and in their clinical manifestations, numerous studies have identified activated myofibroblasts as the common cellular element ultimately responsible for the replacement of normal tissues with nonfunctional fibrotic tissue. Critical signaling cascades, initiated primarily by transforming growth factor-β (TGF-β), but also involving numerous cytokines and signaling molecules which stimulate profibrotic reactions in myofibroblasts, offer potential therapeutic targets. Here, we briefly review the current knowledge of the molecular mechanisms involved in the development of tissue fibrosis and point out some of the most important challenges to research in the fibrotic diseases and to the development of effective therapeutic approaches for this often fatal group of disorders. Efforts to further clarify the complex pathogenetic mechanisms of the fibrotic process should be encouraged to attain the elusive goal of developing effective therapies for these serious, untreatable, and often fatal disorders.

Detecting and Analyzing Genetic Recombination Using RDP4.

Abstract: Recombination between nucleotide sequences is a major process influencing the evolution of most species on Earth. The evolutionary value of recombination has been widely debated and so too has its influence on evolutionary analysis methods that assume nucleotide sequences replicate without recombining. When nucleic acids recombine, the evolution of the daughter or recombinant molecule cannot be accurately described by a single phylogeny. This simple fact can seriously undermine the accuracy of any phylogenetics-based analytical approach which assumes that the evolutionary history of a set of recombining sequences can be adequately described by a single phylogenetic tree. There are presently a large number of available methods and associated computer programs for analyzing and characterizing recombination in various classes of nucleotide sequence datasets. Here we examine the use of some of these methods to derive and test recombination hypotheses using multiple sequence alignments.

NMDA Receptors in the Central Nervous System.

Abstract: NMDA-type glutamate receptors are ligand-gated ion channels that mediate a major component of excitatory neurotransmission in the central nervous system (CNS). They are widely distributed at all stages of development and are critically involved in normal brain functions, including neuronal development and synaptic plasticity. NMDA receptors are also implicated in the pathophysiology of numerous neurological and psychiatric disorders, such as ischemic stroke, traumatic brain injury, Alzheimer's disease, epilepsy, mood disorders, and schizophrenia. For these reasons, NMDA receptors have been intensively studied in the past several decades to elucidate their physiological roles and to advance them as therapeutic targets. Seven NMDA receptor subunits exist that assemble into a diverse array of tetrameric receptor complexes, which are differently regulated, have distinct regional and developmental expression, and possess a wide range of functional and pharmacological properties. The diversity in subunit composition creates NMDA receptor subtypes with distinct physiological roles across neuronal cell types and brain regions, and enables precise tuning of synaptic transmission. Here, we will review the relationship between NMDA receptor structure and function, the diversity and significance of NMDA receptor subtypes in the CNS, as well as principles and rules by which NMDA receptors operate in the CNS under normal and pathological conditions.