Proteinase inhibitors of pigeonpea cv. BSMR 736: Characterization and bioefficacy against Helicoverpa armigera

Pigeonpea is an agriculturally important leguminous crop with high vulnerability to insect pest attack specifically, Helicoverpa armigera. The proteinase inhibitors (PIs) mediated host plant resistance against insect pests is a promising sustainable agricultural research practice. The current study was carried out to perceive biochemical characterization of proteinase inhibitors named PPTI in the pigeonpea (cv. BSMR 736). The purification of PPTI from crude protein seed extract was achieved by acetone precipitation, N-LP-IEF, and trypsin affinity chromatography. It was found to inhibit bovine trypsin and HaGPs in vitro. The optimal conditions for inhibition were pH 8 and temperature 40ºC. The PPTI showed four isoinhibitors bands on native, non-reducing and reducing SDS-PAGE in the range of 26.7–19.3 KDa. Upon resolution on two-dimensional gel electrophoresis (2-DE), PPTI produced nine pI variant spots having isoelectric point (pI) 6.6, 6.6, 6.3, 6.1, 5.9, 5.8, 5.7, 5.6 and 5.6. An artificial diet containing PPTI reduced the H. armigera larval weight about 69%, with 25% mortality. For eco-friendly sustainable agricultural practices, natural compounds like PPTI could be expressed in transgenic crops to prevent the invasion of H. armigera in pigeonpea

The genome of the protozoan parasite Cystoisospora suis and a reverse vaccinology approach to identify vaccine candidates

Vaccine development targeting protozoan parasites remains challenging, partly due to the complex interactions between these eukaryotes and the host immune system. Reverse vaccinology is a promising approach for direct screening of genome sequence assemblies for new vaccine candidate proteins. Here, we applied this paradigm to Cystoisospora suis, an apicomplexan parasite that causes enteritis and diarrhea in suckling piglets and economic losses in pig production worldwide. Using Next Generation Sequencing we produced an ∼84 Mb sequence assembly for the C. suis genome, making it the first available reference for the genus Cystoisospora. Then, we derived a manually curated annotation of more than 11,000 protein-coding genes and applied the tool Vacceed to identify 1,168 vaccine candidates by screening the predicted C. suis proteome. To refine the set of candidates, we looked at proteins that are highly expressed in merozoites and specific to apicomplexans. The stringent set of candidates included 220 proteins, among which were 152 proteins with unknown function, 17 surface antigens of the SAG and SRS gene families, 12 proteins of the apicomplexan-specific secretory organelles including AMA1, MIC6, MIC13, ROP6, ROP12, ROP27, ROP32 and three proteins related to cell adhesion. Finally, we demonstrated in vitro the immunogenic potential of a C. suis-specific 42 kDa transmembrane protein, which might constitute an attractive candidate for further testing

Empirical Investigations OF RNA Fitness Landscapes: Harnessing the Power of High-Throughput Sequencing and Evolutionary Simulations

Fitness landscapes or adaptive landscapes represent the mapping of genotype (sequence) to phenotype (function or fitness). Originally proposed as a metaphor to envision evolutionary processes and mutational interactions, the fitness landscape has recently transitioned from theoretical to empirical. This is due in part to advances in DNA synthesis and high-throughput sequencing. This allows for the construction and analysis of empirical fitness landscapes that encompass thousands of genotypes. These landscapes provide tractable insight into mutational pathways, the predictability of evolution or even the evolution of life. RNA enzymes (ribozymes) are an attractive model system for the construction of empirical fitness landscapes. Ribozymes function as both a genotype (primary RNA sequence) and a phenotype (catalytic function). To construct and characterize empirical RNA fitness landscapes, two high-throughput functional assays (self-cleavage and self-ligation), including a technique to improve data recovery from high-throughput sequencing using phased nucleotide inserts (Appendix A), were developed and implemented. Following fitness landscape construction, a stochastic evolutionary model was developed and employed based on the Wright-Fisher model. This model follows the principles of Darwinian evolution and allows a population to explore the fitness landscape by means of mutation and selection. These newly developed tools allowed for a novel approach to important evolutionary questions. Chapter 1 explored the evolution of innovation at the intersection of two ribozyme functions: self-cleavage and self-ligation. Evolutionary innovations are qualitatively novel traits that emerge through evolution. Theories have suggested that innovations can occur where two genotype networks are in close proximity. However, only isolated examples of intersections have been investigated. The fitness landscape between the two ribozyme functions was explored by determining the ability of numerous neighboring RNA sequences to catalyze two different chemical reactions. This revealed that there was extensive functional overlap, and over half the genotypes can catalyze both functions to some extent. Data-driven evolutionary simulations found that these numerous points of intersection facilitated the discovery of a new function, yet the rate of optimization depended upon the starting location in the genotype network. This study constructed a fitness landscape where genotype networks intersect and uncovered the implications for evolutionary innovations. Chapter 2 determined the effect of higher sequence space complexity and dimensionality on evolutionary adaptation in RNA fitness landscapes. The complexity and dimensionality of landscapes scale with the length of the RNA molecule. For this study, complexity was defined as the size of the genotype space and dimensionality as the number of edges connecting each genotype (node) to other genotypes that differ by a single mutation. Low-dimensional ‘direct’ landscapes consisting of only two possible nucleotides at various positions were compared to higher-dimensional ‘indirect’ landscapes that had all four nucleotides at the same positions. Indirect pathways contributed to the ruggedness and navigability of landscapes. Increased dimensionality in RNA fitness landscapes had the potential to circumvent fitness valleys, however indirect pathways also harbored stasis genotypes isolated by reciprocal sign epistasis. Chapter 3 applied ancestral sequence resurrection and fitness landscape construction to naturally evolved ribozymes. The CPEB3 ribozyme is highly conserved in mammals and has been linked to episodic memory. By predicting, ‘resurrecting’ and functionally characterizing ancient gene sequences, hypotheses about gene function or selection can be empirically tested in an evolutionary context. Using the extant ribozyme sequences found in a range of mammalian species as a basis for inference of ancestral sequences, a phylogenetic fitness landscape was experimentally resurrected and reconstructed. A single high-activity ancestral sequence was found to be highly conserved and purifying selection is expected to have reduced the accumulation of mutations through geologic time. Many of the extant mammalian ribozyme sequences had high ribozyme activity, however a few had relatively low activity. Yet, given the local fitness landscape, a selective pressure for functional ribozyme sequences was seen. A single nucleotide polymorphism (SNP) found in humans, reduced co-transcriptional ribozyme activity in vitro and might alter our understanding of the CPEB3 ribozyme’s biological function. Chapter 4 analyzed epistatic interactions in four published RNA fitness landscapes generated from high-throughput analyses. Two of the landscapes were assessed in vivo and two were assessed in vitro. Epistasis occurs when the effects of some mutations are dependent on the presence or absence of other mutations. The data allowed for an analysis of the distribution of fitness effects of individual mutations as well as combinations of two or more mutations. Two different approaches to measuring epistasis in the data both revealed a predominance of negative epistasis, such that higher combinations of two or more mutations are typically lower in fitness than expected from the effect of each individual mutation. This finding differed from studies using computationally predicted RNA but is similar to mutational experiments in protein enzymes. The work presented here represents a significant contribution to our ability to construct and empirically characterize RNA fitness landscapes. The development of two high-throughput ribozyme assays opens the door for further empirical landscape construction. The implementation of data-driven stochastic evolutionary modeling allows for a clearer evolutionary characterization of the landscape. Understanding the connection between genotype and phenotype in RNA systems is important for designing RNA functions, improving in vitro selections and understanding the origins and evolution of new RNA functions (innovations). Applying these advances yielded valuable information about evolutionary innovations, the effects of higher dimensionality, evolution of extant ribozymes and the prevalence of epistasis in RNA fitness landscapes. Construction and analysis of empirical RNA fitness landscapes provides tractable insight into evolutionary processes, mutational pathways and the predictability of evolution

Spot the difference: Causal contrasts in scientific diagrams

An important function of scientific diagrams is to identify causal relationships. This commonly relies on contrasts that highlight the effects of specific difference-makers. However, causal contrast diagrams are not an obvious and easy to recognize category because they appear in many guises. In this paper, four case studies are presented to examine how causal contrast diagrams appear in a wide range of scientific reports, from experimental to observational and even purely theoretical studies. It is shown that causal contrasts can be expressed in starkly different formats, including photographs of complexly visualized macromolecules as well as line graphs, bar graphs, or plots of state spaces. Despite surface differences, however, there is a measure of conceptual unity among such diagrams. In empirical studies they generally serve not only to infer and communicate specific causal claims, but also as evidence for them. The key data of some studies is given nowhere except in the diagrams. Many diagrams show multiple causal contrasts in order to demonstrate both that an effect exists and that the effect is specific -- that is, to narrowly circumscribe the phenomenon to be explained. In a large range of scientific reports, causal contrast diagrams reflect the core epistemic claims of the researchers

Disease-specific, neurosphere-derived cells as models for brain disorders

There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson's disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson's disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery

Proteomics: Challenges, Techniques and Possibilities to Overcome Biological Sample Complexity

Proteomics is the large-scale study of the structure and function of proteins in complex biological sample. Such an approach has the potential value to understand the complex nature of the organism. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. Advances in protein fractionation and labeling techniques have improved protein identification to include the least abundant proteins. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. However, the major limitation of proteomic investigations remains the complexity of biological structures and physiological processes, rendering the path of exploration paved with various difficulties and pitfalls. The quantity of data that is acquired with new techniques places new challenges on data processing and analysis. This article provides a brief overview of currently available proteomic techniques and their applications, followed by detailed description of advantages and technical challenges. Some solutions to circumvent technical difficulties are proposed

The Bronchoalveolar Lavage Proteome- Phenotypic associations to smoking and divergence towards development of COPD

Proteomic analysis of bronchoalveolar lavage (BAL) fluid from smokers at risk of developing chronic obstructive pulmonary disease (COPD) and never smokers is described. COPD is currently the world's fourth leading cause of death and its prevalence is increasing. The leading cause of COPD is smoking and an estimated 600 million people in the world suffer from COPD which makes it the world's most common chronic disease. The aim of this thesis was to explore and characterize the BAL proteome of never smokers and smokers. The hypotheses were that the BAL proteome reflect smoking habits in subjects, and that smokers susceptible to COPD development have a specific proteome. In order to relate the measurement of protein expression with clinical phenotypes we have developed and utilized an interdisciplinary toolbox that includes protein separation (two-dimensional gel electrophoresis and liquid chromatography), mass spectrometry identification and statistical methods for multivariate analysis. The study material used in this thesis consisted of age matched men all born in 1933, living in one city differing by lifelong smoking history. These were compared by clinical function measurements and histological assessment at the same relative time points. A follow up study after 6-7 years identified a group of subjects who had progressed to COPD GOLD stage 2. Those with COPD shared a distinct protein expression profile in the baseline BAL sample which could be identified using multivariate analysis. This pattern was not observed in BAL samples of asymptomatic smokers free of COPD at the 6-7 year follow-up. The results suggest that specific patterns of protein expression occur in the airways of smokers susceptible to COPD disease progression, before the disease is clinically measurable

GENOMIC, PROTEOMIC AND METABOLOMIC APPROACHES TO STUDY DROUGHT RESPONSES IN AQUILEGIA

Global population is expected to increase 30% by 2040, which will result in an increased need for crop production to feed the growing population. Combined with projected increased drought conditions worldwide, plant genetic research is necessary to gain a deeper knowledge of the molecular factors involved in plant drought response in order to engineer crop species with improved drought tolerance. Aquilegia has been recently developed as a model species for gene exploration based on its ability to thrive in a wide variety of environments including arid locations. An attractive asset of Aquilegia is its evolutionary position, equidistant between rice and Arabidopsis. Multifaceted molecular biology techniques were employed in these studies to identify biological components associated with Aquilegia\u27s response to drought. Techniques utilized were; suppression subtractive hybridization, using drought-stressed and un-stressed tissue-derived mRNAs to selectively amplify differentially expressed genes, metabalomic profiling as a means to examine the identity and possible function of accumulated metabolites, and exploration of proteomes obtained under different levels of drought stress. Subtractive hybridization yielded numerous sequences such as DREB known to be involved with drought and other genes with no known association to drought response. The metabalomics approach yielded vitexin and vitexin 2\u27-O-β-D-glucoside, identified via LCMS and HPLC. Vitexin concentration within Aquilegia leaf tissue increased 357% over the time course of the drought experiment. Previously, vitexin had not been associated with drought response. Proteome analysis identified various proteins known to play some defensive role in drought stress. This parallel initiative yielded numerous possible gene candidates for genetic engineering of crop species. Results from this project confirm Aquilegia\u27s role as an excellent genetic resource for studying responses to a wide range of abiotic stresses, specifically drought

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

<p>Abstract</p> <p>Background</p> <p>Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from <it>M. bovis </it>BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.</p> <p>Results</p> <p>The 2DE proteomic map of <it>M. bovis </it>BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern.</p> <p>Conclusions</p> <p>Here we report the detailed 2DE profile of CFPs from <it>M. bovis </it>BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.</p