VTechWorks Repository :: Browsing by Author "Helm, Richard F."

Browsing by Author "Helm, Richard F."

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Algorithms for Storytelling
Kumar, Deept; Ramakrishnan, Naren; Helm, Richard F.; Potts, Malcolm (Department of Computer Science, Virginia Polytechnic Institute & State University, 2006)
We formulate a new data mining problem called "storytelling" as a generalization of redescription mining. In traditional redescription mining, we are given a set of objects and a collection of subsets defined over these objects. The goal is to view the set system as a vocabulary and identify two expressions in this vocabulary that induce the same set of objects. Storytelling, on the other hand, aims to explicitly relate object sets that are disjoint (and hence, maximally dissimilar) by finding a chain of (approximate) redescriptions between the sets. This problem finds applications in bioinformatics, for instance, where the biologist is trying to relate a set of genes expressed in one experiment to another set, implicated in a different pathway. We outline an efficient storytelling implementation that embeds the CARTwheels redescription mining algorithm in an A* search procedure, using the former to supply next move operators on search branches to the latter. This approach is practical and effective for mining large datasets and, at the same time, exploits the structure of partitions imposed by the given vocabulary. Three application case studies are presented: a study of word overlaps in large English dictionaries, exploring connections between genesets in a bioinformatics dataset, and relating publications in the PubMed index of abstracts.
Analysis of T-DNA alleles of flavonoid biosynthesis genes in Arabidopsis ecotype Columbia
Bowerman, Peter A.; Ramirez, Melissa V.; Price, Michelle B.; Helm, Richard F.; Winkel, Brenda S. J. (2012-09-04)
BACKGROUND: The flavonoid pathway is a long-standing and important tool for plant genetics, biochemistry, and molecular biology. Numerous flavonoid mutants have been identified in Arabidopsis over the past several decades in a variety of ecotypes. Here we present an analysis of Arabidopsis lines of ecotype Columbia carrying T-DNA insertions in genes encoding enzymes of the central flavonoid pathway. We also provide a comprehensive summary of various mutant alleles for these structural genes that have been described in the literature to date in a wide variety of ecotypes. FINDINGS: The confirmed knockout lines present easily-scorable phenotypes due to altered pigmentation of the seed coat (or testa). Knockouts for seven alleles for six flavonoid biosynthetic genes were confirmed by PCR and characterized by UPLC for altered flavonol content. CONCLUSION: Seven mutant lines for six genes of the central flavonoid pathway were characterized in ecotype, Columbia. These lines represent a useful resource for integrating biochemical and physiological studies with genomic, transcriptomic, and proteomic data, much of which has been, and continues to be, generated in the Columbia background.
Aspects of amidization of chitosan
Toffey, Ackah (Virginia Tech, 1996)
The intent of this research was to develop an understanding of an amidized chitosan-from-chitosan regeneration process discovered in our laboratory. In this study several characterization methods including DMTA, TMA, TGA, X-ray diffraction, FTIR, solid state CP-MAS ¹³C NMR, and HPLC were used to study the transformation of various ionic complexes of chitosan (N-acylate) to their respective N-acyl homologs of chitosan; and several properties of these materials were examined. DMTA and TMA provided information on changes in T_g as well as modulus-changes and glass formation underlying the transformation of the N-acylate to the N-acyl derivative. X-ray diffraction and FTIR shed some insights on the morphology of the N-acetyl homolog of chitosan in relation to native chitin. Solid state CP-MAS ¹³C NMR provided evidence of the conversion of N-acylate to N-acetyl. Enzymatic hydrolysis of native chitin and amidized chitosan homologs and subsequent identification of fractions by HPLC allowed a comparison of various amidized chitosan homologs in terms of their recognition and degradation by chitinolytic enzymes. Solid state CP-MAS ¹³C showed that the heat treatment of the ionic complex of chitosan results in thermal dehydration leading to the formation of the N-acetyl group at the C-2 of chitin. The DS of amidized chitosan varied between 0.1 and 0.6. T_g-changes with time and heating temperature were used as a variable to monitor amidization. Kinetics analysis indicated that the amidization of various ionic complexes of chitosan is a first order, two-phase process with activation energies of 14±1 kcal/mol and 21±2 kcal/mol for the first and second phase, respectively. These values did not vary with the type of acid used in the formation of the chitosan complex. This two-phase behavior is explained with the influence of vitrification on chain mobility. In situ DMTA was found to be a suitable technique for monitoring the phase transformation of chitosonium acetate and chitosonium propionate from a rubbery to a glassy phase (vitrification). Consequently, the concept of TTT-cure diagram analysis was used to describe such phase changes and map out vitrification and full cure curves. As in thermosets, the vitrification curve describing glass formation in these materials is S-shaped. The time to full cure decreased with increasing heating temperature. The activation energy for vitrification is the same irrespective of the type of acid used in the preparation of chitosan complex. Thermal analysis revealed that the T_g of N-acyl homologs of chitin displays a stepwise relationship with length of N-acyl substituent. These materials are characterized by two transitions designated as β- and α-relaxation. Additionally, enzymatic hydrolysis of N-acyl homologs of chitosan using an enzyme mixture of chitinase, chitosanase, and β-N-acetylglucosaminidase and subsequent identification of fractions revealed that these enzymes recognize and degrade chitin irrespective of the N-acyl substituent at the C-2 position of chitin at any DS.
Automatic Reconstruction of the Building Blocks of Molecular Interaction Networks
Rivera, Corban G. (Virginia Tech, 2008-08-11)
High-throughput whole-genome biological assays are highly intricate and difficult to interpret. The molecular interaction networks generated from evaluation of those experiments suggest that cellular functions are carried out by modules of interacting molecules. Reverse-engineering the modular structure of cellular interaction networks has the promise of significantly easing their analysis. We hypothesize that: • cellular wiring diagrams can be decomposed into overlapping modules, where each module is a set of coherently-interacting molecules and • a cell responds to a stress or a stimulus by appropriately modulating the activities of a subset of these modules. Motivated by these hypotheses, we develop models and algorithms that can reverse-engineer molecular modules from large-scale functional genomic data. We address two major problems: 1. Given a wiring diagram and genome-wide gene expression data measured after the application of a stress or in a disease state, compute the active network of molecular interactions perturbed by the stress or the disease. 2. Given the active networks for multiple stresses, stimuli, or diseases, compute a set of network legos, which are molecular modules with the property that each active network can be expressed as an appropriate combination of a subset of modules. To address the first problem, we propose an approach that computes the most-perturbed subgraph of a curated pathway of molecular interactions in a disease state. Our method is based on a novel score for pathway perturbation that incorporates both differential gene expression and the interaction structure of the pathway. We apply our method to a compendium of cancer types. We show that the significance of the most perturbed sub-pathway is frequently larger than that of the entire pathway. We identify an association that suggests that IL-2 infusion may have a similar therapeutic effect in bladder cancer as it does in melanoma. We propose two models to address the second problem. First, we formulate a Boolean model for constructing network legos from a set of active networks. We reduce the problem of computing network legos to that of constructing closed biclusters in a binary matrix. Applying this method to a compendium of 13 stresses on human cells, we automatically detect that about four to six hours after treatment with chemicals cause endoplasmic reticulum stress, fibroblasts shut down the cell cycle far more aggressively than fibroblasts or HeLa cells do in response to other treatments. Our second model represents each active network as an additive combination of network legos. We formulate the problem as one of computing network legos that can be used to recover active networks in an optimal manner. We use existing methods for non-negative matrix approximation to solve this problem. We apply our method to a human cancer dataset including 190 samples from 18 cancers. We identify a network lego that associates integrins and matrix metalloproteinases in ovarian adenoma and other cancers and a network lego including the retinoblastoma pathway associated with multiple leukemias.
Bioinformatic Analysis of Wastewater Metagenomes Reveals Microbial Ecological and Evolutionary Phenomena Underlying Associations of Antibiotic Resistance with Antibiotic Use
Brown, Connor L. (Virginia Tech, 2024-01-17)
Antibiotic resistance (AR) is a pervasive crisis that is intricately woven into social and environmental systems. Its escalation is fueled by factors such overuse, poverty, climate change, and the heightened interconnectedness characteristic of our era of globalization. In this dissertation, the impact of antibiotic usage is addressed from the perspective of wastewater-based surveillance (WBS) at the wastewater treatment plant (WWTP) and microbial ecology. Antibiotic usage and contamination was found to influence the prevalence of antibiotic resistance genes (ARGs) and resistant bacteria in both lab-scale and full-scale wastewater treatment settings. Through application of novel bioinformatic approaches developed herein, metagenomics revealed associations between sewage-associated microbes and community antibiotic use that were in part mediated by microbial ecological processes and horizontal gene transfer (HGT). In sum, this dissertation increases the arsenal of bioinformatic tools for AR surveillance in wastewater environments and advances knowledge with respect to the contribution of antibiotic use to the spread of antibiotic resistance at the community-scale. Three studies served to evaluate and/or develop bioinformatic resources for molecular characterization of AR in wastewater. Hybrid assembly combining emerging long read DNA sequencing and short read sequencing was evaluated and found to improve accuracy relative to assembly of long or short reads alone. A novel database of mobile genetic element (MGE) marker genes, mobileOG-db, was compiled in order to address short-comings with pre-existing resources. A pipeline for detecting HGT in metagenomes, Kairos, was created in order to facilitate the detection of HGT in metagenome assemblies which greatly amplified coverage of ARGs. In Chapter 5, a lab-scale study of WWTP bioreactors revealed that elevated antibiotic contamination was correlated with increased prevalence of corresponding ARGs. In addition, multiple in situ HGT events of ARGs encoding resistance to the elevated antibiotics were predicted, including one HGT event likely mediated by a novel bacteriophage. In Chapter 6, influent and effluent from a full-scale municipal WWTP were collected twice-weekly for one year and subjected to deep shotgun metagenomic sequencing. In parallel, collaboration with clinicians enabled statistical modeling of antibiotic usage and resistance, revealing associations between antibiotic prescriptions patterns in the region and resistance at the WWTP. Finally, Chapter 7 details bioinformatic recovery of diverse extended spectrum beta-lactamase gene recovery from the influent and effluent metagenomes, shedding light on the dynamics of circulating resistance genes. In sum, this dissertation identifies bioinformatic evidence for the selection of AR in wastewater environments as a result of antibiotic use in the community and advances hypotheses for explaining the mechanisms of the observed phenomena.
Carbohydrate-Interacting Proteins from Two Nostoc (Cyanobacteria) Species
Jordan, Brian Robert (Virginia Tech, 2004-05-05)
Cyanobacteria of the Nostoc genus are known for the thick, mucilaginous carbohydrate coatings that they produce. In this work, two examples of cyanobacterial glycobiology are considered, each of which involves a cyanobacterium of the Nostoc genus. The first portion of this work details attempts to obtain amino acid sequence information from the enzymes (glycosyltransferases) that are responsible for producing the extracellular polysaccharide (EPS) of Nostoc commune DRH1, ultimately to allow the transfer of this capacity to another organism. Two artificial substrates were synthesized for use in a capillary electrophoresis-based enzyme assay, which was used to look for glycosyltransferase activity in Nostoc commune DRH1 cell extracts. Glucuronosyltransferase activity was detected in association with Nostoc commune membrane material. The active enzyme displayed a divalent cation metal dependence (Mg+2) that is typical of glycosyltransferase enzymes purified from other organisms. Because the enzyme responsible for this activity held the potential to be EPS-related, its purification was attempted. The capillary electrophoresis-based enzyme assay and a 32P-labeled affinity tag were utilized to follow the glucuronosyltransferase enzyme through successive purification steps. The active enzyme was extracted from Nostoc commune membrane material using Triton X-100, and then purified by anion exchange chromatography. The active detergent extract was extremely unstable, and consequently, other purification techniques tested were unsuccessful in enriching activity. Affinity-labeling experiments indicated that the active enzyme was forming protein aggregates during these procedures, which were not amenable to in-gel protease digestion and peptide analysis by tandem mass spectrometry. The second portion of this work describes an investigation of an Anabaena (Nostoc) PCC 7120 soluble cell extract. Upon separation by sodium dodecyl sulfate Â¡V polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent periodic acid-Schiff (PAS) staining of the resulting gel, the components of this cellular fraction produce a ladder-like pattern, which suggests that the extract may contain glycosylated protein. Analyses of several samples that were taken from within the PAS-staining region of such a gel revealed surface layer homology (SLH) domain-containing proteins, likely candidates to be covalently attached to or non-covalently interacting with carbohydrate. Various protein sequence analyses indicated that the detected SLH domain containing proteins belong to a family of (putative) cyanobacterial porins. Proteins in this family possess features that include a N-terminal signal sequence, a single SLH domain motif, followed by a coiled-coil region, and a C-terminal region that is homologous to the b-barrel-forming region of bacterial porins. All of these features were identified in the detected Anabaena (Nostoc) PCC 7120 SLH domain-containing proteins. Smith degradation was performed on a sample that was electroeluted from the PAS-staining region of a preparative-scale SDS-PAGE gel of the soluble cell extract. Subsequent analyses of the resulting sample by SDS-PAGE and mass spectrometry indicated that at least two SLH domain-containing proteins, encoded by all4499 and alr4550, were non-covalently interacting with the PAS-staining material. Following degradation, the PAS-staining material was still of sufficient size to detected by gel electrophoresis, and it continued to migrate in the absence of an interacting protein component. Protease digestion of a similarly prepared sample, and then subsequent analysis by SDS-PAGE and mass spectrometry, revealed that the region between amino acid residues #504 and #536, in the protein encoded by the alr4550 open reading frame, was interacting with the PAS-staining material. Monosaccharide composition analyses of this material revealed more carbohydrate constituents than are found in cyanobacterial primary (peptidoglycan) cell wall polymer alone, indicating that it contained a significant secondary cell wall polymer component as well.
Characterization of a glycerophosphodiester phosphodiesterase in the human malaria parasite Plasmodium falciparum
Denloye, Titilola Ifeoma (Virginia Tech, 2012-04-25)
Active lipid metabolism is a key process required for the intra-erythrocytic development of the malaria parasite, Plasmodium falciparum. Enzymes that hydrolyze host-derived lipids play key roles in parasite growth, virulence, differentiation, cell-signaling and hemozoin formation. Therefore, investigating enzymes involved in lipid degradation could uncover novel drug targets. We have identified in P. falciparum, a glycerophosphodiester phosphodiesterase (PfGDPD), involved in the downstream pathway of phosphatidylcholine degradation. PfGDPD hydrolyzes deacylated phospholipids, glycerophosphodiesters to glycerol-3-phosphate and choline. In this study, we have characterized PfGDPD using bioinformatics, biochemical and genetic approaches. Knockout experiments showed a requirement for PfGDPD for parasite survival. Sequence analysis revealed PfGDPD possesses the unique GDPD insertion domain sharing a cluster of conserved residues present in other GDPD homologues. We generated yellow fluorescent fusion proteins that revealed a complex distribution of PfGDPD within the parasite cytosol, parasitophorous vacuole and food vacuole. To gain insight into the role of PfGDPD, sub-cellular localization was modulated and resulted in a shift in protein distribution, which elicited no growth phenotype. Kinetic analyses suggest PfGDPD activity is Mg₂⁺ dependent and catalytically efficient at the neutral pH environment of the parasitophorous vacuole. Next, our aim was to determine the upstream pathway that provides deacylated glycerophosphodiesters as substrate for PfGDPD. We identified via bioinformatics, a P. falciparum lysophospholipase (PfLPL1) that directly generates the substrate. Knockout clones were generated and genotyped by Southern and PCR analysis. The effects of PfLPL1 knockouts on parasite fitness were studied, and the results showed that PfLPL1was not required for parasite survival and proliferation.
Characterization of Bacillus Spore Membrane Proteomes and Investigation of Their Roles in the Spore Germination Process
Chen, Yan (Virginia Tech, 2014-09-23)
Components of the bacterial spore germination apparatus are crucial for survival and for initiation of infection by some pathogens. While some components of the germination apparatus are well conserved in spore-forming species, such as the spoVA operon, each species may possess a different and possibly unique germinant recognition mechanism. The significance of several individual proteins in the germination process has been characterized. However, the mechanisms of how these proteins perform their functions and the network connecting these proteins in the complete germination process are still a mystery. In this study, we characterized a Bacillus subtilis superdormant spore population and investigated the abundance of 11 germination-related proteins. The relative quantities of these proteins in dormant, germinating and superdormant spores suggested that variation in the levels of proteins, other than germinant receptor proteins may result in superdormancy. Specifically, variation in the abundance of the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Spore membrane proteomes of Bacillus anthracis and B. subtilis were characterized to generate a candidate protein list that can be further investigated. Proteins that were not previously known to be spore-associated were identified, and many of these proteins shared great similarity in both Bacillus species. A significant number of these proteins are implicated in functions that play major roles in spore formation and germination, such as amino acid or inorganic ion transport and protein fate determination. By analyzing the in vivo and in vitro activity of HtrC, we proved that the protease is responsible for YpeB proteolytic processing at specific sites during germination. However, without HtrC present in the spore, other proteases appear to degrade YpeB at a reduced rate. The activity of purified HtrC in vitro was stimulated by a relatively high concentration of Mn²⁺ or Ca²⁺ ions, but the mechanism behind the stimulation is not clear. We also demonstrated that YpeB and SleB, in the absence of their partner protein, were degraded by unknown proteases other than HtrC during spore formation. Identification and characterization of these unknown proteases would be a future direction for revealing the roles of proteases in spore germination.
Characterization of Peptidoglycan, and the Enzymes that Synthesize it, in Borrelia burgdorferi and Insights into the Peptidoglycan of Other Pathogenic Borrelia
DeHart, Tanner Gage (Virginia Tech, 2021-06-03)
Peptidoglycan (PG) is an essential cell-wall biopolymer in virtually all bacteria. It is composed of glycan strands of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) crosslinked by peptide chains of alternating D- and L- amino acids and diamines. PG plays an important role in 1) cell elongation and division, 2) cell strength and morphology, 3) antibiotic susceptibility, and 4) host immune detection and modulation. While differences in peptide chains are common, deviations in glycan strand composition were not previously known to occur. Here, we provide characterization of the first known deviation to bacterial glycan strand composition — GlcNAc-GlcNAc-anhMurNAc (G-G- anhM) in Borrelia burgdorferi, the causative agent of Lyme disease. B. burgdorferi with less G-G-anhM were found to be significantly less motile, flexible, and stress-tolerant while possessing gross morphological defects and less overall PG. Our studies also characterized the muropeptide profile of Borrelia afzelii, Borrelia garinii, and Borrelia hermsii — species of Borrelia associated with causing different disease manifestations of Lyme disease, and relapsing fever, respectively. These species were found to incorporate appreciable amounts of G-G-anhM into their PG, suggesting an evolutionary adaptation to life inside a tick that predates the differentiation of Lyme disease and relapsing fever Borrelia. Finally, we provide partial characterization of a putative penicillin-binding protein in B. burgdorferi — a class of highly conserved PG synthesis enzymes present in the vast majority of bacteria. Collectively, the work in this thesis furthers our understanding of the structure, function, and synthesis of PG in Borrelia.
Characterization of Post-translational Modifications and Resulting Structure/Function Relationships of Recombinant Human Factor IX Produced in the Milk of Transgenic Pigs
Lindsay, Myles (Virginia Tech, 2004-12-15)
Hemophilia B is a debilitating and life-threatening disorder caused by a deficiency in or dysfunction of factor IX (FIX), a complex plasma glycoprotein required for the formation and maintenance of blood clots. Treatment of hemophilia B involves infusion of replacement FIX currently derived from two sources: FIX purified from pools of human plasma (pd-FIX) and a single recombinant FIX product generated in genetically engineered Chinese hamster ovary (CHO) cells. Both of these FIX products are prohibitively expensive, limiting of the treatment options of hemophiliacs worldwide. As a result, a more abundant and affordable FIX product would greatly improve the life prospects for hemophiliacs. The biological activity of FIX is dependent upon its numerous post-translational modifications (PTMs), including gamma-carboxylation, proteolytic maturation, phosphorylation, sulfation, and glycosylation. Of these PTMs, those known to be vital for activity are gamma-carboxylation of multiple glutamate residues near the N-terminus and proteolytic cleavage of the FIX propeptide. When expressed at a high rate in exogenous expression systems, however, the ability of current systems to effect the necessary PTMs is severely rate limited, restricting the production of active FIX. The transgenic pig bioreactor represents a promising source for the production of large quantities biologically active FIX due to its demonstrated ability to perform the required FIX PTMs. It was the goal of this study to characterize the PTM structure and the resulting function of recombinant FIX when expressed at 1-3 mg/ml in the transgenic pig mammary epithelium (tg-FIX). It was found that the expressed tg-FIX is comprised of a heterogeneous mixture of FIX PTM isoforms. This mixture represents a spectrum of tg-FIX molecules of varying gamma-carboxyglutamic acid (Gla) and propeptide content, indicating that rate limitations in effecting these PTMs are present. A purification process was developed utilizing heparin-affinity chromatography to purify the total population of tg-FIX from pig milk, a complex multi-phase feedstock. Subsequently, a process was developed to fractionate the total population of tg-FIX into subpopulations based upon the extent of post-translational modification. Q ion-exchange chromatography was utilized to fractionate tg-FIX based upon molecular acidity which was found to be correlated to both biological activity and Gla content. The resulting biologically active tg-FIX population contained an average of 7 of the 12 Gla residues found in pd-FIX. Immuno-affinity chromatography was subsequently utilized to further fractionate tg-FIX into mature tg-FIX and propeptide-containing tg-FIX populations. The isolated FIX PTM populations were subjected to functional analysis by investigating in vitro clotting activity, activation by factor XIa, and in vivo pharmacokinetics. From this analysis it was found that mature tg-FIX with an average 7 Gla residues, representing approximately 9% of the total tg-FIX produced, exhibits wild-type in vitro clotting activity and normal activation by factor XIa. The remainder of the tg-FIX produced, characterized by either a lower Gla content or the presence of the propeptide, was found to be inactive and displayed less efficient activation by factor IXa. In an in vivo pharmacokinetic study in the hemophilia B mouse model, biologically active tg-FIX was found to possess altered circulating properties. Tg-FIX was characterized by a lower recovery, approximately one-sixth that of pd-FIX, but an extended circulation half-life. From this study it was found that the mean residence time of tg-FIX after injections is approximately twice that observed for pd-FIX. These altered pharmacokinetic properties are likely linked to the unique tg-FIX PTM structure, perhaps through altered endothelial cell binding characteristics caused by the reduced Gla content.
Characterization of the adult Aedes aegypti early midgut peritrophic matrix proteome using LC-MS
Whiten, Shavonn R.; Ray, W. Keith; Helm, Richard F.; Adelman, Zach N. (PLOS, 2018-03-23)
The Aedes aegypti mosquito is the principal vector of arboviruses such as dengue, chikungunya, yellow fever, and Zika virus. These arboviruses are transmitted during adult female mosquito bloodfeeding. While these viruses must transverse the midgut to replicate, the blood meal must also reach the midgut to be digested, absorbed, or excreted, as aggregation of blood meal metabolites can be toxic to the female mosquito midgut. The midgut peritrophic matrix (PM), a semipermeable extracellular layer comprised of chitin fibrils, glycoproteins, and proteoglycans, is one such mechanism of protection for the mosquito midgut. However, this structure has not been characterized for adult female Ae. aegypti. We conducted a mass spectrometry based proteomic analysis to identify proteins that comprise or are associated with the adult female Ae. aegypti early midgut PM. Altogether, 474 unique proteins were identified, with 115 predicted as secreted. GO-term enrichment analysis revealed an abundance of serine-type proteases and several known and novel intestinal mucins. In addition, approximately 10% of the peptides identified corresponded to known salivary proteins, indicating Ae. aegypti mosquitoes extensively swallow their own salivary secretions. However, the physiological relevance of this remains unclear, and further studies are needed to determine PM proteins integral for midgut protection from blood meal derived toxicity and pathogen protection. Finally, we describe substantial discordance between previously described transcriptionally changes observed in the midgut in response to a bloodmeal and the presence of the corresponding protein in the PM. Data are available via ProteomeXchange with identifier PXD007627.
Clean Fractionation of Biomass - Steam Explosion and Extraction
Ibrahim, Mazlan (Virginia Tech, 1998-02-24)
The fractionation of two biomass resources, red oak (Quercus rubra) chips and oil palm (Elaeis guineensis) trunk solids, into constitutive chemical components, cellulose, hemicelluloses (called "other carbohydrates") and non-carbohydrates (includes lignin, tannins, etc.), was studied quantitatively in terms of relative cleanness. Red oak chips were steam exploded using a batch reactor at five different treatment severities, Ro 5,000, 10,000, 15,000, 20,000 and 35,000. Steam exploded fibers (SEF) of each severity were extracted with water and alkali. Mass fractionation and summative analysis data of all solid biomass fractions were determined. These data were interpreted in term of a unifying clean fractionation concept designed to evaluate the effectiveness of the fractionation processes. Within a series of severities applied to a single biomass resource, the quantitative clean fractionation can be used to choose an optimum severity for the isolation of any particular component fraction. The red oak results revealed that 25 % (on average) of biomass solids were lost during steam explosion. Cellulose remained almost unaffected (retained in fibers form) by water and alkali extraction. About 35-55 % of the hemicelluloses can be recovered in the water extracted liquor fraction (WEL). The remaining non-cellulosic carbohydrates were lost during steam explosion, especially at high severity. At Ro 10,000 and above, alkali extracted fibers (AEF) consists almost entirely of cellulose and non-carbohydrates. The majority of the non-carbohydrates component (> 50 %) can be isolated by alkali extraction. The non-carbohydrate component harvested increased with severity to 67% at Ro 35,000.
CMGSDB: integrating heterogeneous Caenorhabditis elegans data sources using compositional data mining
Pati, Amrita; Jin, Ying; Klage, Karsten; Helm, Richard F.; Heath, Lenwood S.; Ramakrishnan, Naren (Oxford University Press, 2008-01-01)
CMGSDB (Database for Computational Modeling of Gene Silencing) is an integration of heterogeneous data sources about Caenorhabditis elegans with capabilities for compositional data mining (CDM) across diverse domains. Besides gene, protein and functional annotations, CMGSDB currently unifies information about 531 RNAi phenotypes obtained from heterogeneous databases using a hierarchical scheme. A phenotype browser at the CMGSDB website serves this hierarchy and relates phenotypes to other biological entities. The application of CDM to CMGSDB produces ‘chains’ of relationships in the data by finding two-way connections between sets of biological entities. Chains can, for example, relate the knock down of a set of genes during an RNAi experiment to the disruption of a pathway or specific gene expression through another set of genes not directly related to the former set. The web interface for CMGSDB is available at https://bioinformatics.cs.vt.edu/cmgs/CMGSDB/, and serves individual biological entity information as well as details of all chains computed by CDM.
Comparative Analysis of Inactivated Wood Surfaces
Sernek, Milan (Virginia Tech, 2002-04-24)
A wood surface, which is exposed to a high temperature condition, can experience inactivation. Surface inactivation results in reduced ability of an adhesive to properly wet, flow, penetrate, and cure. Thus, an inactivated wood surface does not bond well with adhesives. The changes in surface chemistry, wettability, and adhesion of inactivated wood surfaces, including heartwood of yellow-poplar (Liriodendron tulipifera) and southern pine (Pinus taeda), were studied. Wood samples were dried from the green moisture content condition in a convection oven at five different temperature levels ranging from 50 to 200 °C. The comparative characterization of the surface was done by X-ray photoelectron spectroscopy (XPS), sessile drop wettability, and fracture testing of adhesive bonds. The oxygen to carbon ratio (O/C) decreased and the C1/C2 ratio increased with drying temperature. The C1 component is related to carbon-carbon or carbon-hydrogen bonds, and the C2 component represents single carbon-oxygen bond. A low O/C ratio and a high C1/C2 ratio reflected a high concentration of non-polar wood components (extractives/VOCs) on the wood surface, which modified the wood surface from hydrophilic to more hydrophobic. Wettability was directly related to the O/C ratio and inversely related to the C1/C2 ratio. Contact angle decreased with time and increased with the temperature of exposure. Southern pine had a lower wettability than yellow-poplar, which was due to a greater concentration of non-polar hydrocarbon-type extractives and heat-generated volatiles on the surface. Solvent extraction prior to drying did not improved wettability, whereas, extraction after drying improved wettability. A contribution of extractives migration and VOCs generation played a significant role in the heat-induced inactivation process of southern pine. The maximum strain energy release rate (Gmax) showed that surface inactivation was insignificant for yellow-poplar when exposed to drying temperatures < 187°C. The southern pine was most susceptible to inactivation at drying temperatures > 156°C, particularly when bonded with phenol-formaldehyde (PF) adhesive. Chemical treatments improved the wettability of inactivated wood surfaces, but an improvement in adhesion was not evident for specimens bonded with polyvinyl-acetate (PVA) adhesive. NaOH surface treatment was most effective for improving adhesion of the PF adhesive bond.
Comparative Functional Genomics Characterization of Low Phytic Acid Soybeans and Virus Resistant Soybeans
DeMers, Lindsay Carlisle (Virginia Tech, 2020-06-02)
The field of functional genomics aims to understand the complex relationship between genotype and phenotype by integrating genome-wide approaches, such as transcriptomics, proteomics, and metabolomics. Large-scale "-omics" research has been made widely possible by the advent of high-throughput techniques, such as next-generation sequencing and mass-spectrometry. The vast data generated from such studies provide a wealth of information on the biological dynamics underlying phenotypes. Though functional genomics approaches are used extensively in human disease research, their use also spans organisms as miniscule as mycoplasmas to as great as sperm whales. In particular, functional genomics is instrumental in agricultural advancements for the improvement of productivity and sustainability in crop and livestock production. Improvement in soybean production is especially imperative, as soybeans are a primary source of oil and protein for human and livestock consumption, respectively. The research presented here employs functional genomics approaches – transcriptomics and metabolomics – to discern the transcriptional regulation and metabolic events underlying two economically important agronomic traits in soybean: seed phytic acid content and Soybean mosaic virus resistance. At normal levels, seed phytic acid content inhibits mineral absorption in humans and livestock, acting as an antinutrient and contributing to phosphorus pollution; however, the development of low phytic acid soybeans has helped mitigate these issues, as their seeds increase nutrient bioavailability and reduce environmental impact. Despite these desirable qualities, low phytic acid soybeans exhibit poor seed performance, which negatively affects germination rates and yield and has prevented their large-scale commercial production. Thus, part of the focus of this research was investigating the effects of mutations conferring the low phytic acid phenotype on seed germination. Comparative studies between low and normal phytic acid soybean seeds were carried out and revealed distinct differences in metabolite profiles and in the transcriptional regulation of biological pathways that may be vital for successful seed germination. The final part of this research concerns Rsv3-mediated extreme resistance, a unique mode of resistance that is effective against the most virulent strains of Soybean mosaic virus. The molecular mechanisms governing this type of resistance are poorly characterized. Therefore, the research presented here attempts to elucidate the regulatory elements responsible for the induction of the Rsv3-mediated extreme resistance response. Utilizing a comparative transcriptomic time series approach on Soybean mosaic virus-inoculated Rsv3 (resistant) and rsv3 (susceptible) soybean lines, this final study provides gene candidates putatively functioning in the regulation of biological pathways demonstrated to be crucial for Rsv3-mediated resistance.
Comparative Metabolomics of Fruits and Leaves in a Hyperdiverse Lineage Suggests Fruits Are a Key Incubator of Phytochemical Diversification
Schneider, Gerald F.; Salazar, Diego; Hildreth, Sherry B.; Helm, Richard F.; Whitehead, Susan R. (Frontiers, 2021-08-30)
Interactions between plants and leaf herbivores have long been implicated as the major driver of plant secondary metabolite diversity. However, other plant-animal interactions, such as those between fruits and frugivores, may also be involved in phytochemical diversification. Using 12 species of Piper, we conducted untargeted metabolomics and molecular networking with extracts of fruits and leaves. We evaluated organ-specific secondary metabolite composition and compared multiple dimensions of phytochemical diversity across organs, including richness, structural complexity, and variability across samples at multiple scales within and across species. Plant organ identity, species identity, and the interaction between the two all significantly influenced secondary metabolite composition. Leaves and fruit shared a majority of compounds, but fruits contained more unique compounds and had higher total estimated chemical richness. While the relative levels of chemical richness and structural complexity across organs varied substantially across species, fruit diversity exceeded leaf diversity in more species than the reverse. Furthermore, the variance in chemical composition across samples was higher for fruits than leaves. By documenting a broad pattern of high phytochemical diversity in fruits relative to leaves, this study lays groundwork for incorporating fruit into a comprehensive and integrative understanding of the ecological and evolutionary factors shaping secondary metabolite composition at the whole-plant level.
Connecting the Dots between PubMed Abstracts
Hossain, M. Shahriar; Gresock, Joseph; Edmonds, Yvette M.; Helm, Richard F.; Potts, Malcolm; Ramakrishnan, Naren (PLOS, 2012-01-03)
Background There are now a multitude of articles published in a diversity of journals providing information about genes, proteins, pathways, and diseases. Each article investigates subsets of a biological process, but to gain insight into the functioning of a system as a whole, we must integrate information from multiple publications. Particularly, unraveling relationships between extra-cellular inputs and downstream molecular response mechanisms requires integrating conclusions from diverse publications. Methodology We present an automated approach to biological knowledge discovery from PubMed abstracts, suitable for “connecting the dots” across the literature. We describe a storytelling algorithm that, given a start and end publication, typically with little or no overlap in content, identifies a chain of intermediate publications from one to the other, such that neighboring publications have significant content similarity. The quality of discovered stories is measured using local criteria such as the size of supporting neighborhoods for each link and the strength of individual links connecting publications, as well as global metrics of dispersion. To ensure that the story stays coherent as it meanders from one publication to another, we demonstrate the design of novel coherence and overlap filters for use as post-processing steps. Conclusions We demonstrate the application of our storytelling algorithm to three case studies: i) a many-one study exploring relationships between multiple cellular inputs and a molecule responsible for cell-fate decisions, ii) a many-many study exploring the relationships between multiple cytokines and multiple downstream transcription factors, and iii) a one-to-one study to showcase the ability to recover a cancer related association, viz. the Warburg effect, from past literature. The storytelling pipeline helps narrow down a scientist's focus from several hundreds of thousands of relevant documents to only around a hundred stories. We argue that our approach can serve as a valuable discovery aid for hypothesis generation and connection exploration in large unstructured biological knowledge bases.
Differential expression profiling of proteomes of pathogenic and commensal strains of Staphylococcus aureus using SILAC
Manickam, Manisha (Virginia Tech, 2011-11-29)
Staphylococcus aureus (S. aureus) is the etiological agent of food-borne diseases, skin infections in humans and mastitis in bovines. S. aureus is also known to exist as a commensal on skin, nose and other mucosal surfaces of the host. This symbiotic association is a result of immune dampening or tolerance induced in the host by this pathogen. We proposed the variation in protein expression by commensal and pathogenic strain as an important factor behind the difference in pathogenicity. The identification of differentially expressed proteins was carried out using a quantitative mass spectrometry (MS)-based proteomic approach, known as stable isotope labeling of amino acids in cell culture (SILAC). Four commensal and pathogenic strains each were grown in the SILAC minimal media (RPMI 1640), containing light (12C) and heavy (13C) form of lysine, respectively, until early stationary growth phase. Various protein fractions, including cell wall, membrane and secreted, were extracted from the bacterial cultures and mixed in a 1:1 ratio. The relative abundance of proteins present in light and heavy labeled samples was determined using MS analysis. From a total of 151 differentially expressed proteins, 58 were found to be upregulated in the pathogenic strains. These proteins are involved in a variety of cellular functions, including immune modulation, iron-binding, cellular transport, redox reactions, and metabolic enzymes. The differentially expressed proteins can serve as putative candidates to improve current approach towards development of a vaccine against S. aureus.
Discovering contextual connections between biological processes using high-throughput data
Lasher, Christopher Donald (Virginia Tech, 2011-09-12)
Hearkening to calls from life scientists for aid in interpreting rapidly-growing repositories of data, the fields of bioinformatics and computational systems biology continue to bear increasingly sophisticated methods capable of summarizing and distilling pertinent phenomena captured by high-throughput experiments. Techniques in analysis of genome-wide gene expression (e.g., microarray) data, for example, have moved beyond simply detecting individual genes perturbed in treatment-control experiments to reporting the collective perturbation of biologically-related collections of genes, or "processes". Recent expression analysis methods have focused on improving comprehensibility of results by reporting concise, non-redundant sets of processes by leveraging statistical modeling techniques such as Bayesian networks. Simultaneously, integrating gene expression measurements with gene interaction networks has led to computation of response networks--subgraphs of interaction networks in which genes exhibit strong collective perturbation or co-expression. Methods that integrate process annotations of genes with interaction networks identify high-level connections between biological processes, themselves. To identify context-specific changes in these inter-process connections, however, techniques beyond process-based expression analysis, which reports only perturbed processes and not their relationships, response networks, composed of interactions between genes rather than processes, and existing techniques in process connection detection, which do not incorporate specific biological context, proved necessary. We present two novel methods which take inspiration from the latest techniques in process-based gene expression analysis, computation of response networks, and computation of inter-process connections. We motivate the need for detecting inter-process connections by identifying a collection of processes exhibiting significant differences in collective expression in two liver tissue culture systems widely used in toxicological and pharmaceutical assays. Next, we identify perturbed connections between these processes via a novel method that integrates gene expression, interaction, and annotation data. Finally, we present another novel method that computes non-redundant sets of perturbed inter-process connections, and apply it to several additional liver-related data sets. These applications demonstrate the ability of our methods to capture and report biologically relevant high-level trends.
Dual-specific protein phosphatases in the Archaea
Dahche, Hanan Mohamad (Virginia Tech, 2010-04-02)
Three distinct families of PTPs, the conventional (cPTPs), low molecular weight (LMW PTPs), and Cdc25 PTPs, have converged upon a common catalytic mechanism and active site sequence, mainly, the phosphate-binding loop encompassing the PTP signature motif (H/V)C(X)₅R(S/T) and an essential Asp residue on a surface loop. There is little sequence similarity among the three families of phosphatases. All known LMW PTP remove phosphoryl groups esterified to the hydroxyl amino acid: tyrosine, whereas all members of the Cdc25 family are dual-specificity protein phosphatases that dephosphorylate all the hydroxyl amino acids: tyrosine, serine and threonine. The cPTP family primarily functions as tyrosine phosphatases, but it also includes dual-specific members. ORFs encoding potential cPTPs have been identified in five archaeal species: Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Thermococcus kodakaraensis, Pyrococcus horikoshii, and S. solfataricus. Only one has been partially characterized, Tk-PTP from T. kodakaraensis. Hence, our current body of knowledge concerning the functional properties and physiological roles of these enzymes remains fragmented. The genome of S. solfataricus encodes a single conventional protein tyrosine phosphatase, SsoPTP. SsoPTP is the smallest known archaeal PTP (18.3 kDa) with a primary amino acid sequence that conforms to the cPTP protein tyrosine phosphatase paradigm, HCX₅R(S/T). Relatively little is known about its mode of action " whether it follows the conventional PTP mechanism or employs a different route for catalysis " or its physiological role. ORF sso2453 from the genome of Sulfolobus solfataricus, encoding a protein tyrosine phosphatase, was cloned and its recombinant protein product, SsoPTP, was expressed in E. coli and purified by immobilized metal affinity chromatography. SsoPTP displayed the ability to dephosphorylate protein-bound phosphotyrosine as well as protein-bound phosphoserine/phosphothreonine. SsoPTP hydrolyzed both isomers of naphthyl phosphate, an indication of dual specificity. The four conserved residues within the presumed active site sequence: Asp⁶⁹, His⁹⁵, and Arg¹⁰², and the invariant Gln¹³⁹ residue were essential for catalysis, as it was predicted for the established members of the PTP family in both bacteria and eukaryotes. A substrate trapping protein variant, SsoPTP-C96S/D69A, was constructed to isolate possible SsoPTP substrates present in S. solfataricus cell lysates. Several potential substrates were isolated and identified by mass spectroscopy.

Browsing by Author "Helm, Richard F."

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