Comparing methods for metabolic network analysis and an application to metabolic engineering

Bioinformatics tools have facilitated the reconstruction and analysis of cellular metabolism of various organisms based on information encoded in their genomes. Characterization of cellular metabolism is useful to understand the phenotypic capabilities of these organisms. It has been done quantitatively through the analysis of pathway operations. There are several in silico approaches for analyzing metabolic networks, including structural and stoichiometric analysis, metabolic flux analysis, metabolic control analysis, and several kinetic modeling based analyses. They can serve as a virtual laboratory to give insights into basic principles of cellular functions. This article summarizes the progress and advances in software and algorithm development for metabolic network analysis, along with their applications relevant to cellular physiology, and metabolic engineering with an emphasis on microbial strain optimization. Moreover, it provides a detailed comparative analysis of existing approaches under different categories.     

An integrated pathway system modeling of Saccharomyces cerevisiae HOG pathway: a Petri net based approach

Biochemical networks comprise many diverse components and interactions between them. It has intracellular signaling, metabolic and gene regulatory pathways which are highly integrated and whose responses are elicited by extracellular actions. Previous modeling techniques mostly consider each pathway independently without focusing on the interrelation of these which actually functions as a single system. In this paper, we propose an approach of modeling an integrated pathway using an event-driven modeling tool, i.e., Petri nets (PNs). PNs have the ability to simulate the dynamics of the system with high levels of accuracy. The integrated set of signaling, regulatory and metabolic reactions involved in Saccharomyces cerevisiae’s HOG pathway has been collected from the literature. The kinetic parameter values have been used for transition firings. The dynamics of the system has been simulated and the concentrations of major biological species over time have been observed. The phenotypic characteristics of the integrated system have been investigated under two conditions, viz., under the absence and presence of osmotic pressure. The results have been validated favorably with the existing experimental results. We have also compared our study with the study of idFBA (Lee et al., PLoS Comput Biol 4:e1000–e1086, 2008) and pointed out the differences between both studies. We have simulated and monitored concentrations of multiple biological entities over time and also incorporated feedback inhibition by Ptp2 which has not been included in the idFBA study. We have concluded that our study is the first to the best of our knowledge to model signaling, metabolic and regulatory events in an integrated form through PN model framework. This study is useful in computational simulation of system dynamics for integrated pathways as there are growing evidences that the malfunctioning of the interplay among these pathways is associated with disease.     

Design and synthesis of (4E)-4-(4-substitutedbenzylideneamino)-3-substituted-2,3-dihydro-2-thioxothiazole-5-carbonitrile as novel A2A receptor antagonists

Novel 2-thioxothiazole derivatives (6–19) as potential adenosine A_2A receptor (A_2AR) antagonists were synthesized. The strong interaction of the compounds (6–19) with A_2AR in docking study was confirmed by high binding affinity with human A_2AR expressed in HEK293T cells using radioligand-binding assay. The compound 19 demonstrated very high selectivity for A_2AR as compared to standard A_2AR antagonist SCH58261. Decrease in A_2AR-coupled release of endogenous cAMP in treated HEK293T cells demonstrated in vitro A_2AR antagonist potential of the compound 19. Attenuation in haloperidol-induced impairment (catalepsy) in Swiss albino male mice pre-treated with compound 19 is evocative to explore its prospective in therapy of PD.     

Stress fiber growth and remodeling determines cellular morphomechanics under uniaxial cyclic stretch

Biomechanics and Modeling in Mechanobiology - Stress fibers in the cytoskeleton are essential in maintaining cellular shape and influence cellular adhesion and migration. Cyclic uniaxial stretching...     

Quality control and integration of genotypes from two calling pipelines for whole genome sequence data in the Alzheimer's disease sequencing project

The Alzheimer's Disease Sequencing Project (ADSP) performed whole genome sequencing (WGS) of 584 subjects from 111 multiplex families at three sequencing centers. Genotype calling of single nucleotide variants (SNVs) and insertion-deletion variants (indels) was performed centrally using GATK-HaplotypeCaller and Atlas V2. The ADSP Quality Control (QC) Working Group applied QC protocols to project-level variant call format files (VCFs) from each pipeline, and developed and implemented a novel protocol, termed “consensus calling,” to combine genotype calls from both pipelines into a single high-quality set. QC was applied to autosomal bi-allelic SNVs and indels, and included pipeline-recommended QC filters, variant-level QC, and sample-level QC. Low-quality variants or genotypes were excluded, and sample outliers were noted. Quality was assessed by examining Mendelian inconsistencies (MIs) among 67 parent-offspring pairs, and MIs were used to establish additional genotype-specific filters for GATK calls. After QC, 578 subjects remained. Pipeline-specific QC excluded ~12.0% of GATK and 14.5% of Atlas SNVs. Between pipelines, ~91% of SNV genotypes across all QCed variants were concordant; 4.23% and 4.56% of genotypes were exclusive to Atlas or GATK, respectively; the remaining ~0.01% of discordant genotypes were excluded. For indels, variant-level QC excluded ~36.8% of GATK and 35.3% of Atlas indels. Between pipelines, ~55.6% of indel genotypes were concordant; while 10.3% and 28.3% were exclusive to Atlas or GATK, respectively; and ~0.29% of discordant genotypes were. The final WGS consensus dataset contains 27,896,774 SNVs and 3,133,926 indels and is publicly available.     

Deep,Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.Lysine acetylation (Kac)¹ is a well conserved, reversible post-translational modification (PTM) involved in multiple cellular processes (1). Acetylation is regulated by two classes of enzymes: lysine acetyltransferases (KATs) and histone deacetylases (HDACs) (2–4). This modification was originally identified as a nuclear event on histone proteins and has been long appreciated for its role in epigenetic and DNA-dependent processes. With the help of a growing number of large-scale acetylation studies, it has become evident that lysine acetylation is ubiquitous, also occurring on cytoplasmic and mitochondrial proteins and has a role in signaling, metabolism, and immunity (1, 4–6). Therefore, the examination of lysine acetylation on nonhistone proteins has gained a prominent role in PTM analysis.To date, the identification of large numbers of acetylation sites has been challenging because of the substoichiometric nature of this modification (7, 8). Additionally, global acetylation is generally less abundant than phosphorylation and ubiquitylation (1). The introduction of antibodies specific for lysine acetylation has significantly improved the ability to enrich and identify thousands of sites (9–14). A landmark study by Choudhary et al. used anti-Kac antibodies to globally map 3600 lysine acetylation sites on 1750 proteins, thereby demonstrating the feasibility of profiling the acetylome (10). A more recent study by Lundby et al. investigated the function and distribution of acetylation sites in 16 different rat tissues, and identified, in aggregate, 15,474 acetylation sites from 4541 proteins (12).Although anti-acetyl lysine antibodies have been a breakthrough for globally mapping acetylation sites (9–12), it remains a challenge to identify large numbers of lysine acetylation sites from a single sample, as is now routinely possible for phosphorylation and ubiquitylation (13, 15–18). To improve the depth-of-coverage in acetylation profiling experiments there is a clear need for (1) alternative anti-acetyl lysine antibodies with higher specificity, (2) optimized antibody usage parameters, and (3) robust proteomic workflows that permit low to moderate protein input. In this study, we describe a newly commercialized mixture of anti-Kac antibodies and detail a complete proteomic workflow for achieving unprecedented coverage of the acetylome from a single stable isotope labeling by amino acids in cell culture (SILAC) labeled sample as well as isobaric tags for relative and absolute quantitation (iTRAQ)- and tandem mass tag (TMT)-labeled samples.     

Experimental Investigation of Mechanical and Thermal Properties of Silica Nanoparticle-Reinforced Poly(acrylamide) Nanocomposite Hydrogels

Current studies investigating properties of nanoparticle-reinforced polymers have shown that nanocomposites often exhibit improved properties compared to neat polymers. However, over two decades of research, using both experimental studies and modeling analyses, has not fully elucidated the mechanistic underpinnings behind these enhancements. Moreover, few studies have focused on developing an understanding among two or more polymer properties affected by incorporation of nanomaterials. In our study, we investigated the elastic and thermal properties of poly(acrylamide) hydrogels containing silica nanoparticles. Both nanoparticle concentration and size affected hydrogel properties, with similar trends in enhancements observed for elastic modulus and thermal diffusivity. We also observed significantly lower swellability for hydrogel nanocomposites relative to neat hydrogels, consistent with previous work suggesting that nanoparticles can mediate pseudo crosslinking within polymer networks. Collectively, these results indicate the ability to develop next-generation composite materials with enhanced mechanical and thermal properties by increasing the average crosslinking density using nanoparticles.