Key Biochemical Pathways Research Articles

Transcriptomic Effects of the Cell Cycle Regulator LGO in Arabidopsis Sepals.

Endoreduplication is a specialized cell cycle in which DNA replication occurs, but mitosis is skipped creating enlarged polyploid cells. Endoreduplication is associated with the differentiation of many specialized cell types. In the Arabidopsis thaliana sepal epidermis endoreduplicated giant cells form interspersed between smaller cells. Both the transcription factor Arabidopsis thaliana MERISTEM LAYER1 (ATML1) and the plant-specific cyclin dependent kinase inhibitor LOSS OF GIANT CELLS FROM ORGANS (LGO)/SIAMESE RELATED1 (SMR1) are required for the formation of giant cells. Overexpression of LGO is sufficient to produce sepals covered in highly endoreduplicated giant cells. Here we ask whether overexpression of LGO changes the transcriptome of these mature sepals. We show that overexpression of LGO in the epidermis (LGOoe) drives giant cell formation even in atml1 mutant sepals. Using RNA-seq we show that LGOoe has significant effects on the mature sepal transcriptome that are primarily ATML1-independent changes of gene activity. Genes activated by LGOoe, directly or indirectly, predominantly encode proteins involved in defense responses, including responses to wounding, insects (a predator of Arabidopsis), and fungus. They also encode components of the glucosinolate biosynthesis pathway, a key biochemical pathway in defense against herbivores. LGOoe-activated genes include previously known marker genes of systemic acquired resistance such as PR1 through PR5. The defensive functions promoted by LGOoe in sepals overlap with functions recently shown to be transcriptionally activated by hyperimmune cpr5 mutants in a LGO-dependent manner. Our findings show that the cell cycle regulator LGO can directly or indirectly drive specific states of gene expression; in particular, they are consistent with recent findings showing LGO to be necessary for transcriptional activation of many defense genes in Arabidopsis.

Abstract 4215: Hyperpolarized magnetic resonance metabolic imaging and NMR metabolomics to assess the progression and aggressiveness of patient-derived pancreatic cancer xenografts

Abstract Pancreatic cancer, the most lethal of solid tumors is an aggressive disease that develops relatively symptom-free. The absence of early symptoms has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic intervention to improve the survival rate of pancreatic cancer patients. Our goal is to identify metabolic biomarkers for early detection with an understanding of pancreatic cancer progression from benign lesions to a malignant state. Hyperpolarized magnetic resonance imaging (HP-MRI) provides a >10,000-fold signal enhancement for detecting of endogenous metabolic substrates to monitor metabolic fluxes through the multiple key biochemical pathways including glycolysis and citric acid cycle. In particular, conversion between hyperpolarized 13C-labeled pyruvate and lactate, catalyzed by lactate dehydrogenase (LDH), has been shown to have a number of potential applications such as diagnosis, staging tumor grade and monitoring therapy response. Genetic mutation is evident in causing tumorigenesis and often time these mutations trigger the signaling pathways that are associated with “metabolic transformations. There is currently tremendous research interest in dissecting the mechanisms of metabolic transformation as the cancers progress. In this effort, we are applying high-resolution NMR (nuclear magnetic resonance) metabolomics and the real-time hyperpolarized metabolic imaging of patient-derived xenograft tumors for probing the underlying mechanism of altered-metabolism and correlate with pancreatic cancer progression and its aggressiveness. The protocol detailing heterotopic engraftment of pancreatic cancer patient tumors into immunodeficient mice and expansion of direct xenograft tumors was recently reported elsewhere. These patient-derived xenografts (PDX) were well annotated with original patient tumors. For the NMR metabolomics study, the fresh-frozen tumor samples were homogenized, lyophilized, and resuspended in D2O and 1-D proton NMR spectroscopy was employed to characterize the water-soluble portion of the metabolome. These data suggest that elevated glycolysis (production of high lactate) and alterations in choline metabolism may arise in pancreatic cancer development. Also, the metabolite concentrations were significantly higher in aggressive tumors compare to non-aggressive one. The real-time metabolic imaging shows the immediate conversion of lactate after injection (i.v.) of hyperpolarized 13C pyruvate and probe the upregulated LDH activity as the cancer progress. The basic genomic profiling of these tumors is in progress. This model may provide an excellent platform where mutational status of these patient tumors can be correlated with both high resolution and hyperpolarized dynamical metabolomics data for characterizing individual tumor phenotypes. Citation Format: Prasanta Dutta, Mayrim Rios Perez, Travis Cole Salzillo, Michael Pratt, Yaan Kang, Niki Zacharias, Anirban Maitra, Jason B. Fleming, Pratip Bhattacharya. Hyperpolarized magnetic resonance metabolic imaging and NMR metabolomics to assess the progression and aggressiveness of patient-derived pancreatic cancer xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4215.

Artemisinin ‘Bomb’ Attacks Many Proteins

First isolated in the 1970s, artemisinin is an effective drug against malaria, but questions remain about how it works. With the help of chemical proteomics, researchers have now shown that artemisinin kills the malaria parasite Plasmodium falciparum by indiscriminately binding to proteins in many of the organism’s key biochemical pathways. To identify the drug’s binding partners, a team led by Qingsong Lin, Kevin S. W. Tan, and Jigang Wang of National University of Singapore synthesized an alkyne-tagged artemisinin analog (Nat. Commun. 2015, DOI: 10.1038/ncomms10111). After incubating the probe with live malaria parasites and attaching biotin to its alkyne tag, the team fished out all the proteins to which artemisinin binds. The researchers did this by incubating the mixture with streptavidin-labeled beads that strongly attract biotin. They then identified the purified proteins with mass spectrometry. The researchers were unsurprised to find that the probe bound to a whopping 124 different proteins.

Development of a method for enhancing metabolomics coverage of human sweat by gas chromatography–mass spectrometry in high resolution mode

Sweat has recently gained popularity as clinical sample in metabolomics analysis as it is a non-invasive biofluid the composition of which could be modified by certain pathologies, as is the case with cystic fibrosis that increases chloride levels in sweat. However, the whole composition of sweat is still unknown and there is a lack of analytical strategies for sweat analysis. The aim of the present study was to develop and validate a method for metabolomic analysis of human sweat by gas chromatography–time of flight/mass spectrometry (GC–TOF/MS) in high resolution mode. Thus, different sample preparation strategies were compared to check their effect on the profile of sweat metabolites. Sixty-six compounds were tentatively identified by the obtained MS information. Amino acids, dicarboxylic acids and other interesting metabolites such as myo-inositol and urocanic acid were identified. Among the tested protocols, methyoxiamination plus silylation after deproteinization was the most suited option to obtain a representative snapshot of sweat metabolome. The intra-day repeatability of the method ranged from 0.60 to 16.99% and the inter-day repeatability from 2.75 to 31.25%. As most of the identified metabolites are involved in key biochemical pathways, this study opens new possibilities to the use of sweat as a source of metabolite biomarkers of specific disorders.

Characterizing autism spectrum disorders by key biochemical pathways.

The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions.

Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity.

Imazethapyr (IM) is a widely used chiral herbicide that inhibits the synthesis of branched-chain amino acids (BCAAs). IM is thought to exert its toxic effects on amino acid synthesis mainly through inhibition of acetolactate synthase activity, but little is known about the potential effects of IM on other key biochemical pathways. Here, we exposed the model plant Arabidospsis thaliana to trace S- and R-IM enantiomer concentrations and examined IM toxicity effects on the root proteome using iTRAQ. Conventional analyses of root carbohydrates, organic acids, and enzyme activities were also performed. We discovered several previously unknown key biochemical pathways targeted by IM in Arabidospsis. 1,322 and 987 proteins were differentially expressed in response to R- and S-IM treatments, respectively. Bioinformatics and physiological analyses suggested that IM reduced the BCAA tissue content not only by strongly suppressing BCAA synthesis but also by increasing BCAA catabolism. IM also affected sugar and starch metabolism, changed the composition of root cell walls, increased citrate production and exudation, and affected the microbial community structure of the rhizosphere. The present study shed new light on the multiple toxicity mechanisms of a selective herbicide on a model plant.

Transcript dynamics at early stages of molecular interactions of MYMIV with resistant and susceptible genotypes of the leguminous host, Vigna mungo.

Initial phases of the MYMIV- Vigna mungo interaction is crucial in determining the infection phenotype upon challenging with the virus. During incompatible interaction, the plant deploys multiple stratagems that include extensive transcriptional alterations defying the virulence factors of the pathogen. Such molecular events are not frequently addressed by genomic tools. In order to obtain a critical insight to unravel how V. mungo respond to Mungbean yellow mosaic India virus (MYMIV), we have employed the PCR based suppression subtractive hybridization technique to identify genes that exhibit altered expressions. Dynamics of 345 candidate genes are illustrated that differentially expressed either in compatible or incompatible reactions and their possible biological and cellular functions are predicted. The MYMIV-induced physiological aspects of the resistant host include reactive oxygen species generation, induction of Ca2+ mediated signaling, enhanced expression of transcripts involved in phenylpropanoid and ubiquitin-proteasomal pathways; all these together confer resistance against the invader. Elicitation of genes implicated in salicylic acid (SA) pathway suggests that immune response is under the regulation of SA signaling. A significant fraction of modulated transcripts are of unknown function indicating participation of novel candidate genes in restricting this viral pathogen. Susceptibility on the other hand, as exhibited by V. mungo Cv. T9 is perhaps due to the poor execution of these transcript modulation exhibiting remarkable repression of photosynthesis related genes resulting in chlorosis of leaves followed by penalty in crop yield. Thus, the present findings revealed an insight on the molecular warfare during host-virus interaction suggesting plausible signaling mechanisms and key biochemical pathways overriding MYMIV invasion in resistant genotype of V. mungo. In addition to inflate the existing knowledge base, the genomic resources identified in this orphan crop would be useful for integrating MYMIV-tolerance trait in susceptible cultivars of V. mungo.

Endogenous gradients of resting potential instructively pattern embryonic neural tissue via Notch signaling and regulation of proliferation.

Biophysical forces play important roles throughout embryogenesis, but the roles of spatial differences in cellular resting potentials during large-scale brain morphogenesis remain unknown. Here, we implicate endogenous bioelectricity as an instructive factor during brain patterning in Xenopus laevis. Early frog embryos exhibit a characteristic hyperpolarization of cells lining the neural tube; disruption of this spatial gradient of the transmembrane potential (Vmem) diminishes or eliminates the expression of early brain markers, and causes anatomical mispatterning of the brain, including absent or malformed regions. This effect is mediated by voltage-gated calcium signaling and gap-junctional communication. In addition to cell-autonomous effects, we show that hyperpolarization of transmembrane potential (Vmem) in ventral cells outside the brain induces upregulation of neural cell proliferation at long range. Misexpression of the constitutively active form of Notch, a suppressor of neural induction, impairs the normal hyperpolarization pattern and neural patterning; forced hyperpolarization by misexpression of specific ion channels rescues brain defects induced by activated Notch signaling. Strikingly, hyperpolarizing posterior or ventral cells induces the production of ectopic neural tissue considerably outside the neural field. The hyperpolarization signal also synergizes with canonical reprogramming factors (POU and HB4), directing undifferentiated cells toward neural fate in vivo. These data identify a new functional role for bioelectric signaling in brain patterning, reveal interactions between Vmem and key biochemical pathways (Notch and Ca(2+) signaling) as the molecular mechanism by which spatial differences of Vmem regulate organogenesis of the vertebrate brain, and suggest voltage modulation as a tractable strategy for intervention in certain classes of birth defects.

Metabolic phenotyping reveals a lipid mediator response to ionizing radiation.

Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here, we screened mouse serum for metabolic alterations following an acute exposure to γ radiation using a multiplatform mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated fatty acids (PUFAs) in the two groups of animals. Exposure to γ radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature that could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine.

SWEET CORN CAROTENOID CONCENTRATIONS INFLUENCED BY HERBICIDE APPLICATIONS

Carotenoids serve antioxidant functions in plant photosynthetic processes, as well as in actions of disease reduction in mammalian systems. Lutein and zeaxanthin are important dietary carotenoids in suppressing aging eye diseases. Age-related macular degeneration now affects more than 1.75 million individuals in the US. Sweet corn (Zea mays var. rugosa) is one of only a few vegetable sources high in zeaxanthin. Mesotrione herbicide is currently labeled for selective pre- and post-emergence weed control in sweet corn production. Mesotrione competitively inhibits phytoene desaturase, a critical enzyme in carotenoid biosynthesis, which results in bleaching of leaf tissues in susceptible species and eventual plant death. Sweet corn is tolerant to mesotrione applications; however, differing sensitivity exists among genotypes. What remains unclear is the impact of mesotrione on carotenoid concentrations in mature sweet corn kernels following post-emergent applications to young corn plants. Our research objective was to measure mature kernel carotenoid concentrations in response to post-emergence applications of mesotrione to genotypes of different sensitivities. Post-emergence treatments included mesotrione applied alone, or in mixtures with the photosystem II inhibitor atrazine applied to corn plants of either 5-10 or 15-20 cm in height. Kernels were harvested from mature plants and measured for carotenoid concentrations. Mesotrione applied alone, or in mixtures with atrazine acted to increase concentrations of kernel antheraxanthin, lutein, and zeaxanthin in several sweet corn genotypes. Mesotrione applications resulted in greater pools of kernel carotenoids once the sweet corn genotypes overcame initial herbicidal photo-oxidative stress. This is the first report of herbicides directly up regulating a key biochemical pathway linked to the nutritional quality of a vegetable crop.

Optimization study for metabolomics analysis of human sweat by liquid chromatography–tandem mass spectrometry in high resolution mode

Sweat has recently gained popularity as a potential tool for diagnostics and biomarker monitoring as it is a non-invasive biofluid the composition of which could be modified by certain pathologies, as is the case with cystic fibrosis, which increases chloride levels in sweat. The aim of the present study was to develop an analytical method for analysis of human sweat by liquid chromatography–mass spectrometry (LC–Q-TOF MS/MS) in high resolution mode. Thus, different sample preparation strategies and different chromatographic modes (HILIC and C18 reverse modes) were compared to check their effect on the profile of sweat metabolites. Forty-one compounds were identified by the MS/MS information obtained with a mass tolerance window below 4ppm. Amino acids, dicarboxylic acids and other interesting metabolites such as inosine, choline, uric acid and tyramine were identified. Among the tested protocols, direct analysis after dilution was a suited option to obtain a representative snapshot of sweat metabolome. In addition, sample clean up by C18 SpinColumn SPE cartridges improved the sensitivity of most identified compounds and reduced the number of interferents. As most of the identified metabolites are involved in key biochemical pathways, this study opens new possibilities to the use of sweat as a source of metabolite biomarkers of specific disorders.

Exome sequencing identifies NFS1 deficiency in a novel Fe‐S cluster disease, infantile mitochondrial complex II/III deficiency

Iron-sulfur (Fe-S) clusters are a class of highly conserved and ubiquitous prosthetic groups with unique chemical properties that allow the proteins that contain them, Fe-S proteins, to assist in various key biochemical pathways. Mutations in Fe-S proteins often disrupt Fe-S cluster assembly leading to a spectrum of severe disorders such as Friedreich's ataxia or iron-sulfur cluster assembly enzyme (ISCU) myopathy. Herein, we describe infantile mitochondrial complex II/III deficiency, a novel autosomal recessive mitochondrial disease characterized by lactic acidemia, hypotonia, respiratory chain complex II and III deficiency, multisystem organ failure and abnormal mitochondria. Through autozygosity mapping, exome sequencing, in silico analyses, population studies and functional tests, we identified c.215G>A, p.Arg72Gln in NFS1 as the likely causative mutation. We describe the first disease in man likely caused by deficiency in NFS1, a cysteine desulfurase that is implicated in respiratory chain function and iron maintenance by initiating Fe-S cluster biosynthesis. Our results further demonstrate the importance of sufficient NFS1 expression in human physiology.

Abstract B210: Novel inhibitors of nicotinamide phosphoribosyl transferase and their evaluation in combination with bortezomib.

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD for a number of key biochemical pathways, inhibition of NAMPT has been shown to result in antitumor efficacy in preclinical models. NAMPT inhibitors have also been reported to enhance sensitivity to a number of targeted agents and overcome resistance to available therapies such as bortezomib resistance in multiple myeloma. In view of the sub-optimal profile of the reported first generation NAMPT inhibitors with respect to pharmacokinetics and drug-drug interaction, we sought to develop NAMPT inhibitors with the "best-in-class" profile for overcoming mechanism-based toxicities and/or resistance to current therapies. Utilizing structure-guided drug design and SAR-based approaches, we have optimized two chemical series of inhibitors of NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT. Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion of cell lines derived from multiple myeloma, prostate and breast cancers. The anti-proliferative activities were rescued in NAPRT- proficient cell lines with the addition of NA due to the NAMPT independent salvage pathway for biosynthesis of NAD, confirming the mechanism of action through NAD depletion. Lead compounds exhibited desirable drug-like properties including solubility, permeability, metabolic stability, lack of CYP inhibition and pharmacokinetic exposure. In a xenograft model of pancreatic cancer, treatment with lead compounds resulted in regression of tumors with no signs of toxicity. Recent reports demonstrating the overexpression of NAMPT in bortezomib-resistant cells and a synergistic efficacy with a combination of NAMPT inhibitor and bortezomib against the resistant cells prompted us to evaluate the combination with our optimized leads. The combination of lead NAMPT inhibitors with bortezomib showed synergistic killing of cultured multiple myeloma cell lines. Evaluation of efficacy of the lead compounds as a single agent or in combination with bortezomib in xenograft models of multiple myeloma is currently underway. Modulation of NAD levels at lower doses of NAMPT inhibitors in combination with bortezomib could overcome the limitations such as mechanism-based toxicity and/or resistance of both these therapies and provide an effective treatment option for multiple myeloma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B210. Citation Format: Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, S Karthikeyan, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra, Hosahalli Subramanya, Chetan Pandit, Murali Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase and their evaluation in combination with bortezomib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B210.

Mechanism on differential gene expression and heterosis formation

Despite the rediscovery of heterosis about a century ago and the suggestion of various genetic models to explain this phenomenon, little consensus has yet been reached about the genetic basis of heterosis. Following the genome organization variation and gene effects, an understanding of gene differential expression in hybrids and its parents provides a new opportunity to speculate on mechanisms that might lead to heterosis. Investigation on allele-specific gene expression in hybrid and gene differential expression between hybrids and its parents might contribute to improve our understanding of the molecular basis of heterosis and eventually guide breeding practices. In this review, we discussed the recent researches on allelic-specific expression in hybrid which was frequently observed in recent studies and analyzed its regulatory mechanism. All possible modes of gene action, including additivity, high- and low-parent dominance, underdominance, and over-dominance, were observed when investigating gene differential expression between hybrids and its parents. Data from transcriptomic studies screened several heterosis-associated genes and highlighted the importance of certain key biochemical pathways that may prove to be quintessential for the manifestation of heterosis. So far, no uniform global expression pat-terns were observed in these gene expression studies. Most heterosis-associated gene expression analyses have not revealed a predominant functional category to which differentially expressed genes belong. However, these gene expression profiling studies represent a first step towards the definition of the complex gene expression networks that might be relevant in the context of heterosis. New technique on gene expression profile and advancements in bioinformatics will facilitate our understanding of the genetic basis of heterosis at the gene-expression level.

Family-based exome-sequencing approach identifies rare susceptibility variants for lithium-responsive bipolar disorder

Bipolar disorder (BD) is a psychiatric condition characterized by the occurrence of at least two episodes of clinically disturbed mood including mania and depression. A vast literature describing BD studies suggests that a strong genetic contribution likely underlies this condition; heritability is estimated to be as high as 80%. Many studies have identified BD susceptibility loci, but because of the genetic and phenotypic heterogeneity observed across individuals, very few loci were subsequently replicated. Research in BD genetics to date has consisted of classical linkage or genome-wide association studies, which have identified candidate genes hypothesized to present common susceptibility variants. Although the observation of such common variants is informative, they can only explain a small fraction of the predicted BD heritability, suggesting a considerable contribution would come from rare and highly penetrant variants. We are seeking to identify such rare variants, and to increase the likelihood of being successful, we aimed to reduce the phenotypic heterogeneity factor by focusing on a well-defined subphenotype of BD: excellent response to lithium monotherapy. Our group has previously shown positive response to lithium therapy clusters in families and has a consistent clinical presentation with minimal comorbidity. To identify such rare variants, we are using a targeted exome capture and high-throughput DNA sequencing approach, and analyzing the entire coding sequences of BD affected individuals from multigenerational families. We are prioritizing rare variants with a frequency of less than 1% in the population that segregate with affected status within each family, as well as being potentially highly penetrant (e.g., protein truncating, missense, or frameshift) or functionally relevant (e.g., 3'UTR, 5'UTR, or splicing). By focusing on rare variants in a familial cohort, we hope to explain a significant portion of the missing heritability in BD, as well as to narrow our current insight on the key biochemical pathways implicated in this complex disorder.

A xenogeneic-free protocol for isolation and expansion of human adipose stem cells for clinical uses.

Human adipose stem cells (hASCs) play a crucial role in the fields of regenerative medicine and tissue engineering for different reasons: the abundance of adipose tissue, their easy harvesting, the ability to multipotent differentiation and the fact that they do not trigger allogeneic blood response or secrete cytokines that act as immunosuppressants. The vast majority of protocols use animal origin reagents, with the underlying risk of transmitting infections by non-human pathogens. We have designed a protocol to isolate and maintain the properties of hASCs avoiding xenogeneic reagents. These changes not only preserve hASCs morphology, but also increase cell proliferation and maintain their stem cell marker profile. On the other hand, human serum albumin (HSA), Tryple® and human Serum (HS), do not affect hASCs multipotent differentiation ability. The amendments introduced do not trigger modifications in the transcriptional profile of hASCs, alterations in key biochemical pathways or malignization. Thus, we have proven that it is possible to isolate and maintain hASCs avoiding animal reagents and, at the same time, preserving crucial culture parameters during long term culture. Thereby we have revealed a novel and effective tool for the improvement of clinical, cell-based therapies.

Abstract 5389: Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT).

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD as a co-factor or substrate for a number of key biochemical pathways including those catalyzed by PARP1, Sirtuins and ADP-ribosyl cyclase, inhibition of NAMPT has been shown to result in anti-tumor efficacy in preclinical models. Two NAMPT Inhibitors FK866/APO866 and GMX1778 are currently in clinical trials for oncology indications. In the presence of these clinical agents, cultured cell lines show development of resistance due to mutations underscoring the potential need for inhibitors from distinct chemical series. Here, we report a structure-guided drug design based approach for identification of lead compounds from two chemical series selectively targeting NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT against both wild type and resistance mutants (G217R and H191R) Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion in several cancer cell lines. Normal cells and selected cancer cell lines have an NAMPT independent salvage pathway for biosynthesis of NAD, which is dependent on nicotinic acid phosphoribosyltransferase (NAPRT) and Nicotinic acid (NA). The anti-proliferative activities were fully rescued in NAPRT- proficient cell lines with the addition of NA, confirming the mechanism of action through specific NAD depletion. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability and permeability, and desired exposure in pharmacokinetic studies. Anti-tumor activities of these compounds including NA rescue in NAPRT-proficient tumor models are currently being evaluated in preclinical models. Citation Format: Murali Ramachandra, Chetan Pandit, Hosahalli Subramanya, Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, Arnab Bera, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5389. doi:10.1158/1538-7445.AM2013-5389

Molecular Platforms Utilized to Detect BRAF V600E Mutation in Melanoma

Metastatic melanoma (MM) is a deadly skin disease refractory to standard chemotherapy. Despite numerous clinical and pathological parameters derived to guide patient management, clinical outcomes in melanoma patients remain difficult to predict. There is a critical need to delineate the important biomarkers typical of this disease. These biomarkers will ideally illuminate those key biochemical pathways responsible for the aggressive behavior of melanoma and, in the process, unveil new opportunities for the design of rational therapeutic interventions in high-risk patients. The most common recurring mutation in cutaneous melanoma is the prooncogenic BRAF V600E mutation that drives melanoma cell proliferation. The development of RAF inhibitors targeted against BRAF V600E mutant melanoma cells has revolutionized the treatment of MM. Clinical trials with BRAF inhibitor vemurafenib have shown objective clinical response and improved survival in patients with MM; therefore, knowledge of the molecular signature of melanoma in patients will be important in directing management decisions. Several molecular platforms exist to analyze the mutation status of melanoma. These include Sanger sequencing, pyrosequencing, allele-specific reverse transcriptase polymerase chain reaction, mass spectrometry base sequencing (Sequenom), high-resolution melting curve analysis, and next-generation sequencing methods using microfluidics technology. The Food and Drug Administration has approved the cobas BRAF V600 Mutation Test developed by Roche to analyze BRAF mutation status in formalin-fixed paraffin-embedded tumor samples. The cobas Mutation Test has been designed specifically to detect BRAF V600E mutations, and the analytic performance of this assay has demonstrated >99% sensitivity in the detection of BRAF V600E mutation when compared with the Sanger sequencing method and confirmed with the next-generation sequencing 454-pyrosequencing technology. The lower limit of detection of the percentage of mutant alleles in a tissue sample for the cobas test is less than 4%-5%. Some cross-reactivity with other variants of mutant BRAF was seen with the cobas V600 platform; however, this clinical test offers highly sensitive reproducible BRAF V600E mutation analysis in formalin-fixed paraffin-embedded tumor samples.

Homology modeling, simulation and molecular docking studies of catechol-2, 3-Dioxygenase from Burkholderia cepacia: Involved in degradation of Petroleum hydrocarbons.

Catechol 2, 3-dioxygenase is present in several types of bacteria and undergoes degradation of environmental pollutants through an important key biochemical pathways. Specifically, this enzyme cleaves aromatic rings of several environmental pollutants such as toluene, xylene, naphthalene and biphenyl derivatives. Hence, the importance of Catechol 2, 3-dioxygenase and its role in the degradation of environmental pollutants made us to predict the three-dimensional structure of Catechol 2, 3-dioxygenase from Burkholderia cepacia. The 10ns molecular dynamics simulation was carried out to check the stability of the modeled Catechol 2, 3- dioxygenase. The results show that the model was energetically stable, and it attains their equilibrium within 2000 ps of production MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3-dioxygenase reveals that the benzene, O-xylene, Toluene, Fluorene, Naphthalene, Carbazol, Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl makes strong hydrogen bond and Van der waals interaction with the active site residues of H150, L152, W198, H206, H220, H252, I254, T255, Y261, E271, L276 and F309. Free energy of binding and estimated inhibition constant of these compounds demonstrates that they are energetically stable in their binding cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except Pyrene all the substrates made close contact with Fe atom by the distance ranges from 1.67 to 2.43 Å. In addition to that, the above mentioned substrate except pyrene all other made π-π stacking interaction with H252 by the distance ranges from 3.40 to 3.90 Å. All these docking results reveal that, except Chrysene all other substrate has good free energy of binding to hold enough in the active site and makes strong VdW interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of catalyzing the above-mentioned substrate.

Mitochondrial Proteome Heterogeneity between Tissues from the Vegetative and Reproductive Stages of Arabidopsis thaliana Development

Specialization of the mitochondrial proteome in Arabidopsis has the potential to underlie the roles of these organelles at different developmental time points and in specific organs; however, most research to date has been limited to studies of mitochondrial composition from a few vegetative tissue types. To provide further insight into the extent of mitochondrial heterogeneity in Arabidopsis, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, bolt stem, and silique, were analyzed. Of the 286 protein spots on a 2-D gel of the mitochondrial proteome, the abundance of 237 spots was significantly varied between different samples. Identification of these spots revealed a nonredundant set of 83 proteins which were differentially expressed between organ/cell types, and the protein identification information can be analyzed in an integrated manner in an interactive fashion online. A number of mitochondrial protein spots were identified as being derived from the same genes in Arabidopsis but differed in their pI, indicating organ-specific variation in the post-translational modifications, or in their MW, suggesting differences in truncated mitochondrial products accumulating in different tissues. Comparisons of the proteomic data for the major isoforms with microarray analysis showed a positive correlation between mRNA and mitochondrial protein abundance and 60-90% concordance between changes in protein and transcript abundance. These analyses demonstrate that, while mitochondrial proteins are controlled transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the state or regulation of proteins in key biochemical pathways in plant mitochondria for specific functions. The integration of protein abundance and protein modification data with respiratory measurements, enzyme assays, and transcript data sets has allowed the identification of organ-enhanced differences in central carbon and amino acid metabolism pathways and provides ranked lists of mitochondrial proteins that are strongly transcriptionally regulated vs those whose abundance or activity is strongly influenced by a variety of post-transcriptional processes.