Genetic Networks of Liver Metabolism Revealed by Integration of Metabolic and Transcriptional Profiling

Although numerous quantitative trait loci (QTL) influencing disease-related phenotypes have been detected through gene mapping and positional cloning, identification of the individual gene(s) and molecular pathways leading to those phenotypes is often elusive. One way to improve understanding of genetic architecture is to classify phenotypes in greater depth by including transcriptional and metabolic profiling. In the current study, we have generated and analyzed mRNA expression and metabolic profiles in liver samples obtained in an F2 intercross between the diabetes-resistant C57BL/6 leptinob/ob and the diabetes-susceptible BTBR leptinob/ob mouse strains. This cross, which segregates for genotype and physiological traits, was previously used to identify several diabetes-related QTL. Our current investigation includes microarray analysis of over 40,000 probe sets, plus quantitative mass spectrometry-based measurements of sixty-seven intermediary metabolites in three different classes (amino acids, organic acids, and acyl-carnitines). We show that liver metabolites map to distinct genetic regions, thereby indicating that tissue metabolites are heritable. We also demonstrate that genomic analysis can be integrated with liver mRNA expression and metabolite profiling data to construct causal networks for control of specific metabolic processes in liver. As a proof of principle of the practical significance of this integrative approach, we illustrate the construction of a specific causal network that links gene expression and metabolic changes in the context of glutamate metabolism, and demonstrate its validity by showing that genes in the network respond to changes in glutamine and glutamate availability. Thus, the methods described here have the potential to reveal regulatory networks that contribute to chronic, complex, and highly prevalent diseases and conditions such as obesity and diabetes

HIGH RESOLUTION STUDY OF THE CONFORMERS OF 3-AMINOPHENOL.aAMINOPHENOL.^{a}

$^{a}$Work supported by NSF.Author Institution: Department of Chemistry, University of PittisburghExtensive study of monosubstituted aromatic rings like phenol and aniline has prompted the investigation of molecules containing two different substituents, such as the aminophenols. The vibrationally resolved $S_{1} \leftarrow S_{0}$ electronic spectrum of 3-aminophenol exhibits two origin bands, separated by $353 cm^{-1}$. A spectrum of each origin band was recorded at full rotational resolution. Analyses of these spectra show that the bands correspond to the cis and trans conformers of 3-aminophenol. Evidence for the identification of the conformers, which differ only in the position of the hydroxy hydrogen atom, will be discussed

Congenital Microstomia in a Neonate with Impending Respiratory Compromise

Microstomia is the term used to describe a reduction in the size of the oral aperture that is severe enough to compromise quality of life, nutrition, and cosmesis. Few cases of congenital microstomia have been reported as most microstomia cases are due to burn injuries. We are presenting a case of a neonate who was found to be in respiratory distress with severe congenital microstomia from no known cause. This case illustrates the rarity of this type of pathologic anatomy as well as the teamwork and tools necessary to treat these patients

Neuronal calcium sensor-1 gene ncs-1a is essential for semicircular canal formation in zebrafish inner ear.

International audienceWe have analyzed the functional role of neuronal calcium sensor-1 (Ncs-1) in zebrafish development. We identified two orthologs of the mammalian NCS-1 gene. Full-length cDNAs encoding zebrafish Ncs-1a and Ncs-1b polypeptides were cloned and characterized. Whole-mount in situ hybridization revealed that ncs-1a mRNA was expressed beginning at early somitogenesis. As development progressed, ncs-1a mRNA was present throughout the embryo with expression detected in ventral hematopoietic mesoderm, pronephric tubules, CNS nuclei, and otic vesicle. By 4.5 days post fertilization (dpf), ncs-1a expression was detected primarily in the brain. Expression of ncs-1b mRNA was first detected at 36 hours post fertilization (hpf) and was restricted to the olfactory bulb. By 4.5 dpf, ncs-1b was expressed at low levels throughout the brain. Knockdown of ncs-1a mRNA translation with antisense morpholinos blocked formation of semicircular canals. These studies identify a novel function for ncs-1a in inner ear development and suggest that this calcium sensor plays an important role in vestibular function

Preparation and Analysis of Multiple Hydrates of Simple Salts

We have developed a laboratory project in which the student prepares a series of hydrates of simple salts and then determines the extent of hydration of the product(s). We believe this provides a good introduction to the concepts of solubility, saturation, recrystallization, relative compound stability (e.g., a dihydrate vs tetrahydrate at elevated temperature), and simple gravimetric analysis. Moreover, the project lends itself to many variations. For example, a student could be given a starting hydrated salt and asked to prepare another hydrate within a specified temperature range. Or students could be given the formulas of several hydrates stable over different temperature ranges and be asked to discover a method of preparation. If it is deemed desirable to extend the project, the cation and/or anion could be determined quantitatively. The preparation of ionic hydrates is accomplished by four methods: (i) slow evaporation of the solvent from a near saturated solution of the starting hydrate at a temperature within the stability range of the desired hydrate, (ii) crystallization within the temperature range of the target hydrate from a saturated solution prepared at higher temperatures, (iii) crystallization within the temperature stability range from mixed solvents, and (iv) heating a higher hydrate to the temperature range of the desired lower hydrate. Analysis for water of hydration content is performed gravimetrically by gently heating the sample in a Bunsen flame. Students were able to produce results generally within 1-5% of the theoretical

Preparation and Analysis of Multiple Hydrates of Simple Salts

We have developed a laboratory project in which the student prepares a series of hydrates of simple salts and then determines the extent of hydration of the product(s). We believe this provides a good introduction to the concepts of solubility, saturation, recrystallization, relative compound stability (e.g., a dihydrate vs tetrahydrate at elevated temperature), and simple gravimetric analysis. Moreover, the project lends itself to many variations. For example, a student could be given a starting hydrated salt and asked to prepare another hydrate within a specified temperature range. Or students could be given the formulas of several hydrates stable over different temperature ranges and be asked to discover a method of preparation. If it is deemed desirable to extend the project, the cation and/or anion could be determined quantitatively. The preparation of ionic hydrates is accomplished by four methods: (i) slow evaporation of the solvent from a near saturated solution of the starting hydrate at a temperature within the stability range of the desired hydrate, (ii) crystallization within the temperature range of the target hydrate from a saturated solution prepared at higher temperatures, (iii) crystallization within the temperature stability range from mixed solvents, and (iv) heating a higher hydrate to the temperature range of the desired lower hydrate. Analysis for water of hydration content is performed gravimetrically by gently heating the sample in a Bunsen flame. Students were able to produce results generally within 1-5% of the theoretical

Polynitroxylated-Pegylated Hemoglobin Attenuates Fluid Requirements and Brain Edema in Combined Traumatic Brain Injury Plus Hemorrhagic Shock in Mice

Polynitroxylated-pegylated hemoglobin (PNPH), a bovine hemoglobin decorated with nitroxide and polyethylene glycol moieties, showed neuroprotection vs. lactated Ringer’s (LR) in experimental traumatic brain injury plus hemorrhagic shock (TBI+HS). Hypothesis: Resuscitation with PNPH will reduce intracranial pressure (ICP) and brain edema and improve cerebral perfusion pressure (CPP) vs. LR in experimental TBI+HS. C57/BL6 mice (n=20) underwent controlled cortical impact followed by severe HS to mean arterial pressure (MAP) of 25 to 27 mm Hg for 35 minutes. Mice (n=10/group) were then resuscitated with a 20 mL/kg bolus of 4% PNPH or LR followed by 10 mL/kg boluses targeting MAP\u3e70 mm Hg for 90 minutes. Shed blood was then reinfused. Intracranial pressure was monitored. Mice were killed and %brain water (%BW) was measured (wet/dry weight). Mice resuscitated with PNPH vs. LR required less fluid (26.0±0.0 vs. 167.0±10.7 mL/kg, PPP=0.002), higher CPP during resuscitation (69.2±0.46 vs. 45.5±0.68 mm Hg, PP=0.003). After TBI+HS, resuscitation with PNPH lowers fluid requirements, improves ICP and CPP, and reduces brain edema vs. LR, supporting its development