Shotgun microbial profiling of fossil remains.

Millions to billions of DNA sequences can now be generated from ancient skeletal remains thanks to the massive throughput of next-generation sequencing platforms. Except in cases of exceptional endogenous DNA preservation, most of the sequences isolated from fossil material do not originate from the specimen of interest, but instead reflect environmental organisms that colonized the specimen after death. Here, we characterize the microbial diversity recovered from seven c. 200- to 13 000-year-old horse bones collected from northern Siberia. We use a robust, taxonomy-based assignment approach to identify the microorganisms present in ancient DNA extracts and quantify their relative abundance. Our results suggest that molecular preservation niches exist within ancient samples that can potentially be used to characterize the environments from which the remains are recovered. In addition, microbial community profiling of the seven specimens revealed site-specific environmental signatures. These microbial communities appear to comprise mainly organisms that colonized the fossils recently. Our approach significantly extends the amount of useful data that can be recovered from ancient specimens using a shotgun sequencing approach. In future, it may be possible to correlate, for example, the accumulation of postmortem DNA damage with the presence and/or abundance of particular microbes.

What we know that could influence future treatment of phenylketonuria.

Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype (OMIM 261600), is an inborn error of metabolism that can result in impaired postnatal cognitive development. The phenotypic outcome is multifactorial in origin, based both in nature, the mutations in the gene encoding the L-phenylalanine hydroxylase enzyme, and nurture, the nutritional experience introducing L-phenylalanine into the diet. The PKU story contains many messages including a framework to appreciate the complexity of this disease where phenotype reflects both locus-specific and genomic components. This knowledge is now being applied in the development of patient-specific therapies.

Measurement of phenyllactate, phenylacetate, and phenylpyruvate by negative ion chemical ionization-gas chromatography/mass spectrometry in brain of mouse genetic models of phenylketonuria and non-phenylketonuria hyperphenylalaninemia.

Phenylketonuria (PKU) (OMIM 261600) is the first Mendelian disease to have an identified chemical cause of impaired cognitive development. The disease is accompanied by hyperphenylalaninemia (HPA) and elevated levels of phenylalanine metabolites (phenylacetate (PAA), phenyllactate (PLA), and phenylpyruvate (PPA)) in body fluids. Here we describe a method to determine the concentrations of PAA, PPA, and PLA in the brain of normal and mutant orthologous mice, the latter being models of human PKU and non-PKU HPA. Stable isotope dilution techniques are employed with the use of [(2)H(5)]-phenylacetic acid and [2,3, 3-(2)H(3)]-3-phenyllactic acid as internal standards. Negative ion chemical ionization (NICI)-GC/MS analyses are performed on the pentafluorobenzyl ester derivatives formed in situ in brain homogenates. Unstable PPA in the homogenate is reduced by NaB(2)H(4) to stable PLA, which is labeled with a single deuterium and discriminated from endogenous PLA in the mass spectrometer on that basis. The method demonstrates that these metabolites are easily measured in normal mouse brain and are elevated moderately in HPA mice and greatly in PKU mice. However, their concentrations are not sufficient in PKU to be "toxic"; phenylalanine itself remains the chemical candidate causing impaired cognitive development.

A heteroallelic mutant mouse model: A new orthologue for human hyperphenylalaninemia.

Hyperphenylalaninemias (HPA) are Mendelian disorders resulting from deficiencies in the conversion of phenylalanine to tyrosine. The vast majority are explained by a primary deficiency of phenylalanine hydroxylase (PAH) activity. The majority of untreated patients experience irreversible impairment of cognitive development. Although it is one of the best known hereditary metabolic disorders, mechanisms underlying the pathophysiology of the disease are still not fully understood; to this end, the availability of an orthologous animal model is relevant. Various mutant hyperphenylalaninemic mouse models with an HPA phenotype, generated by N-ethyl-N'-nitrosourea (ENU) mutagenesis at the Pah locus, have become available. Here we report a new hybrid strain, ENU1/2, with primary enzyme deficiency, produced by cross breeding. The ENU1, ENU1/2, and ENU2 strains display mild, moderate, and severe phenotypes, respectively, relative to the control strain (BTBR/Pas). The Pah enzyme activities of the various models correlate inversely with the corresponding phenylalanine levels in plasma and brain and the delay in plasma clearance response following a phenylalanine challenge. The maternal HPA effect on the fetus correlates directly with the degree of hyperphenylalaninemia, but only the ENU2 strain has impaired learning.

PAHdb: a locus-specific knowledgebase.

PAHdb is an online relational locus-specific "mutation database" (http://www.mcgill.ca/pahdb) for the human phenylalanine hydroxylase gene (symbol PAH) and its associated phenotypes (protein, metabolic, clinical). When combined with associated information (population distribution of allele, haplotype association, etc.) PAHdb functions as a knowledgebase. From the outset, and in the absence of raw data (e.g., sequence gels), PAHdb has instead been an annotated repository of information about mutations maintained by a team of curators. It is also disease-oriented, being focused on a variant phenotype (hyperphenylalaninemia (HPA) and its most important form of disease, phenylketonuria (PKU)) resulting from primary dysfunction of the PAH enzyme (EC 1.14.16.1); it is "patient friendly" in that it contains information for those personally involved with HPA/PKU (MIM# 261600). PAHdb also serves its community through direct interaction.

A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase.

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N'-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)-both pharmacologic (with a clear dose-response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.

Effects of fluoxetine or D-fenfluramine on serotonin release from, and levels in, rat frontal cortex.

Using in vivo microdialysis of frontal cortex in anesthetized rats, as well as analysis of frontal cortex homogenates, we examined the effects of chronic administration of fluoxetine (30 mg/kg, i.p.) or D-fenfluramine (7.5 mg/kg, i.p.), administered daily for 3 days, on serotonin and 5-HIAA levels a day later. Measurements were also taken after 3-, 7- , and 21-day recovery periods. Neither chronic fluoxetine nor D-fenfluramine changed basal serotonin release. Both treatments, however, transiently decreased the release of serotonin evoked by an acute dose of D-fenfluramine (10 mg/kg, i.p.). Release initially was completely suppressed in fluoxetine-pretreated animals but returned to normal by the 21st day of washout; following D-fenfluramine pretreatment, normal release was attained by the 7th day of washout. Both fluoxetine and D-fenfluramine transiently decreased 5-HIAA levels in the dialysates and tissues. Both drugs also caused prolonged changes in frontal cortex serotonin levels, D-fenfluramine lowering them but fluoxetine elevating them. These results suggest that, at comparable dosage levels relative to their ED50s, fluoxetine and D-fenfluramine cause comparable reversible effects on brain serotonin release. The drugs also cause prolonged but opposite changes in brain serotonin levels, probably reflecting differences in the extents to which they or their principal metabolites release serotonin and block its reuptake.