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Flood loss modelling is associated with considerable uncertainty. If prediction uncertainty of flood loss models is large, the reliability of model outcomes is questionable, and thus challenges the practical usefulness. A key problem in flood loss estimation is the transfer of models to geographical regions and to flood events that may differ from the ones used for model development. Variations in local characteristics and continuous system changes require regional adjustments and continuous updating with current evidence. However, acquiring data on damage influencing factors is usually very costly. Therefore, it is of relevance to assess the value of additional data in terms of model performance improvement. We use empirical flood loss data on direct damage to residential buildings available from computer aided telephone interviews that were compiled after major floods in Germany. This unique data base allows us to trace the changes in predictive model performance by incrementally extending the data base used to derive flood loss models. Two models are considered: a uni-variable stage damage function and RF-FLEMO, a multi-variable probabilistic model approach using Random Forests. Additional data are useful to improve model predictive performance and increase model reliability, however the gains also seem to depend on the model approach.

Vigabatrin (VGB; γ-vinyl-GABA) is an antiepileptic drug that elevates CNS GABA via irreversible inactivation of the GABA catabolic enzyme GABA-transaminase. VGB's clinical utility, however, can be curtailed by peripheral visual field constriction (pVFC) and thinning of the retinal nerve fiber layer (RNFL). Earlier studies from our laboratory revealed disruptions of autophagy by VGB. Here, we tested the hypothesis that VGB administration to animals would reveal alterations of gene expression in VGB-treated retina that associated with autophagy. VGB (140 mg/kg/d; subcutaneous minipump) was continuously administered to mice (n = 6 each VGB/vehicle) for 12 days, after which animals were euthanized. Retina was isolated for transcriptome (RNAseq) analysis and further validation using qRT-PCR and immunohistochemistry (IHC). For 112 differentially expressed retinal genes (RNAseq), two databases (Gene Ontology; Kyoto Encyclopedia of Genes and Genomes) were used to identify genes associated with visual function. Twenty four genes were subjected to qRT-PCR validation, and five (Gb5, Bdnf, Cplx9, Crh, Sox9) revealed significant dysregulation. IHC of fixed retinas verified significant down-regulation of Gb5 in photoreceptor cells. All of these genes have been previously shown to play a role in retinal function/circuitry signaling. Minimal impact of VGB on retinal autophagic gene expression was observed. This is the first transcriptome analysis of retinal gene expression associated with VGB intake, highlighting potential novel molecular targets potentially related to VGB's well known ocular toxicity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: Vigabatrin (VGB) is an effective antiepileptic that increases concentrations of inhibitory γ-aminobutyric acid (GABA) by inhibiting GABA transaminase. Reports of VGB-associated visual field loss limit its clinical usefulness, and retinal toxicity studies in laboratory animals have yielded conflicting results., Methods: We examined the functional and morphologic effects of VGB in C57BL/6J mice that received either VGB or saline IP from 10 to 18 weeks of age. Retinal structure and function were assessed in vivo by optical coherence tomography (OCT), ERG, and optomotor response. After euthanasia, retinas were processed for immunohistochemistry, and retinal GABA, and VGB quantified by mass spectrometry., Results: No significant differences in visual acuity or total retinal thickness were identified between groups by optomotor response or optical coherence tomography, respectively. After 4 weeks of VGB treatment, ERG b-wave amplitude was enhanced, and amplitudes of oscillatory potentials were reduced. Dramatic rod and cone bipolar and horizontal cell remodeling, with extension of dendrites into the outer nuclear layer, was observed in retinas of VGB-treated mice. VGB treatment resulted in a mean 3.3-fold increase in retinal GABA concentration relative to controls and retinal VGB concentrations that were 20-fold greater than brain., Conclusions: No evidence of significant retinal thinning or ERG a- or b-wave deficits were apparent, although we describe significant alterations in ERG b-wave and oscillatory potentials and in retinal cell morphology in VGB-treated C57BL/6J mice. The dramatic concentration of VGB in retina relative to the target tissue (brain), with a corresponding increase in retinal GABA, offers insight into the pathophysiology of VGB-associated visual field loss.

We present an update to the status of research on succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD), a rare disorder of GABA metabolism. This is an unusual disorder featuring the accumulation of both GABA and its neuromodulatory analog, gamma-hydroxybutyric acid (GHB), and recent studies have advanced the potential clinical application of NCS-382, a putative GHB receptor antagonist. Animal studies have provided proof-of-concept that enzyme replacement therapy could represent a long-term therapeutic option. The characterization of neuronal stem cells (NSCs) derived from aldehyde dehydrogenase 5a1 -/- (aldh5a1 -/- ) mice, the murine model of SSADHD, has highlighted NSC utility as an in vitro system in which to study therapeutics and associated toxicological properties. Gene expression analyses have revealed that transcripts encoding GABA A receptors are down-regulated and may remain largely immature in aldh5a1 -/- brain, characterized by excitatory as opposed to inhibitory outputs, the latter being the expected action in the mature central nervous system. This indicates that agents altering chloride channel activity may be therapeutically relevant in SSADHD. The most recent therapeutic prospects include mTOR (mechanistic target of rapamycin) inhibitors, drugs that have received attention with the elucidation of the effects of elevated GABA on autophagy. The outlook for novel therapeutic trials in SSADHD continues to improve.

We report the in vitro assessment of pharmacotoxicity for the high-affinity GHB receptor ligand, NCS-382, using neuronal stem cells derived from mice with a targeted deletion of the aldehyde dehydrogenase 5a1 gene (succinic semialdehyde dehydrogenase(SSADH)-deficient mice). These animals represent a phenocopy of the human disorder of GABA metabolism, SSADH deficiency, that metabolically features accumulation of both GABA and the GABA-analog γ-hydroxybutyric acid in conjunction with a nonspecific neurological phenotype. We demonstrate for the first time using MDCK cells that NCS-382 is actively transported and capable of inhibiting GHB transport. Following these in vitro assays with in vivo studies in aldh5a1 -/- mice, we found the ratio of brain/liver GHB to be unaffected by chronic NCS-382 administration (300mg/kg; 7 consecutive days). Employing a variety of cellular parameters (reactive oxygen and superoxide species, ATP production and decay, mitochondrial and lysosomal number, cellular viability and necrosis), we demonstrate that up to 1mM NCS-382 shows minimal evidence of pharmacotoxicity. As well, studies at the molecular level indicate that the effects of NCS-382 at 0.5mM are minimally toxic as evaluated using gene expression assay. The cumulative data provides increasing confidence that NCS-382 could eventually be considered in the therapeutic armament for heritable SSADH deficiency., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We explored the utility of neural stem cells (NSCs) as an in vitro model for evaluating preclinical therapeutics in succinic semialdehyde dehydrogenase-deficient (SSADHD) mice. NSCs were obtained from aldh5a1+/+ and aldh5a1-/- mice (aldh5a1 = aldehyde dehydrogenase 5a1 = SSADH). Multiple parameters were evaluated including: (1) production of GHB (γ-hydroxybutyrate), the biochemical hallmark of SSADHD; (2) rescue from cell death with the dual mTOR (mechanistic target of rapamycin) inhibitor, XL-765, an agent previously shown to rescue aldh5a1-/- mice from premature lethality; (3) mitochondrial number, total reactive oxygen species, and mitochondrial superoxide production, all previously documented as abnormal in aldh5a1-/- mice; (4) total ATP levels and ATP consumption; and (5) selected gene expression profiles associated with epilepsy, a prominent feature in both experimental and human SSADHD. Patterns of dysfunction were observed in all of these parameters and mirrored earlier findings in aldh5a1-/- mice. Patterns of dysregulated gene expression between hypothalamus and NSCs centered on ion channels, GABAergic receptors, and inflammation, suggesting novel pathomechanisms as well as a developmental ontogeny for gene expression potentially associated with the murine epileptic phenotype. The NSC model of SSADHD will be valuable in providing a first-tier screen for centrally-acting therapeutics and prioritizing therapeutic concepts of preclinical animal studies applicable to SSADHD.

Background: The mainstay of therapy for phenylketonuria (PKU) remains dietary protein restriction. Developmental and neurocognitive outcomes for patients, however, remain suboptimal. We tested the hypothesis that mice with PKU receiving protein-restricted diets would reveal disruptions of brain amino acids that shed light on these neurocognitive deficits., Method: Phenylalanine hydroxylase-deficient (PKU) mice and parallel controls (both wild-type and heterozygous) were fed custom diets containing 18, 6, and 4 % protein for 3 weeks, after which tissues (brain, liver, sera) were collected for amino acid analysis profiling., Results: Phenylalanine (phe) was increased in all tissues (p < 0.0001) of PKU mice and improved with protein restriction. In sera, decreased tyrosine (p < 0.01) was corrected (defined as not significantly different from the level in control mice receiving 18 % chow) with protein restriction, whereas protein restriction significantly increased many other amino acids. A similar trend for increased amino acid levels with protein restriction was also observed in liver. In brain, the effects of protein restriction on large neutral amino acids (LNAAs) were variable, with some deficit correction (threonine, methionine, glutamine) and no correction of tyrosine under any dietary paradigm. Protein restriction (4 % diet) in PKU mice significantly decreased lysine, arginine, taurine, glutamate, asparagine, and serine which had been comparable to control mice under 18 % protein intake., Conclusion: Depletion of taurine, glutamate, and serine in the brain of PKU mice with dietary protein restriction may provide new insight into neurocognitive deficits of PKU.

We investigated the mechanisms of mushroom toxin resistance in the Drosophila Genetic Reference Panel (DGRP) fly lines, using genome-wide association studies (GWAS). While Drosophila melanogaster avoids mushrooms in nature, some lines are surprisingly resistant to α-amanitin-a toxin found solely in mushrooms. This resistance may represent a pre-adaptation, which might enable this species to invade the mushroom niche in the future. Although our previous microarray study had strongly suggested that pesticide-metabolizing detoxification genes confer α-amanitin resistance in a Taiwanese D. melanogaster line Ama-KTT, none of the traditional detoxification genes were among the top candidate genes resulting from the GWAS in the current study. Instead, we identified Megalin, Tequila, and widerborst as candidate genes underlying the α-amanitin resistance phenotype in the North American DGRP lines, all three of which are connected to the Target of Rapamycin (TOR) pathway. Both widerborst and Tequila are upstream regulators of TOR, and TOR is a key regulator of autophagy and Megalin-mediated endocytosis. We suggest that endocytosis and autophagy of α-amanitin, followed by lysosomal degradation of the toxin, is one of the mechanisms that confer α-amanitin resistance in the DGRP lines.

Background: Gamma-vinyl-γ-aminobutyric acid (GABA) (vigabatrin) is an antiepileptic drug and irreversible GABA transaminase inhibitor associated with visual field impairment, which limits its clinical utility. We sought to relate altered visual evoked potentials associated with vigabatrin intake to transcriptional changes in the mechanistic target of rapamycin (mTOR) pathway and GABA receptors to expose further mechanisms of vigabatrin-induced visual field loss., Methods: Vigabatrin was administered to mice via an osmotic pump for two weeks to increase GABA levels. Visual evoked potentials were examined, eye samples were collected, and gene expression was measured by quantitative reverse transcription-polymerase chain reaction. Similarly, human retinal pigment epithelial cells (ARPE19) were exposed to vigabatrin and treated with mTOR inhibitors for mTOR pathway analysis and to assess alterations in organelle accumulation by microscopy., Results: Dysregulated expression of transcripts in the mTOR pathway, GABA A/B receptors, metabotropic glutamate (Glu) receptors 1/6, and GABA/glutamate transporters in the eye were found in association with visual evoked potential changes during vigabatrin administration. Rrag genes were upregulated in both mouse eye and ARPE19 cells. Immunoblot of whole eye revealed greater than three fold upregulation of a 200 kDa band when immunoblotted for ras-related guanosine triphosphate binding D. Microscopy of ARPE19 cells revealed selective reversal of vigabatrin-induced organelle accumulation by autophagy-inducing drugs, notably Torin 2. Changes in the mTOR pathway gene expression, including Rrag genes, were corrected by Torin 2 in ARPE19 cells., Conclusions: Our studies, indicating GABA-associated augmentation of RRAG and mTOR signaling, support further preclinical evaluation of mTOR inhibitors as a therapeutic strategy to potentially mitigate vigabatrin-induced ocular toxicity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aldehyde dehydrogenase 5a1-deficient (aldh5a1 -/- ) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts autophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypothesis that aldh5a1 -/- mice would show altered levels of mRNA for genes associated with mTOR signaling and oxidative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1 -/- mice. These metabolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1 -/- mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2-nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1 -/- mice, and several were significantly improved with the administration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxidative stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Gamma-hydroxybutyrate (GHB) is a drug of abuse, an approved therapeutic for narcolepsy, an agent employed for facilitation of sexual assault, as well as a biomarker of succinic semialdehyde dehydrogenase deficiency (SSADHD). Our laboratory seeks to identify surrogate biomarkers in SSADHD that can shed light on the developmental course of this neurometabolic disease. Since GHB may be quantified in hair as a potential surrogate to identify victims of drug-related assault, we have opted to examine its level in SSADHD. We quantified GHB in hair derived from ten patients with SSADHD, and documented a significant negative age correlation. These findings are consistent with recent results in patient biological fluids, including plasma and red blood cells. These findings may provide additional insight into the developmental course of SSADHD (Jansen et al., J Inherit Metab Dis 39:795-800, 2016).

We hypothesized that blood levels of γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB), biomarkers of succinic semialdehyde dehydrogenase deficiency (SSADHD), would correlate with age. GABA and GHB were quantified in plasma and red blood cells (RBCs) from 18 patients (age range 5-41 years; median 8). Both metabolites negatively correlated with age (P < 0.05). Plasma and RBC GHB declined with age, reaching a nadir and approximate steady state by 10 years. Declining plasma GABA achieved this approximate steady state at 30-40 years of age. These biomarker relationships may reflect further GABA- and GHB-ergic neurotransmission imbalances that correlate with the onset of adolescent/adulthood neuropsychiatric morbidity and epilepsy in SSADHD., Competing Interests: The authors cumulatively declare they have no, and no perceived, conflicts of interest.

Recent studies have identified a role for supraphysiological gamma-aminobutyric acid (GABA) in the regulation of mechanistic target of rapamycin (mTOR), a protein kinase with pleiotropic roles in cellular development and homeostasis, including integration of growth factors and nutrient sensing and synaptic input in neurons (Lakhani et al. 2014; Vogel et al. 2015). Aldehyde dehydrogenase 5a1-deficient (aldh5a1 -/- ) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA that disrupts mitophagy and increases mitochondria number with enhanced oxidant stress. Treatment with the mTOR inhibitor, rapamycin, significantly attenuates these GABA-related anomalies. We extend those studies through characterization of additional rapamycin analog (rapalog) agents including temsirolimus, dual mTOR inhibitors [Torin 1 and 2 (Tor 1/ Tor 2), Ku-0063794, and XL-765], as well as mTOR-independent autophagy inducers [trehalose, tat-Beclin 1, tacrolimus (FK-506), and NF-449) in aldh5a1 -/- mice. Rapamycin, Tor 1, and Tor 2 rescued these mice from premature lethality associated with status epilepticus. XL-765 extended lifespan significantly and induced weight gain in aldh5a1 -/- mice; untreated aldh5a1 -/- mice failed to increase body mass. Expression profiling of animals rescued with Tor 1/Tor 2 and XL-765 revealed multiple instances of pharmacological compensation and/or correction of GABAergic and glutamatergic receptors, GABA/glutamate transporters, and GABA/glutamate-associated proteins, with Tor 2 and XL-765 showing optimal outcomes. Our studies lay the groundwork for further evaluation of mTOR inhibitors in aldh5a1 -/- mice, with therapeutic ramifications for heritable disorders of GABA and glutamate neurotransmission., Competing Interests: Kara R. Vogel declares that she has no conflict of interest. Garrett R. Ainslie declares that he has no conflict of interest K. Michael Gibson declares that he has no conflict of interest.

Gamma-aminobutyric acid (GABA) is an endogenous inhibitory neurotransmitter and precursor of gamma-hydroxybutyric acid (GHB). NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid), a known GHB receptor antagonist, has shown significant efficacy in a murine model of succinic semialdehyde dehydrogenase deficiency (SSADHD), a heritable neurological disorder featuring chronic elevation of GHB that blocks the final step of GABA degradation. NCS-382 exposures and elimination pathways remain unknown; therefore, the goal of the present work was to obtain in vivo pharmacokinetic data in a murine model and to identify the NCS-382 metabolites formed by mouse and human. NCS-382 single-dose mouse pharmacokinetics were established following an intraperitoneal injection (100, 300, and 500 mg/kg body weight) and metabolite identification was conducted using HPLC-MS/MS. Kinetic enzyme assays employed mouse and human liver microsomes. Upon gaining an understanding of the NCS-382 clearance mechanisms, a chemical inhibitor was used to increase NCS-382 brain exposure in a pharmacokinetic/pharmacodynamic study. Two major metabolic pathways of NCS-382 were identified as dehydrogenation and glucuronidation. The K m for the dehydrogenation pathway was determined in mouse ( K m  = 29.5 ± 10.0  μ mol/L) and human ( K m  = 12.7 ± 4.8  μ mol/L) liver microsomes. Comparable parameters for glucuronidation were >100  μ mol/L in both species. Inhibition of NCS-382 glucuronidation, in vivo, by diclofenac resulted in increased NCS-382 brain concentrations and protective effects in gamma-butyrolactone-treated mice. These initial evaluations of NCS-382 pharmacokinetics and metabolism inform the development of NCS-382 as a potential therapy for conditions of GHB elevation (including acute intoxication & SSADHD).

Discovered some 35 years ago, succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a rare, autosomal recessively-inherited defect in the second step of the GABA degradative pathway. Some 200 patients have been reported, with broad phenotypic and genotypic heterogeneity. SSADHD represents an unusual neurometabolic disorder in which two neuromodulatory agents, GABA (and the GABA analogue, 4-hydroxybutyrate), accumulate to supraphysiological levels. The unexpected occurrence of epilepsy in several patients is counterintuitive in view of the hyperGABAergic state, in which sedation might be expected. However, the epileptic status of some patients is most likely represented by broader imbalances of GABAergic and glutamatergic neurotransmission. Cumulative research encompassing decades of basic and clinical study of SSADHD reveal a monogenic disease with broad pathophysiological and clinical phenotypes. Numerous metabolic perturbations unmasked in SSADHD include alterations in oxidative stress parameters, dysregulation of autophagy and mitophagy, dysregulation of both inhibitory and excitatory neurotransmitters and gene expression, and unique subsets of SNP alterations of the SSADH gene (so-called ALDH5A1, or aldehyde dehydrogenase 5A1 gene) on the 6p22 chromosomal arm. While seemingly difficult to collate and interpret, these anomalies have continued to open novel pathways for pharmacotherapeutic considerations. Here, we present an update on selected aspects of SSADHD, the ALDH5A1 gene, and future avenues for research on this rare disorder of GABA metabolism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hypothesis: An enzymatic assay for quantification of γ-hydroxybutyric acid (GHB) in biofluids can be employed for targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) in selected populations., Rationale: We used a two-tiered study approach, in which the first study (proof of concept) examined 7 urine samples derived from patients with SSADHD and 5 controls, and the second study (feasibility study) examined a broader sample population of patients and controls, including plasma., Objective: Split samples of urine and plasma (anonymized) were evaluated by enzymatic assay, gas chromatography alone (proof of concept) and gas chromatography-mass spectrometry, and the results compared., Method: Multiple detection methods have been developed to detect GHB. We evaluated an enzymatic assay which employs recombinant GHB dehydrogenase coupled to NADH production, the latter quantified on a Cobas Integra 400 Plus. Results: In our proof of concept study, we analyzed 12 urine samples (5 controls, 7 SSADHD), and in the feasibility study we evaluated 33 urine samples (23 controls, 10 SSADHD) and 31 plasma samples (14 controls, 17 SSADHD). The enzymatic assay carried out on a routine clinical chemistry analyzer was robust, revealing excellent agreement with instrumental methods in urine (GC-FID: r = 0.997, p ≤ 0.001; GC-MS: r = 0.99, p ≤ 0.001); however, the assay slightly over-estimated GHB levels in plasma, especially those in which GHB levels were low. Conversely, correlations for the enzymatic assay with comparator methods for higher plasma GHB levels were excellent (GC-MS; r = 0.993, p ≤ 0.001)., Conclusion: We have evaluated the capacity of this enzymatic assay to identify patients with SSADHD via quantitation of GHB. The data suggests that the enzymatic assay may be a suitable screening method to detect SSADHD in selected populations using urine. In addition, the assay can be used in basic research the elucidate the mechanism of the underlying disease or monitor GHB- levels for the evaluation of drug candidates., Synopsis: An enzymatic assay for GHB in biofluids was evaluated as a screening method for SSADHD and found to be reliable in urine, but in need of refinement for application to plasma.

Recent findings in mice with targeted deletion of the GABA-metabolic enzyme succinic semialdehyde dehydrogenase revealed a new role for supraphysiological GABA (4-aminobutyric acid) in the activation of the mechanistic target of rapamycin (mTOR) that results in disruption of endogenous mitophagy. Employing biochemical and electron microscopic methodology, we examined the hypothesis that similar outcomes would be observed during intervention with vigabatrin, whose antiepileptic capacity hinges on central nervous system GABA elevation. Vigabatrin intervention was associated with significantly enhanced mitochondrial numbers and areas in normal mice that could be selectively normalized with the rapalog and mechanistic target of rapamycin inhibitor, Torin 1. Moreover, short-term administration of vigabatrin induced apoptosis and enhanced phosphorylation of mechanistic target of rapamycin Ser 2448 in liver. Our results provide new insight into adverse outcomes associated with vigabatrin intervention, and the first evidence that its administration is associated with increased mitochondrial number in central and peripheral tissues that may associate with mechanistic target of rapamycin function and enhanced cell death.

Objective: Initial studies on the use of non-physiological amino acids (NPAAs) to block the accretion of Phe in the brain Pah enu2-/- mice revealed that 2-aminoisobutyrate (AIB) and N -methyl-2-aminoisobutyrate (MAIB) were promising lead compounds whose pharmacokinetic parameters warranted investigation., Methods: Control and Pah enu2-/- mice received intraperitoneal NPAA treatments as test compounds (150, 300 and 500 mg/kg, 1 or 7 days;) followed by collection of sera, liver and brain. LC-MS analysis was developed to quantify both AIB and MAIB in all matrices, and pharmacokinetic parameters for distribution, partitioning, accumulation and MAIB demethylation were determined., Results: MAIB was partially converted to AIB in vivo . AIB and MAIB partitioned similarly from sera to brain and liver, with an approximate 10-fold higher accumulation in liver compared to brain. In comparison to MAIB, AIB accumulated to approximately 3 to 7-fold higher concentration in brain. Analysis of brain and liver revealed a trend toward decreased Phe with increased MAIB sera concentration., Conclusions: Our data support further pharmacokinetic characterization of MAIB and AIB in preparation for further preclinical safety, toxicity and tolerability studies of both AIB and MAIB.

Insect resistance to toxins exerts not only a great impact on our economy, but also on the ecology of many species. Resistance to one toxin is often associated with cross-resistance to other, sometimes unrelated, chemicals. In this study, we investigated mushroom toxin resistance in the fruit fly Drosophila melanogaster (Meigen). This fruit fly species does not feed on mushrooms in nature and may thus have evolved cross-resistance to α-amanitin, the principal toxin of deadly poisonous mushrooms, due to previous pesticide exposure. The three Asian D. melanogaster stocks used in this study, Ama-KTT, Ama-MI, and Ama-KLM, acquired α-amanitin resistance at least five decades ago in their natural habitats in Taiwan, India, and Malaysia, respectively. Here we show that all three stocks have not lost the resistance phenotype despite the absence of selective pressure over the past half century. In response to α-amanitin in the larval food, several signs of developmental retardation become apparent in a concentration-dependent manner: higher pre-adult mortality, prolonged larva-to-adult developmental time, decreased adult body size, and reduced adult longevity. In contrast, female fecundity nearly doubles in response to higher α-amanitin concentrations. Our results suggest that α-amanitin resistance has no fitness cost, which could explain why the resistance has persisted in all three stocks over the past five decades. If pesticides caused α-amanitin resistance in D. melanogaster, their use may go far beyond their intended effects and have long-lasting effects on ecosystems.

Background: Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain., Methods: To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses., Results: LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU., Conclusions: Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders.

In addition to key roles in embryonic neurogenesis and myelinogenesis, γ-aminobutyric acid (GABA) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6K1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm these processes in mammals, we examined the succinic semialdehyde dehydrogenase (SSADH)-deficient mouse model that accumulates supraphysiological GABA in the central nervous system and other tissues. Mutant mice displayed increased mitochondrial numbers in the brain and liver, expected with a defect in mitophagy, and morphologically abnormal mitochondria. Administration of rapamycin to these mice reduced mTOR activity, reduced the elevated mitochondrial numbers, and normalized aberrant antioxidant levels. These results confirm a novel role for GABA in cell signaling and highlight potential pathomechanisms and treatments in various human pathologies, including SSADH deficiency, as well as other diseases characterized by elevated levels of GABA.

The applications, outcomes and future strategies of hepatocyte transplantation (HTx) as a corrective intervention for inherited metabolic disease (IMD) are described. An overview of HTx in IMDs, as well as preclinical evaluations in rodent and other mammalian models, is summarized. Current treatments for IMDs are highlighted, along with short- and long-term outcomes and the potential for HTx to supplement or supplant these treatments. Finally, the advantages and disadvantages of HTx are presented, highlighted by long-term challenges with interorgan engraftment and expansion of transplanted cells, in addition to the future prospects of stem cell transplants. At present, the utility of HTx is represented by the potential to bridge patients with life-threatening liver disease to organ transplantation, especially as an adjuvant intervention where severe organ shortages continue to pose challenges.

This review summarizes a presentation made at the retirement Symposium of Prof. Dr. Cornelis Jakobs in November of 2011, highlighting the progress toward clinical trials in succinic semialdehyde dehydrogenase (SSADH) deficiency, a disorder first recognized in 1981. Active and potential clinical interventions, including vigabatrin, L-cycloserine, the GHB receptor antagonist NCS-382, and the ketogenic diet, are discussed. Several biomarkers to gauge clinical efficacy have been identified, including cerebrospinal fluid metabolites, neuropsychiatric testing, MRI, EEG, and measures of GABAergic function including (11 C)flumazenil positron emission tomography (PET) and transcranial magnetic stimulation (TMS). Thirty years after its discovery, encompassing extensive studies in both patients and the corresponding murine model, we are now running an open-label trial of taurine intervention, and are poised to undertake a phase II trial of the GABAB receptor antagonist SGS742.

Transport of large neutral amino acids (LNAA) across the blood brain barrier (BBB) is facilitated by the L-type amino acid transporter, LAT1. Peripheral accumulation of one LNAA (e.g., phenylalanine (phe) in PKU) is predicted to increase uptake of the offending amino acid to the detriment of others, resulting in disruption of brain amino acid homeostasis. We hypothesized that selected non-physiological amino acids (NPAAs) such as DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), 2-aminoisobutyrate (AIB), and N-methyl-aminoisobutyrate (MAIB), acting as competitive inhibitors of various brain amino acid transporters, could reduce brain phe in Pah (enu2) mice, a relevant murine model of PKU. Oral feeding of 5 % NL, 5 % AIB, 0.5 % NB and 3 % MAIB reduced brain phe by 56 % (p < 0.01), -1 % (p = NS), 27 % (p < 0.05) and 14 % (p < 0.01), respectively, compared to untreated subjects. Significant effects on other LNAAs (tyrosine, methionine, branched chain amino acids) were also observed, however, with MAIB displaying the mildest effects. Of interest, MAIB represents an inhibitor of the system A (alanine) transporter that primarily traffics small amino acids and not LNAAs. Our studies represent the first in vivo use of these NPAAs in Pah (enu2) mice, and provide proof-of-principle for their further preclinical development, with the long-term objective of identifying NPAA combinations and concentrations that selectively restrict brain phe transport while minimally impacting other LNAAs and downstream intermediates.

Background: Our laboratory seeks a pharmacotherapeutic intervention for PKU that utilizes non-physiological amino acids (NPAAs) to block the accumulation of phenylalanine (Phe) in the brain. In previous studies (Vogel et al. 2013), methylation of the amino group of 2-aminoisobutyrate (AIB) provided an enhanced degree of selectivity for Phe restriction into the brain of Pah(enu2) mice in comparison to unmethylated AIB, leading to the hypothesis that 2-(methylamino)alkanoic acid analogs of AIB might represent targeted inhibitors of Phe accretion into the brain., Methods: Pah(enu2) and control mice were intraperitoneally administered (500-750 mg/kg body weight, once daily; standard 19% protein diet) AIB, methyl AIB (MAIB), isovaline, and two MAIB analogs, 2-methyl-2-(methylamino)butanoic (MeVal) and 3-methyl-2-(methylamino)pentanoic (MePent) acids for one week, followed by brain and blood isolation for amino acid analyses using UPLC., Results: In the brain, AIB significantly reduced Phe accretion in Pah(enu2) mice, while MeVal significantly improved glutamine and aspartic acids. Four of five test compounds improved brain threonine and arginine levels. AIB, MAIB and IsoVal significantly reduced blood Phe, with no effect of any drug intervention on other sera amino acids., Conclusions: Further evaluation of AIB and the 2-(methylamino)alkanoic acids as inhibitors of brain Phe accumulation in Pah(enu2) mice is warranted, with more detailed evaluations of route of administration, combinatorial intervention, and detailed toxicity studies., (© 2013.)

Microwave atomic clocks have been the de facto standards for precision time and frequency metrology over the past 50 years, finding widespread use in basic scientific studies, communications, and navigation. However, with its higher operating frequency, an atomic clock based on an optical transition can be much more stable. We demonstrate an all-optical atomic clock referenced to the 1.064-petahertz transition of a single trapped 199Hg+ ion. A clockwork based on a mode-locked femtosecond laser provides output pulses at a 1-gigahertz rate that are phase-coherently locked to the optical frequency. By comparison to a laser-cooled calcium optical standard, an upper limit for the fractional frequency instability of 7 x 10(-15) is measured in 1 second of averaging-a value substantially better than that of the world's best microwave atomic clocks.

The frequency comb created by a femtosecond mode-locked laser and a microstructured fiber is used to phase coherently measure the frequencies of both the Hg+ and Ca optical standards with respect to the SI second. We find the transition frequencies to be f(Hg) = 1 064 721 609 899 143(10) Hz and f(Ca) = 455 986 240 494 158(26) Hz, respectively. In addition to the unprecedented precision demonstrated here, this work is the precursor to all-optical atomic clocks based on the Hg+ and Ca standards. Furthermore, when combined with previous measurements, we find no time variations of these atomic frequencies within the uncertainties of the absolute value of( partial differential f(Ca)/ partial differential t)/f(Ca) < or =8 x 10(-14) yr(-1) and the absolute value of(partial differential f(Hg)/ partial differential t)/f(Hg) < or =30 x 10(-14) yr(-1).

With a fiber-broadened, femtosecond-laser frequency comb, the 76-THz interval between two laser-cooled optical frequency standards was measured with a statistical uncertainty of 2x10(-13) in 5 s , to our knowledge the best short-term instability thus far reported for an optical frequency measurement. One standard is based on the calcium intercombination line at 657 nm, and the other, on the mercury ion electric-quadrupole transition at 282 nm. By linking this measurement to the known Ca frequency, we report a new frequency value for the Hg(+) clock transition with an improvement in accuracy of ~10(5) compared with its best previous measurement.