Reducing attractiveness of e-liquids: proposal for a restrictive list of tobacco-related flavourings.

OBJECTIVE: Electronic cigarettes are addictive and harmful, and flavour is a key factor determining their abuse liability. Both adult smokers and young non-smokers like sweet and fruity flavours in particular. In order to discourage e-cigarette use among youth, the Dutch government announced in 2020 to only allow tobacco flavours in e-liquids. We propose a restrictive list of flavourings that will only enable the production of e-liquids with a tobacco flavour. METHODS: We used e-liquid ingredient data notified via the European Common Entry Gate system before the government's announcement. First, we classified all e-liquids into flavour categories, and continued with the set of flavourings present in tobacco e-liquids. Five selection criteria related to prevalence of use, chemical composition, flavour description and health effects were defined to compile a restrictive list of tobacco flavourings. RESULTS: E-liquids marketed as having tobacco flavour contained 503 different flavourings, some with tobacco flavour, but also other (such as sweet) flavours. We excluded (1) 330 flavourings used in <0.5% of e-liquids, (2) 77 used less frequently in tobacco than in all e-liquids, (3) 13 plant extracts, (4) 60 that are sweet or not associated with a tobacco flavour and (5) 7 flavourings with hazardous properties. This resulted in a final list of 16 flavourings. CONCLUSIONS: Implementing this restrictive list will likely discourage e-cigarette use among youth, but could also make e-cigarettes less attractive as smoking cessation aid.

Sensory appeal and puffing intensity of e-cigarette use: Influence of nicotine salts versus free-base nicotine in e-liquids.

BACKGROUND: In the US, nicotine salts (with protonated nicotine instead of free-based nicotine) have been reported to lower the harshness and bitterness of e-cigarette aerosols, making it easier to inhale high levels of nicotine. This study aimed to determine whether nicotine salts also increase sensory appeal at lower concentrations (< 20mg/mL). Moreover, and novel, inhalation intensity of both types of e-liquids was compared. METHODS: In a randomized, double-blinded, within-participants design, healthy adults who use e-cigarettes (n=68) vaped tobacco-flavored e-liquids containing 12mg/mL of free-based nicotine or nicotine salt ad libitum, using their own device, during two online sessions (June-July 2021, Utrecht, The Netherlands). The sensory parameters perceived liking, nicotine intensity, harshness, and pleasantness were rated on a 100-unit visual analog scale. The intensity of use was determined by the recorded puff number, duration and interval. RESULTS: Test scores on appeal, harshness and puffing behavior parameters showed no significant differences between the nicotine salt and the free-base condition. The average inhalation time was 2.5seconds. Additional analyses found no significant effect of liquid order, age, gender, smoking status, vaping frequency and familiarity with nicotine salts. Significant positive correlations were found between the sensory parameters except for harshness. CONCLUSIONS: Contrary to a previous study that used higher nicotine concentrations and standardized puffing conditions in a laboratory setting, we did not observe the effects of nicotine salts on sensory appeal in our real-life study paradigm. Moreover, we did not see effects on study parameters related to puffing intensity.

Comprehensive Dutch market data analysis shows that e-liquids with nicotine salts have both higher nicotine and flavour concentrations than those with free-base nicotine.

OBJECTIVES: Recent years have seen an increase in e-liquids containing nicotine salts. Nicotine salts are less harsh and bitter than free-base nicotine and therefore can facilitate inhalation. Because inhalation-facilitating ingredients are banned in the European Union, we assessed the occurrence and characteristics of nicotine salt-containing e-liquids notified for the Netherlands. METHODS: We analysed data for 39 030 products, submitted by manufacturers in the European Union Common Entry Gate system, as extracted on 30 June 2020. RESULTS: Nicotine salts were present in 13% of e-liquids, especially in pod-related e-liquids (73%) and e-liquids registered from 2018 onwards (over 25%). We found six nicotine salt ingredients (NSIs): nicotine lactate, salicylate, benzoate, levulinate, ditartrate and malate. Nicotine salts also occurred as nicotine-organic acid ingredient combination (NAIC), like nicotine and benzoic acid. Nicotine concentrations were twofold higher in e-liquids with NSI (median 14 mg/mL) and NAIC (11 mg/mL) than for free-base nicotine (6 mg/mL). E-liquids with NSI contained a fourfold higher number (median n=17) and concentration (median 31.0 mg/mL) of flavour ingredients than e-liquids with free-base nicotine (n=4, 7.4 mg/mL). In NAIC-containing e-liquids, these were threefold higher (n=12, 21.5 mg/mL). E-liquids with nicotine salts were less often tobacco flavoured but more often had fruity or sweet flavours. CONCLUSIONS: A substantial and increasing share of e-liquids in the Netherlands contains nicotine salts. Their characteristics can make such e-liquids more addictive and more attractive, especially to young and beginning users. Policymakers are advised to consider regulating products containing nicotine salts.

Sex-specific newborn screening for X-linked adrenoleukodystrophy.

Males with X-linked adrenoleukodystrophy (ALD) are at high risk for developing adrenal insufficiency and/or progressive leukodystrophy (cerebral ALD) at an early age. Pathogenic variants in ABCD1 result in elevated levels of very long-chain fatty acids (VLCFA), including C26:0-lysophosphatidylcholine (C26:0-LPC). Newborn screening for ALD enables prospective monitoring and timely therapeutic intervention, thereby preventing irreversible damage and saving lives. The Dutch Health Council recommended to screen only male newborns for ALD without identifying untreatable conditions associated with elevated C26:0-LPC, like Zellweger spectrum disorders and single peroxisomal enzyme defects. Here, we present the results of the SCAN (Screening for ALD in the Netherlands) study which is the first sex-specific newborn screening program worldwide. Males with ALD are identified based on elevated C26:0-LPC levels, the presence of one X-chromosome and a variant in ABCD1, in heel prick dried bloodspots. Screening of 71 208 newborns resulted in the identification of four boys with ALD who, following referral to the pediatric neurologist and confirmation of the diagnosis, enrolled in a long-term follow-up program. The results of this pilot show the feasibility of employing a boys-only screening algorithm that identifies males with ALD without identifying untreatable conditions. This approach will be of interest to countries that are considering ALD newborn screening but are reluctant to identify girls with ALD because for girls there is no direct health benefit. We also analyzed whether gestational age, sex, birth weight and age at heel prick blood sampling affect C26:0-LPC concentrations and demonstrate that these covariates have a minimal effect.

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.

Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

Conceptual model for the evaluation of attractiveness, addictiveness and toxicity of tobacco and related products: The example of JUUL e-cigarettes.

Many new tobacco and related products (nTRP) have emerged on the market, with unknown health risks. Here, we present a conceptual model containing the factors and relations between them that contribute to the nTRP's health effects. Factors that determine attractiveness, addictiveness and toxicity of nTRP were defined based on previous assessments, literature, and expert discussions. Our model will aid in identifying key risk factors contributing to increased risk of adverse health effects for a product in a qualitative manner. Additionally, it can gauge attractiveness for specific user groups, as a determinant for population prevalence of use. Our model can be used to identify aspects of nTRP that require attention for public information or product regulation. As an example, we applied this to JUUL, a popular e-cigarette in the US. Aspects of concern for JUUL are its attractive and discrete shape, user-friendly prefilled pods, flavors, high aerosol nicotine levels, and liquids containing nicotine salts instead of free-based nicotine. The addictiveness and especially attractiveness are sufficiently high to have a large potential impact on population health due to its contribution to use and hence exposure. Products and their use can change over time; therefore market research and monitoring are crucial.

Improving the Analysis of E-Cigarette Emissions: Detecting Human "Dry Puff" Conditions in a Laboratory as Validated by a Panel of Experienced Vapers.

INTRODUCTION: E-cigarette product regulation requires accurate analyses of emissions. User behavior, including device power setting selection, should be mimicked closely when generating e-cigarette emissions in a laboratory. Excessively high power settings result in an adverse burnt off-taste, called "dry puff flavor". This should be avoided because it results in an overestimation of toxicant levels (especially certain carbonyls). This study presents a human volunteer-validated approach to detect excessively high e-cigarette power settings by HPLC-DAD (high-performance liquid chromatography-diode array detection) carbonyl analysis. METHODS: Thirteen experienced e-cigarette users evaluated whether the "dry puff flavor" was present at different power settings (10 W-25 W), recording their assessment on a 100-unit visual analog scale (VAS). They assessed e-cigarettes equipped with 1.2 Ω or 1.6 Ω coils containing menthol, vanilla or fruit-flavored e-liquids. In a machine-vaping experiment, emissions from the same liquid/coil/power setting combinations were subjected to HPLC-DAD analysis of dinitrophenol hydrazine (DNPH)-derivatized carbonyls, such as lactaldehyde and formaldehyde. A simple algorithm, based on the cutoff values for each marker, was applied to relate the dry puff flavor (as assessed by the human volunteers) to the laboratory measurements. RESULTS: Eleven carbonyl compounds were found to agree with the human assessments. Based on the amounts of these compounds in the emissions, the dry-puff flavor did match at all combinations of e-liquids and coils examined. Dry-puff flavor was observed at different power levels with the different liquids tested. CONCLUSIONS: The described method can detect dry puff conditions and is therefore a useful tool to ensure user-relevant conditions in laboratory analyses of e-cigarette emissions. IMPLICATIONS: This study improves the chemical analysis of e-cigarette emissions. It offers a method to select an appropriate (i.e., user-relevant) power setting for e-cigarettes, which is a critical parameter for emission analysis and therefore important for regulatory purposes and risk assessments. Compared to the approach of using human volunteers to select appropriate power settings for different products by taste, the described method is cheaper, faster, more practical and more ethical.

Corrigendum: Adrenoleukodystrophy Newborn Screening in the Netherlands (SCAN Study): The X-Factor.

[This corrects the article DOI: 10.3389/fcell.2020.00499.].

Adrenoleukodystrophy Newborn Screening in the Netherlands (SCAN Study): The X-Factor.

X-linked adrenoleukodystrophy (ALD) is a devastating metabolic disorder affecting the adrenal glands, brain and spinal cord. Males with ALD are at high risk for developing adrenal insufficiency or progressive cerebral white matter lesions (cerebral ALD) at an early age. If untreated, cerebral ALD is often fatal. Women with ALD are not at risk for adrenal insufficiency or cerebral ALD. Newborn screening for ALD in males enables prospective monitoring and timely therapeutic intervention, thereby preventing irreparable damage and saving lives. The Dutch Ministry of Health adopted the advice of the Dutch Health Council to add a boys-only screen for ALD to the newborn screening panel. The recommendation made by the Dutch Health Council to only screen boys, without gathering any unsolicited findings, posed a challenge. We were invited to set up a prospective pilot study that became known as the SCAN study (SCreening for ALD in the Netherlands). The objectives of the SCAN study are: (1) designing a boys-only screening algorithm that identifies males with ALD and without unsolicited findings; (2) integrating this algorithm into the structure of the Dutch newborn screening program without harming the current newborn screening; (3) assessing the practical and ethical implications of screening only boys for ALD; and (4) setting up a comprehensive follow-up that is both patient- and parent-friendly. We successfully developed and validated a screening algorithm that can be integrated into the Dutch newborn screening program. The core of this algorithm is the "X-counter." The X-counter determines the number of X chromosomes without assessing the presence of a Y chromosome. The X-counter is integrated as second tier in our 4-tier screening algorithm. Furthermore, we ensured that our screening algorithm does not result in unsolicited findings. Finally, we developed a patient- and parent-friendly, multidisciplinary, centralized follow-up protocol. Our boys-only ALD screening algorithm offers a solution for countries that encounter similar ethical considerations, for ALD as well as for other X-linked diseases. For ALD, this alternative boys-only screening algorithm may result in a more rapid inclusion of ALD in newborn screening programs worldwide.

Characteristic Human Individual Puffing Profiles Can Generate More TNCO than ISO and Health Canada Regimes on Smoking Machine When the Same Brand Is Smoked.

Human smoking behavior influences exposure to smoke toxicants and is important for risk assessment. In a prospective observational study, the smoking behavior of Marlboro smokers was measured for 36 h. Puff volume, duration, frequency, flow and inter-puff interval were recorded with the portable CReSSmicro™ device, as has often been done by other scientists. However, the use of the CReSSmicro™ device may lead to some registration pitfalls since the method of insertion of the cigarette may influence the data collection. Participants demonstrated consistent individual characteristic puffing behavior over the course of the day, enabling the creation of a personalized puffing profile. These puffing profiles were subsequently used as settings for smoking machine experiments and tar, nicotine and carbon monoxide (TNCO) emissions were generated. The application of human puffing profiles led to TNCO exposures more in the range of Health Canada Intense (HCI)-TNCO emissions than for those of the International Standardization Organization (ISO). Compared to the ISO regime, which applies a low puff volume relative to human smokers, the generation of TNCO may be at least two times higher than when human puffing profiles were applied on the smoking machine. Human smokers showed a higher puffing intensity than HCI and ISO because of higher puffing frequency, which resulted in more puffs per cigarette, than both HCI and ISO.

The Health Risks of Electronic Cigarette Use to Bystanders.

This works aimed to assess the health risks of e-cigarette use to bystanders. The exhaled breath of 17 volunteers was collected while they were vaping, and the levels of nicotine, propylene glycol, glycerol, formaldehyde, acetaldehyde, acrolein, tobacco-specific nitrosamines (TSNAs), and heavy metals were analyzed. Increased levels of nicotine, propylene glycol, TSNAs and copper were found in the exhaled breath of the volunteers. From these measurements, bystander exposure was estimated for two different scenarios: (1) A non-ventilated car with two e-cigarette users and (2) a ventilated office with one e-cigarette user. Our results show that bystanders may experience irritation of the respiratory tract as a result of exposure to propylene glycol and glycerol. Systemic effects of nicotine should also be expected if nicotine-containing e-liquid is used, including palpitations, and an increase of the systolic blood pressure. Furthermore, due to the presence of TSNAs in some e-liquids, an increased risk of tumors could not be excluded for the 'car' scenario. While e-cigarette use can clearly have effects on the health of bystanders, the risks depend on the rate of ventilation, dimensions of the room, and vaping behavior of the e-cigarette user. The presence of TSNAs in e-liquids can be avoided, which will prevent the most serious effect identified (increased risk of tumors).

Identification of flavour additives in tobacco products to develop a flavour library.

OBJECTIVES: This study combines chemical analysis and flavour descriptions of flavour additives used in tobacco products, and provides a starting point to build an extensive library of flavour components, useful for product surveillance. METHODS: Headspace gas chromatography-mass spectrometry (GC-MS) was used to compare 22 commercially available tobacco products (cigarettes and roll-your-own) expected to have a characterising flavour and 6 commercially available products not expected to have a characterising flavour with 5 reference products (natural tobacco leaves and research cigarettes containing no flavour additives). The flavour components naturally present in the reference products were excluded from components present in commercially available products containing flavour additives. A description of the remaining flavour additives was used for categorisation. RESULTS: GC-MS measurements of the 33 tobacco products resulted in an overview of 186 chemical compounds. Of these, 144 were solely present in commercially available products. These 144 flavour additives were described using 62 different flavour descriptors extracted from flavour databases, which were categorised into eight groups largely based on the definition of characterising flavours from the European Tobacco Product Directive: fruit, spice, herb, alcohol, menthol, sweet, floral and miscellaneous. CONCLUSIONS: We developed a method to identify and describe flavour additives in tobacco products. Flavour additives consist of single flavour compounds or mixtures of multiple flavour compounds, and different combinations of flavour compounds can cause a certain flavour. A flavour library helps to detect flavour additives that are characteristic for a certain flavour, and thus can be useful for regulation of flavours in tobacco and related products.

The Sensory Difference Threshold of Menthol Odor in Flavored Tobacco Determined by Combining Sensory and Chemical Analysis.

Cigarettes are an often-used consumer product, and flavor is an important determinant of their product appeal. Cigarettes with strong nontobacco flavors are popular among young people, and may facilitate smoking initiation. Discriminating flavors in tobacco is important for regulation purposes, for instance to set upper limits to the levels of important flavor additives. We provide a simple and fast method to determine the human odor difference threshold for flavor additives in a tobacco matrix, using a combination of chemical and sensory analysis. For an example, the human difference threshold for menthol odor, one of the most frequently used tobacco flavors, was determined. A consumer panel consisting of 20 women compared different concentrations of menthol-flavored tobacco to unflavored cigarette tobacco using the 2-alternative forced choice method. Components contributing to menthol odor were quantified using headspace GC-MS. The sensory difference threshold of menthol odor corresponded to a mixture of 43 (37-50)% menthol-flavored tobacco, containing 1.8 (1.6-2.1) mg menthol, 2.7 (2.3-3.1) µg menthone, and 1.0 (0.9-1.2) µg neomenthyl acetate per gram of tobacco. Such a method is important in the context of the European Tobacco Product Directive, and the US Food and Drug Administration Tobacco Control Act, that both prohibit cigarettes and roll-your-own tobacco with a characterizing flavor other than tobacco. Our method can also be adapted for matrices other than tobacco, such as food.

A novel bile acid biosynthesis defect due to a deficiency of peroxisomal ABCD3.

ABCD3 is one of three ATP-binding cassette (ABC) transporters present in the peroxisomal membrane catalyzing ATP-dependent transport of substrates for metabolic pathways localized in peroxisomes. So far, the precise function of ABCD3 is not known. Here, we report the identification of the first patient with a defect of ABCD3. The patient presented with hepatosplenomegaly and severe liver disease and showed a striking accumulation of peroxisomal C27-bile acid intermediates in plasma. Investigation of peroxisomal parameters in skin fibroblasts revealed a reduced number of enlarged import-competent peroxisomes. Peroxisomal beta-oxidation of C26:0 was normal, but beta-oxidation of pristanic acid was reduced. Genetic analysis revealed a homozygous deletion at the DNA level of 1758bp, predicted to result in a truncated ABCD3 protein lacking the C-terminal 24 amino acids (p.Y635NfsX1). Liver disease progressed and the patient required liver transplantation at 4 years of age but expired shortly after transplantation. To corroborate our findings in the patient, we studied a previously generated Abcd3 knockout mouse model. Abcd3-/- mice accumulated the branched chain fatty acid phytanic acid after phytol loading. In addition, analysis of bile acids revealed a reduction of C24 bile acids, whereas C27-bile acid intermediates were significantly increased in liver, bile and intestine of Abcd3-/- mice. Thus, both in the patient and in Abcd3-/- mice, there was evidence of a bile acid biosynthesis defect. In conclusion, our studies show that ABCD3 is involved in transport of branched-chain fatty acids and C27 bile acids into the peroxisome and that this is a crucial step in bile acid biosynthesis.

D-amino acid aberrations in cerebrospinal fluid and plasma of smokers.

The glutamatergic neurotransmission system and the N-methyl-D-aspartate receptor (NMDAR) have been implicated in smoking and alcohol consumption behavior. Preclinical studies have demonstrated that nicotine and ethanol influence NMDAR functionality, which may have a role in tendencies to consume these substances. Nonetheless, little is known about concentrations of NMDAR coagonists in the cerebrospinal fluid (CSF) and plasma of individuals who smoke or consume alcohol. Glycine and L- and D-stereoisomers of alanine, serine, and proline were therefore measured using ultra-high-performance liquid chromatography-tandem mass spectrometry in 403 healthy subjects. Nicotine and alcohol consumption were quantified using questionnaires. Possible differences in NMDAR coagonist concentrations in plasma and CSF were investigated using ANCOVA with age, body mass index, and storage duration as covariates. The significance threshold was Bonferroni corrected (α=0.00625). Compared with non-smokers, smokers displayed lower levels of D-proline in plasma (p=0.0027, Cohen's d=-0.41) and D-proline in CSF (p=0.0026, Cohen's d=-0.43). D-Serine in CSF was higher in smokers than in non-smokers (p=0.0052, Cohen's d=0.41). After subdividing participants based on smoking quantity, dose-dependent decreases were demonstrated in smokers for D-proline in plasma (F=5.65, p=0.0039) and D-proline in CSF (F=5.20, p=0.0060). No differences in NMDAR coagonist levels between alcohol consumption groups were detected. To our knowledge, this is the first report to implicate D-amino acids in smoking behavior of humans. Whether such concentration differences lie at the root of or result from smoking habits may be addressed in prospective studies.

The intestine plays a substantial role in human vitamin B6 metabolism: a Caco-2 cell model.

BACKGROUND: Vitamin B6 is present in various forms (vitamers) in the diet that need to be metabolized to pyridoxal phosphate (PLP), the active cofactor form of vitamin B6. In literature, the liver has been reported to be the major site for this conversion, whereas the exact role of the intestine remains to be elucidated. OBJECTIVE: To gain insight into the role of the intestine in human vitamin B6 metabolism. MATERIALS AND METHODS: Expression of the enzymes pyridoxal kinase (PK), pyridox(am)ine phosphate oxidase (PNPO) and PLP-phosphatase was determined in Caco-2 cells and in lysates of human intestine. Vitamin B6 uptake, conversion and excretion were studied in polarized Caco-2 cell monolayers. B6 vitamer concentrations (pyridoxine (PN), pyridoxal (PL), PLP, pyridoxamine (PM), pyridoxamine phosphate (PMP)) and pyridoxic acid (PA) were quantified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using stable isotope-labeled internal standards. RESULTS: The enzymatic system involved in vitamin B6 metabolism (PK, PNPO and PLP-phosphatase) is fully expressed in Caco-2 cells as well as in human intestine. We show uptake of PN, PM and PL by Caco-2 cells, conversion of PN and PM into PL and excretion of all three unphosphorylated B6 vitamers. CONCLUSION: We demonstrate, in a Caco-2 cell model, that the intestine plays a substantial role in human vitamin B6 metabolism.

Vitamin B6 vitamer concentrations in cerebrospinal fluid differ between preterm and term newborn infants.

BACKGROUND AND OBJECTIVE: Vitamin B(6) plays a pivotal role in brain development and functioning. Differences in vitamin B(6) homeostasis between preterm and term newborn infants have been reported. The authors sought to investigate whether B(6) vitamers in cerebrospinal fluid (CSF) of preterm and term newborn infants are different. METHODS: B(6) vitamer concentrations were determined in 69 CSF samples of 36 newborn infants (26 born preterm and 10 born term) by ultra performance liquid chromatography-tandem mass spectrometry. CSF samples, taken from a subcutaneous intraventricular reservoir, were bedside frozen and protected from light. RESULTS: Concentrations of pyridoxal (PL), pyridoxal phosphate (PLP), pyridoxic acid (PA), and pyridoxamine (PM) in preterm newborns (postmenstrual age 30-37 weeks) were at least twice as high as in older newborns (postmenstrual age ≥ 42 weeks). Pyridoxine and pyridoxamine phosphate concentrations were below limits of quantification in all newborns. In CSF of 2 very preterm newborns (postmenstrual age <30 weeks), significant amounts of pyridoxine were present besides high concentrations of PL, PA, and PM, whereas PLP concentrations were relatively low. B(6) vitamers in CSF were positively correlated, especially PA, PLP, and PL. CONCLUSIONS: In CSF of newborn infants, PL, PLP, PA, and PM are present, and concentrations are strongly dependent on postmenstrual age. Our results indicate that vitamin B(6) homeostasis in brain differs between preterm and term newborns. These results should be taken into account for diagnosis and treatment of epilepsy and vitamin B(6) deficiency in newborn infants.

A common variant in ERBB4 regulates GABA concentrations in human cerebrospinal fluid.

The neuregulin 1 (NRG1) receptor ErbB4 is involved in the development of cortical inhibitory GABAergic circuits and NRG1-ErbB4 signaling has been implicated in schizophrenia (SCZ). A magnetic resonance spectroscopy ((1)H-MRS) study has demonstrated that a single-nucleotide polymorphism in ERBB4, rs7598440, influences human cortical GABA concentrations. Other work has highlighted the significant impact of this genetic variant on expression of ERBB4 in the hippocampus and dorsolateral prefrontal cortex in human post mortem tissue. Our aim was to examine the association of rs7598440 with cerebrospinal fluid (CSF) GABA levels in healthy volunteers (n=155). We detected a significant dose-dependent association of the rs7598440 genotype with CSF GABA levels (G-allele standardized ß=-0.23; 95% CIs: -0.39 to -0.07; P=0.0066). GABA concentrations were highest in A homozygous, intermediate in heterozygous, and lowest in G homozygous subjects. When excluding subjects on psychotropic medication (three subjects using antidepressants), the results did not change (G-allele standardized ß=-0.23; 95% CIs: -0.40 to -0.07; P=0.0051). The explained variance in CSF GABA by rs7598440 in our model is 5.2% (P=0.004). The directionality of our findings agrees with the aforementioned (1)H-MRS and gene expression studies. Our observation therefore strengthens the evidence that the A-allele of rs7598440 in ERBB4 is associated with increased GABA concentrations in the human central nervous system (CNS). To our knowledge, our finding constitutes the first confirmation that CSF can be used to study genotype-phenotype correlations of GABA levels in the CNS. Such quantitative genetic analyses may be extrapolated to other CSF constituents relevant to SCZ in future studies.

Identification of a human trans-3-hydroxy-L-proline dehydratase, the first characterized member of a novel family of proline racemase-like enzymes.

A family of eukaryotic proline racemase-like genes has recently been identified. Several members of this family have been well characterized and are known to catalyze the racemization of free proline or trans-4-hydroxyproline. However, the majority of eukaryotic proline racemase-like proteins, including a human protein called C14orf149, lack a specific cysteine residue that is known to be critical for racemase activity. Instead, these proteins invariably contain a threonine residue at this position. The function of these enzymes has remained unresolved until now. In this study, we demonstrate that three enzymes of this type, including human C14orf149, catalyze the dehydration of trans-3-hydroxy-L-proline to Δ(1)-pyrroline-2-carboxylate (Pyr2C). These are the first enzymes of this subclass of proline racemase-like genes for which the enzymatic activity has been resolved. C14orf149 is also the first human enzyme that acts on trans-3-hydroxy-L-proline. Interestingly, a mutant enzyme in which the threonine in the active site is mutated back into cysteine regained 3-hydroxyproline epimerase activity. This result suggests that the enzymatic activity of these enzymes is dictated by a single residue. Presumably, human C14orf149 serves to degrade trans-3-hydroxy-L-proline from the diet and originating from the degradation of proteins that contain this amino acid, such as collagen IV, which is an important structural component of basement membrane.

Alternative splicing directs dual localization of Candida albicans 6-phosphogluconate dehydrogenase to cytosol and peroxisomes.

The pentose phosphate pathway (PPP) is the main source of NADPH in the cell and therefore essential for the maintenance of the redox balance and anabolic reactions. NADPH is produced by the two dehydrogenases in the oxidative branch of the PPP: glucose-6-phosphate dehydrogenase (Zwf1) and 6-phosphogluconate dehydrogenase (Gnd1). We observed that in the commensal fungus Candida albicans these two enzymes contain putative peroxisomal targeting signals (PTSs): Zwf1 has a putative PTS1, while the annotated intron of GND1 encodes a PTS2. By subcellular fractionation and fluorescence microscopy, we show that both enzymes have a dual localization in which the majority is cytosolic, but a small fraction is peroxisome associated. Analysis of GND1 transcripts revealed that dual targeting of Gnd1 is directed by alternative splicing resulting in two Gnd1 isoforms, one without targeting signals localized to the cytosol and one with an N-terminal PTS2 targeted to peroxisomes. To our knowledge, Gnd1 is the first example of dual targeting of a protein by alternative splicing in C. albicans. In silico analysis suggests that PTS-mediated peroxisomal targeting of Zwf1 and Gnd1 is conserved across closely related Candida species. We discuss putative functions of the peroxisomal oxidative PPP in these organisms.