Gamma-Ray-Induced Amino Acid Formation during Aqueous Alteration in Small Bodies: The Effects of Compositions of Starting Solutions.

Organic compounds, such as amino acids, are essential for the origin of life, and they may have been delivered to the prebiotic Earth from extra-terrestrial sources, such as carbonaceous chondrites. In the parent bodies of carbonaceous chondrites, the radioactive decays of short-lived radionuclides, such as 26Al, cause the melting of ice, and aqueous alteration occurs in the early stages of solar system formation. Many experimental studies have shown that complex organic matter, including amino acids and high-molecular-weight organic compounds, is produced by such hydrothermal processes. On the other hand, radiation, particularly gamma rays from radionuclides, can contribute to the formation of amino acids from simple molecules such as formaldehyde and ammonia. In this study, we investigated the details of gamma-ray-induced amino acid formation, focusing on the effects of different starting materials on aqueous solutions of formaldehyde, ammonia, methanol, and glycolaldehyde with various compositions, as well as hexamethylenetetramine. Alanine and glycine were the most abundantly formed amino acids after acid hydrolysis of gamma-ray-irradiated products. Amino acid formation increased with increasing gamma-ray irradiation doses. Lower amounts of ammonia relative to formaldehyde produced more amino acids. Glycolaldehyde significantly increased amino acid yields. Our results indicated that glycolaldehyde formation from formaldehyde enhanced by gamma rays is key for the subsequent production of amino acids.

Incorporation of five common hypnotics into hair after a single dose and application to a forensic case of drug facilitated crimes.

In order to obtain fundamental information on the disposition of hypnotics into hair after a single oral dose the quantitative hair analysis of triazolam (TZ), etizolam (EZ), flunitrazepam (FNZ), nitrazepam (NZ) and zolpidem (ZP) have been performed using a validated LC-MS/MS procedure. Hair specimens (straight, black) were collected from three subjects about one month and three months after a single 0.25 mg dose of TZ, 1 mg of EZ, 2 mg of FNZ, 5 mg of NZ and 10 mg of ZP tartrate. The subjects ingested just one out of five different hypnotics on each day, each of five days in turn. All ingested hypnotics have been detected in hair from each subject both one month and three months after intake, and their concentrations were in the range of 0.023-0.043 pg/hair strand (0.077-0.36 pg/mg) for TZ, 0.11-0.63 pg/hair strand (0.44-5.2 pg/mg) for EZ, 0.14-2.6 pg/hair strand (0.56-22 pg/mg) for FNZ, 0.33-1.7 pg/hair strand (1.3-17 pg/mg) for NZ and 20-40 pg/hair strand (120-270 pg/mg) for ZP. For FNZ and NZ, not only the parent drugs but also their metabolites, 7-amino-FNZ and 7-amino-NZ, were detected in the range of 2.3-9.2 pg/hair strand (9.2-82 pg/mg) and 2.4-9.1 pg/hair strand (8.0-55 pg/mg), respectively. The calculated incorporation ratios into hair against the dose were found to exhibit similarity between the four benzodiazepines. This finding suggests the ability to apply these quantitative data to approximately estimating the amounts of other benzodiazepines, which have similar chemical structures, in hair although it should be noted that the amounts of drugs in hair varies considerably depending on the hair color. On the other hand, the incorporation ratio of ZP showed 15-29 times higher than that of TZ, indicating that lipophilic ZP was more likely to incorporate into hair than benzodiazepines. In addition, the application of the present data to a drug-facilitated sexual assault was shown.

Oestrogen receptor α in T cells controls the T cell immune profile and glucose metabolism in mouse models of gestational diabetes mellitus.

AIMS/HYPOTHESIS: The imbalance between maternal insulin resistance and a relative lack of insulin secretion underlies the pathogenesis of gestational diabetes mellitus (GDM). Alterations in T cell subtypes and increased levels of circulating proinflammatory cytokines have been proposed as potential mechanisms underlying the pathophysiology of insulin resistance in GDM. Since oestrogen modulates T cell immunity, we hypothesised that oestrogen plays a homeostatic role in visceral adipose tissue by coordinating T cell immunity through oestrogen receptor α (ERα) in T cells to prevent GDM. METHODS: Female CD4-cre ERαfl/fl (KO) mice on a C57BL/6 background with ERα ablation specifically in T cells, and ERαfl/fl (ERα-floxed [FL]) mice were fed 60 kJ% high-fat diet (HFD) for 4 weeks. Female mice mated with male BALB/c mice to achieve allogenic pregnancy and were maintained on an HFD to generate the GDM model. Mice were divided into four experimental groups: non-pregnant FL, non-pregnant KO, pregnant FL (FL-GDM) and pregnant KO (KO-GDM). GTTs and ITTs were performed on day 12.5 or 13.5 and 16.5 after breeding, respectively. On day 18.5 after breeding, mice were killed and T cell subsets in the gonadal white adipose tissue (gWAT) and spleen were analysed using flow cytometry. Histological examination was also conducted and proinflammatory gene expression in gWAT and the liver was evaluated. RESULTS: KO mice that mated with BALB/c mice showed normal fertility rates and fetal weights as compared with FL mice. Body and tissue weights were similar between FL and KO mice. When compared with FL-GDM mice, KO-GDM mice showed decreased insulin secretion (serum insulin concentration 15 min after glucose loading: 137.3 ± 18.3 pmol/l and 40.1 ± 36.5 pmol/l, respectively; p < 0.05), impaired glucose tolerance (glucose AUC in GTT: 2308.3 ± 54.0 mmol/l × min and 2620.9 ± 122.1 mmol/l × min, respectively; p < 0.05) and increased numbers of T helper (Th)17 cells in gWAT (0.4 ± 0.0% vs 0.8 ± 0.1%; p < 0.05). However, the contents of Th1 and regulatory T cells (Tregs) in gWAT remained similar between FL-GDM and KO-GDM. Glucose-stimulated insulin secretion was similar between isolated islets derived from FL and KO mice, but was reduced by IL-17A treatment. Moreover, the levels of proinflammatory gene expression, including expression of Emr1 and Tnfa in gWAT, were significantly higher in KO-GDM mice than in FL-GDM mice (5.1-fold and 2.7-fold, respectively; p < 0.01 for both). Furthermore, KO-GDM mice showed increased expression of genes encoding hepatokines, Ahsg and Fgf21 (both were 2.4-fold higher vs FL-GDM mice; p < 0.05 and p = 0.09, respectively), with no changes in inflammatory gene expression (e.g., Tnfa and Ifng) in the liver compared with FL-GDM mice. CONCLUSIONS/INTERPRETATION: Deletion of ERα in T cells caused impaired maternal adaptation of insulin secretion, changes in hepatokine profiles, and enhanced chronic inflammation in gWAT alongside an abnormal increase in Th17 cells. These results suggest that the ERα-mediated oestrogen signalling effects in T cells regulate T cell immunity and contribute to glucose homeostasis in pregnancy.

Incorporation of Methoxyphenamine into Hair in Early Stage after Intake.

In order to investigate the incorporation behavior of drugs into hair in early stage (within 24 h) after intake, time-course changes in drug distribution in black hair were carefully analyzed after a single oral administration of methoxyphenamine (MOP), a non-regulated analog of methamphetamine. Single-hair specimens collected by plucking with the roots intact at appropriate intervals post-intake were each divided into 1-mm segments from the proximal end, and MOP in each segment was determined by a validated liquid chromatography-tandem mass spectrometry procedure. At 10 min after intake, MOP was not detected in any of the segments. MOP became detectable 30 min after intake in the hair bulb (0-1-mm segment from the proximal end) and 1 h after intake in the upper dermis zone (1-2-mm to 4-5-mm segments). The amount of MOP in the hair bulb increased rapidly over 3 h after intake and reached a maximum concentration of ∼100-900 pg/1-mm single hair (11-95 ng/mg) around 3-10 h after intake, whereas that in the upper dermis zone increased at a more gradual pace over 24 h and reached a plateau at ∼30-100 pg/1-mm hair (3-11 ng/mg). These differences can be attributed to the different incorporation mechanisms of the drug. Results from this study can further elucidate the drug incorporation mechanism, which is crucial for accurately interpreting results in hair analyses. Our findings also suggest that hair drug analysis with special attention to the hair root can serve as a useful complementary approach to urine- and blood-based testing in the field of forensic toxicology.

White matter abnormalities across different epilepsy syndromes in adults: an ENIGMA-Epilepsy study.

The epilepsies are commonly accompanied by widespread abnormalities in cerebral white matter. ENIGMA-Epilepsy is a large quantitative brain imaging consortium, aggregating data to investigate patterns of neuroimaging abnormalities in common epilepsy syndromes, including temporal lobe epilepsy, extratemporal epilepsy, and genetic generalized epilepsy. Our goal was to rank the most robust white matter microstructural differences across and within syndromes in a multicentre sample of adult epilepsy patients. Diffusion-weighted MRI data were analysed from 1069 healthy controls and 1249 patients: temporal lobe epilepsy with hippocampal sclerosis (n = 599), temporal lobe epilepsy with normal MRI (n = 275), genetic generalized epilepsy (n = 182) and non-lesional extratemporal epilepsy (n = 193). A harmonized protocol using tract-based spatial statistics was used to derive skeletonized maps of fractional anisotropy and mean diffusivity for each participant, and fibre tracts were segmented using a diffusion MRI atlas. Data were harmonized to correct for scanner-specific variations in diffusion measures using a batch-effect correction tool (ComBat). Analyses of covariance, adjusting for age and sex, examined differences between each epilepsy syndrome and controls for each white matter tract (Bonferroni corrected at P < 0.001). Across 'all epilepsies' lower fractional anisotropy was observed in most fibre tracts with small to medium effect sizes, especially in the corpus callosum, cingulum and external capsule. There were also less robust increases in mean diffusivity. Syndrome-specific fractional anisotropy and mean diffusivity differences were most pronounced in patients with hippocampal sclerosis in the ipsilateral parahippocampal cingulum and external capsule, with smaller effects across most other tracts. Individuals with temporal lobe epilepsy and normal MRI showed a similar pattern of greater ipsilateral than contralateral abnormalities, but less marked than those in patients with hippocampal sclerosis. Patients with generalized and extratemporal epilepsies had pronounced reductions in fractional anisotropy in the corpus callosum, corona radiata and external capsule, and increased mean diffusivity of the anterior corona radiata. Earlier age of seizure onset and longer disease duration were associated with a greater extent of diffusion abnormalities in patients with hippocampal sclerosis. We demonstrate microstructural abnormalities across major association, commissural, and projection fibres in a large multicentre study of epilepsy. Overall, patients with epilepsy showed white matter abnormalities in the corpus callosum, cingulum and external capsule, with differing severity across epilepsy syndromes. These data further define the spectrum of white matter abnormalities in common epilepsy syndromes, yielding more detailed insights into pathological substrates that may explain cognitive and psychiatric co-morbidities and be used to guide biomarker studies of treatment outcomes and/or genetic research.

Dehydration-fragmentation mechanism of cathinones and their metabolites in ESI-CID.

Various cathinone-derived designer drugs (CATs) have recently appeared on the drug market. This study examined the mechanism for the generation of dehydrated ions for CATs during electrospray ionization collision-induced dissociation (ESI-CID). The generation mechanism of dehydrated ions is dependent on the amine classification in the cathinone skeleton, which is used in the identification of CATs. The two hydrogen atoms eliminated during the dehydration of cathinone (primary amine) and methcathinone (secondary amine) were determined, and the reaction mechanism was elucidated through the deuterium labeling experiments. The hydrogen atom bonded to the amine nitrogen was eliminated with the proton added during ESI, in both of the tested compounds. This provided evidence that CATs with tertiary amine structures (such as dimethylcathinone and α-pyrrolidinophenones [α-PPs]) do not undergo dehydration. However, it was shown that the two major tertiary amine metabolites (1-OH and 2″-oxo) of CATs generate dehydrated ions in ESI-CID. The dehydration mechanisms of the metabolites of α-pyrrolidinobutiophenone (α-PBP) belongs to α-PPs were also investigated. Stable-isotope labeling showed the dehydration of the 1-OH metabolite following a simple mechanism where the hydroxy group was eliminated together with the proton added during ESI. In contrast, the dehydration mechanism of the 2″-oxo metabolite involved hydrogen atoms in three or more locations along with the carbonyl group oxygen, indicating that dehydration occurred via multiple mechanisms likely including the rearrangement reaction of hydrogen atoms. These findings presented herein indicate that the dehydrated ions in ESI-CID can be used for the structural identification of CATs.

Estrogen regulates sex-specific localization of regulatory T cells in adipose tissue of obese female mice.

Regulatory T cells (Treg) play essential roles in maintaining immune homeostasis. Resident Treg in visceral adipose tissue (VAT-Treg) decrease in male obese mice, which leads to the development of obesity-associated chronic inflammations and insulin resistance. Although gender differences in immune responses have been reported, the effects of the difference in metabolic environment on VAT-Treg are unclear. We investigated the localization of VAT-Treg in female mice in comparison with that in male mice. On a high-fat diet (HFD), VAT-Treg decreased in male mice but increased in female mice. The increase was abolished in ovariectomized and HFD-fed mice, but was restored by estrogen supplementation. The IL33 receptor ST2, which is important for the localization and maturation of VAT-Treg in males, was reduced in CD4+CD25+ T cells isolated from gonadal fat of obese mice of both genders, suggesting that a different system exists for VAT-Treg localization in females. Extensive analysis of chemokine expression in gonadal fat and adipose CD4+CD25+T cells revealed several chemokine signals related to female-specific VAT-Treg accumulation such as CCL24, CCR6, and CXCR3. Taken together, the current study demonstrated sexual dimorphism in VAT-Treg localization in obese mice. Estrogen may attenuate obesity-associated chronic inflammation partly through altering chemokine-related VAT-Treg localization in females.

Effects of lipophilicity and functional groups of synthetic cannabinoids on their blood concentrations and urinary excretion.

The influence of lipophilicity and functional groups of synthetic cannabinoids (SCs) on their blood concentrations and urinary excretion has been studied by analyzing blood and urine specimens sampled from drivers who were involved in a car crashes under the influence of SCs. A total of 58 specimens (26 urine and 31 blood specimens), sampled within 13h of the occurrence, were analyzed by liquid chromatography-tandem mass spectrometry. Fifteen SCs were detected in those specimens; the SCs detected were categorized as follows: Class 1, Naphthoyl/Benzoyl indole (EAM2201 and three other analogs); Class 2, Indole-3-carboxylate/carboxamide containing naphthol/quinol (5F-PB-22 and four other analogs); and Class 3, Indazole-3-carboxamide containing valine/tert-leucine derivative (5F-AMB and five other analogs). The calculated lipophilicity index log P, the octanol/water participation coefficient, of those SCs in Classes 1, 2, and 3 ranged between 5.01-8.14, 5.80-6.74 and 2.29-3.81, respectively. Class 3 SCs were detectable in 12 out of 13 urine specimens, but those in Classes 1 and 2 were not detected in urine. Our analytical results indicated that the boundary line for their detectability in urine lies between log P 4 and 5. The blood concentrations of Class 3 SCs varied widely (0.0036-31ng/ml) depending on their log P, while much smaller variation was observed among those in Class 2 (0.10-5.0ng/ml).

Incorporation of zolpidem and methoxyphenamine into white hair strands after single administrations: Influence of hair pigmentation on drug incorporation.

In order to investigate the influence of pigmentation on the incorporation of drugs into hair, time-course changes in drug distribution along non-pigmented (white) hairs as well as pigmented (black) hairs plucked from the same subject was observed following single administrations of two basic drugs with different properties, zolpidem and methoxyphenamine. These drugs in 1-mm sections of single hair specimens were each determined by a liquid chromatography-tandem mass spectrometric procedure. During the early stage (12-36 h) after intake, for black hairs, both drugs were detected over the entire area of hair root (4-5 mm in length), in which notable concentration of these drugs in the hair bulb (0-1-mm segment from the bottom of hair root, Region 1) and lower concentrations in the upper dermis zone (1-2-mm to 3-4-mm or to 4-5-mm segments, Region 2) were commonly observed. Meanwhile, for white hairs, high drug concentrations in Region 1 as detected in black hairs were not observed although only small amounts of these drugs were detected over Region 2. Subsequent time-course changes in the concentration of drugs in hair demonstrated that the drugs once incorporated into white hair via Region 2 decreased gradually over the period from 24 h to 35 days after intake, but those of black hairs remained almost unchanged. These findings revealed here suggest that hair pigments have two important roles in the distribution of drugs: (1) incorporation of drugs into hair via Region 1, and (2) retention of already incorporated drugs in the hair tissue. These findings would be useful for discussing individual drug-use history based on hair analysis in the forensic fields.

Anxiety and depression symptoms disrupt resting state connectivity in patients with genetic generalized epilepsies.

OBJECTIVE: To analyze the lifetime trajectories in genetic generalized epilepsies (GGEs) and investigate the impact of symptoms of anxiety and depression on resting state functional connectivity (FC). METHODS: Seventy-four GGE patients were classified according to the pharmacological response as seizure-free (12 patients), pharmacoresistant (PhR; 14 patients), and fluctuating (FL; 48 patients). Fifty-four subjects completed both the Beck Depression Inventory (BDI) and Beck Anxiety Inventory (BAI), and 38 also underwent 3-T resting state functional magnetic resonance imaging. These 38 patients were subdivided into a positive group (13 patients with concurrent symptoms of depression and anxiety) and a negative group (21 asymptomatic patients and four with mild anxiety or depression symptoms). For FC analysis of resting state networks, we matched 38 healthy asymptomatic volunteers and used the UF2C toolbox running on MATLAB2017/SPM12. RESULTS: The PhR group presented shorter duration of epilepsy (P = 0.016) and follow-up (P < 0.001) compared to the FL group. The PhR group showed higher levels (median = 20) on the BAI and BDI. Myoclonic seizures were the most difficult to control, as 50% of subjects persisted with them at last appointment, compared to generalized tonic-clonic seizures and absence seizures (<40%). Patients with concurrent anxiety and depression symptoms were 7.7 times more likely to exhibit pharmacoresistant seizures, although an increase of 1 year of epilepsy duration was associated with a decrease in the odds of presenting pharmacoresistance by a factor of 0.9. Overall, FC was altered between default mode network (DMN) and visuospatial/dorsal attention. However, only the positive group displayed abnormal FC between DMN and left executive control network, and between salience and visuospatial/dorsal attention. SIGNIFICANCE: Our findings may help clinicians to have a better understanding of GGE clinical course and increase attention to the potential relationship of psychopathologies and brain connectivity.

Synthetic Glycopeptides Allow for the Quantitation of Scarce Nonfucosylated IgG Fc N-Glycans of Therapeutic Antibody.

Glycans attached to the IgG Fc domain affect strongly biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of therapeutic antibodies. However, molecular mechanism in the glycoform-dependent functional modulation of the IgGs remains elusive. The present study communicates that selected reaction monitoring (SRM)-based assay of tryptic IgG Fc glycopeptides is a promising approach for the characterization of antibodies when combined with structure-defined synthetic Fc peptides having a focused N-glycoform as a calibration standard. We describe a novel synthetic approach to the human IgG1 Fc peptide having a bisected decasaccharide and its nonbisected counterpart compound, the signatures of antibodies involving Fc domain with rare N-glycans expected to show much higher ADCC/CDC than abundant IgG N-glycans, and their application to the SRM-based quantitative glycoproteomics. Use of a key intermediate, phenyl (2-O-benzyl-4,6-O-benzylidine-ß-d-mannopyranosyl)-(1 → 4)-3,6-di-O-benzyl-2-azido-2-deoxy-1-thio-ß-d-glucopyranoside, derived from locust bean gum galactomannan, facilitated greatly the synthesis of a bisected nonasaccharide as a stable precursor of oxazoline derivative needed for the enzymatic trans-glycosylation with Fc nonapeptide carrying a GlcNAc at Asn297 residue, while the coupling reaction catalyzed by mutant endo-M-N175Q proceeded very slowly. Strikingly, SRM assay using the synthetic Fc glycopeptides as calibration standards uncovered the occurrence of the targeted IgG1 Fc fragment carrying a nonfucosylated and bisected (315 fmol, 0.20%) and its nonbisected counterpart (1154 fmol, 0.73%) in the tryptic digests from 158 pmol of anticancer antibody Herceptin (trastuzumab). The results suggest that aberrantly glycosylated IgG Fc variants may contribute to the total biological activities of the therapeutic antibodies.

Eplerenone prevented obesity-induced inflammasome activation and glucose intolerance.

Obesity-associated activation of the renin-angiotensin-aldosterone system is implicated in the pathogenesis of insulin resistance; however, influences of mineralocorticoid receptor (MR) inhibition remain unclear. Therefore, we aimed to clarify the anti-inflammatory mechanisms of MR inhibition using eplerenone, a selective MR antagonist, in C57BL/6 mice fed a high-fat diet (HFD) for 12 weeks. Eplerenone prevented excessive body weight gain and fat accumulation, ameliorated glucose intolerance and insulin resistance and enhanced energy metabolism. In the epididymal white adipose tissue (eWAT), eplerenone prevented obesity-induced accumulation of F4/80+CD11c+CD206--M1-adipose tissue macrophage (ATM) and reduction of F4/80+CD11c-CD206+-M2-ATM. Interestingly, M1-macrophage exhibited lower expression levels of MR, compared with M2-macrophage, in the ATM of eWAT and in vitro-polarized bone marrow-derived macrophages (BMDM). Importantly, eplerenone and MR knockdown attenuated the increase in the expression levels of proIl1b, Il6 and Tnfa, in the eWAT and liver of HFD-fed mice and LPS-stimulated BMDM. Moreover, eplerenone suppressed IL1b secretion from eWAT of HFD-fed mice. To reveal the anti-inflammatory mechanism, we investigated the involvement of NLRP3-inflammasome activation, a key process of IL1b overproduction. Eplerenone suppressed the expression of the inflammasome components, Nlrp3 and Caspase1, in the eWAT and liver. Concerning the second triggering factors, ROS production and ATP- and nigericin-induced IL1b secretion were suppressed by eplerenone in the LPS-primed BMDM. These results indicate that eplerenone inhibited both the priming and triggering signals that promote NLRP3-inflammasome activation. Therefore, we consider MR to be a crucial target to prevent metabolic disorders by suppressing inflammasome-mediated chronic inflammation in the adipose tissue and liver under obese conditions.