Developmental vitamin D and autism spectrum disorders: findings from the Stockholm Youth Cohort.

Animal studies indicate that early life vitamin D is crucial for proper neurodevelopment. Few studies have examined whether maternal and neonatal vitamin D concentrations influence risk of autism spectrum disorders (ASD). Participants were sampled from the Stockholm Youth Cohort, a register-based cohort in Sweden. Concentrations of total 25-hydroxyvitamin D (25OHD) were assessed from maternal and neonatal biosamples using a highly sensitive liquid chromatography tandem mass spectrometry method. The maternal sample consisted of 449 ASD cases and 574 controls, the neonatal sample: 1399 ASD cases and 1607 controls; and the paired maternal-neonatal sample: 340 ASD cases and 426 controls. Maternal 25OHD was not associated with child ASD in the overall sample. However, in Nordic-born mothers, maternal 25OHD insufficiency (25 - <50 nmol/L) at ~11 weeks gestation was associated with 1.58 times higher odds of ASD (95% CI: 1.00, 2.49) as compared with 25OHD sufficiency (≥50 nmol/L). Neonatal 25OHD < 25 nmol/L was associated with 1.33 times higher odds of ASD (95% CI: 1.02, 1.75) as compared with 25OHD ≥ 50 nmol/L. Sibling-matched control analyses indicated these associations were not likely due to familial confounding. Children with both maternal 25OHD and neonatal 25OHD below the median had 1.75 (95% CI: 1.08, 2.86) times the odds of ASD compared with children with maternal and neonatal 25OHD both below the median. Our results are consistent with an increasing body of evidence suggesting that vitamin D concentrations in early life may be associated with increased risk of neurodevelopmental disorders including ASD.

When a "Dimroth Rearrangement" Is Not a Dimroth Rearrangement.

In the Dimroth rearrangement of heterocycles, often pyrimidines, an exocyclic and a ring substituent are interchanged. However, the term Dimroth rearrangement is frequently used even when there is no knowledge of the reaction mechanism and alternatives are likely. Here, we have employed density functional theory (DFT) calculations at the M06-2X/6-311+G(d,p) level to determine the most plausible rearrangement pathways of 3-aminothiocarbonylquinazoline 5, tetrahydrofuranylpyrimidine 21, and 5-allyltriazocine 30. For the rearrangement of quinazoline 5 to 9, the [1,3]-sigmatropic shift of the thioamido group with an activation barrier of 26.7 kcal/mol is much preferred over the Dimroth rearrangement (∼46 kcal/mol). An even lower barrier of 21.6 kcal/mol applies to a stepwise [1,3]-shift. The migration of the tetrahydrofuranyl unit in pyrimidines like 21 → 23 can take place by means of a [1,3]-sigmatropic shift with a low barrier (≤17.5 kcal/mol) rather than a Dimroth rearrangement under acidic conditions and most likely also under neutral conditions (∼30 kcal/mol). In the rearrangement of 5-allyl-6-iminotriazocine 30 to 32, the [3,3]-sigmatropic shift (aza-Cope rearrangement) is preferred over the Dimroth mechanism under neutral conditions, but in the presence of acid, the azonia-Cope rearrangement of an allyl group and the true Dimroth rearrangement have comparable activation energies.

25-Hydroxyvitamin D concentration and all-cause mortality: the Melbourne Collaborative Cohort Study.

OBJECTIVE: To investigate relationships between mortality and circulating 25-hydroxyvitamin D (25(OH)D), 25-hydroxycholecalciferol (25(OH)D3) and 25-hydroxyergocalciferol (25(OH)D2). DESIGN: Case-cohort study within the Melbourne Collaborative Cohort Study (MCCS). We measured 25(OH)D2 and 25(OH)D3 in archived dried blood spots by LC-MS/MS. Cox regression was used to estimate mortality hazard ratios (HR), with adjustment for confounders. SETTING: General community. SUBJECTS: The MCCS included 29 206 participants, who at recruitment in 1990-1994 were aged 40-69 years, had dried blood spots collected and no history of cancer. For the present study we selected participants who died by 31 December 2007 (n 2410) and a random sample (sub-cohort, n 2996). RESULTS: The HR per 25 nmol/l increment in concentration of 25(OH)D and 25(OH)D3 were 0·86 (95 % CI 0·78, 0·96; P=0·007) and 0·85 (95 % CI 0·77, 0·95; P=0·003), respectively. Of 5108 participants, sixty-three (1·2 %) had detectable 25(OH)D2; their mean 25(OH)D concentration was 11·9 (95 % CI 7·3, 16·6) nmol/l higher (P<0·001). The HR for detectable 25(OH)D2 was 1·80 (95 % CI 1·09, 2·97; P=0·023); for those with detectable 25(OH)D2, the HR per 25 nmol/l increment in 25(OH)D was 1·06 (95 % CI 0·87, 1·29; P interaction=0·02). HR were similar for participants who reported being in good, very good or excellent health four years after recruitment. CONCLUSIONS: Total 25(OH)D and 25(OH)D3 concentrations were inversely associated with mortality. The finding that the inverse association for 25(OH)D was restricted to those with no detectable 25(OH)D2 requires confirmation in populations with higher exposure to ergocalciferol.

Minimizing Matrix Effects for the Accurate Quantification of 25-Hydroxyvitamin D Metabolites in Dried Blood Spots by LC-MS/MS.

BACKGROUND: The noncalcemic actions of vitamin D in multiple organs are now widely recognized. Vitamin D status has been linked with a wide variety of conditions, which has led to an increasing demand for vitamin D screening. In particular, there is intense interest in the impact of vitamin D on a variety of developmental conditions. The most readily accessible pediatric samples are dried blood spots, and health organizations are increasingly archiving such samples for later assessment of the antecedents of disease. METHODS: In 2009, we developed a method to quantify the major circulatory form of vitamin D, 25-hydroxyvitamin D, in archived dried blood spots. Over the last 6 years, we have made substantial alterations to the published method to enhance throughput, sensitivity, and assay robustness. RESULTS: With the alterations, the assay was 3 times faster than the previously published assay and had a >10-fold increase in signal strength. Intraassay imprecision decreased from 13.4% to 6.9%, and there was a 5-fold reduction in interfering phospholipids. In actual use over 2 years, the assay showed an interassay imprecision of 11.6%. CONCLUSIONS: This assay has performed reliably over the past 6 years. The practical changes we have made should allow clinical chemists to successfully adapt this method.

Rearrangements of 4-Quinolylcarbene, 3-Quinolylcarbene, and 2-Quinolylcarbene to 1-Naphthylnitrene and Cyanoindenes by Falling Solid Flash Vacuum Pyrolysis.

The relationship between 4-quinolylcarbene 17, 3-quinolylcarbene 21, 2-quinolylcarbene 25, and 1-naphthylnitrene 35 has been explored experimentally and computationally. The diazomethylquinolines generated from (5-tetrazolyl)quinolines or 1,2,3-triazolo[1,5-a]quinoline by conventional flash vacuum pyrolysis (FVP) were observed by IR spectroscopy. The carbenes were generated by falling solid flash vacuum pyrolysis (FS-FVP). 4-Quinolylcarbene 17 was found to rearrange to 3-quinolylcarbene 21 and then to 2-quinolylcarbene 25, and finally via 1-naphthylnitrene 35 to 1-cyanoindene 36, which then isomerizes to 3- and 2-cyanoindenes 12 and 13. The thermal rearrangement of 2-quinolylcarbene to 1-naphthylnitrene was verified by ESR spectroscopy. The reaction mechanism has been elucidated with the help of calculations of the structures and energies of the quinolylcarbenes and 1-naphthylnitrene and the intervening aza-benzobicyclo[4.1.0]heptatrienes, aza-benzocycloheptatetraenes, and aza-benzocycloheptatrienylidenes and the transition states connecting them at the B3LYP/6-31G* level. The nonobserved 1,2-hydrogen shifts in aza-benzocycloheptatetraenes/aza-benzocycloheptatrienylidenes are found to have very high activation barriers.

Ring Contraction in Arylcarbenes and Arylnitrenes; Rearrangements of 1- and 3-Isoquinolylcarbenes and 2-Naphthylnitrene to Cyanoindenes.

Flash vacuum pyrolysis (FVP) of 1-(5-(13)C-5-tetrazolyl)isoquinoline 18 generates 1-((13)C-diazomethyl)isoquinoline 19 and 1-isoquinolyl-((13)C-carbene) 22, which undergoes carbene-nitrene rearrangement to 2-naphthylnitrene 23. The thermally generated nitrene 23 is observed directly by matrix-isolation ESR spectroscopy, but undergoes ring contraction to a mixture of 3- and 2-cyanoindenes 26 and 27 under the FVP conditions. The (13)C label distribution in the cyanoindenes was determined by (13)C NMR spectroscopy and indicates the occurrence of two parallel paths of ring contraction starting from 1-isoquinolylcarbene; path a via ring expansion to 3-aza-benzo[c]cyclohepta-1,2,4,6-tetraene 32 bifurcating to 2-naphthylnitrene 23 and 2-aza-benzobicyclo[3.2.0]heptatriene 39 (paths a1 and a2); and path b via ring closure of the carbene onto the ring nitrogen, yielding 1-aza-benzo[d]bicyclo[4.1.0]hepta-2,4,6-triene 34 and 3-aza-benzo[d]cyclohepta-2,3,5,7-tetraene 35. Product studies demand that the major path is route a1 via 2-naphthylnitrene 23, which then undergoes direct ring contraction to 1-cyanoindene; but the (13)C label distribution requires that the non-nitrene route b contributes significantly. The two reaction paths are modeled at the B3LYP/6-31G* level. The initially formed carbene 22 is estimated to carry chemical activation of some 40 kcal/mol. This allows both reaction channels to proceed simultaneously under low-pressure FVP conditions. FVP of 3-(5-tetrazolyl)isoquinoline 28 similarly generates 3-diazomethylisoquinoline 29 and 3-isoquinolylcarbene 30, which rearranges to 3- and 2-cyanoindenes 26 and 27.

Transcriptome and venom proteome of the box jellyfish Chironex fleckeri.

BACKGROUND: The box jellyfish, Chironex fleckeri, is the largest and most dangerous cubozoan jellyfish to humans. It produces potent and rapid-acting venom and its sting causes severe localized and systemic effects that are potentially life-threatening. In this study, a combined transcriptomic and proteomic approach was used to identify C. fleckeri proteins that elicit toxic effects in envenoming. RESULTS: More than 40,000,000 Illumina reads were used to de novo assemble ∼ 34,000 contiguous cDNA sequences and ∼ 20,000 proteins were predicted based on homology searches, protein motifs, gene ontology and biological pathway mapping. More than 170 potential toxin proteins were identified from the transcriptome on the basis of homology to known toxins in publicly available sequence databases. MS/MS analysis of C. fleckeri venom identified over 250 proteins, including a subset of the toxins predicted from analysis of the transcriptome. Potential toxins identified using MS/MS included metalloproteinases, an alpha-macroglobulin domain containing protein, two CRISP proteins and a turripeptide-like protease inhibitor. Nine novel examples of a taxonomically restricted family of potent cnidarian pore-forming toxins were also identified. Members of this toxin family are potently haemolytic and cause pain, inflammation, dermonecrosis, cardiovascular collapse and death in experimental animals, suggesting that these toxins are responsible for many of the symptoms of C. fleckeri envenomation. CONCLUSIONS: This study provides the first overview of a box jellyfish transcriptome which, coupled with venom proteomics data, enhances our current understanding of box jellyfish venom composition and the molecular structure and function of cnidarian toxins. The generated data represent a useful resource to guide future comparative studies, novel protein/peptide discovery and the development of more effective treatments for jellyfish stings in humans. (Length: 300).

Applying SWATH Mass Spectrometry to Investigate Human Cervicovaginal Fluid During the Menstrual Cycle.

Inherent interindividual and intraindividual variation in the length of the menstrual cycle limits the accuracy of predicting days of peak fertility. To improve detection of days of peak fertility, a more detailed understanding of longitudinal changes in cervicovaginal fluid (CVF) biomarkers during the normal menstrual cycle is needed. The aim of this study, therefore, was to characterize longitudinal changes in CVF proteins during the menstrual cycle using a quantitative, data-independent acquisition mass spectrometry approach. Six serial samples were collected from women (n = 10) during the menstrual cycle. Samples were obtained at two time points for each phase of the cycle: early and late preovulatory, ovulatory, and postovulatory. Information-dependent acquisition (IDA) of mass spectra from all individual CVF samples was initially performed and identified 278 total proteins. Samples were then pooled by time of collection (n = 6 pools) and analyzed using IDA and information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]). The IDA library generated contained 176 statistically significant protein identifications (P < 0.000158). The variation in the relative abundance of CVF proteins across the menstrual cycle was established by comparison with the SWATH profile against the IDA library. Using time-series, pooled samples obtained from 10 women, quantitative data were obtained by SWATH analysis for 43 CVF proteins. Of these proteins, 28 displayed significant variation in relative abundance during the menstrual cycle (assessed by ANOVA). Statistical significant changes in the relative expression of CVF proteins during preovulatory, ovulatory, and postovulatory phases of menstrual cycle were identified. The data obtained may be of utility not only in elucidating underlying physiological mechanisms but also as clinically useful biomarkers of fertility status.

Azulenylcarbene and naphthylcarbene isomerizations. Falling solid flash vacuum pyrolysis.

1-Azulenylcarbene 18 has been generated from 5-(1-azulenyl)tetrazole and the sodium salt of azulene-1-carbaldehyde tosylhydrazone using the falling solid flash vacuum pyrolysis (FS-FVP) method. The principal products, which are also formed from both 1- and 2-naphthylcarbenes, cyclobuta[de]naphthalene 6, cyclopenta[cd]indene 16, and benzofulvenallene 17, are explained in terms of two reaction paths, (a) a rearrangement to benzofulvenyl-7-carbene 13 and (b) a rearrangement to 1-naphthylcarbene 1. Moreover, 16 is also formed from 2-azulenylcarbene 30, thereby indicating the occurrence of a 2-azulenylcarbene-1-azulenylcarbene rearrangement. The reaction mechanisms are supported by density functional theory calculations at the B3LYP/6-31G** level, which indicate that all the rearrangements have activation barriers of <35 kcal/mol, thus making them readily achievable under FVP conditions.

C9H8 Pyrolysis. o-Tolylacetylene, Indene, 1-Indenyl, and Biindenyls and the Mechanism of Indene Pyrolysis.

o-Tolylacetylene 5 is obtained by flash vacuum pyrolysis (FVP) of the isoxazolone 13a at 800 °C/10(-4) hPa. At 900-1000 °C the acetylene 5 isomerizes to indene 1, which reacts further by elimination of a hydrogen atom and dimerization of the 1-indenyl radical 9 to 1,1'-biindenyl 10. The latter undergoes partial isomerization to 3,3'-biindenyl 16, and further pyrolysis of the biindenyls yields higher polycyclic aromatic hydrocarbons (PAHs), particularly chrysene 2. C-H bond breakage in indene, which occurs with an activation energy of 80 ± 5 kcal/mol with formation of the 1-indenyl radical 9, has been the subject of much investigation in relation to hydrocarbon combustion, in particular the formation of chrysene and other PAHs from indene, which itself is formed in the combustion of toluene and other hydrocarbons. However, C-C bond breakage also needs to be considered. Calculations at the B3LYP/6-311+G(d,p) level indicate that key C-C bond breakages in indene have free energies of activation of ca. 80 kcal/mol. Positive entropies of activation make all these reactions more facile at high temperatures relevant to hydrocarbon combustion chemistry. C1-C2 bond breakage results in the formation of o-tolylvinylidene 6 and o-tolylacetylene 5. The reversible 1,2-shift interconverting 5 and 6 (the Roger Brown rearrangement) can lead to carbon scrambling in C3-labeled indene 1a, resulting in indene 1d carrying the label in positions 1, 2, and 3 and explaining the (14)C-labeling pattern observed by Badger et al. in the derived chrysene 2d. o-Tolylacetylene 5 and o-tolylvinylidene 6 should be considered as intermediates in models of the fuel-rich combustion of toluene, indene, and other hydrocarbons.

Molecular cleft or tweezer compounds derived from trioxabicyclo[3.3.1]nonadiene diisocyanate and diacid dichloride.

The structures of two derivatives of the bisdioxine diisocyanate 1, the bisurea 4 and the biscarbamate 5, are established by X-ray crystallography and DFT calculations. These compounds possess endo,endo structures, in the case of the bisurea 4 with two nearly parallel pendant chains. The X-ray structures are reproduced very well by DFT calculations. Similar endo,endo conformations are calculated for the bisamide crown ether derivatives 7, where two proximate and nearly parallel crown ether units endow the molecules with a claw-like molecular cleft or tweezer structure as evidenced by an enhanced ability to extract some alkali, alkaline earth and rare earth metal ions.

Method Development for the Detection of Human Myostatin by High-Resolution and Targeted Mass Spectrometry.

Myostatin, a highly conserved secretory protein, negatively regulates muscle development, affecting both the proliferation and differentiation of muscle cells. Proteolytic processing of the myostatin precursor protein generates a myostatin pro-peptide and mature protein. Dimerization of the mature myostatin protein creates the active form of myostatin. Myostatin dimer activity can be inhibited by noncovalent binding of two monomeric myostatin pro-peptides. This ability for myostatin to self-regulate as well as the altered expression of myostatin in states of abnormal health (e.g., muscle wasting) support the need for specific detection of myostatin forms. Current protein detection methods (e.g., Western blot) rely greatly on antibodies and are semiquantitative at best. Tandem mass spectometry (as in this study) provides a highly specific method of detection, enabling the characterization of myostatin protein forms through the analysis of discrete peptides fragments. Utilizing the scheduled high-resolution multiple reaction monitoring paradigm (sMRMHR; AB SCIEX 5600 TripleTOF) we identified the lower limit of quantitation (LLOQ) of both mature (DFGLDCDEHSTESR) and pro-peptide regions (ELIDQYDVQR) as 0.19 nmol/L. Furthermore, scheduled multiple reaction monitoring (sMRM; AB SCIEX QTRAP 5500) identified a LLOQ for a peptide of the pro-peptide region (LETAPNISK) as 0.16 nmol/L and a peptide of the mature region (EQIIYGK) as 0.25 nmol/L.

Phenylnitrene, phenylcarbene, and pyridylcarbenes. Rearrangements to cyanocyclopentadiene and fulvenallene.

Flash vacuum thermolysis (FVT) of phenyl azide 29 as well as precursors of 2-pyridylcarbene 34 and 4-pyridylcarbene 25 affords phenylnitrene 30 (labeled or unlabeled), as revealed by matrix isolation electron spin resonance spectroscopy. FVT of 1-(13)C-phenyl azide 29 affords 1-cyanocyclopentadiene (cpCN) 32, which is exclusively labeled on the CN carbon, thus demonstrating direct ring contraction in phenylnitrene 30 without the intervention of cycloperambulation and 1,3-H shifts. However, the cpCN obtained by rearrangement of pyridyl-2-((13)C-carbene) 34 carries (13)C label on all carbon atoms, including the CN carbon. Calculations at the B3LYP/6-31G* level and in part at the CASSCF/6-31G* and CASPT2/cc-pVDZ//CASSCF(8,8)/cc-pVDZ levels support a new mechanism whereby 2-pyridylcarbene rearranges in part via 1-azacyclohepta-1,2,4,6-tetraene 36 to phenylnitrene, which then undergoes direct ring contraction to cpCN. Another portion of 2-pyridylcarbene undergoes ring expansion to 4-azacyclohepta-1,2,4,6-tetraene 42, which then by trans-annular cyclization affords 6-azabicyclo[3.2.0]cyclohepta-1,3,5-triene 43. Further rearrangement of 43 via the spiroazirine 44 and biradical/vinylnitrene 45 affords cpCN with the label on the CN group. An analogous mechanisms accounts for the labeling pattern in fulvenallene 60 formed by ring contraction of 1-(13)C-phenylcarbene 59 in the FVT of 1-(13)C-phenyldiazomethane 58.

3-Pyridazinylnitrenes and 2-pyrimidinylnitrenes.

Mild flash vacuum thermolysis of tetrazolo[1,5-b]pyridazines 8T generates small amounts of 3-azidopyridazines 8A (8aA, IR 2145, 2118 cm(-1); 8bA, 2142 cm(-1)). Photolysis of the tetrazoles/azides 8T/8A in Ar matrix generates 3-pyridazinylnitrenes 9, detected by ESR spectroscopy (9a: D/hc = 1.006; E/hc = 0.003 cm(-1)). Cyanovinylcarbenes 11, derived from 4-diazobut-2-enenitriles 10, are also detected by ESR spectroscopy (11a: D/hc = 0.362; E/hc = 0.021 cm(-1)). Carbenes 11 rearrange to cyanoallenes 12 and 3-cyanocyclopropenes 13. Triazacycloheptatetraenes 20 were not observed in the photolyses of 8. Photolysis of tetrazolo[1,5-a]pyrimidines/2-azidopyridmidines 18T/18A in Ar matrices at 254 nm yields 2-pyrimidinylnitrenes 19, observable by ESR, UV, and IR spectroscopy (19a: ESR: D/hc = 1.217; E/hc = 0.0052 cm(-1)). Excellent agreement with the calculated IR spectrum identifies the 1,2,4-triazacyclohepta-1,2,4,6-tetraenes 20 (20a, 1969 cm(-1); 20b, 1979 cm(-1)). Compounds 20 undergo photochemical ring-opening to 1-isocyano-3-diazopropenes 23. Further irradiation also causes Type II ring-opening of pyrimidinylnitrenes 19 to 2-(cyanimino)vinylnitrenes 21 (21a: D/hc = 0.875; E/hc = 0.00 cm(-1)), isomerization to cyaniminoketenimine 25 (2044 cm(-1)), and cyclization to 1-cyanopyrazoles 22. The reaction mechanisms are discussed and supported by DFT calculations on key intermediates and pathways. There is no evidence for the interconversion of 3-pyridazinylnitrenes 9 and 2-pyrimidinylnitrenes 19.

Nitrene-carbene-carbene rearrangement. Photolysis and thermolysis of tetrazolo[5,1-a]phthalazine with formation of 1-phthalazinylnitrene, o-cyanophenylcarbene, and phenylcyanocarbene.

1-Azidophthalazine 9A is generated in trace amount by mild FVT of tetrazolo[5,1-a]phthalazine 9T and is observable by its absorption at 2121 cm(-1) in the Ar matrix IR spectrum. Ar matrix photolysis of 9T/9A at 254 nm causes ring opening to generate two conformers of (o-cyanophenyl)diazomethane 11 (2079 and 2075 cm(-1)), followed by (o-cyanophenyl)carbene (3)12, cyanocycloheptatetraene 13, and finally cyano(phenyl)carbene (3)14 as evaluated by IR spectroscopy. The two carbenes (3)12 and (3)14 were observed by ESR spectroscopy (D|hc = 0.5078, E|hc = 0.0236 and D|hc = 0.6488, E|hc = 0.0195 cm(-1), respectively). The rearrangement of 12 ⇄ 13 ⇄ 14 constitutes a carbene-carbene rearrangement. 1-Phthalazinylnitrene (3)10 is observed by means of its UV-vis spectrum in Ar matrix following FVT of 9 above 550 °C. Rearrangement to cyanophenylcarbenes also takes place on FVT of 9 as evidenced by observation of the products of ring contraction, viz., fulvenallenes and ethynylcyclopentadienes 16-18. Thus the overall rearrangement 10 → 11 → 12 ⇄ 13 ⇄ 14 can be formulated.

mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

4-Pyridylnitrene and 2-pyrazinylcarbene.

Both flash vacuum thermolysis (FVT) and matrix photolysis generate 2-diazomethylpyrazine (22) from 1,2,3-triazolo[1,5-a]pyrazine (24). FVT of 4-azidopyridine (18) as well as of 24 or 2-(5-tetrazolyl)pyrazine (23) affords the products expected from the nitrene, i.e., 4,4'-azopyridine and 2- and 3-cyanopyrroles. Matrix photolyses of both 18 and 24 result in ring expansion of 4-pyridylnitrene/2-pyrazinylcarbene to 1,5-diazacyclohepta-1,2,4,6-tetraene (20). Further photolysis causes ring opening to the ketenimine 27.

3-Pyridylnitrene, 2- and 4-pyrimidinylcarbenes, 3-quinolylnitrenes, and 4-quinazolinylcarbenes. Interconversion, ring expansion to diazacycloheptatetraenes, ring opening to nitrile ylides, and ring contraction to cyanopyrroles and cyanoindoles.

Precursors of 3-pyridylnitrene and 2- and 4-pyrimidinylcarbenes all afford mixtures of 2- and 3-cyanopyrroles on flash vacuum thermolysis, but 3-cyanopyrroles are the first-formed products. 3-Quinolylnitrenes and 4-quinazolinylcarbenes similarly afford 3-cyanoindoles. 2-Pyrimidinylcarbenes rearrange to 3-pyridylnitrenes, but 4-pyrimidinylcarbenes and 4-quinazolinylcarbenes do not necessarily rearrange to the corresponding 3-pyridylnitrenes or 3-quinolylnitrenes. The ring contraction reactions are interpreted in terms of ring opening of either the nitrenes or the diazacycloheptatetraenes to nitrile ylides.

Label-free high-throughput screening via acoustic ejection mass spectrometry put into practice.

Acoustic droplet ejection-open port interface-mass spectrometry (ADE-OPI-MS) is a novel label-free analytical technique, promising to become a versatile readout for high-throughput screening (HTS) applications. The recent introduction of ADE-OPI-MS devices to the laboratory equipment market, paired with their compatibility with laboratory automation platforms, should facilitate the adoption of this technology by a broader community. Towards this goal, instrument robustness in the context of HTS campaigns - where up to millions of samples in complex matrices are tested in a short time frame - represents a major challenge, which explains the absence of detailed literature reports on this subject. Here, we present the results of our first fully automated HTS campaign, based on the ADE-OPI-MS technology, aiming to identify inhibitors of a metabolic enzyme in a >1 million compound library. The report encompasses the assay development and validation steps, as well as the adaptation for HTS requirements, where refinement of the capillary cleaning concept was crucial for final success. Altogether, our study unequivocally demonstrates the applicability of the ADE-OPI-MS technology for HTS-based drug discovery.

2-quinoxalinylnitrenes and 4-quinazolinylnitrenes: rearrangement to cyclic and acyclic carbodiimides and ring-opening to nitrile ylides.

This work was undertaken with the aim to obtain direct evidence for the interrelationships between hetarylnitrenes, their ring-expanded cyclic carbodiimide isomers, and ring-opened nitrile ylides. Tetrazolo[1,5-a]quinoxaline 11T and tetrazolo[5.1-c]quinazoline 13T undergo valence tautomerization to the corresponding azides 11A and 13A on mild flash vacuum thermolysis (FVT). Photolysis in Ar matrixes at ca. 15 K affords the triplet nitrenes 12 and 14, identified by ESR, UV, and IR spectroscopy. The nitrenes are converted photochemically to the seven-membered ring carbodiimide 15 followed by the open-chain carbodiimide 22. The 3-methoxy- and 3-chloro-2-quinoxalinylnitrenes 24 yield the ring-expanded carbodiimides 26 very cleanly on matrix photolysis, whereas FVT affords N-cyanobenzimidazoles 28. The ring-opened nitrile ylides 36 and 49 are identified as intermediates in the photolyses of 2-phenyl-4-quinazolinylnitrene 32 and 7-nitro-2-phenyl-4- quinazolinylnitrene 47. In these systems, a photochemically reversible interconversion of the seven-membered ring carbodiimides 35 and 48 and the nitrile ylides 36 and 49 is established. Recyclization of open-chain nitrile ylides is identified as an important mechanism of formation of ring contraction products (N-cyanobenzimidazoles).