Transition Metal-Free Catalytic C-H Zincation and Alumination.

C-H metalation is the most efficient method to prepare aryl-zinc and -aluminium complexes that are ubiquitous nucleophiles. Virtually all C-H metalation routes to form Al/Zn organometallics require stoichiometric, strong Brønsted bases with no base-catalyzed reactions reported. Herein we present a catalytic in amine/ammonium salt (Et3N/[(Et3N)H]+) C-H metalation process to form aryl-zinc and aryl-aluminium complexes. Key to this approach is coupling an endergonic C-H metalation step with a sufficiently exergonic dehydrocoupling step between the ammonium salt by-product of C-H metalation ([(Et3N)H]+) and a Zn-H or Al-Me containing complex. This step, forming H2/MeH, makes the overall cycle exergonic while generating more of the reactive metal electrophile. Mechanistic studies supported by DFT calculations revealed metal-specific dehydrocoupling pathways, with the divergent reactivity due to the different metal valency (which impacts the accessibility of amine-free cationic metal complexes) and steric environment. Notably, dehydrocoupling in the zinc system proceeds through a ligand-mediated pathway involving protonation of the ß-diketiminate Cγ position. Given this process is applicable to two disparate metals (Zn and Al), other main group metals and ligand sets are expected to be amenable to this transition metal-free, catalytic C-H metalation.

LncRNA Tuna is activated in cadmium-induced placental insufficiency and drives the NRF2-mediated oxidative stress response.

Cadmium (Cd) is a toxic heavy metal found throughout the environment and one of the top ten toxicants of major public health concern identified by the World Health Organization. In utero Cd exposure causes fetal growth restriction, malformation, and spontaneous abortion; however, the mechanisms by which Cd impacts these outcomes are poorly understood. Cd accumulates in the placenta, suggesting that these negative outcomes may be a consequence of disrupted placental function and placental insufficiency. To understand the impact of Cd on gene expression within the placenta, we developed a mouse model of Cd-induced fetal growth restriction through maternal consumption of CdCl2 and performed RNA-seq on control and CdCl2 exposed placentae. The top differentially expressed transcript was the Tcl1 Upstream Neuron-Associated (Tuna) long non-coding RNA, which was upregulated over 25-fold in CdCl2 exposed placentae. Tuna has been shown to be critical for neural stem cell differentiation. However, within the placenta, there is no evidence that Tuna is normally expressed or functional at any developmental stage. To determine the spatial expression of Cd-activated Tuna within the placenta, we used in situ hybridization as well as placental layer-specific RNA isolation and analysis. Both methods confirmed the absence of Tuna expression in control samples and determined that Cd-induced Tuna expression is specific to the junctional zone. Since many lncRNAs regulate gene expression, we hypothesized that Tuna forms part of the mechanism of Cd-induced transcriptomic changes. To test this, we over-expressed Tuna in cultured choriocarcinoma cells and compared gene expression profiles to those of control and CdCl2 exposed cells. We demonstrate significant overlap between genes activated by Tuna overexpression and genes activated by CdCl2 exposure, with enrichment in the NRF2-mediated oxidative stress response. Herein we analyze the NRF2 pathway and show that Tuna increases NRF2/NRF2 both at the transcript and protein levels. Tuna drives increased NRF2 target gene expression, a result that is abrogated with the use of an NRF2 inhibitor, confirming that Tuna activates oxidative stress response genes through this pathway. This work identifies the lncRNA Tuna as a potential novel player in Cd-induced placental insufficiency.

Epigenetic control of the imprinted growth regulator Cdkn1c in cadmium-induced placental dysfunction.

Cadmium (Cd) is a toxic metal ubiquitous in the environment. In utero, Cd is inefficiently transported to the foetus but causes foetal growth restriction (FGR), likely through impairment of the placenta where Cd accumulates. However, the underlying molecular mechanisms are poorly understood. Cd can modulate the expression of imprinted genes, defined by their transcription from one parental allele, which play critical roles in placental and foetal growth. The expression of imprinted genes is governed by DNA methylation at Imprinting Control Regions (ICRs), which are susceptible to environmental perturbation. The imprinted gene Cdkn1c/CDKN1C is a major regulator of placental development, is implicated in FGR, and shows increased expression in response to Cd exposure in mice. Here, we use a hybrid mouse model of in utero Cd exposure to determine if the increase in placental Cdkn1c expression is caused by changes to ICR DNA methylation and loss of imprinting (LOI). Consistent with prior studies, Cd causes FGR and impacts placental structure and Cdkn1c expression at late gestation. Using polymorphisms to distinguish parental alleles, we demonstrate that increased Cdkn1c expression is not driven by changes to DNA methylation or LOI. We show that Cdkn1c is expressed primarily in the placental labyrinth which is proportionally increased in size in response to Cd. We conclude that the Cd-associated increase in Cdkn1c expression can be fully explained by alterations to placental structure. These results have implications for understanding mechanisms of Cd-induced placental dysfunction and, more broadly, for the study of FGR associated with increased Cdkn1c/CDKN1C expression.

The imprinted gene Zac1 regulates steatosis in developmental cadmium-induced nonalcoholic fatty liver disease.

Cadmium (Cd) exposure in adulthood is associated with nonalcoholic fatty liver disease (NAFLD), characterized by steatosis, inflammation, and fibrosis. The prevalence of NAFLD in children is increasing, suggesting a role for the developmental environment in programming susceptibility. However, the role of developmental Cd exposure in programming NAFLD and the underlying mechanisms remain unclear. We have proposed that imprinted genes are strong candidates for connecting the early life environment and later life disease. In support of this, we previously identified roles for the Imprinted Gene Network (IGN) and its regulator Zac1 in programming NAFLD in response to maternal metabolic dysfunction. Here, we test the hypothesis that developmental Cd exposure is sufficient to program NAFLD, and further, that this process is mediated by Zac1 and the IGN. Using mice, we show that developmental cadmium chloride (CdCl2) exposure leads to histological, biochemical, and molecular signatures of steatosis and fibrosis in juveniles. Transcriptomic analyses comparing livers of CdCl2-exposed and control mice show upregulation of Zac1 and the IGN coincident with disease presentation. Increased hepatic Zac1 expression is independent of promoter methylation and imprinting statuses. Finally, we show that over-expression of Zac1 in cultured hepatocytes is sufficient to induce lipid accumulation in a Pparγ-dependent manner and demonstrate direct binding of Zac1 to the Pparγ promoter. Our findings demonstrate that developmental Cd exposure is sufficient to program NAFLD in later life, and with our previous work, establish Zac1 and the IGN as key regulators of prosteatotic and profibrotic pathways, two of the major pathological hallmarks of NAFLD.

Genomic map of candidate human imprint control regions: the imprintome.

Imprinted genes - critical for growth, metabolism, and neuronal function - are expressed from one parental allele. Parent-of-origin-dependent CpG methylation regulates this expression at imprint control regions (ICRs). Since ICRs are established before tissue specification, these methylation marks are similar across cell types. Thus, they are attractive for investigating the developmental origins of adult diseases using accessible tissues, but remain unknown. We determined genome-wide candidate ICRs in humans by performing whole-genome bisulphite sequencing (WGBS) of DNA derived from the three germ layers and from gametes. We identified 1,488 hemi-methylated candidate ICRs, including 19 of 25 previously characterized ICRs (https://humanicr.org/). Gamete methylation approached 0% or 100% in 332 ICRs (178 paternally and 154 maternally methylated), supporting parent-of-origin-specific methylation, and 65% were in well-described CTCF-binding or DNaseI hypersensitive regions. This draft of the human imprintome will allow for the systematic determination of the role of early-acquired imprinting dysregulation in the pathogenesis of human diseases and developmental and behavioural disorders.

Dapagliflozin partially restores reproductive function in MC4R KO obese female mice.

Obese women often have certain degree of reproductive dysfunction with infertility. Although the clinical impact of obesity on female infertility has been extensively studied, the effective and targeted treatment is still lacking. Melanocortin-4-receptor knock-out (MC4R KO) mouse is an over-eating obese model with hyperphagia, hyperinsulinemia, reduced growth hormone (GH), and insulin resistance. Dapagliflozin improved the metabolic and hormonal parameters in MC4R KO mice. MC4R KO female mice were treated with dapagliflozin for 14 weeks from 14-week age. Age-matched WT littermates and non-treated MC4R KO mice were used as control groups. Food intake was measured daily. Body weight was measured twice a week. Estrous cycles, GH, and luteinizing hormone (LH) profiles were measured. Selected tissues were collected at the end of experiments for gene expression profiles and hematoxylin-eosin staining. Regularity and mode of hormonal profiles were restored by the dapagliflozin treatment. Estrous cycle was partially normalized, number of CL was significantly increased, and the expression of Kiss1 and Gnrh1 in the hypothalamus and LH in the pituitary was markedly increased by the dapagliflozin treatment. It is conclsuded that dapagliflozin may recover LH and GH profiles partially through modification of relevant gene expression in the hypothalamus and pituitary, and result in an improved ovulation rate in obese mouse model. Dapagliflozin may therefore improve fertility in obese patients.

Zac1 and the Imprinted Gene Network program juvenile NAFLD in response to maternal metabolic syndrome.

BACKGROUND AND AIMS: Within the next decade, NAFLD is predicted to become the most prevalent cause of childhood liver failure in developed countries. Predisposition to juvenile NAFLD can be programmed during early life in response to maternal metabolic syndrome (MetS), but the underlying mechanisms are poorly understood. We hypothesized that imprinted genes, defined by expression from a single parental allele, play a key role in maternal MetS-induced NAFLD, due to their susceptibility to environmental stressors and their functions in liver homeostasis. We aimed to test this hypothesis and determine the critical periods of susceptibility to maternal MetS. APPROACH AND RESULTS: We established a mouse model to compare the effects of MetS during prenatal and postnatal development on NAFLD. Postnatal but not prenatal MetS exposure is associated with histological, biochemical, and molecular signatures of hepatic steatosis and fibrosis in juvenile mice. Using RNA sequencing, we show that the Imprinted Gene Network (IGN), including its regulator Zac1, is up-regulated and overrepresented among differentially expressed genes, consistent with a role in maternal MetS-induced NAFLD. In support of this, activation of the IGN in cultured hepatoma cells by overexpressing Zac1 is sufficient to induce signatures of profibrogenic transformation. Using chromatin immunoprecipitation, we demonstrate that Zac1 binds the TGF-ß1 and COL6A2 promoters, forming a direct pathway between imprinted genes and well-characterized pathophysiological mechanisms of NAFLD. Finally, we show that hepatocyte-specific overexpression of Zac1 is sufficient to drive fibrosis in vivo. CONCLUSIONS: Our findings identify a pathway linking maternal MetS exposure during postnatal development to the programming of juvenile NAFLD, and provide support for the hypothesis that imprinted genes play a central role in metabolic disease programming.

Time-Restricted Feeding Restored Insulin-Growth Hormone Balance and Improved Substrate and Energy Metabolism in MC4RKO Obese Mice.

BACKGROUND: Dysregulation of metabolic regulatory hormones often occurs during the progress of obesity. Key regulatory hormone insulin-growth hormone (GH) balance has recently been proposed to maintain metabolism profiles. Time-restricted feeding (TRF) is an effective strategy against obesity without detailed research on pulsatile GH releasing patterns. METHODS: TRF was performed in an over-eating melanocortin 4 receptor-knockout (MC4RKO) obese mouse model using normal food. Body weight and food intake were measured. Series of blood samples were collected for 6-h pulsatile GH profile, glucose tolerance test, and insulin tolerance test at 5, 8, and 9 weeks of TRF, respectively. Indirect calorimetric recordings were performed by the Phenomaster system at 6 weeks for 1 week, and body composition was measured by nuclear magnetic resonance spectroscopy (NMR). Substrate- and energy metabolism-related gene expressions were measured in terminal liver and subcutaneous white adipose tissues. RESULTS: TRF increased pulsatile GH secretion in dark phase and suppressed hyperinsulinemia in MC4RKO obese mice to reach a reduced insulin/GH ratio. This was accompanied by the improvement in insulin sensitivity, metabolic flexibility, glucose tolerance, and decreased glucose fluctuation, together with appropriate modification of gene expression involved in substrate metabolism and adipose tissue browning. NMR measurement showed that TRF decreased fat mass but increased lean mass. Indirect calorimeter recording indicated that TRF decreased the respiratory exchange ratio (RER) reflecting consumption of more fatty acid in energy production in light phase and increased the oxygen consumption during activities in dark phase. CONCLUSIONS: TRF effectively decreases hyperinsulinemia and restores pulsatile GH secretion in the overeating obese mice with significant improvement in substrate and energy metabolism and body composition without reducing total caloric intake.

Reversible Dissociation of a Dialumene*.

Dialumenes are neutral AlI compounds with Al=Al multiple bonds. We report the isolation of an amidophosphine-supported dialumene. Our X-ray crystallographic, spectroscopic, and computational DFT analyses reveal a long and extreme trans-bent Al=Al bond with a low dissociation energy and bond order. In solution, the dialumene can dissociate into monomeric AlI species. Reactivity studies reveal two modes of reaction: as dialumene or as aluminyl monomers.

Transcriptomic, proteomic, and metabolomic analyses identify candidate pathways linking maternal cadmium exposure to altered neurodevelopment and behavior.

Cadmium (Cd) is a ubiquitous toxic heavy metal of major public concern. Despite inefficient placental transfer, maternal Cd exposure impairs fetal growth and development. Increasing evidence from animal models and humans suggests maternal Cd exposure negatively impacts neurodevelopment; however, the underlying molecular mechanisms are unclear. To address this, we utilized multiple -omics approaches in a mouse model of maternal Cd exposure to identify pathways altered in the developing brain. Offspring maternally exposed to Cd presented with enlarged brains proportional to body weights at birth and altered behavior at adulthood. RNA-seq in newborn brains identified exposure-associated increases in Hox gene and myelin marker expression and suggested perturbed retinoic acid (RA) signaling. Proteomic analysis showed altered levels of proteins involved in cellular energy pathways, hypoxic response, and RA signaling. Consistent with transcriptomic and proteomic analyses, we identified increased levels of retinoids in maternally-exposed newborn brains. Metabolomic analyses identified metabolites with significantly altered abundance, supportive of changes to cellular energy pathways and hypoxia. Finally, maternal Cd exposure reduced mitochondrial DNA levels in newborn brains. The identification of multiple pathways perturbed in the developing brain provides a basis for future studies determining the mechanistic links between maternal Cd exposure and altered neurodevelopment and behavior.

The Phospha-Bora-Wittig Reaction.

We report the phospha-bora-Wittig reaction for the direct preparation of phosphaalkenes from aldehydes, ketones, esters, or amides. The transient phosphaborene Mes*P═B-NR2 reacts with carbonyl compounds to form 1,2,3-phosphaboraoxetanes, analogues of oxaphosphetane intermediates in the classical Wittig reaction. 1,2,3-Phosphaboraoxetanes undergo thermal or Lewis acid-promoted cycloreversion, yielding phosphaalkenes. Experimental and density functional theory studies reveal far-reaching similarities between classical and phospha-bora-Wittig reactions.

Imprinted Gene Expression and Function of the Dopa Decarboxylase Gene in the Developing Heart.

Dopa decarboxylase (DDC) synthesizes serotonin in the developing mouse heart where it is encoded by Ddc_exon1a, a tissue-specific paternally expressed imprinted gene. Ddc_exon1a shares an imprinting control region (ICR) with the imprinted, maternally expressed (outside of the central nervous system) Grb10 gene on mouse chromosome 11, but little else is known about the tissue-specific imprinted expression of Ddc_exon1a. Fluorescent immunostaining localizes DDC to the developing myocardium in the pre-natal mouse heart, in a region susceptible to abnormal development and implicated in congenital heart defects in human. Ddc_exon1a and Grb10 are not co-expressed in heart nor in brain where Grb10 is also paternally expressed, despite sharing an ICR, indicating they are mechanistically linked by their shared ICR but not by Grb10 gene expression. Evidence from a Ddc_exon1a gene knockout mouse model suggests that it mediates the growth of the developing myocardium and a thinning of the myocardium is observed in a small number of mutant mice examined, with changes in gene expression detected by microarray analysis. Comparative studies in the human developing heart reveal a paternal expression bias with polymorphic imprinting patterns between individual human hearts at DDC_EXON1a, a finding consistent with other imprinted genes in human.

Aluminium-Catalyzed C(sp)-H Borylation of Alkynes.

Historically used in stoichiometric hydroalumination chemistry, recent advances have transformed aluminium hydrides into versatile catalysts for the hydroboration of unsaturated multiple bonds. This catalytic ability is founded on the defining reactivity of aluminium hydrides with alkynes and alkenes: 1,2-hydroalumination of the unsaturated π-system. This manuscript reports the aluminium hydride catalyzed dehydroborylation of terminal alkynes. A tethered intramolecular amine ligand controls reactivity at the aluminium hydride centre, switching off hydroalumination and instead enabling selective reactions at the alkyne C-H σ-bond. Chemoselective C-H borylation was observed across a series of aryl- and alkyl-substituted alkynes (21 examples). On the basis of kinetic and density functional theory studies, a mechanism in which C-H borylation proceeds by σ-bond metathesis between pinacolborane (HBpin) and alkynyl aluminium intermediates is proposed.

The melanocortin pathway and energy homeostasis: From discovery to obesity therapy.

BACKGROUND: Over the past 20 years, insights from human and mouse genetics have illuminated the central role of the brain leptin-melanocortin pathway in controlling mammalian food intake, with genetic disruption resulting in extreme obesity, and more subtle polymorphic variations influencing the population distribution of body weight. At the end of 2020, the U.S. Food and Drug Administration (FDA) approved setmelanotide, a melanocortin 4 receptor agonist, for use in individuals with severe obesity due to either pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency. SCOPE OF REVIEW: Herein, we chart the melanocortin pathway's history, explore its pharmacology, genetics, and physiology, and describe how a neuropeptidergic circuit became an important druggable obesity target. MAJOR CONCLUSIONS: Unravelling the genetics of the subset of severe obesity has revealed the importance of the melanocortin pathway in appetitive control; coupling this with studying the molecular pharmacology of compounds that bind melanocortin receptors has brought a new obesity drug to the market. This process provides a drug discovery template for complex disorders, which for setmelanotide took 25 years to transform from a single gene into an approved drug.

Stimulation of endogenous pulsatile growth hormone secretion by activation of growth hormone secretagogue receptor reduces the fat accumulation and improves the insulin sensitivity in obese mice.

Obese individuals often show low growth hormone (GH) secretion, which leads to reduced lipid mobilization and further fat accumulation. Pharmacological approaches to increase GH levels in obese individuals by GH injection or GH-releasing hormone receptor agonist showed promising effects on fat reduction. However, side effects on glucose metabolism and the heavy costs on making large peptides hindered their clinical application. Here, we tested whether stimulation of endogenous GH secretion by a synthetic GH secretagogue receptor (GHSR) agonist, hexarelin, improved the metabolism in a hyperphagic obese mouse model. Male melanocortin 4 receptor knockout mice (MC4RKO) were pair-fed and received continuous hexarelin (10.56 µg/day) or vehicle infusion by an osmotic pump for 3-4 weeks. Hexarelin treatment significantly increased the pulsatile GH secretion without detectable alteration on basal GH secretion in MC4RKO mice. The treated mice showed increased lipolysis and lipid oxidation in the adipose tissue, and reduced de novo lipogenesis in the liver, leading to reduced visceral fat mass, reduced triglyceride content in liver, and unchanged circulating free fatty acid levels. Importantly, hexarelin treatment improved the whole-body insulin sensitivity but did not alter glucose tolerance, insulin levels, or insulin-like growth factor 1 (IGF-1) levels. The metabolic effects of hexarelin were likely through the direct action of GH, as indicated by the increased expression level of genes involved in GH signaling pathways in visceral adipose tissues and liver. In conclusion, hexarelin treatment stimulated the pulsatile GH secretion and reduced the fat accumulation in visceral depots and liver in obese MC4RKO mice with improved insulin sensitivity without altered levels of insulin or IGF-1. It provides evidence for managing obesity by enhancing pulsatile GH secretion through activation of GHSR in the pituitary gland.

Reversible Reductive Elimination in Aluminum(II) Dihydrides.

Oxidative addition and reductive elimination are defining reactions of transition-metal organometallic chemistry. In main-group chemistry, oxidative addition is now well-established but reductive elimination reactions are not yet general in the same way. Herein, we report dihydrodialanes supported by amidophosphine ligands. The ligand serves as a stereochemical reporter for reversible reductive elimination/oxidative addition chemistry involving AlI and AlIII intermediates.

Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing.

Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome.

Characterization of the Zwitterionic Intermediate in 1,1-Carboboration of Alkynes.

The reaction of a Lewis acidic borane with an alkyne is a key step in a diverse range of main group transformations. Alkyne 1,1-carboboration, the Wrackmeyer reaction, is an archetypal transformation of this kind. 1,1-Carboboration has been proposed to proceed through a zwitterionic intermediate. We report the isolation and spectroscopic, structural and computational characterization of the zwitterionic intermediates generated by reaction of B(C6 F5 )3 with alkynes. The stepwise reactivity of the zwitterion provides new mechanistic insight for 1,1-carboboration and wider B(C6 F5 )3 catalysis. Making use of intramolecular stabilization by a ferrocene substituent, we have characterized the zwitterionic intermediate in the solid state and diverted reactivity towards alkyne cyclotrimerization.

Silyl Anion Initiated Hydroboration of Aldehydes and Ketones.

Hydroboration is an emerging method for mild and selective reduction of carbonyl compounds. Typically, transition-metal or reactive main-group hydride catalysts are used in conjunction with a mild reductant such as pinacolborane. The reactivity of the main-group catalysts is a consequence of the nucleophilicity of their hydride ligands. Silicon hydrides are significantly less reactive and are therefore not efficient hydroboration catalysts. Here, a readily prepared silyl anion is reported to be an effective initiator for the reduction of aldehydes and ketones requiring mild conditions, low catalyst loadings and with a good substrate scope. The silyl anion it is shown to activate HBpin to generate a reactive borohydride in situ which reacts with aldehydes and ketones to afford the hydroboration product.