Dinitrogen Cleavage and Multicoupling with Isocyanides in a Dititanium Dihydride Framework.

Dinitrogen (N2) activation and functionalization through N-N bond cleavage and N-C bond formation are of great interest and importance but remain highly challenging. We report here for the first time N2 cleavage and selective multicoupling with isocyanides in a dititanium dihydride framework. The reaction of a dinitrogen dititanium dihydride complex [{(acriPNP)Ti}2(µ-η1:η2-N2)(µ-H)2] (1) with an excess (four or more equivalents) of p-methoxyphenyl isocyanide at room temperature gave a novel amidoamidinatoguanidinate complex [(acriPNP)Ti{NC(═NR)NC(═NR)CH2NR}Ti(acriPNP)(CNR)] (2, acriPNP = 4,5-bis(diisopropylphosphino)-2,7,9,9-tetramethyl-9H-acridin-10-ide; R = p-MeOC6H4) through N2 splitting and coupling with three isocyanide molecules. When 1 equiv of p-methoxyphenyl isocyanide was used to react with 1 at -30 °C, the hydrogenation of the isocyanide unit by the two hydride ligands in 1 took place, affording an amidomethylene-bridged dititanium dinitrogen complex [{(acriPNP)Ti}2(µ-η1:η2-N2){µ-η1:η2-CH2N(p-MeOC6H4)}] (3), which upon reaction with another equivalent of p-methoxyphenyl isocyanide at room temperature gave an amidomethylene/nitrido/carbodiimido complex [(acriPNP)Ti(N═C═NR)(µ-N)(µ-η1:η2-CH2NR)Ti(acriPNP)] (4) through N2 cleavage and N═C bond formation. Further reaction of 4 with 1 equiv of p-methoxyphenyl isocyanide led to an unprecedented four-component (carbodiimido, nitrido, isocyanide, and amidomethylene) coupling, yielding an amidoamidinatoguanidinate complex [{(acriPNP)Ti}2{NC(═NR)NC(═NR)CH2NR}] (5), which on reaction with another equivalent of p-methoxyphenyl isocyanide afforded the isocyanide-coordinated analogue 2. The reaction of 1 with 2-naphthyl isocyanide also took place in a similar multicoupling fashion. Moreover, the cross-coupling reactions of the p-methoxyphenyl isocyanide-derived amidomethylene/nitrido/carbodiimido complex 4 with 2-naphthyl isocyanide, cyclohexyl isocyanide, and tert-butyl isocyanide were also achieved, which afforded the corresponding amidoamidinatoguanidinate products consisting of two different isocyanides. Density functional theory (DFT) calculations further elucidated the mechanistic details.

Copper Difluorocarbene Enables Catalytic Difluoromethylation.

Despite the synthetic versatility of difluorocarbene, its high reactivity severely regulates widespread applications of difluorocarbene in organic synthesis. Here, we report a copper difluorocarbene-involved catalytic coupling, representing a new mode of the difluoromethylation reaction. This method allows difluoromethylation of a wide range of readily available allyl/propargyl electrophiles with NaBH3CN and low-cost difluorocarbene precursor BrCF2CO2K, featuring high cost-efficiency, high stereo- and regioselectivities, and high functional group tolerance, even with complex drug-like molecules. Applying the method led to the efficient synthesis of deuterated difluoromethylated compounds of medicinal interest. The resulting difluoromethylated allyl and allenyl products can serve as versatile synthons for diverse transformations, rendering the approach attractive for synthesizing complex fluorinated structures. Experimental mechanistic studies and computational calculations reveal that the formation of a difluoromethylcopper(I) intermediate through the nucleophilic attack of boron hydride on the copper(I) difluorocarbene is the key step in the reaction.

Self-signal electrochemical identification of circulating tumor DNA employing poly-xanthurenic acid assembled on black phosphorus nanosheets.

A self-signal electrochemical identification interface was prepared for the determination of circulating tumor DNA (ctDNA) in peripheral blood based on poly-xanthurenic acid (PXTA) assembled on black phosphorus nanosheets (BPNSs) acquired through simple ultrasonication method. The BPNSs with large surface area could be integrated with the xanthurenic acid (XTA) monomers by right of physisorption, and hence improved the electropolymerization efficiency and was beneficial to the enlargement of the signal response of PXTA. The assembled PXTA/BPNSs composite with attractive electrochemical activity was adopted as a platform for the recognition of DNA immobilization and hybridization. The probe ssDNA was covalently fixed onto the PXTA/BPNSs composite with plentiful carboxyl groups through the terminate free amines of DNA probes by use of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydrosulfosuccinimide cross-linking reaction, accompanied with the decline of the self-signal response. When the hybridization between the probe ssDNA and the target DNA was accomplished, the self-signal response of the composite interface reproduced by virtue of the shaping of helix construction. The determination limit of the assembled DNA identification interface was 2.1 × 10-19 mol/L, and the complementary target DNA concentrations varied from 1.0 × 10-18 mol/L to 1.0 × 10-12 mol/L. The DNA identification platform displayed magnificent sensitivity, specificity and stability, and was efficaciously implemented to the mensuration of ctDNA derived from colorectal cancer.

Superior Adsorptive Removal of Anionic Azo Dyes from Aqueous Solutions Using Sulfonic Acid Group-Modified MIL-101@Graphene Oxide Composite.

It is critical to remove organic contaminants from wastewater released by the printing and dyeing industry for addressing water pollution issue. Therefore, the fabrication of new adsorbents with excellent removal efficiencies is an urgent task. A composite of MIL-101 partially functionalized with -SO3H (MIL-101-SO3H) and graphene oxide (GO) was prepared by assembling MIL-101-SO3H truncated octahedrons on the GO framework. The synthesized MIL-101-SO3H@GO has a superior adsorption efficiency for anionic azo dyes. The maximum adsorption capacities of MIL-101-SO3H@GO-1 for Congo red, methyl orange, acid orange 7, and acid orange G reached 2711.3, 818.8, 551.2, and 319.8 mg/g, respectively, which are considerably higher than those obtained using unmodified MIL-101. This is because additional interactions that promote azo dye adsorption, such as hydrogen bonding between the dye and the sulfonic acid groups of MIL-101-SO3H or the carboxyl groups of GO, were induced, and agglomerate pores that accommodated the dye were formed in the composite. The ultrahigh removal efficiency of the composite for azo dyes is mainly driven by hydrogen bonding, electrostatic interactions, π-π stacking between the MIL-101-SO3H@GO and dye molecules, synergistic interactions at the interface of GO and MIL-101-SO3H microcrystals, and the pore-filling effect. Understanding these driving forces for dye adsorption can contribute to the development of sustainable and functionally modified metal-organic framework composite adsorbents.

Intake of B vitamins and the risk of developing islet autoimmunity and type 1 diabetes in the TEDDY study.

PURPOSE: The aim was to study the association between dietary intake of B vitamins in childhood and the risk of islet autoimmunity (IA) and progression to type 1 diabetes (T1D) by the age of 10 years. METHODS: We followed 8500 T1D-susceptible children born in the U.S., Finland, Sweden, and Germany in 2004 -2010 from the Environmental Determinants of Diabetes in the Young (TEDDY) study, which is a prospective observational birth cohort. Dietary intake of seven B vitamins was calculated from foods and dietary supplements based on 24-h recall at 3 months and 3-day food records collected regularly from 6 months to 10 years of age. Cox proportional hazard models were adjusted for energy, HLA-genotype, first-degree relative with T1D, sex, and country. RESULTS: A total of 778 (9.2) children developed at least one autoantibody (any IA), and 335 (3.9%) developed multiple autoantibodies. 280 (3.3%) children had IAA and 319 (3.8%) GADA as the first autoantibody. 344 (44%) children with IA progressed to T1D. We observed that higher intake of niacin was associated with a decreased risk of developing multiple autoantibodies (HR 0.95; 95% CI 0.92, 0.98) per 1 mg/1000 kcal in niacin intake. Higher intake of pyridoxine (HR 0.66; 95% CI 0.46, 0.96) and vitamin B12 (HR 0.87; 95% CI 0.77, 0.97) was associated with a decreased risk of IAA-first autoimmunity. Higher intake of riboflavin (HR 1.38; 95% CI 1.05, 1.80) was associated with an increased risk of GADA-first autoimmunity. There were no associations between any of the B vitamins and the outcomes "any IA" and progression from IA to T1D.  CONCLUSION: In this multinational, prospective birth cohort of children with genetic susceptibility to T1D, we observed some direct and inverse associations between different B vitamins and risk of IA.

Aza-Michael Addition of Dinitrogen to α,ß-Unsaturated Carbonyl Compounds in a Dititanium Framework.

The direct use of dinitrogen (N2) as a building block for the synthesis of NN-containing organic compounds is of fundamental interest and practical importance but has remained a formidable challenge to date. Here, we report an unprecedented 1,4-conjugate (aza-Michael) addition of N2 to α,ß-unsaturated carbonyl compounds in a dititanium framework. The resulting hydrazinopropenolate products could be easily converted to diverse NN-containing organic compounds such as ß-hydrazine-functionalized esters and amides, pyrazolidinones, and pyrazolines depending on the types of Michael acceptors through protonation with MeOH. Further transformations of a hydrazinopropenolate titanium complex through C-C and N-C bond formations with electrophiles such as CO2 and benzaldehyde have also been achieved. The mechanistic details of the N2 addition reaction have been elucidated by computational studies, revealing the importance of redox-active metal centers in this event. This work showcases the potential of using N2 as a building block for the synthesis of NN-containing organic compounds through activation and functionalization in a molecular metal framework.

Dietary Intake and Body Mass Index Influence the Risk of Islet Autoimmunity in Genetically At-Risk Children: A Mediation Analysis Using the TEDDY Cohort.

Background/Objective: Growth and obesity have been associated with increased risk of islet autoimmunity (IA) and progression to type 1 diabetes. We aimed to estimate the effect of energy-yielding macronutrient intake on the development of IA through BMI. Research Design and Methods: Genetically at-risk children (n = 5,084) in Finland, Germany, Sweden, and the USA, who were autoantibody negative at 2 years of age, were followed to the age of 8 years, with anthropometric measurements and 3-day food records collected biannually. Of these, 495 (9.7%) children developed IA. Mediation analysis for time-varying covariates (BMI z-score) and exposure (energy intake) was conducted. Cox proportional hazard method was used in sensitivity analysis. Results: We found an indirect effect of total energy intake (estimates: indirect effect 0.13 [0.05, 0.21]) and energy from protein (estimates: indirect effect 0.06 [0.02, 0.11]), fat (estimates: indirect effect 0.03 [0.01, 0.05]), and carbohydrates (estimates: indirect effect 0.02 [0.00, 0.04]) (kcal/day) on the development of IA. A direct effect was found for protein, expressed both as kcal/day (estimates: direct effect 1.09 [0.35, 1.56]) and energy percentage (estimates: direct effect 72.8 [3.0, 98.0]) and the development of GAD autoantibodies (GADA). In the sensitivity analysis, energy from protein (kcal/day) was associated with increased risk for GADA, hazard ratio 1.24 (95% CI: 1.09, 1.53), p = 0.042. Conclusions: This study confirms that higher total energy intake is associated with higher BMI, which leads to higher risk of the development of IA. A diet with larger proportion of energy from protein has a direct effect on the development of GADA.

Photoredox Nickel-Catalysed Stille Cross-Coupling Reactions.

The Stille cross-coupling reaction is one of the most common strategies for the construction of C-C bonds. Despite notable strides in the advancement of the Stille reaction, persistent challenges persist in hindering its greener evolution. These challenges encompass multiple facets, such as the high cost of precious metals and ligands, the demand for various additives, and the slow reaction rate. In comparison to the dominant palladium-catalysed Stille reactions, cost-effective nickel-catalysed systems lag behind, and enantioconvergent Stille reactions of racemic stannanes remain undeveloped. Herein, we present a pioneering instance of nickel-catalysed enantioconvergent Stille cross-coupling reactions of racemic stannane reagents, resulting in the formation of C-C bonds in good to high yields with excellent stereoselectivity. This strategy provides a practical, scalable, and operationally straightforward method for the synthesis of C(sp3 )-C(sp3 ), C(sp3 )-C(sp2 ), and C(sp3 )-C(sp) bonds under exceptionally mild conditions (without additives and bases, ambient temperature). The innovative use of synergistic photoredox/nickel catalysis enables a novel single-electron transmetalation process of stannane reagents, providing a new research paradigm of Stille reactions.

Dinitrogen Cleavage and Functionalization with Carbon Dioxide in a Dititanium Dihydride Framework.

The activation and functionalization of dinitrogen (N2) with carbon dioxide (CO2) are of great interest and importance but highly challenging. We report here for the first time the reaction of N2 with CO2 in a dititanium dihydride framework, which leads to N-C bond formation and N-N and C-O bond cleavage. Exposure of a dinitrogen dititanium hydride complex {[(acriPNP)Ti]2(µ2-η1:η2-N2)(µ2-H)2} (1) (acriPNP = 4,5-bis(diisopropylphosphino)-2,7,9,9-tetramethyl-9H-acridin-10-ide) to a CO2 atmosphere at room temperature rapidly yielded a nitrido/N,N-dicarboxylamido complex {[(acriPNP)Ti]2(µ2-N)[µ2-N(CO2)2]} (2, 28%) and a diisocyanato/dioxo complex {[(acriPNP)Ti]2(NCO)2(µ2-O)2} (3, 52%) with release of H2. When the reaction of 1 with CO2 (1 atm) was carried out at -50 °C, complex 2 was selectively formed in 82% yield within 5 min. Heating 2 at 80 °C under 1 atm CO2 for 30 min afforded 3 in 67% yield. When 1 was allowed to react with 1.5 equiv of CO2 at room temperature, an isocyanato/nitrido/oxo complex {[(acriPNP)Ti]2(NCO)(µ2-N)(µ2-O)} (4) was exclusively formed in 89% yield within 5 min. The reaction of 4 with CO2 at room temperature almost quantitatively yielded the dioxo/diisocyanato complex 3 within 5 min. The mechanistic details were clarified by the 15N- and 13C-labeled experiments and density functional theory (DFT) calculations, providing unprecedented insights into the reaction of N2 with CO2. A titanium-mediated cycle for the synthesis of trimethylsilyl isocyanate Me3SiNCO from N2, CO2, and Me3SiCl using H2 as a reducing agent was also established.

Long Range Endocrine Delivery of Circulating miR-210 to Endothelium Promotes Pulmonary Hypertension.

RATIONALE: Unproven theories abound regarding the long-range uptake and endocrine activity of extracellular blood-borne microRNAs into tissue. In pulmonary hypertension (PH), microRNA-210 (miR-210) in pulmonary endothelial cells promotes disease, but its activity as an extracellular molecule is incompletely defined. OBJECTIVE: We investigated whether chronic and endogenous endocrine delivery of extracellular miR-210 to pulmonary vascular endothelial cells promotes PH. METHODS AND RESULTS: Using miR-210 replete (wild-type [WT]) and knockout mice, we tracked blood-borne miR-210 using bone marrow transplantation and parabiosis (conjoining of circulatory systems). With bone marrow transplantation, circulating miR-210 was derived predominantly from bone marrow. Via parabiosis during chronic hypoxia to induce miR-210 production and PH, miR-210 was undetectable in knockout-knockout mice pairs. However, in plasma and lung endothelium, but not smooth muscle or adventitia, miR-210 was observed in knockout mice of WT-knockout pairs. This was accompanied by downregulation of miR-210 targets ISCU (iron-sulfur assembly proteins)1/2 and COX10 (cytochrome c oxidase assembly protein-10), indicating endothelial import of functional miR-210. Via hemodynamic and histological indices, knockout-knockout pairs were protected from PH, whereas knockout mice in WT-knockout pairs developed PH. In particular, pulmonary vascular engraftment of miR-210-positive interstitial lung macrophages was observed in knockout mice of WT-knockout pairs. To address whether engrafted miR-210-positive myeloid or lymphoid cells contribute to paracrine miR-210 delivery, we studied miR-210 knockout mice parabiosed with miR-210 WT; Cx3cr1 knockout mice (deficient in myeloid recruitment) or miR-210 WT; Rag1 knockout mice (deficient in lymphocytes). In both pairs, miR-210 knockout mice still displayed miR-210 delivery and PH, thus demonstrating a pathogenic endocrine delivery of extracellular miR-210. CONCLUSIONS: Endogenous blood-borne transport of miR-210 into pulmonary vascular endothelial cells promotes PH, offering fundamental insight into the systemic physiology of microRNA activity. These results also describe a platform for RNA-mediated crosstalk in PH, providing an impetus for developing blood-based miR-210 technologies for diagnosis and therapy in this disease.

The association of physical activity to oral glucose tolerance test outcomes in multiple autoantibody positive children: The TEDDY Study.

OBJECTIVE: To examine the association of physical activity (PA), measured by accelerometry, to hemoglobin AIC (HbA1c) and oral glucose tolerance test (OGTT) outcomes in children who were multiple persistent confirmed autoantibody positive for type 1 diabetes (T1D). METHODS: The Environmental Determinants of Diabetes in the Young (TEDDY) multinational study followed children from birth. Children ≥3 years of age who were multiple persistent confirmed autoantibody positive were monitored by OGTTs every 6 months. TEDDY children's PA was measured by accelerometry beginning at 5 years of age. We examined the relationship between moderate plus vigorous (mod + vig) PA, HbA1c, and OGTT in 209 multiple autoantibody children who had both OGTT and PA measurements. RESULTS: Mod + vig PA was associated with both glucose and C-peptide measures (fasting, 120-min, and AUC); higher mod + vig PA was associated with a better OGTT response primarily in children with longer duration of multiple autoantibody positivity. Mod + vig PA also interacted with child age; lower mod + vig PA was associated with a greater increase in C-peptide response across age. Mod + vig PA was not related to fasting insulin, HOMA-IR or HbA1c. CONCLUSIONS: The OGTT is the gold standard for diabetes diagnosis and is used to monitor those at high risk for T1D. We found higher levels of mod + vig PA were associated with better OGTT outcomes in children ≥5 years of age who have been multiple autoantibody positive for longer periods of time. Physical activity should be the focus of future efforts to better understand the determinants of disease progression in high-risk children.

Maternal food consumption during late pregnancy and offspring risk of islet autoimmunity and type 1 diabetes.

AIMS/HYPOTHESIS: We aimed to investigate the association between maternal consumption of gluten-containing foods and other selected foods during late pregnancy and offspring risk of islet autoimmunity (IA) and type 1 diabetes in The Environmental Determinants of Diabetes in the Young (TEDDY) study. METHODS: The TEDDY study recruited children at high genetic risk for type 1 diabetes at birth, and prospectively follows them for the development of IA and type 1 diabetes (n = 8556). A questionnaire on the mother's diet in late pregnancy was completed by 3-4 months postpartum. The maternal daily intake was estimated from a food frequency questionnaire for eight food groups: gluten-containing foods, non-gluten cereals, fresh milk, sour milk, cheese products, soy products, lean/medium-fat fish and fatty fish. For each food, we described the distribution of maternal intake among the four participating countries in the TEDDY study and tested the association of tertile of maternal food consumption with risk of IA and type 1 diabetes using forward selection time-to-event Cox regression. RESULTS: By 28 February 2019, 791 cases of IA and 328 cases of type 1 diabetes developed in TEDDY. There was no association between maternal late-pregnancy consumption of gluten-containing foods or any of the other selected foods and risk of IA, type 1 diabetes, insulin autoantibody-first IA or GAD autoantibody-first IA (all p ≥ 0.01). Maternal gluten-containing food consumption in late pregnancy was higher in Sweden (242 g/day), Germany (247 g/day) and Finland (221 g/day) than in the USA (199 g/day) (pairwise p < 0.05). CONCLUSIONS/INTERPRETATION: Maternal food consumption during late pregnancy was not associated with offspring risk for IA or type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00279318.

Selective Macrocycle Formation in Cavitands.

The traditional end-to-end cyclization of long-chain linear precursors is difficult and often unpredictable because the unfavorable entropy of macrocyclic closure allows undesired intermolecular reactions to compete. Here, we apply cavitands to the selective intramolecular aldol/dehydration reaction of long-chain α,ω-dialdehydes in aqueous solution. Hydrophobic forces drive the dialdehydes into the cavitands in folded conformations and favor macrocyclization reactions over intermolecular reactions observed in bulk solution. The macrocyclic aldol reaction products are isolated in good yields (30-85%) over a wide range (11 to 17-membered rings). Unlike conventional templates that become guests inside their assembled hosts, cavitands reverse the roles and resemble the situation in biological catalysis-the templates are hosts for guests undergoing the assisted reaction processes.

Rigidified Cavitand Hosts in Water: Bent Guests, Shape Selectivity, and Encapsulation.

We report the synthesis and characterization of two water-soluble container compounds (cavitand hosts) with rigidified open ends. One cavitand uses four (CH2)4's as spacers to bridge the adjacent walls, while another cavitand uses four CH2CH2OCH2CH2's bridges and features a wider open end. The spacers preorganize the deep cavitands into vase-like, receptive shapes and prevent their unfolding to the unreceptive kite-like conformation. Cycloalkane guests (C6-C8) and small n-alkanes (C5-C7) form 1:1 complexes with the cavitands and move freely in the cavitands' spaces. Hydrophilic compounds 1,4-dioxane, tetrahydrofuran, tetrahydropyran, pyridine, and 1-methylimidazole also showed good binding affinity to the new cavitands. Longer alkanes (C11-C14) and n-alcohols (C11-C16) are taken up with a -CH3 group fixed at the bottom of the cavity and the groups near the rim in compressed conformations. The methylene bridges appear to divide the cavitand into a narrow hydrophobic compartment and a broader space with exposure to the aqueous medium. Longer alkane guests (C15-C18), N,N-dimethyldioctylammonium, and dioctylamine induce the formation of capsules (2:1 host:guest complexes). The new cavitands showed selectivity for p/m-cresol isomers and xylene isomers. The cavitand with CH2CH2OCH2CH2 bridges bound long-chain α,ω-diols (C13-C15) and diamines in folded, U-shaped conformations with polar functions exposed to the aqueous medium. It was used to separate o-xylene from its isomers by using simple extraction procedures.

Theoretical Studies of Rare-Earth-Catalyzed [3 + 2] Annulation of Aromatic Aldimine with Styrene: Mechanism and Origin of Diastereoselectivity.

The synthesis of multisubstituted 1-aminoindanes through catalyst-controlled diastereodivergent [3 + 2] annulation of aromatic imines with alkenes is of great interest and importance. An understanding of the exact reaction mechanism, especially for the origin of diastereoselectivity, is an essential aspect for further development of such reactions. In this study, density functional theory calculations have been carried out on the rare-earth-catalyzed diastereodivergent [3 + 2] annulation of benzaldimine with styrene. The results show that the reaction mainly involves generation of active species, olefin insertion, cyclization, and protonation steps. The noncovalent interactions, such as C-H···π and metal···π interactions, play an important role in stabilizing the key transition state or intermediate. Both steric and electronic factors account for the diastereoselectivity. The preferred cis-diastereoselectivity could be ascribed to more efficient orbital interaction, while the crowded space will induce the formation of a C-H···π interaction between the NtBu group and benzene ring in a trans-diastereoselectivity manner, thus stabilizing the trans-selective transition state. Therefore, the stereospecific product could be obtained by fine-tuning the ligand/metal combination of the catalysts.

The MicroRNA-92a/Sp1/MyoD Axis Regulates Hypoxic Stimulation of Myogenic Lineage Differentiation in Mouse Embryonic Stem Cells.

Hypoxic microenvironments exist in developing embryonic tissues and determine stem cell fate. We previously demonstrated that hypoxic priming plays roles in lineage commitment of embryonic stem cells. In the present study, we found that hypoxia-primed embryoid bodies (Hyp-EBs) efficiently differentiate into the myogenic lineage, resulting in the induction of the myogenic marker MyoD, which was not mediated by hypoxia-inducible factor 1α (HIF1α) or HIF2α, but rather by Sp1 induction and binding to the MyoD promoter. Knockdown of Sp1 in Hyp-EBs abrogated hypoxia-induced MyoD expression and myogenic differentiation. Importantly, in the cardiotoxin-muscle injury mice model, Hyp-EB transplantation facilitated muscle regeneration in vivo, whereas transplantation of Sp1-knockdown Hyp-EBs failed to do. Moreover, we compared microRNA (miRNA) expression profiles between EBs under normoxia versus hypoxia and found that hypoxia-mediated Sp1 induction was mediated by the suppression of miRNA-92a, which directly targeted the 3' untranslated region (3' UTR) of Sp1. Further, the inhibitory effect of miRNA-92a on Sp1 in luciferase assay was abolished by a point mutation in specific sequence in the Sp1 3' UTR that is required for the binding of miRNA-92a. Collectively, these results suggest that hypoxic priming enhances EB commitment to the myogenic lineage through miR-92a/Sp1/MyoD regulatory axis, suggesting a new pathway that promotes myogenic-lineage differentiation.

Cyclase-associated protein 1 is a binding partner of proprotein convertase subtilisin/kexin type-9 and is required for the degradation of low-density lipoprotein receptors by proprotein convertase subtilisin/kexin type-9.

AIMS: Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. METHODS AND RESULTS: The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. CONCLUSION: We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.

Dinitrogen Activation and Hydrogenation by C5Me4SiMe3-Ligated Di- and Trinuclear Chromium Hydride Complexes.

Activation of dinitrogen (N2) by well-defined metal hydrides is of much interest and importance, but studies in this area have remained limited to date. We report here N2 activation and hydrogenation by C5Me4SiMe3-ligated di- and trinuclear chromium polyhydride complexes. Hydrogenolysis of [Cp'Cr(µ-Me)2CrCp'] (Cp' = C5Me4SiMe3) (1) with H2 in a dilute hexane solution under N2-free conditions affords the dichromium dihydride complex [Cp'Cr(µ-H)2CrCp'] (2), while hydrogenolysis of 1 in a concentrated solution or without solvent provides the trinuclear chromium tetrahydride complex [(Cp'Cr)3(µ3-H)(µ-H)3] (3). When the reaction is carried out in the presence of N2 in a dilute hexane solution, the tetranuclear diimide/dihydride complex [(Cp'Cr)4(µ3-NH)2(µ3-H)2] (4) is formed via N-N bond cleavage and N-H bond formation. The reaction of 2 with N2 at room temperature gives the tetranuclear imide/nitride/dihydride complex [(Cp'Cr)3(C5Me3(CH2)SiMe3)Cr(µ3-NH)(µ3-N)(µ-H)2] (5) via N2 cleavage and hydrogenation and C-H bond activation of a Cp methyl group. At -30 °C, the reaction of 2 with N2 affords the dinitride intermediate [(Cp'Cr)4(µ3-N)2(µ3-H)2] (6), which is quantitatively transformed to 5 at room temperature. Complex 5 reversibly converts to the stereoisomer 5'. The hydrogenation of a mixture of 5 and 5' with H2 affords 4. The reaction of 3 with N2 proceeds at 100 °C to afford [(Cp'Cr)3(µ3-NH)2] (7). This transformation has also been investigated by DFT calculations. Both experimental and computational studies suggest that N2 incorporation into the chromium hydride cluster is involved in the rate-determining step. This work represents the first example of N2 cleavage and hydrogenation by well-defined chromium hydride complexes.

Insertion of Alkylidene Carbenes into B-H Bonds.

We have developed a protocol for insertion of alkylidene carbenes into the B-H bonds of amine-borane adducts, enabling, for the first time, the construction of C(sp2)-B bonds by means of carbene-insertion reactions. Various acyclic and cyclic alkenyl borane-amine adducts were prepared from readily accessible starting materials in good to high yields and were subsequently subjected to a diverse array of functional group transformations. The unprecedented spiro B-N heterocycles prepared in this study have potential utility as building blocks for the synthesis of pharmaceuticals. Preliminary mechanistic studies suggest that insertion of the alkylidene carbenes into the B-H bonds of the amine-borane adducts proceeds via a concerted process involving a three-membered-ring transition state.

The LAMMER kinase is involved in morphogenesis and response to cell wall- and DNA-damaging stresses in Candida albicans.

Dual specificity LAMMER kinase has been reported to be conserved across species ranging from yeasts to animals and has multiple functions. Candida albicans undergoes dimorphic switching between yeast cells and hyphal growth forms as its key virulence factors. Deletion of KNS1, which encodes for LAMMER kinase in C. albicans, led to pseudohyphal growth on YPD media and defects in filamentous growth both on spider and YPD solid media containing 10% serum. These cells exhibited expanded central wrinkled regions and specifically reduced peripheral filaments. Among the several stresses tested, the kns1Δ strains showed sensitivity to cell-wall and DNA-replicative stress. Under fluorescent microscopy, an increase in chitin decomposition was observed near the bud necks and septa in kns1Δ cells. When the expression levels of genes for cell wall integrity (CWI) and the DNA repair mechanism were tested, the kns1 double-deletion cells showed abnormal patterns compared to wild-type cells; The transcript levels of genes for glycosylphosphatidylinositol (GPI)-anchored proteins were increased upon calcofluor white (CFW) treatment. Under DNA replicative stress, the expression of MluI-cell cycle box binding factor (MBF)-targeted genes, which are expressed during the G1/S transition in the cell cycle, was not increased in the kns1 double-deletion cells. This strain showed increased adhesion to the surface of an agar plate and zebrafish embryo. These results demonstrate that Kns1 is involved in dimorphic transition, cell wall integrity, response to DNA replicative stress, and adherence to the host cell surface in C. albicans.