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Using fully internally contracted (FIC)-CASPT2 analytical gradients, geometry optimizations of spin-crossover complexes are reported. This approach is tested on a series of Fe(II) complexes with different sizes, ranging from 13 to 61 atoms. A combination of active space and basis set choices are employed to investigate their role in determining reliable molecular geometries. The reported strategy demonstrates that a wave function-based level of theory can be used to optimize the geometries of metal complexes in reasonable times and enables one to treat the molecular geometry and electronic structure of the complexes using the same level of theory. For a series of smaller Fe(II) SCO complexes, strong field ligands in the LS state result in geometries with the largest differences between DFT and CASPT2; however, good agreement overall is observed between DFT and CASPT2. For the larger complexes, moderate sized basis sets yield geometries that compare well with DFT and available experimental data. We recommend using the (10 e ,12 o ) active space since convergence to a minimum structure was more efficient than with truncated active spaces despite having similar Fe-ligand bond distances.

The spin state in heterobimetallic complexes heavily influences both reactivity and magnetism. Exerting control over spin states in main group-based heterobimetallics requires a different approach as the orbital interactions can differ substantially from that of classic coordination complexes. By deliberately engendering an energetic mismatch within the two metals in a bimetallic complex we can mimic the electronic structure of lanthanides. Towards this end, we report a new family of complexes, [ Ph,Me TpMSnPh 3 ] where M = Mn ( 3 ), Fe ( 4 ), Co ( 5 ), Ni ( 6 ), Zn ( 7 ), featuring unsupported bonding between a transition metal and Sn which represent an unusual high spin electronic structure. Analysis of the frontier orbitals reveal the desired orbital mismatch with Sn 5s/5p primarily interacting with 4s/4p M orbitals yielding localized, non-bonding d orbitals. This approach offers a mechanism to design and control spin states in bimetallic complexes., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

A relativistic coupled-cluster version of the Feller-Peterson-Dixon composite method has been used to accurately calculate the first ionization potentials (IPs) and electron affinities (EAs) of the post-d, p-block elements Ga-Rn. Complete basis set extrapolations including outer-core correlation at the CCSD(T) level of theory were combined with contributions from higher order electron correlation up to CCSDTQ, quantum electrodynamic effects (Lamb shift), and spin-orbit (SO) coupling including the Gaunt contribution. Several methods for including SO were investigated, in which all involved the four-component (4c) Dirac-Coulomb (DC) Hamiltonian. The treatment of SO coupling was the contribution that limited the final accuracy of the present results. In the cases where 4c-DC-CCSD(T) could be reliably used for the SO contributions, the final composite IPs and EAs agreed with the available experimental values to within an unsigned average error of just 0.16 and 0.20 kcal/mol, respectively. In all cases, the final IPs and EAs were within 1 kcal/mol of the available experimental values, except for the EAs of the group 13 elements (Ga, In, and Tl), where the currently accepted experimental values appear to be too large by as much as 4 kcal/mol. The values predicted in this work, which have estimated uncertainties of ±0.5 kcal/mol, are 5.25 (Ga), 7.69 (In), and 7.39 (Tl) kcal/mol. For the EAs of Po and At, which do not have experimental values, the current calculations predict values of 34.2 and 55.8 kcal/mol with estimated uncertainties of ±0.6 and ±0.3 kcal/mol, respectively.

High level Brueckner doubles with triples correction method-based ab initio calculations have been used to investigate the nature of intramolecular hydrogen bonding and intramolecular hydrogen atom transfer in cis-malonaldehyde (MA) and its radical analogues. The radicals considered here are the ones that correspond to the homolytic cleavage of C-H bonds in cis-MA. The results suggest that cis-MA and its radical analogues, cis-MA RS , and cis-MA RA , both exist in planar geometry. The calculated intramolecular O-H⋯O=C bond in cis-MA is shorter than that in the radical analogues. The intramolecular hydrogen bond in cis-MA is stronger than in its radicals by at least 3.0 kcal/mol. The stability of a cis-malonaldehyde radical correlates with the extent of electron spin delocalization; cis-MA RA , in which the radical spin is more delocalized, is the most stable MA radical, whereas cis-MA RS , in which the radical spin is strongly localized, is the least stable radical. The natural bond orbital analysis indicates that the intramolecular hydrogen bonding (O⋯H⋯O) in cis-malonaldehyde radicals is stabilized by the interaction between the lone pair orbitals of donor oxygen and the σ * orbital of acceptor O-H bond (n → σ * OH ). The calculated barriers indicate that the intramolecular proton transfer in cis-MA involves 2.2 kcal/mol lower barrier than that in cis-MA RS .

An ab initio investigation has been employed to determine the structural and spectroscopic parameters, such as rotational constants, vibrational frequencies, vertical excitation energies, and the stability of the ethyl-water complex. The ethyl-water complex has a binding energy of 1.15 kcal⋅mol -1 . The interaction takes place between the hydrogen of water and the unpaired electron of the radical. This interaction is found to produce a red shift in the OH stretching bands of water of ca. 84 cm -1 , and a shift of all UV absorption bands to higher energies.

Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplanin(fl/fl) mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage., (© 2015 by The American Society of Hematology.)

We have used a novel knockin mouse to investigate the effect of disruption of phosphotyrosine binding of the N-terminal SH2 domain of Syk on platelet activation by GPVI, CLEC-2, and integrin αIIbβ3. The Syk(R41Afl/fl) mouse was crossed to a PF4-Cre(+) mouse to induce expression of the Syk mutant in the megakaryocyte/platelet lineage. Syk(R41Afl/fl;PF4-Cre) mice are born at approximately 50% of the expected frequency and have a similar phenotype to Syk(fl/fl;PF4-Cre) mice, including blood-lymphatic mixing and chyloascites. Anastomosis of the venous and lymphatic vasculatures can be seen in the mesenteric circulation accounting for rapid and continuous mixing of the 2 vasculatures. Platelet activation by CLEC-2 and GPVI is abolished in Syk(R41Afl/fl;PF4-Cre) platelets. Syk phosphorylation on Tyr519/20 is blocked in CLEC-2-stimulated platelets, suggesting a model in which binding of Syk via its N-terminal SH2 domain regulates autophosphorylation. In contrast, outside-in signaling by integrin αIIbβ3 is not altered, but it is inhibited in the presence of inhibitors of Src and Syk tyrosine kinases. These results demonstrate that αIIbβ3 regulates Syk through an ITAM-independent pathway in mice and provide novel insight into the course of events underlying Syk activation and hemITAM phosphorylation by CLEC-2., (© 2015 by The American Society of Hematology.)

The importance of CLEC-2, a natural ligand/receptor for Gp38/Podoplanin, in the formation of the lymphatic vasculature has recently been demonstrated. As the development and maintenance of lymph nodes (LNs) is dependent on the formation of the lymphatic vasculature and the differentiation of Gp38/Podoplanin(+) stromal cells, we investigated the role of CLEC-2 in lymphoneogenesis and LN homeostasis. Using constitutive Clec1b(-/-) mice, we showed that while CLEC-2 was not necessary for initiation of the LN anlage, it was required at late stages of development. Constitutive deletion of CLEC-2 induced a profound defect in lymphatic endothelial cell proliferation, resulting in lack of LNs at birth. In contrast, conditional deletion of CLEC-2 in the megakaryocyte/platelet lineage in Clec1b(fl/fl)PF4-Cre mice led to the development of blood-filled LNs and fibrosis, in absence of a proliferative defect of the lymphatic endothelial compartment. This phenotype was also observed in chimeric mice reconstituted with Clec1b(fl/fl)PF4-Cre bone marrow, indicating that CLEC-2 expression in platelets was required for LN integrity. We demonstrated that LNs of Clec1b(fl/fl)PF4-Cre mice are able to sustain primary immune responses but show a defect in immune cell recirculation after repeated immunizations, thus suggesting CLEC-2 as target in chronic immune response., (© 2014 by The American Society of Hematology.)

Blood platelets have recently been proposed to play a critical role in the development and repair of the lymphatic system. The platelet C-type lectin receptor CLEC-2 and its ligand, the transmembrane protein Podoplanin, which is expressed at high levels on lymphatic endothelial cells (LECs), are required to prevent mixing of the blood and lymphatic vasculatures during mid-gestation. A similar defect is seen in mice deficient in the tyrosine kinase Syk, which plays a vital role in mediating platelet activation by CLEC-2. Furthermore, blood-lymphatic mixing is also present in mice with platelet-/megakaryocyte-specific deletions of CLEC-2 and Syk, suggesting that the phenotype is platelet in origin. The molecular basis of this effect is not known, but it is independent of the major platelet receptors that support hemostasis, including integrin αIIbβ3 (GPIIb-IIIa). Radiation chimeric mice reconstituted with CLEC-2-deficient or Syk-deficient bone marrow exhibit blood-lymphatic mixing in the intestines, illustrating a role for platelets in repair and growth of the lymphatic system. In this review, we describe the events that led to the identification of this novel role of platelets and discuss possible molecular mechanisms and the physiological and pathophysiological significance.

Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E)11.5-16.5 in mouse) in a hypercalcaemic environment (~1.7 in the fetus vs. ~1.1-1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca(2+) channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.

Objective: Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif-bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice., Approach and Results: We investigated whether both (hem)immunoreceptor tyrosine activation motif-bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2-depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2., Conclusions: These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti-CLEC-2-based agents in the prevention of thrombotic diseases.

Background: In comparison to the classical isoforms of protein kinase C (PKC), the novel isoforms are thought to play minor or inhibitory roles in the regulation of platelet activation and thrombosis., Objectives: To measure the levels of PKCθ and PKCε and to investigate the phenotype of mice deficient in both novel PKC isoforms., Methods: Tail bleeding and platelet activation assays were monitored in mice and platelets from mice deficient in both PKCθ and PKCε., Results: PKCε plays a minor role in supporting aggregation and secretion following stimulation of the collagen receptor GPVI in mouse platelets but has no apparent role in spreading on fibrinogen. PKCθ, in contrast, plays a minor role in supporting adhesion and filopodial generation on fibrinogen but has no apparent role in aggregation and secretion induced by GPVI despite being expressed at over 10 times the level of PKCε. Platelets deficient in both novel isoforms have a similar pattern of aggregation downstream of GPVI and spreading on fibrinogen as the single null mutants. Strikingly, a marked reduction in aggregation on collagen under arteriolar shear conditions is observed in blood from the double but not single-deficient mice along with a significant increase in tail bleeding., Conclusions: These results reveal a greater than additive role for PKCθ and PKCε in supporting platelet activation under shear conditions and demonstrate that, in combination, the two novel PKCs support platelet activation., (© 2012 International Society on Thrombosis and Haemostasis.)

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.

Endochondral bone formation requires new blood vessel formation, and endothelial progenitor cells (EPCs) may play a role in this process. Endothelial colony-forming cells (ECFCs), one subtype of EPCs, isolated from the microvasculature of rat lungs, exhibited cell surface antigen markers and gene products characteristic of endothelial cells and displayed high proliferative potential and an ability to form vessel-like network structures in vitro. The aim of this study was to evaluate whether ECFCs facilitate bone healing during fracture repair and stimulate bone regeneration. When type I collagen sponge containing ECFCs were surgically wrapped around the fractured femurs of rats, newly formed bone mineral at the site of fracture was 13% greater (P = 0.01) and energy to failure was 46% greater (P = 0.01) compared to sponge-wrapped fractures without ECFCs. When ECFCs in type I collagen sponge were surgically implanted into the bone defective area, more new vessels formed locally in comparison with sponge-alone controls and new bone tissues were seen. Further, co-implantation of ECFCs and hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds at the bone defective sites stimulated more new bone tissues than HA/TCP scaffold alone. These results show that cell therapy with vessel wall-derived ECFCs can induce new vessel formation, stimulate new bone formation, and facilitate bone repair and could be a useful approach to treat non-union fractures and bone defects.

The capacity to sense and adapt to changes in environmental cues is of paramount importance for every living organism. From yeast to man, cells must be able to match cellular activities to growth environment and nutrient availability. Key to this process is the development of membrane-bound systems that can detect modifications in the extracellular environment and to translate these into biological responses. Evidence gathered over the last 15 years has demonstrated that many of these cell surface "sensors" belong to the G protein-coupled receptor superfamily. Crucial to our understanding of nutrient sensing in mammalian species has been the identification of the extracellular Ca(2+)/cation-sensing receptor, CaR. CaR was the first ion-sensing molecule identified in man and genetic studies in humans have revealed the importance of the CaR in mineral ion metabolism. Latter, it has become apparent that the CaR also plays an important role outside the Ca(2+) homeostatic system, as an integrator of multiple environmental signals for the regulation of many vital cellular processes, from cell-to-cell communication to secretion and cell survival/cell death. Recently, novel aspects of receptor function reveal an unexpected role for the CaR in the regulation of growth and development in utero.

Postnatal lung function is critically dependent upon optimal embryonic lung development. As the free ionized plasma calcium concentration ([Ca(2+)](o)) of the fetus is higher than that of the adult, the process of lung development occurs in a hypercalcaemic environment. In the adult, [Ca(2+)](o) is monitored by the G-protein coupled, extracellular calcium-sensing receptor (CaR), but neither its ontogeny nor its potential role in lung development are known. Here, we demonstrate that CaR is expressed in the mouse lung epithelium, and that its expression is developmentally regulated, with a peak of expression at embryonic day 12.5 (E12.5) and a subsequent decrease by E18, after which the receptor is absent. Experiments carried out using the lung explant culture model in vitro show that lung branching morphogenesis is sensitive to [Ca(2+)](o), being maximal at physiological adult [Ca(2+)](o) (i.e. 1.0-1.3 mM) and lowest at the higher, fetal (i.e. 1.7 mM) [Ca(2+)](o). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high [Ca(2+)](o) on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances intracellular calcium signalling, lung distension and fluid secretion. Conditions which are restrictive either to branching or to secretion can be rescued by manipulating [Ca(2+)](o) in the culture medium. In conclusion, fetal Ca(2+)(o), acting through a developmentally regulated CaR, is an important extrinsic factor that modulates the intrinsic lung developmental programme. Our observations support a novel role for the CaR in preventing hyperplastic lung disease in utero.

Expression of the human epidermal growth factor receptor (EGFR) in murine Schwann cells results in loss of axon-Schwann cell interactions and collagen deposition, modeling peripheral nerve response to injury and tumorigenesis. Mast cells infiltrate nerves in all three situations. We show that mast cells are present in normal mouse peripheral nerve beginning at 4 weeks of age, and that the number of mast-cells in EGFR(+) nerves increases abruptly at 5-6 weeks of age as axons and Schwann cells dissociate. The increase in mast cell number is preceded and accompanied by elevated levels of mRNAs encoding the mast-cell chemoattractants Rantes, SCF and VEGF. Genetic ablation of mast cells and bone marrow reconstitution in W(41) x EGFR(+) mice indicate a role for mast cells in loss of axon-Schwann cell interactions and collagen deposition. Pharmacological stabilization of mast cells by disodium cromoglycate administration to EGFR(+) mice also diminished loss of axon-Schwann cell interaction. Together these three lines of evidence support the hypothesis that mast cells can contribute to alterations in peripheral nerves.