Stellated cuboctahedron of FeIII.

The solvothermal reaction of FeCl2·4H2O and H4TBC[4] in a basic dmf/EtOH solution affords an [FeIII18] Keplerate conforming to a stellated cuboctahedron. Magnetic measurements reveal spin frustration effects arising from the high symmetry.

MITRAL REGURGITATION IN SADDLE-BILLED STORKS (EPHIPPIORHYNCHUS SENEGALENSIS) IN HUMAN CARE: DIAGNOSIS, ECHOCARDIOGRAPHIC MEASUREMENTS, AND MANAGEMENT.

The asymptomatic and slow progressive nature of cardiopathies represents a risk to the welfare of avian species in human care. Diagnosis and treatment of cardiac disease in birds pose a challenge due to unique anatomic and physiologic characteristics. Comprehensive cardiac assessments with diagnostic tools such as echocardiography, color-Doppler, the biomarker cardiac troponin I (cTn1), and cholesterol serum concentrations have been utilized in different bird species with varying success. Saddle-billed storks (Ephippiorhynchus senegalensis) have been maintained in human care for over 80 yrs and several institutions have noted heart murmurs and cardiomegaly. Despite these findings, peer-reviewed literature describing cardiopathies is lacking for this species. This case series documents the identification of mitral valve regurgitation in saddle-billed storks in a breeding center. Transcoelomic echocardiography using a ventromedial approach with a two-chambered view and color Doppler was utilized. Echocardiographic measurements were taken and compared 1 yr later in most of the birds. There was left atrial enlargement and worsened mitral regurgitation in one geriatric patient, and no progression of the disease in two young birds. Serum samples showed that cTn1 had different concentrations depending on the severity of the disease, whereas cholesterol was within reference range for all birds. Treatment with digoxin and pimobendan was recommended in one bird, serum concentrations of digoxin were tested in a 6-mon span, results were within therapeutic range, and there were no overt adverse effects. There was a suspected genetic component in this population, as four of the five birds with confirmed mitral regurgitation were related.

Odd and Even Numbered Ferric Wheels.

The structurally related odd and even numbered wheels [FeIII 11 ZnII 4 (tea)10 (teaH)1 (OMe)Cl8 ] (1) and [FeIII 12 ZnII 4 (tea)12 Cl8 ] (2) can be synthesized under ambient conditions by reacting FeIII and ZnII salts with triethanolamine (teaH3 ), the change in nuclearity being dictated by the solvents employed. An antiferromagnetic exchange between nearest neighbors, J = -10.0 cm-1 for 1 and J = -12.0 cm-1 for 2, leads to a frustrated S = 1/2 ground state in the former and an S = 0 ground state in the latter.

BARIUM SULFATE GELATIN AS A NOVEL APPROACH TO FACILITATE GASTROINTESTINAL TRACT POSITIVE CONTRAST STUDIES IN A CAPTIVE HARBOR SEAL (PHOCA VITULINA) AND CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS) WITHOUT RESTRAINT.

Gastrointestinal tract contrast medium studies are a valuable diagnostic modality to evaluate gastrointestinal anatomy, motility, and pathology. Four positive contrast medium studies were performed twice on a harbor seal (Phoca vitulina) and once each on two California sea lions (Zalophus californianus) to evaluate for gastrointestinal abnormalities by using barium-impregnated gelatin. Because marine mammals in human care routinely receive plain gelatin as a component of their diet and as secondary reinforcement, feeding a barium gelatin mitigates the need for tube feeding or restraint. Operant conditioning was used to position the pinnipeds for voluntary radiographs. The barium gelatin permitted adequate evaluation of gastric emptying and intestinal transit times and good evaluation of the structure of the intestinal tract. Full evaluation of gastric anatomy was limited due to the gelatin blocks not conforming to the stomach in their solid form and the barium quickly exiting the stomach as barium gelatin blocks disintegrated and because orthogonal views could not always be acquired. Even with these limitations, barium gelatin resulted in diagnostically valuable contrast imaging in a stress-free patient setting with reduced risk of aspiration and eliminated the effects that anesthesia can have on gastric motility as well as other anesthesia-associated risks.

Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor.

In response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel-Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls actomyosin ring function, aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

An [FeIII8] molecular oxyhydroxide.

An [FeIII8] hexagonal bipyramid displays antiferromagnetic exchange between the two capping tetrahedral ions and the six ring octahedral ions resulting in a spin ground state of S = 10.

Structure modeling hints at a granular organization of the Golgi ribbon.

BACKGROUND: In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this "ribbon" architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses" whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. RESULTS: WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a "breathing" arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. CONCLUSIONS: Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.

Shrinking Weibel-Palade bodies prevents high platelet recruitment in assays using thrombotic thrombocytopenic purpura plasma.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), caused by a genetic or autoimmune-driven lack of ADAMTS-13 activity, leads to high levels of the ultra-large von Willebrand factor (VWF) multimers produced by endothelial cells, causing excess platelet recruitment into forming thrombi, often with mortal consequences. Treatments include plasma infusion or replacement to restore ADAMTS-13 activity, or prevention of platelet recruitment to VWF. OBJECTIVES: We tested a different approach, exploiting the unique cell biology of the endothelium. Upon activation, the VWF released by exocytosis of Weibel-Palade bodies (WPBs), transiently anchored to the cell surface, unfurls as strings into flowing plasma, recruiting platelets. Using plasma from patients with TTP increases platelet recruitment to the surface of cultured endothelial cells under flow. WPBs are uniquely plastic, and shortening WPBs dramatically reduces VWF string lengths and the recruitment of platelets. We wished to test whether the TTP plasma-driven increase in platelet recruitment would be countered by reducing formation of the longest WPBs that release longer strings. METHODS: Endothelial cells grown in flow chambers were treated with fluvastatin, one of 37 drugs shown to shorten WPBs, then activated under flow in the presence of platelets and plasma of either controls or patients with TTP. RESULT: We found that the dramatic increase in platelet recruitment caused by TTP plasma is entirely countered by treatment with fluvastatin, shortening the WPBs. CONCLUSIONS: This potential approach of ameliorating the endothelial contribution to thrombotic risk by intervening far upstream of hemostasis might prove a useful adjunct to more conventional and direct therapies.

An [FeIII30] molecular metal oxide.

Dissolution of FeBr3 in a mixture of acetonitrile and 3,4-lutidine in the presence of an amine results in the formation of an [Fe30] molecular metal oxide containing alternating layers of tetrahedral and octahedral FeIII ions. Mass spectrometry suggests the cluster is formed quickly and remains stable in solution, while magnetic measurements and DFT calculations reveal competing antiferromagnetic exchange interactions.

[Fe15]: a frustrated, centred tetrakis hexahedron.

The combination of two different FeIII salts in a solvothermal reaction with triethanolamine results in the formation of a high symmetry [FeIII15] cluster whose structure conforms to a centred, tetrakis hexahedron.

Synthesis and Characterization of Symmetrically versus Unsymmetrically Proton-Bridged Hexa-Iron Clusters.

Syntheses and magnetic and structural characterization of hexa-iron complexes of derivatized salicylaldoximes are discussed. Complexation of Fe(BF4)2·6H2O with each ligand (H2 L1 and H4 L2) in a methanolic-pyridine solution resulted in hexa-iron compounds (C1 and C2, respectively), which each contain two near-parallel metal triangles of [Fe3-µ3-O], linked by six fluoride bridges and stabilized by a hydrogen-bonded proton between the µ3-O groups. Within each metal triangle of C2, Fe(III) ions are connected via the amine "straps" of (H4 L2-2H). Variable-temperature magnetic susceptibility and Mössbauer data of C1 and C2 indicate the presence of dominant antiferromagnetic interactions between the high-spin (S = 5/2) Fe(III) centers. For C1, two quadrupole doublets are observed at room temperature and 5 K, consistent with structural data from which discrete but disordered [Fe3-µ3-O] and [Fe3-µ3-OH] species were inferred. For C2, a single sharp quadrupole doublet with splitting intermediate between those determined for C1 was observed, consistent with the symmetric [Fe3-µ3-O···H···µ3-O-Fe3] species inferred crystallographically from the very short µ3-O···µ3-O separation. The differences in the physical properties of the complexes, as seen in the Mössbauer, X-ray, and magnetic data, are attributed to the conformational flexibility imparted by the nature of the linkages between the closely related ligands.

Magneto-structural studies of an unusual [MnIIIMnIIGdIII(OR)4]4- partial cubane from 2,2'-bis-p-tBu-calix[4]arene.

Reaction of 2,2'-bis-p-tBu-calix[4]arene (H8L) with MnCl2·4H2O, GdCl3·6H2O and 2,6-pyridinedimethanol (H2pdm) affords [MnIIIMnIIGdIII(H3L)(pdmH)(pdm)(MeOH)2(dmf)]·3MeCN·dmf (3·3MeCN·dmf) upon vapour diffusion of MeCN into the basic dmf/MeOH mother liquor. 3 crystallises in the tetragonal space group P41212 with the asymmetric unit comprising the entire cluster. The highly unusual core contains a triangular arrangement of MnIIIMnIIGdIII ions housed within a [MnIIIMnIIGdIII(OR)4]4- partial cubane. Magnetic susceptibility and magnetisation data reveal best fit parameters JMn(II)-Mn(III) = +0.415 cm-1, JMn(III)-Gd(III) = +0.221 cm-1, JMn(II)-Gd(III) = -0.258 cm-1 and DMn(III) = -4.139 cm-1. Theoretically derived magnetic exchange interactions, anisotropy parameters, and magneto-structural correlations for 3 are in excellent agreement with the experimental data.

Modulation of endothelial organelle size as an antithrombotic strategy.

BACKGROUND: It is long established that von Willebrand factor (VWF) is central to hemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), organelles generated in a wide range of lengths (0.5-5.0 µm). WPB size responds to physiological cues and pharmacological treatment, and VWF secretion from shortened WPBs dramatically reduces platelet and plasma VWF adhesion to an endothelial surface. OBJECTIVE: We hypothesized that WPB-shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist. METHODS: Using a microscopy approach coupled to automated image analysis, we measured the size of WPB bodies in primary human endothelial cells treated with licensed compounds for 24 hours. RESULTS AND CONCLUSIONS: A novel approach to identification of antithrombotic compounds generated a significant number of candidates with the ability to shorten WPBs. In vitro assays of two selected compounds confirm that they inhibit the pro-hemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the hemostatic process could well prove to be a useful adjunct to current antithrombotic therapeutics. Further, in the current SARS-CoV-2 pandemic, with a considerable fraction of critically ill COVID-19 patients affected by hypercoagulability, these WPB size-reducing drugs might also provide welcome therapeutic leads for frontline clinicians and researchers.

The first amino acid bound manganese-calcium clusters: a {[MnCa]2} methylalanine complex, and a [MnCa] trigonal prism.

An amino acid containing octanuclear heterometallic {[MnCa]2} cluster has been synthesized, alongside a structurally-related trigonal prismatic [MnCa]2+ cage.

Investigation for correction formulas on the basis of packed cell volume for blood glucose concentration measurements obtained with portable glucometers when used in rabbits.

OBJECTIVE: To determine effects of PCV on blood glucose (BG) concentration measurements obtained with a human portable blood glucometer (HPBG) and a veterinary portable blood glucometer (VPBG) on canine (cVPBG) and feline (fVPBG) settings (test methods) when used in rabbits and to develop correction formulas to mitigate effects of PCV on such measurements. SAMPLE: 48 resuspended blood samples with known PVCs (range, 0% [plasma] to 92% [plasma and packed RBCs]) from 6 healthy research rabbits (experimental sample set) and 252 historic measurements of BG concentration and PCV in 84 client-owned rabbits evaluated at a veterinary hospital (validation data set). PROCEDURES: Duplicate measurements of BG concentration with each test method and of PCV were obtained for each sample in the experimental sample set, and the mean results for each variable for each test method and sample were compared with results from a clinical laboratory analyzer (reference method) used to determine the true BG concentration for each sample. Mean ± SD differences in measurements between the reference and test methods were calculated. Linear regression and modified Clarke error grid analysis were used to develop correction formulas for the test methods given known PCVs, and these formulas were evaluated on the validation data set with linear regression and a modified Clarke error grid. RESULTS: Blood glucose concentrations were falsely low for cVPBG and fVPBG used on samples with PCV < 31% and were falsely high for all test methods used on samples with PCV > 43%. Compared with original measurements, formula-corrected measurements overall had better agreement with reference method measurements for the experimental sample set; however, only the formula-corrected HPBG measurements had improved agreement for the validation data set. CONCLUSIONS AND CLINICAL RELEVANCE: Findings indicated that, in rabbits, HPBG measurements had improved accuracy with the use of the correction formula HPBG measurement of BG concentration + ([0.75 × PCV] - 15); however, the correction formulas did not improve the accuracy of VPBG measurements, and we believe that neither the cVPBG nor fVPBG should be used in rabbits.

RGS4 controls secretion of von Willebrand factor to the subendothelial matrix.

The haemostatic protein von Willebrand factor (VWF) exists in plasma and subendothelial pools. The plasma pools are secreted from endothelial storage granules, Weibel-Palade bodies (WPBs), by basal secretion with a contribution from agonist-stimulated secretion, and the subendothelial pool is secreted into the subendothelial matrix by a constitutive pathway not involving WPBs. We set out to determine whether the constitutive release of subendothelial VWF is actually regulated and, if so, what functional consequences this might have. Constitutive VWF secretion can be increased by a range of factors, including changes in VWF expression, levels of TNF and other environmental cues. An RNA-seq analysis revealed that expression of regulator of G protein signalling 4 (RGS4) was reduced in endothelial cells (HUVECs) grown under these conditions. siRNA RGS4 treatment of HUVECs increased constitutive basolateral secretion of VWF, probably by affecting the anterograde secretory pathway. In a simple model of endothelial damage, we show that RGS4-silenced cells increased platelet recruitment onto the subendothelial matrix under flow. These results show that changes in RGS4 expression alter levels of subendothelial VWF, affecting platelet recruitment. This introduces a novel control over VWF function.

Human endothelial cells size-select their secretory granules for exocytosis to modulate their functional output.

BACKGROUND: The secretory granules of endothelial cells, Weibel-Palade bodies, are released in response to numerous extracellular signals. Their cargo is critical to many vascular functions including hemostasis and inflammation. This presents a fundamental problem: how can these cells initiate tailor-made responses from the release of a single type of organelle, each with similar cargo? Each cell contains Weibel-Palade bodies in a wide range of sizes, and we have shown that experimentally shortening these organelles disproportionately reduces their ability to initiate hemostasis in vitro, leaving leukocyte recruitment unaffected. Could the production of this range of sizes underpin differential responses? OBJECTIVES: To determine whether different agonists drive the exocytosis of different sizes of Weibel-Palade bodies. METHODS: We used a high-throughput automated unbiased imaging workflow to analyze the sizes of Weibel-Palade bodies within human umbilical vein endothelial cells (HUVECs) before and after agonist activation to determine changes in organelle size distributions. RESULTS: We found that a subset of agonists differentially evoke the release of the longest, most pro-hemostatic organelles. Inhibiting the release of these longest organelles by just 15% gives a fall of 60% in an assay of secreted von Willebrand factor (vWF) function. CONCLUSIONS: The size-selection of granules for exocytosis represents a novel layer of control, allowing endothelial cells to provide diverse responses to different signals via the release of a single type of organelle.

An [FeIII 34 ] Molecular Metal Oxide.

The dissolution of anhydrous iron bromide in a mixture of pyridine and acetonitrile, in the presence of an organic amine, results in the formation of an [Fe34 ] metal oxide molecule, structurally characterised by alternate layers of tetrahedral and octahedral FeIII ions connected by oxide and hydroxide ions. The outer shell of the complex is capped by a combination of pyridine molecules and bromide ions. Magnetic data, measured at temperatures as low as 0.4 K and fields up to 35 T, reveal competing antiferromagnetic exchange interactions; DFT calculations showing that the magnitudes of the coupling constants are highly dependent on both the Fe-O-Fe angles and Fe-O distances. The simplicity of the synthetic methodology, and the structural similarity between [Fe34 ], bulk iron oxides, previous FeIII -oxo cages, and polyoxometalates (POMs), hints that much larger molecular FeIII oxides can be made.

New salicylaldoximato-borate ligands resulting from anion hydrolysis and their respective copper and iron complexes.

Anion hydrolysis reactions between salicylaldoximato ligands (L'-L''') and copper and iron BF4- metal salts, have resulted in the formation of new salicylaldoximato borate containing transition metal complexes: [Fe2(L' + 2H)2](BF4)2(MeOH)4 (C1), [Fe3(L'' + 4H)(OH)2(Py)2](BF4)2(H2O)2(Py)2 (C2), and [Cu2(L''' + H)2Cl2] (C3). Each of the complexes have been structurally characterised, revealing the indirect role boron plays in the formation of these complexes. For complexes C1 and C2, Mössbauer spectroscopy confirmed the existence of Fe(iii) oxidation states. SQUID magnetometry measurements were performed on complexes C2 and C3, revealing the presence of two competing exchange pathways between the three Fe(iii) centres in C2, with antiferromagnetic exchange dominating. For C3 weak antiferromagnetic exchange dominated between the two Cu(ii) centres.

A GBF1-Dependent Mechanism for Environmentally Responsive Regulation of ER-Golgi Transport.

How can anterograde membrane trafficking be modulated by physiological cues? A screen of Golgi-associated proteins revealed that the ARF-GEF GBF1 can selectively modulate the ER-Golgi trafficking of prohaemostatic von Willebrand factor (VWF) and extracellular matrix (ECM) proteins in human endothelial cells and in mouse fibroblasts. The relationship between levels of GBF1 and the trafficking of VWF into forming secretory granules confirmed GBF1 is a limiting factor in this process. Further, GBF1 activation by AMPK couples its control of anterograde trafficking to physiological cues; levels of glucose control GBF1 activation in turn modulating VWF trafficking into secretory granules. GBF1 modulates both ER and TGN exit, the latter dramatically affecting the size of the VWF storage organelles, thereby influencing the hemostatic capacity of the endothelium. The role of AMPK as a central integrating element of cellular pathways with intra- and extra-cellular cues can now be extended to modulation of the anterograde secretory pathway.