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Background: Percutaneous left-atrial appendage closure (LAAC) is an established method for preventing strokes in patients with atrial fibrillation, offering an alternative to oral anticoagulation. Various occluder devices have been developed to cater to individual anatomical needs and ensure a safe and effective procedure. In this retrospective, monocentric cohort study, we compare different LAAO devices with respect to clinical outcomes, LAA sealing properties, and device-related complications., Methods: We conducted a retrospective analysis of 270 patients who underwent percutaneous LAA closure in our center between 2009 and 2023. Patient data were extracted from medical records, including gender, age at implantation, indication, device type and size, laboratory values, LAA anatomy, periprocedural complications, ECG parameters, transthoracic and transesophageal echocardiography parameters (TTE and TEE), as well as medication at discharge. Moreover, fluoroscopy time and implantation duration, as well as post-implantation clinical events up to 1 year, were collected. Endpoints were bleeding events, recurrent stroke, thrombi on devices, and death., Results: The implanted devices were the Watchman 2.5, Watchman FLX, Amplatzer Cardiac Plug (ACP), and Amulet. The procedural success rate was 95.7% ( n = 265), with cactus anatomy posing the most challenges across all devices. The mean patient age was 75.5 ± 7.7 years, with 64.5% being male. The median CHA2DS2-VASc score was 4.8 ± 1.5 and the median HAS-BLED score was 3.8 ± 1.0. Indications for LAA closure included past bleeding events and elevated bleeding risk. Periprocedural complications were most commonly bleeding at the puncture site, particularly after ACP implantation ( p = 0.014). Significant peridevice leaks (PDL) were observed in 21.4% of simple sealing mechanism devices versus 0% in double sealing mechanism devices ( p = 0.004). Thrombi were detected on devices in six patients, with no subsequent ischemic stroke or thromboembolic event. Comparative analysis revealed no significant differences in the occurrence of stroke, transient ischemic attack (TIA), thromboembolic events, device-related thrombi, or mortality among different device types. A 62.3% relative risk reduction in thromboembolic events and 38.6% in major bleedings could be observed over 568.2 patient years., Conclusions: In summary, our study highlights the efficacy and safety of LAA closure using various occluder devices despite anatomical challenges. Our long-term follow-up findings support LAA closure as a promising option for stroke prevention in selected patient cohorts. Further research is needed to refine patient selection criteria and optimize outcomes in LAA closure procedures.

Colony stimulating factor-1 receptor (CSF1R or c-FMS), a class III receptor tyrosine kinase expressed on members of the mononuclear phagocyte system (MPS), plays a key role in the proper functioning of macrophages, microglia, and related cells. Aberrant signaling through CSF1R has been associated with a variety of disease states, including cancer, inflammation, and neurodegeneration. In this Letter, we detail our efforts to develop novel CSF1R inhibitors. Drawing on previously described compounds, including GW2580 ( 4 ), we have discovered a novel series of compounds based on the imidazo[4,5- b ]pyridine scaffold. Initial structure-activity relationship studies culminated in the identification of 36 , a lead compound with potent CSF1R biochemical and cellular activity, acceptable in vitro ADME properties, and oral exposure in rat., Competing Interests: The authors declare the following competing financial interest(s): All authors are or were employees of Sanofi and may have stock and/or stock options., (© 2024 American Chemical Society.)

Clostridium difficile toxins are the primary causative agents for hospital-acquired diarrhea and pseudomembranous colitis. Numerous monoclonal antibodies (mAbs) targeting different domains of Clostridium difficile toxin have been reported. Here we report the crystal structures of two mAbs, B1 and B2, in complex with the glycosyltransferase domain (GTD) of the Clostridium difficile toxin B (TcdB). B2 bound to the N-terminal 4 helix bundle of the GTD, a conserved membrane localization domain (MLD) found in the large clostridial glycosylating toxin family implicated in targeting plasma membrane. B1 bound to a distinct epitope at the hinge region between the MLD and the catalytic subdomain of the GTD. Functional studies revealed the potency of these mAbs in vitro and in vivo to be synergistic when given in combination., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Microglia serve as the innate immune cells of the central nervous system (CNS) by providing continuous surveillance of the CNS microenvironment and initiating defense mechanisms to protect CNS tissue. Upon injury, microglia transition into an activated state altering their transcriptional profile, transforming their morphology, and producing pro-inflammatory cytokines. These activated microglia initially serve a beneficial role, but their continued activation drives neuroinflammation and neurodegeneration. Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the CNS, and activated microglia and macrophages play a significant role in mediating disease pathophysiology and progression. Colony-stimulating factor-1 receptor (CSF1R) and its ligand CSF1 are elevated in CNS tissue derived from MS patients. We performed a large-scale RNA-sequencing experiment and identified CSF1R as a key node of disease progression in a mouse model of progressive MS. We hypothesized that modulating microglia and infiltrating macrophages through the inhibition of CSF1R will attenuate deleterious CNS inflammation and reduce subsequent demyelination and neurodegeneration. To test this hypothesis, we generated a novel potent and selective small-molecule CSF1R inhibitor (sCSF1R inh ) for preclinical testing. sCSF1R inh blocked receptor phosphorylation and downstream signaling in both microglia and macrophages and altered cellular functions including proliferation, survival, and cytokine production. In vivo, CSF1R inhibition with sCSF1R inh attenuated neuroinflammation and reduced microglial proliferation in a murine acute LPS model. Furthermore, the sCSF1R inh attenuated a disease-associated microglial phenotype and blocked both axonal damage and neurological impairments in an experimental autoimmune encephalomyelitis (EAE) model of MS. While previous studies have focused on microglial depletion following CSF1R inhibition, our data clearly show that signaling downstream of this receptor can be beneficially modulated in the context of CNS injury. Together, these data suggest that CSF1R inhibition can reduce deleterious microglial proliferation and modulate microglial phenotypes during neuroinflammatory pathogenesis, particularly in progressive MS.

Hepatocellular carcinoma (HCC) accounts for a majority of primary liver cancer and is one of the most common forms of cancer worldwide. Aberrant signaling of the FGF19-FGFR4 pathway leads to HCC in mice and is hypothesized to be a driver in FGF19 amplified HCC in humans. Multiple small molecule inhibitors have been pursued as targeted therapies for HCC in recent years, including several selective FGFR4 inhibitors that are currently being evaluated in clinical trials. Herein, we report a novel series of highly selective, covalent 2-amino-6,8-dimethyl-pyrido[2,3- d ]pyrimidin-7(8 H )-ones that potently and selectively inhibit FGFR4 signaling through covalent modification of Cys552, which was confirmed by X-ray crystallography. Correlative target occupancy and pFGFR4 inhibition were observed in vivo, as well as tumor regression in preclinical models of orthotopic and sorafenib-resistant HCC., Competing Interests: The authors declare no competing financial interest.

Although metal phthalocyanines are widely used in optoelectronic devices, e.g., as hole-transport and electron-blocking layers, or as UV-stable dyes, their multilayer growth on metal substrates has surprisingly not been studied very systematically. Even for CuPc, one of the most widely studied representatives of phthalocyanines, contradictory structures are reported for films grown on gold, a common electrode material, suggesting that the influence of actual substrate surface properties on film growth has not been sufficiently considered. In this study, we analyze the growth of CuPc films on gold substrates for thicknesses ranging from the initial seed layer to thick multilayers (50 nm) by combining near-edge X-ray absorption spectroscopy with atomic force microscopy and X-ray diffraction. To study the influence of surface roughness, we compare the formation of CuPc films on well-ordered Au(111) and sputter-deposited polycrystalline gold substrates and also investigate the influence of surface contamination by exposing these gold surfaces to air before film growth. While on clean gold substrates, CuPc molecules exclusively adopt a recumbent orientation and form (112̅)-oriented films, they also grow in an upright orientation on contaminated gold surfaces. On Au(111), this leads to the coexistence of (112̅)- and (100)-oriented regions, whereas only (100)-oriented films are formed on contaminated polycrystalline gold. Remarkably, the (112̅)-oriented films consist of extended but isolated crystalline islands, resulting in large overall roughness, whereas the (100)-oriented films consist of rather small domains but have significantly lower film roughness.

Fossil evidence indicates that numerous catarrhine clades of African origin expanded or shifted their ranges into Eurasia, among them macaques Macaca Lacépède, 1799. Macaques represent the sister taxon of African papionins and can thus be used as a model comparing an 'out-of-Africa' with an intra-African, e.g., baboons-Papio Erxleben, 1777 evolutionary history. The first step for such a comparison is to establish a well-resolved phylogeny of macaques with reliably estimated divergence times and to compare it with that of baboons and the fossil record. Therefore, we used mitochondrial (mtDNA) genome data deposited in GenBank of 16 out of 23 extant macaque species and of all six baboon taxa. We reconstructed phylogenetic trees using maximum-likelihood and Bayesian inferences and dated differentiation events using three fossil-based calibration sets. The obtained tree topology is in agreement with findings from earlier mtDNA studies, but yielded stronger nodal supports. We observed some para- and polyphylies in macaques and baboons, suggesting that ancient gene flow among divergent lineages has been common in both genera. Our divergence time estimates are in broad agreement with earlier findings and with the fossil record. Macaques started to diversify 7.0-6.7 Ma, followed by a stepwise radiation into several species groups in Asia, whereas baboons commenced diversification around 2.2 Ma. Accordingly, divergence of species groups and species in macaques clearly predates divergences in baboons. Based on our phylogenetic results with estimated divergence times and the recorded chronostratigraphic ranges of extinct macaque and baboon taxa, we compare the evolutionary radiations of both genera from paleobiogeographic and adaptive viewpoints., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Recent studies suggest that closely related species can accumulate substantial genetic and phenotypic differences despite ongoing gene flow, thus challenging traditional ideas regarding the genetics of speciation. Baboons (genus Papio ) are Old World monkeys consisting of six readily distinguishable species. Baboon species hybridize in the wild, and prior data imply a complex history of differentiation and introgression. We produced a reference genome assembly for the olive baboon ( Papio anubis ) and whole-genome sequence data for all six extant species. We document multiple episodes of admixture and introgression during the radiation of Papio baboons, thus demonstrating their value as a model of complex evolutionary divergence, hybridization, and reticulation. These results help inform our understanding of similar cases, including modern humans, Neanderthals, Denisovans, and other ancient hominins.

Background: As Short Interspersed Elements (SINEs), human-specific Alu elements can be used for population genetic studies. Very recent inserts are polymorphic within and between human populations. In a sample of 30 elements originating from three different Alu subfamilies, we investigated whether they are preserved in prehistorical skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. In the present study, we examined a prehistoric triad of father, mother and daughter., Results: For 26 of the 30 Alu loci investigated, definite results were obtained. We were able to demonstrate that presence/absence analyses of Alu elements can be conducted on individuals who lived 3,000 years ago. The preservation of the ancient DNA (aDNA) is good enough in two out of three ancient individuals to routinely allow the amplification of 500 bp fragments. The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures. We here present an alternative molecular approach to deal with these degradation phenomena by using internal Alu subfamily specific primers producing short fragments of approximately 150 bp., Conclusions: Our data clearly show the possibility of presence/absence analyses of Alu elements in individuals from the Lichtenstein cave. Thus, we demonstrate that our method is reliably applicable for aDNA samples with good or moderate DNA preservation. This method will be very useful for further investigations with more Alu loci and larger datasets. Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.

Aberrant activation of the phosphoinositide 3-kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3-phosphoinositide-dependent kinase-1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N-terminal lobe of the 3-phosphoinositide-dependent kinase-1 catalytic domain contains a docking site which recognizes the non-catalytic C-terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so-called PDK1 Interacting Fragment pocket allows interaction with 3-phosphoinositide-dependent kinase-1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3-phosphoinositide-dependent kinase-1 domain construct against a library of chemically diverse fragments in order to identify small, ligand-efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP-site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand-induced changes in 1H-15N TROSY spectra acquired using uniformly 15N-enriched PDK1 provided evidence to distinguish ATP-site from PDK1 Interacting Fragment-site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.

An approach and preliminary results for utilizing legacy MEK inhibitors as templates for a reiterative structural based design and synthesis of novel, type III NCKIs (non-classical kinase inhibitors) is described. Evidence is provided that the MEK-pocket or pockets closely related to it may exist in kinases other than MEK.

Polo-like kinase 1 is an important regulator of cell cycle progression whose over-expression is often associated with oncogenesis. Polo-like kinase 1 hence represents an attractive target for cancer intervention. BI 2536 (Boehringer Ingelheim, Ingelheim, Germany), a Polo-like kinase 1 inhibitor currently in clinical trials, exhibits nanomolar potency against Polo-like kinase isoforms and high selectivity against other kinases. We have previously published the crystal structures of the Polo-like kinase 1 domain in complex with AMPPNP and an Aurora A inhibitor. In this work, we present the co-crystal structure of Polo-like kinase 1 with BI 2536. The structure, in combination with selectivity data for BI 2536 and related compounds, illustrates important features for potency and selectivity. In particular, we show that the methoxy group of BI 2536 is an important specificity determinant against non-Polo-like kinases by taking advantage of a small pocket generated by Leu 132 in the hinge region of Polo-like kinase 1. The work presented here provides a framework for structure-based drug design of Polo-like kinase 1-specific inhibitors.

Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.

The Morel-Lavallée lesion is a rare condition that was first described by the French physician Maurice Morel-Lavallée in 1853. The lesion is caused by forces of pressure and shear stress at the borders of subcutaneous tissue to the muscle fascia or bone as they are seen in run-over accidents. It leads to a shear of skin and subcutaneous tissue from the neighboring fascia followed by the development of a blood-filled hollow space at predestined regions of the body. If therapy is insufficient, large areas of necrosis can form, which will negatively influence operative measures. We report about three patients with the diagnosis of a Morel-Lavallée lesion. The history and the impressive clinical findings are demonstrated as well as the differential operative therapy performed, partially with osteosynthesis of accompanying bone injuries. According to the recommendations of the literature known to us, an adjusted therapeutic regime suited to the particular findings was carried out and in all three cases uncomplicated healing can be reported. Our results are in line with the existing recommendations, which are to relieve the soft tissue hematoma in time and sufficiently, and secondly to carry out débridements initially as well as planned second-look operations.

The enzymatic A1 chain of cholera toxin retrotranslocates across the endoplasmic reticulum membrane into the cytosol, where it induces toxicity. Almost all other retrotranslocation substrates are modified by the attachment of polyubiquitin chains and moved into the cytosol by the ubiquitin-interacting p97 ATPase complex. The cholera toxin A1 chain, however, can induce toxicity in the absence of ubiquitination, and the motive force that drives retrotranslocation is not known. Here, we use adenovirus expressing dominant-negative mutants of p97 to test whether p97 is required for toxin action. We find that cholera toxin still functions with only a small decrease in potency in cells that cannot retrotranslocate other substrates at all. These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function.

Phosphodiesterases (PDEs) modulate signaling by cyclic nucleotides in diverse processes such as cardiac contractility, platelet aggregation, lipolysis, glycogenolysis, and smooth muscle contraction. Cyclic guanosine monophosphate (cGMP) stimulated human phosphodiesterase 2 (PDE2) is expressed mainly in brain and heart tissues. PDE2A is involved in the regulation of blood pressure and fluid homeostasis by the atrial natriuretic peptide (ANP), making PDE2-type enzymes important targets for drug discovery. The design of more potent and selective inhibitors of PDE2A for the treatment of heart disease would be greatly aided by the identification of active site residues in PDE2A that determine substrate and inhibitor selectivity. The identification of active site residues through traditional mutational studies involves the time-consuming and tedious purification of a large number of mutant proteins from overexpressing cells. Here we report an alternative approach to rapidly produce active site mutants of human PDE2A and identify their enzymatic properties using a wheat germ in vitro translation (IVT, also known as cell-free translation) system. We also present the crystal structure of the catalytic domain of human PDE2A determined at 1.7 A resolution, which provided a framework for the rational design of active site mutants. Using a rapid IVT approach for expression of human PDE2A mutants, we identified the roles of active site residues Asp811, Gln812, Ile826, and Tyr827 in inhibitor and substrate selectivity for PDE2A.

Carbamoyl phosphate synthetase synchronizes the utilization of two ATP molecules at duplicated ATP-grasp folds to catalyze carbamoyl phosphate formation. To define the dedicated functional role played by each of the two ATP sites, we have carried out pulse/labeling studies using the synthetases from Aquifex aeolicus and Methanococcus jannaschii, hyperthermophilic organisms that encode the two ATP-grasp folds on separate subunits. These studies allowed us to differentially label each active site with [gamma-(32)P]ATP and determine the fate of the labeled gamma-phosphate in the synthetase reaction. Our results provide the first direct demonstration that enzyme-catalyzed transfer of phosphate from ATP to carbamate occurs on the more C-terminal of the two ATP-grasp folds. These findings rule out one mechanism proposed for carbamoyl phosphate synthetase, where one ATP acts as a molecular switch, and provide additional support for a sequential reaction mechanism where the gamma-phosphate groups of both ATP molecules are transferred to reactants. CP synthesis by subunit C in our single turnover pulse/chase assays did not require subunit N, but subunit N was required for detectable CP synthesis in the traditional continuous assay. These findings suggest that cross-talk between domain N and C is required for product release from subunit C.

Carbamoyl-phosphate synthetases (CPSs) utilize two molecules of ATP at two internally duplicated domains, B and C. Domains B and C have recently been shown to be structurally [Thoden, J. B., Holden, H. M., Wesenberg, G., Raushel, F. M. & Rayment, I. (1997) Biochemistry 36, 6305-6316] and functionally [Guy, H. I. & Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769] equivalent. We have carried out a site-directed mutagenic analysis that is consistent with ATP binding to a palmate motif rather than to a Walker A/B motif in domains B and C. To accommodate our present findings, as well as the other recent findings of structural and functional equivalence, we are proposing a novel mechanism for CPS. In this mechanism utilization of ATP bound to domain C is coupled to carbamoyl-phosphate synthesis at domain B via a nucleotide switch, with the energy of ATP hydrolysis at domain C allowing domain B to cycle between two alternative conformations.

To investigate the importance of amino acid infusions in immediate postoperative parenteral nutrition, cardiac patients were randomly allocated into two groups. Applicating identical carbohydrate calories (2000 kcal/day) group 1 received only essential amino acids while in group 2 a combined pattern of essential and non essential amino acids was infused. In addition to routine laboratory data several parameters of protein metabolism including nitrogen balance were evaluated. Although nitrogen balance was positive only in group 2 the differences between the two groups concerning other parameters measured were minimal. The different infusion regimes are discussed revealing the significance of additional parenteral nutrition in these patients.