A novel TASK channel antagonist nasal spray reduces sleep apnea severity in physiological responders: a randomized, blinded, trial.

Preclinical and human physiological studies indicate that topical, selective TASK 1/3 K+ channel antagonism increases upper airway dilator muscle activity and reduces pharyngeal collapsibility during anesthesia and nasal breathing during sleep. The primary aim of this study was to determine the effects of BAY2586116 nasal spray on obstructive sleep apnea (OSA) severity and whether individual responses vary according to differences in physiological responses and route of breathing. Ten people (5 females) with OSA [apnea-hypopnea index (AHI) = 47 ± 26 events/h (means ± SD)] who completed previous sleep physiology studies with BAY2586116 were invited to return for three polysomnography studies to quantify OSA severity. In random order, participants received either placebo nasal spray (saline), BAY2586116 nasal spray (160 µg), or BAY2586116 nasal spray (160 µg) restricted to nasal breathing (chinstrap or mouth tape). Physiological responders were defined a priori as those who had improved upper airway collapsibility (critical closing pressure ≥2 cmH2O) with BAY2586116 nasal spray (NCT04236440). There was no systematic change in apnea-hypopnea index (AHI3) from placebo versus BAY2586116 with either unrestricted or nasal-only breathing versus placebo (47 ± 26 vs. 43 ± 27 vs. 53 ± 33 events/h, P = 0.15). However, BAY2586116 (unrestricted breathing) reduced OSA severity in physiological responders compared with placebo (e.g., AHI3 = 28 ± 11 vs. 36 ± 12 events/h, P = 0.03 and ODI3 = 18 ± 10 vs. 28 ± 12 events/h, P = 0.02). Morning blood pressure was also lower in physiological responders after BAY2586116 versus placebo (e.g., systolic blood pressure = 137 ± 24 vs. 147 ± 21 mmHg, P < 0.01). In conclusion, BAY2586116 reduces OSA severity during sleep in people who demonstrate physiological improvement in upper airway collapsibility. These findings highlight the therapeutic potential of this novel pharmacotherapy target in selected individuals.NEW & NOTEWORTHY Preclinical findings in pigs and humans indicate that blocking potassium channels in the upper airway with topical nasal application increases pharyngeal dilator muscle activity and reduces upper airway collapsibility. In this study, BAY2586116 nasal spray (potassium channel blocker) reduced sleep apnea severity in those who had physiological improvement in upper airway collapsibility. BAY2586116 lowered the next morning's blood pressure. These findings highlight the potential for this novel therapeutic approach to improve sleep apnea in certain people.

A Fluoroponytailed NHC-Silver Complex Formed from Vinylimidazolium/AgNO3 under Aqueous-Ammoniacal Conditions.

3-(1H,1H,2H,2H-Perfluorooctyl)-1-vinylimidazolium chloride [2126844-17-3], a strong fluorosurfactant with remarkably high solubility in water, was expediently converted into the respective doubly NHC-complexed silver salt with nitrate as counter ion in quantitative yield. Due to its vinyl substituents, [bis(3-(1H,1H,2H,2H-perfluorooctyl)-1-vinylimidazol-2-ylidene)silver(I)] nitrate, Ag(FNHC)2NO3, represents a polymerizable N-heterocyclic carbene transfer reagent, thus potentially offering simple and robust access to coordination polymers with crosslinking metal bridges. The compound was characterized by infrared and NMR spectroscopy, mass spectrometry as well as elemental analysis, and supplemented by X-ray single-crystal structure determination. It crystallizes in the monoclinic crystal system in the space group P21/c. With 173.3°, the geometry of the Ag-carbene bridge deviates slightly from linearity. The disordered perfluoroalkyl side chains exhibit a helical conformation.

Novel PCNT variants in MOPDII with attenuated growth restriction and pachygyria.

Biallelic loss-of-function mutations in the centrosomal pericentrin gene (PCNT) cause microcephalic osteodysplastic primordial dwarfism type II (MOPDII), which is characterized by extreme growth retardation, microcephaly, skeletal dysplasia, and dental anomalies. Life expectancy is reduced due to a high risk of cerebral vascular anomalies. Here, we report two siblings with MOPDII and attenuated growth restriction, and pachygyria. Compound heterozygosity for two novel truncated PCNT variants was identified. Both truncated PCNT proteins were expressed in patient's fibroblasts, with a reduced total protein amount compared to control. Patient's fibroblasts showed impaired cell cycle progression. As a novel finding, 20% of patient's fibroblasts were shown to express PCNT comparable to control. This was associated with normal mitotic morphology and normal co-localization of mutated PCNT with centrosome-associated proteins γ-tubulin and centrin 3, suggesting some residual function of truncated PCNT proteins. These data expand the clinical and molecular spectrum of MOPDII and indicate that residual PCNT function might be associated with attenuated growth restriction in MOPDII.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

Microfluidic Diffusion Platform for Characterizing the Sizes of Lipid Vesicles and the Thermodynamics of Protein-Lipid Interactions.

Elucidation of the fundamental interactions of proteins with biological membranes under native conditions is crucial for understanding the molecular basis of their biological function and malfunction. Notably, the large surface to volume ratio of living cells provides a molecular landscape for significant interactions of cellular components with membranes, thereby potentially modulating their function. However, such interactions can be challenging to probe using conventional biophysical methods due to the heterogeneity of the species and processes involved. Here, we use direct measurements of micron scale molecular diffusivity to detect and quantify the interactions of α-synuclein, associated with the etiology of Parkinson's disease, with negatively charged lipid vesicles. We further demonstrate that this microfluidic approach enables the characterization of size distributions of different binary mixtures of vesicles, which are not readily accessible using conventional light scattering techniques. Finally, the size distributions of the two α-synuclein conformations, free α-synuclein and membrane-bound α-synuclein, were resolved under varying lipid:protein ratios, thus, allowing the determination of the dissociation constant and the binding stoichiometry associated with this protein-lipid system. The microfluidic diffusional sizing platform allows these measurements to be performed on a time scale of minutes using microlitre volumes, thus, establishing the basis for an approach for the study of molecular interactions of heterogeneous systems under native conditions.

Highly Fluorescent Merocyanine and Cyanine PMMA Copolymers.

Polymerizable merocyanine and cyanine dye monomers are synthesized and applied in a statistical copolymerization with methylmethacrylate, giving a series of highly fluorescent poly(methyl methacrylate) (PMMA) copolymers. Photophysical properties of yellow to red merocyanine- and of pink to dark purple cyanine-containing copolymers are studied by fluorescence spectroscopy in solid state as well as in different solvents. The highest quantum yield measured in the solid state is observed for copolymers with the lowest dye content: 16% for merocyanine-based and 13% for cyanine-based copolymers, respectively. Fluorescence properties in solution show positive solvatochromism for both merocyanine monomer and copolymer. Copolymer, in comparison to monomer, is hypsochromically shifted to lower wavelengths which point toward H-aggregation of the chromophores in the copolymer matrix.

Accumulation of Multipotent Progenitor Cells on Polymethylpentene Membranes During Extracorporeal Membrane Oxygenation.

Multipotent progenitor cells were mobilized during pediatric extracorporeal membrane oxygenation (ECMO). We hypothesize that these cells also adhered onto polymethylpentene (PMP) fibers within the membrane oxygenator (MO) during adult ECMO support. Mononuclear cells were removed from the surface of explanted PMP-MOs (n = 16). Endothelial-like outgrowth and mesenchymal-like cells were characterized by flow cytometric analysis using different surface markers. Spindle-shaped attaching cells were identified early, but without proliferative activity. After long-term cultivation palisading type or cobblestone-type outgrowth cells with high proliferative activity appeared and were characterized as (i) leukocytoid CD45+/CD31+ (CD133+/VEGFR-II+/CD90+/CD14+/CD146dim/CD105dim); (ii) endothelial-like CD45-/CD31+ (VEGF-RII+/CD146+/CD105+/CD133-/CD14-/CD90-); and (iii) mesenchymal-like cells CD45-/CD31- (CD105+/CD90+/CD133dim/VEGFR-II-/CD146-/CD14-). The distribution of the cell populations depended on the MO and cultivation time. Endothelial-like cells formed capillary-like structures and did uptake Dil-acetylated low-density lipoprotein. Endothelial- and mesenchymal-like cells adhered on the surface of PMP-MOs. Further research is needed to identify the clinical relevance of these cells.

Instructive even after a decade: Complete results of initial virological diagnostics and re-evaluation of molecular data in the German rabies virus "outbreak" caused by transplantations.

In 2005, six patients in Germany received solid organs and both corneas from a donor with an unrecognized rabies infection. Initial virological diagnostics with the machinery available at the two national reference laboratories could quickly clarify the situation. Rabies virus antigen was detected in the organ donor's brain. In two of the three recipients with neurological alterations, intra vitam diagnosis was achieved by conventional RT-PCRs. Comparison of the phylogenetic relatedness of the different viral isolates proved transmission from the donor and, consequently, also established the diagnosis for the third patient. As indicated by the titre of neutralizing antibodies, the liver transplant recipient was protected from the lethal infection due to a vaccination against rabies virus, which he had received more than 15 years ago. All samples from the recipients of the corneas were invariably negative. Re-evaluation of the molecular data by real-time PCR did not lead to an improvement of intra vitam diagnosis but provided intriguing insights regarding the relative amounts of rabies virus RNA in different body fluids and peripheral organs. In saliva and skin, they were 250-200,000 times lower than in the infected patient's brains. Furthermore, in saliva samples taken serially from the same patient fluctuations by a factor of 160-500 were recorded. These findings highlight the problems of intra vitam diagnosis of rabies virus infections and make understandable why the virus can escape from all diagnostic attempts. Finally, in this context one should recall an almost trivial fact: Simple and appropriate postexposure prophylaxis could not only have saved the young organ donor's life but would also have prevented the whole transplantation-associated rabies "outbreak" in Germany.

Oral and dental health in Huntington's disease - an observational study.

BACKGROUND: Only a few case reports and case series dealing with oral and dental health care are available in literature until now. The aim of the present pilot study was to determine the status of dental health in comparison to matched controls and to heighten the neurologists' and dentists' awareness of the oral aspects of the disease. METHODS: 42 Huntington's disease (HD) participants were scored according to the Unified Huntington's Disease Rating Scale. The dental status was assessed by using the well established score for decayed, missing, and filled teeth (DMFT) and the dental plaque score (Silness-Loe plaque index). RESULTS: Compared to controls HD participants showed significantly more decayed teeth and more plaques in both plaque indices. A higher motor impairment and a lower functional status of the patients lead to a worsening in dental status. CONCLUSION: Possible reasons for our findings are discussed. Apart from local oral complications general complications may also occur. Thus, as a consequence, we would encourage patients, caregivers, neurologists, and the dentists to ensure regular preventive dental examinations and dental treatments of individuals with Huntington's disease even in the premanifest stage of this disease.

Reduced dose and duration of peginterferon alfa-2b and weight-based ribavirin in patients with genotype 2 and 3 chronic hepatitis C.

BACKGROUND & AIMS: There is increasing interest in identifying patients with chronic hepatitis C genotype 2 or 3 infection in whom it is possible to lower the burden of therapy while retaining high levels of efficacy. METHODS: Treatment-naive patients with chronic hepatitis C genotype 2/3 infection were randomized to receive peginterferon alfa-2b (1.5µg/kg/wk) for 24weeks (group A); peginterferon alfa-2b (1.0µg/kg/wk) for 24weeks (group B); or peginterferon alfa-2b (1.5µg/kg/wk) for 16weeks (group C), each in combination with weight-based ribavirin (800-1200mg/d). The study population comprised two cohorts: the Hep-Net cohort enrolled in Germany and an International cohort enrolled at study sites throughout Europe and Asia. The primary end point was sustained virological response (SVR). RESULTS: The study included 682 patients; 80.2% had genotype 3 infection. In the intent-to-treat population, SVR rates were 66.5%, 64.3%, and 56.6% in groups A, B, and C, and were similar in Asian and white patients. Treatment differences (A vs. B and A vs. C) failed to reach the predefined margin for noninferiority of -10%; and thus groups B and C failed to show noninferiority relative to group A. Among patients with undetectable HCV RNA at week 4, SVR rates were 75.3%, 75.9%, and 72.4%, respectively. Relapse rates were 17.8%, 16.3%, and 29.3%, respectively. Treatment-emergent serious adverse events were highest in group A and lowest in group C, and adverse events leading to discontinuation were similar across treatment arms. CONCLUSIONS: For patients with chronic hepatitis C genotype 2/3 infection, 24weeks of peginterferon alfa-2b (1.5µg/kg/wk) plus weight-based ribavirin remains a standard-of-care therapy; however, treatment for 16weeks may be considered for patients with undetectable HCV RNA at week 4 of the treatment.

Direct plant tissue analysis and imprint imaging by desorption electrospray ionization mass spectrometry.

The ambient mass spectrometry technique, desorption electrospray ionization mass spectrometry (DESI-MS), is applied for the rapid identification and spatially resolved relative quantification of chlorophyll degradation products in complex senescent plant tissue matrixes. Polyfunctionalized nonfluorescent chlorophyll catabolites (NCCs), the "final" products of the chlorophyll degradation pathway, are detected directly from leaf tissues within seconds and structurally characterized by tandem mass spectrometry (MS/MS) and reactive-DESI experiments performed in situ. The sensitivity of DESI-MS analysis of these compounds from degreening leaves is enhanced by the introduction of an imprinting technique. Porous polytetrafluoroethylene (PTFE) is used as a substrate for imprinting the leaves, resulting in increased signal intensities compared with those obtained from direct leaf tissue analysis. This imprinting technique is used further to perform two-dimensional (2D) imaging mass spectrometry by DESI, producing well-resolved images of the spatial distribution of NCCs in senescent leaf tissues.

The effect of chin-to-chest contact on upper neck axial force in UN Regulation No. 129 frontal impact tests of child restraint systems.

OBJECTIVE: The objective of this study was to investigate the effect of chin-to-chest contact on upper neck axial force in United Nations (UN) Regulation No. 129 frontal impact tests of child restraint systems. METHODS: Frontal impact pilot experiments were carried out according to the test procedure in UN Regulation No. 129. Q-Series child dummies were seated in a small convenience sample of forward-facing child restraint systems. The timing and duration of chin-to-chest contact were determined using the procedure for calculating external head impact force in SAE J2052. RESULTS: Chin-to-chest contact was observed in all of our experiments and generated a tensile peak in the upper neck axial force of the Q-Series dummies. Prior to chin-to-chest contact, a purely inertial, noncontact peak was observed in the axial tension force. The tensile peak due to chin-to-chest contact was often greater than the inertial, noncontact peak force. CONCLUSIONS: Chin-to-chest contact can increase axial neck tension force beyond the level it would reach under purely inertial loading. Adopting upper neck axial force in regulation, without considering how the force is generated, might encourage child restraint designs that mitigate only chin-to-chest contact, rather than the true inertial neck loading.

Targeting interleukin-4 to the arthritic joint.

Anti-inflammatory cytokines are a promising class of therapeutics for treatment of rheumatoid arthritis (RA), but their use is currently limited by a rapid clearance and systemic toxicity. Interleukin-4 is a small cytokine with potential for RA therapy. To increase its pharmacokinetic features, we engineered a murine IL4 conjugate by incorporating an unnatural amino acid through genetic code expansion to which PEG-folate, as a targeting moiety and PEG alone as control, were site-specifically bound. Both IL4 conjugates retained bioactivity and induced primary murine macrophage polarization into an alternatively activated (M2) related phenotype. The PEGylated conjugates had a terminal half-life of about four hours in healthy mice compared to unPEGylated IL4 (0.76 h). We showed that both conjugates successfully accumulated into arthritic joints in an antigen-induced arthritis (AIA) mouse model, as assessed by non-invasive fluorescence imaging. The modular nature of the IL4 conjugate chemistry presented herein facilitates easy adaption of PEG chain length and targeting moieties for further improvement of half-life and targeting function for future efficacy studies.

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry.

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

A DFT Study on the Co-polymerization of CO2 and Ethylene: Feasibility Analysis for the Direct Synthesis of Polyethylene Esters.

The co-polymerization of CO2 with the non-polar monomer ethylene, though highly desirable, still presents a challenge whereas the palladium-catalyzed CO/C2 H4 co-polymerization is well understood. Building on this analogy, the goal of this study was to elucidate the feasibility of developing suitable catalysts for co-polymerizing CO2 with ethylene to polyethylene esters. Computational methods based on density functional theory were hereby employed. In the search for new catalyst lead structures, a closed catalytic cycle was identified for the palladium-catalyzed CO2 /C2 H4 co-polymerization reaction. The computational study on palladium complexes with a substituted anionic 2-[bis(2,4-dimethoxyphenyl)-phosphine]-benzene-2-hydroxo ligand revealed key aspects that need to be considered when designing ligand sets for potential catalysts for the non-alternating co-polymerization of CO2 and ethylene.

Analysis of thrombotic deposits in extracorporeal membrane oxygenators by multidetector computed tomography.

Oxygenator thrombosis is a serious complication in extracorporeal membrane oxygenation (ECMO) and may necessitate a system exchange. Coagulation and fibrinolysis parameters, flow dynamics, and gas transfer performance are currently used to evaluate the degree of oxygenator thrombosis, but there is no technical approach for direct visualization and quantification of thrombotic deposits within the membrane oxygenator (MO). We used multidetector computed tomography (MDCT) with three-dimensional postprocessing to assess the incidence of oxygenator thrombosis, to quantify thrombus extent, and to localize clot distribution. Twenty heparin-coated MOs after successful weaning were analyzed. Mean ECMO support time was 7 ± 4 days, mean activated partial thromboplastin time (aPTT) during ECMO was 59 ± 20 seconds. Thrombotic deposits were detected in all MOs. The mean clot volume was 51.7 ± 22.3 cm. All thrombotic deposits were located in the venous, i.e., inlet part of the device, without apparent evidence of embolization in patients. There was no correlation between clot volume and ECMO support time or aPTT. Clot formation within the MO is a common finding in ECMO despite adequate systemic anticoagulation. The clinical significance of thrombus formation and its influence on gas exchange capacity and hemostatic complications have to be addressed in further studies.

Caries and periodontal disease in patients with Parkinson's disease.

Parkinson's disease (PD) has been associated with aging, reduced fine motor skills, and malnutrition caused by eating soft sticky foods and a decreased liquid intake, which may contribute to the onset of caries, periodontal disease, and tooth loss. The objective of this study was to investigate the oral health of 101 patients with PD (mean age: 66.2 ± 10.5 years) and compare them to 75 control subjects (CO) (mean age 71 ± 10.53). Patients with PD had poorer oral health than the control group (papilla bleeding index: PD 6.97 ± 8.34; CO 2.12 ± 2.73). Lower frequencies of daily toothbrushing (PD: 1.69 ± 0.83; CO: 2.08 ± 0.80), longer time since the last dentist visit (PD: 1.94 ± 1.49; CO: 1.21 ± 0.60 years), and reduced salivary flow (PD: 2.69 ± 0.94; CO: 3.53 ± 1.11 ml). All of these factors may be related to the gingival recession and tooth mobility found in our patients with PD. Individuals with PD, their caregivers, and their physicians need to focus more on their oral health and quality of oral hygiene.

Antigenic characterisation of yeast-expressed lyssavirus nucleoproteins.

In Europe, three genotypes of the genus Lyssavirus, family Rhabdoviridae, are present, classical rabies virus (RABV, genotype 1), European bat lyssavirus type 1 (EBLV-1, genotype 5) and European bat lyssavirus type 2 (EBLV-2, genotype 6). The entire authentic nucleoprotein (N protein) encoding sequences of RABV (challenge virus standard, CVS, strain), EBLV-1 and EBLV-2 were expressed in yeast Saccharomyces cerevisiae at high level. Purification of recombinant N proteins by caesium chloride gradient centrifugation resulted in yields between 14-17, 25-29 and 18-20 mg/l of induced yeast culture for RABV-CVS, EBLV-1 and EBLV-2, respectively. The purified N proteins were evaluated by negative staining electron microscopy, which revealed the formation of nucleocapsid-like structures. The antigenic conformation of the N proteins was investigated for their reactivity with monoclonal antibodies (mAbs) directed against different lyssaviruses. The reactivity pattern of each mAb was virtually identical between immunofluorescence assay with virus-infected cells, and ELISA and dot blot assay using the corresponding recombinant N proteins. These observations lead us to conclude that yeast-expressed lyssavirus N proteins share antigenic properties with naturally expressed virus protein. These recombinant proteins have the potential for use as components of serological assays for lyssaviruses.