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Abstract Background Surgical aortic valve replacement (SAVR) is an established therapy for severe calcific aortic stenosis. Enhanced recovery after cardiac surgery (ERACS) protocols have been shown to improve outcomes for elective cardiac procedures. The COVID-19 pandemic prompted early extubation post-elective surgeries to preserve critical care resources. Aim of this study To investigate the effects of extubating patients within 6 h post-elective SAVR on hospital and ICU length of stay, mortality rates, ICU readmissions, and postoperative pneumonia. Study Design and methods The retrospective analysis at the University Hospital Aachen, Germany, includes data from 2017 to 2022 and compares a total of 73 elective SAVR patients. Among these, 23 patients were extubated within 6 h (EXT group), while 50 patients remained intubated for over 6 h (INT group). Results The INT group experienced longer postoperative ventilation, needed more vasopressor support, had a higher incidence of postoperative pneumonia, and longer ICU length of stay. No significant differences were noted in overall hospital length of stay, mortality, or ICU readmission rates between the groups. Conclusion This study demonstrates that early extubation in high-risk, multimorbid surgical aortic valve replacement patients is safe, and is associated with a reduction of pneumonia rates, and with shorter ICU and hospital length of stays, reinforcing the benefits of ERACS protocols, especially critical during the COVID-19 pandemic to optimize intensive care use.

The central amygdala inhibitory microcircuits mediate fear extinction by reversible, stimulus- and context-specific changes in neuronal responses. These alterations are absent when extinction is deficient and selective silencing of PKCδ neurons impairs fear extinction.

Hepcidin regulates iron homeostasis by controlling the level of ferroportin, the only membrane channel that facilitates export of iron from within cells. Binding of hepcidin to ferroportin induces the ubiquitination of ferroportin at multiple lysine residues and subsequently causes the internalization and degradation of the ligand-channel complex within lysosomes. The objective of this study was to identify components of the ubiquitin system that are involved in ferroportin degradation. A HepG2 cell line, which inducibly expresses ferroportingreen fluorescent protein (FPN-GFP), was established to test the ability of small interfering (siRNA) directed against components of the ubiquitin system to prevent BMP6- and exogenous hepcidin-induced ferroportin degradation. Of the 88 siRNA directed against components of the ubiquitin pathway that were tested, siRNA-mediated depletion of the alternative E1 enzyme UBA6 as well as the adaptor protein NDFIP1 prevented BMP6- and hepcidin-induced degradation of ferroportin in vitro. A third component of the ubiquitin pathway, ARIH1, indirectly inhibited ferroportin degradation by impairing BMP6-mediated induction of hepcidin. In mice, the AAV-mediated silencing of Ndfip1 in the murine liver increased the level of hepatic ferroportin and increased circulating iron. The results suggest that the E1 enzyme UBA6 and the adaptor protein NDFIP1 are involved in iron homeostasis by regulating the degradation of ferroportin. These specific components of the ubiquitin system may be promising targets for the treatment of iron-related diseases, including iron overload and anemia of inflammation.

Abstract Chronic hepatitis B virus (HBV) infection may follow four different consecutive phases, which are defined by virology as well as biochemical markers and differ in terms of prognosis and need for antiviral treatment. Currently, host responses reflected by immune markers are not considered in this definition. We aimed to study soluble immune markers and their distribution in different phases of chronic HBV infection. In this cross-sectional retrospective study, we investigated a panel of 14 soluble immune markers (SIM) including CXCL10 in 333 patients with chronic HBV infection. In a small cohort of HBeAg positive patients we analyzed SIM before and after HBeAg seroconversion and compared seroconverters to patients with unknown outcome. Significant differences were documented in the levels of several SIM between the four phases of chronic HBV infection. The most pronounced difference among all investigated SIM was observed for CXCL10 concentrations with highest levels in patients with hepatitis. TGF-β and IL-17 revealed different levels between HBeAg negative patients. HBeAg positive patients with HBeAg seroconversion presented higher amounts of IL-12 before seroconversion compared to HBeAg positive patients with unknown follow up. SIM such as CXCL10 but also IL-12, TGF-β and IL-17 may be useful markers to further characterize the phase of chronic HBV infection.

Rationale: Inhalation of nitric oxide (NO) exerts selective pulmonary vasodilation. Nitric oxide also has an antimicrobial effect on a broad spectrum of pathogenic viruses, bacteria and fungi. Objectives: The aim of this study was to investigate the effect of inhaled NO on bacterial burden and disease outcome in a murine model of Klebsiella pneumonia. Methods: Mice were infected with Klebsiella pneumoniae and inhaled either air alone, air mixed with constant levels of NO (at 80, 160, or 200 parts per million (ppm)) or air intermittently mixed with high dose NO (300 ppm). Forty-eight hours after airway inoculation, the number of viable bacteria in lung, spleen and blood was determined. The extent of infiltration of the lungs by inflammatory cells and the level of myeloperoxidase activity in the lungs were measured. Atomic force microscopy was used to investigate a possible mechanism by which nitric oxide exerts a bactericidal effect. Measurements and main results: Compared to control animals infected with K. pneumoniae and breathed air alone, intermittent breathing of NO (300 ppm) reduced viable bacterial counts in lung and spleen tissue. Inhaled NO reduced infection-induced lung inflammation and improved overall survival of mice. NO destroyed the cell wall of K. pneumoniae and killed multiple-drug resistant K. pneumoniae in-vitro. Conclusions: Intermittent administration of high dose NO may be an effective approach to the treatment of pneumonia caused by K. pneumoniae.

Abstract Lesion verification and quantification is traditionally done via histological examination of sectioned brains, a time-consuming process that relies heavily on manual estimation. Such methods are particularly problematic in posterior cortical regions (e.g. visual cortex), where sectioning leads to significant damage and distortion of tissue. Even more challenging is the post hoc localization of micro-electrodes, which relies on the same techniques, suffers from similar drawbacks and requires even higher precision. Here, we propose a new, simple method for quantitative lesion characterization and electrode localization that is less labor-intensive and yields more detailed results than conventional methods. We leverage staining techniques standard in electron microscopy with the use of commodity micro-CT imaging. We stain whole rat and zebra finch brains in osmium tetroxide, embed these in resin and scan entire brains in a micro-CT machine. The scans result in 3D reconstructions of the brains with section thickness dependent on sample size (12–15 and 5–6 microns for rat and zebra finch respectively) that can be segmented manually or automatically. Because the method captures the entire intact brain volume, comparisons within and across studies are more tractable, and the extent of lesions and electrodes may be studied with higher accuracy than with current methods.

South American camelids (SAC) are being more and more presented at the veterinary Clinics in Germany. A bad nutritional condition, which can be easily categorized using a body condition score (BCS) of the animals, is often not noticed by the owners. Further anaemia is also often only detected in an advanced stage in SAC. Clinical detection of anaemia can be performed by assessing the FAMACHA©-score (FS), that is adapted from small ruminants. So far, there is only little information available about BCS and FS in SAC. In this study, both clinical scores were assessed in alpacas and llamas presented at the veterinary clinic and compared with the haematological parameters from the animals. The data were extracted retrospectively from the animals’ medical records and compared statistically. More than half of the alpacas (60%) and llamas (70%) had a BCS < 3, while 12% of the alpacas and 21% of the llamas had a FS > 2. A decreased BCS was associated with a decrease in haematocrit, haemoglobin, lymphocytes, and eosinophils, as well as an increase in FS and neutrophils. BCS and FS should be assessed regularly in SAC to detect emaciation and anaemia in time.

Pyrazole, a member of the structural class of azoles, exhibits molecular properties of interest in pharmaceuticals and materials chemistry, owing to the two adjacent nitrogen atoms in the five-membered ring system. The weakly basic nitrogen atoms of deprotonated pyrazoles have been applied in coordination chemistry, particularly to access coordination polymers and metal-organic frameworks, and homocoupling reactions can in principle provide facile access to bipyrazole ligands. In this context, we summarize recent advances in homocoupling reactions of pyrazoles and other types of azoles (imidazoles, triazoles and tetrazoles) to highlight the utility of homocoupling reactions in synthesizing symmetric bi-heteroaryl systems compared with traditional synthesis. Metal-free reactions and transition-metal catalyzed homocoupling reactions are discussed with reaction mechanisms in detail.

Cortical gamma oscillations are associated with cognitive processes and are altered in several neuropsychiatric conditions such as schizophrenia and Alzheimer’s disease. Since dopamine D3 receptors are possible targets in treatment of these conditions, it is of great importance to understand their role in modulation of gamma oscillations. The effect of D3 receptors on gamma oscillations and the underlying cellular mechanisms were investigated by extracellular local field potential and simultaneous intracellular sharp micro-electrode recordings in the CA3 region of the hippocampus in vitro. D3 receptors decreased the power and broadened the bandwidth of gamma oscillations induced by acetylcholine or kainate. Blockade of the D3 receptors resulted in faster synchronization of the oscillations, suggesting that endogenous dopamine in the hippocampus slows down the dynamics of gamma oscillations by activation of D3 receptors. Investigating the underlying cellular mechanisms for these effects showed that D3 receptor activation decreased the rate of action potentials during gamma oscillations and reduced the precision of the action potential phase coupling to the gamma cycle in CA3 pyramidal cells. The results may offer an explanation how selective activation of D3 receptors may impair cognition and how, in converse, D3 antagonists may exert pro-cognitive and antipsychotic effects.

The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm) showed a plasmon absorption band located within the near-infrared range (650-900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N-formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.

BACKGROUND:SNPs near the interferon lambda (IFNL) 3 gene are predictors for sustained virological response (SVR) in patients with chronic hepatitis C genotype (GT) 1. In addition, a dinucleotide frame shift in ss469415590 was described, which generates IFNL4. In this study, we compared the role of IFNL4 variants with IFNL3-(rs12979860) and IFNL3-(rs8099917) on response to pegylated (PEG)-IFN and Ribavirin (RBV) in patients with chronic hepatitis C GT2/3. METHODS:We recruited 1006 patients with chronic hepatitis C and GT2/3 in a large German registry. A treatment with PEG-IFN and Ribavirin was started by 959 patients. We performed genotyping of IFNL3 (rs12979860, n = 726; rs8099917, n = 687) and of IFNL4 (ss469415590; n = 631). RESULTS:Both preferable IFNL3 genotypes were associated with RVR (both p

Evidence based clinical guidelines are implemented to treat patients efficiently that include efficacy, tolerability but also health economic considerations. This is of particular relevance to the new direct acting antiviral agents that have revolutionized treatment of chronic hepatitis C. For hepatitis C genotypes 2/3 interferon free treatment is already available with sofosbuvir plus ribavirin. However, treatment with sofosbuvir-based regimens is 10-20 times more expensive compared to pegylated interferon alfa and ribavirin (PegIFN/RBV). It has to be discussed if PegIFN/RBV is still an option for easy to treat patients. We assessed the treatment of patients with chronic hepatitis C genotypes 2/3 with PegIFN/RBV in a real world setting according to the latest German guidelines. Overall, 1006 patients were recruited into a prospective patient registry with 959 having started treatment. The intention-to-treat analysis showed poor SVR (GT2 61%, GT3 47%) while patients with adherence had excellent SVR in the per protocol analysis (GT2 96%, GT3 90%). According to guidelines, 283 patients were candidates for shorter treatment duration, namely a treatment of 16 weeks (baseline HCV-RNA

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies.

The protein structure is a cumulative result of interactions between amino acid residues interacting with each other through space and/or chemical bonds. Despite the large number of high resolution protein structures, the "protein structure code" has not been fully identified. Our manuscript presents a novel approach to protein structure analysis in order to identify rules for spatial packing of amino acid pairs in proteins. We have investigated 8706 high resolution non-redundant protein chains and quantified amino acid pair interactions in terms of solvent accessibility, spatial and sequence distance, secondary structure, and sequence length. The number of pairs found in a particular environment is stored in a cell in an 8 dimensional data tensor. When plotting the cell population against the number of cells that have the same population size, a scale free organization is found. When analyzing which amino acid paired residues contributed to the cells with a population above 50, pairs of Ala, Ile, Leu and Val dominate the results. This result is statistically highly significant. We postulate that such pairs form "structural stability points" in the protein structure. Our data shows that they are in buried α-helices or β-strands, in a spatial distance of 3.8-4.3Å and in a sequence distance >4 residues. We speculate that the scale free organization of the amino acid pair interactions in the 8D protein structure combined with the clear dominance of pairs of Ala, Ile, Leu and Val is important for understanding the very nature of the protein structure formation. Our observations suggest that protein structures should be considered as having a higher dimensional organization.

Background. The rise of highly resistant bacteria creates a persistent urge to develop new antimicrobial agents. This paper investigates the application of the lipopeptide antibiotic daptomycin in infections involving the human bone. Methods. Compressive and tensile strength testing of daptomycin-laden PMMA was performed referring to the ISO 5833. The microstructure of the antibiotic-laden PMMA was evaluated by scanning electron microscopy. Intracellular activity of daptomycin was determined by a human osteoblast infection model. Elution kinetics of the antibiotic-laden bone cement was measured by using a continuous flow chamber setup. Results. There was no significant negative effect of adding 1.225% and 7.5% per weight of daptomycin to the PMMA. There was no significant difference in intracellular activity comparing gentamicin to daptomycin. Elution of daptomycin from PMMA showed within the first-hour initial peak values of 15–20 μg/mL. Conclusion. Daptomycin has a certain degree of activity in the intracellular environment of osteoblasts. Daptomycin admixed to PMMA remains bactericidal and does not significantly impair structural characteristics of the PMMA. The results of this paper suggest that daptomycin might be a potent alternative for treating osteomyelitis and implant-associated infection in trauma and orthopedic surgery caused by multiresistant strains.

In this work we report the effects of continuous UV-light (276 nm, ~2.20 W.m(-2)) excitation of human insulin on its absorption and fluorescence properties, structure and functionality. Continuous UV-excitation of the peptide hormone in solution leads to the progressive formation of tyrosine photo-product dityrosine, formed upon tyrosine radical cross-linkage. Absorbance, fluorescence emission and excitation data confirm dityrosine formation, leading to covalent insulin dimerization. Furthermore, UV-excitation of insulin induces disulphide bridge breakage. Near- and far-UV-CD spectroscopy shows that UV-excitation of insulin induces secondary and tertiary structure losses. In native insulin, the A and B chains are held together by two disulphide bridges. Disruption of either of these bonds is likely to affect insulin's structure. The UV-light induced structural changes impair its antibody binding capability and in vitro hormonal function. After 1.5 and 3.5 h of 276 nm excitation there is a 33.7% and 62.1% decrease in concentration of insulin recognized by guinea pig anti-insulin antibodies, respectively. Glucose uptake by human skeletal muscle cells decreases 61.7% when the cells are incubated with pre UV-illuminated insulin during 1.5 h. The observations presented in this work highlight the importance of protecting insulin and other drugs from UV-light exposure, which is of outmost relevance to the pharmaceutical industry. Several drug formulations containing insulin in hexameric, dimeric and monomeric forms can be exposed to natural and artificial UV-light during their production, packaging, storage or administration phases. We can estimate that direct long-term exposure of insulin to sunlight and common light sources for indoors lighting and UV-sterilization in industries can be sufficient to induce irreversible changes to human insulin structure. Routine fluorescence and absorption measurements in laboratory experiments may also induce changes in protein structure. Structural damage includes insulin dimerization via dityrosine cross-linking or disulphide bond disruption, which affects the hormone's structure and bioactivity.

Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis.

Background: Radiotherapy of the prostate and the pelvic lymph nodes (LN) is a part of the standard of care treatment for high‐risk prostate cancer. The independent translational and rotational (i.e., six‐degrees‐of‐freedom, [6DoF]) motion of the prostate and LN target during and between fractions can perturb the dose distribution. However, no standard dose reconstruction method accounting for differential 6DoF target motion is available. Purpose: We present a framework for monitoring motion‐induced dose perturbations for two independently moving target volumes in 6DoF. The framework was used to determine the dose perturbation for the prostate and the LN target caused by differential 6DoF motion for a cohort of high‐risk prostate cancer patients. As a potential first step toward real‐time dose‐guided high‐risk prostate radiotherapy, we furthermore investigated if the dose reconstruction was fast enough for real‐time application for both targets. Methods: Twenty high‐risk prostate cancer patients were treated with 3‐arc volumetric modulated arc therapy (VMAT). Kilovoltage intrafraction monitoring (KIM) with triggered kilovoltage (kV) images acquired every 3 throughout 7–10 fractions per patient was used for retrospective 6DoF intrafraction prostate motion estimation. The 6DoF interfraction LN motion was determined from a pelvic bone match between the planning CT and a post‐treatment cone beam CT (CBCT). Using the retrospectively extracted motion, real‐time 6DoF motion‐including dose reconstruction was simulated using the in‐house developed software DoseTracker. A data stream with the 6DoF target positions and linac parameters was broadcasted at a 3‐Hz frequency to DoseTracker. In a continuous loop, DoseTracker calculated the target dose increments including the specified motion and, for comparison, without motion. The motion‐induced change in D99.5% for the prostate CTV (ΔD99.5%) and in D98% for the LN CTV (ΔD98%) was calculated using the final cumulative dose of each fraction and averaged over all imaged fractions. The real‐time reconstructed dose distribution of DoseTracker was benchmarked against a clinical treatment planning system (TPS) and it was investigated whether the calculation speed was fast enough to keep up with the incoming data stream. Results: Translational motion was largest in cranio‐caudal (CC) direction (prostate: [‐5.9, +8.4] mm; LN: [‐9.9; +11.0] mm) and anterior–posterior (AP) direction (prostate:[‐5.6; +6.9] mm; LN: [‐9.6; +11.0] mm). The pitch was the largest rotation (prostate: [‐22.5; +25.2] deg; LN: [‐3.9; +5.5] deg). The prostate CTV ΔD99.5% was [‐16.2; +2.5]% for single fractions and [‐3.0; +1.7]% when averaged over all imaged fractions. The LN CTV ΔD98% was [‐19.8; +1.2]% for single fractions and [‐3.1; +0.9]% after averaging. Mean (Standard deviation) absolute dose errors in DoseTracker of 107.8% (Std: 1.9%) for the prostate and 105.5% (Std:1.4%) for the LN were corrected during dose reconstruction by automatically calculated normalization factors. It resulted in accurate calculation of the motion‐induced dose errors with relative differences between DoseTracker and TPS dose calculations of ‐0.1% (Std: 0.5%) (prostate CTV ΔD99.5%) and ‐0.2% (Std: 0.5%) (LN CTV ΔD98%). The DoseTracker calculation was fast enough to keep up with the incoming inputs for all but two out of 107 184 dose calculations. Conclusion: Using the developed framework for dose perturbation monitoring, we found that the differential 6DoF target motion caused substantial dose perturbation for individual fractions, which largely averaged out after several fractions. The framework was shown to provide reliable dose calculations and a sufficiently high‐dose reconstruction speed to be applicable in real‐time. [ABSTRACT FROM AUTHOR]