Status quo after one year of COVID-19 pandemic in otolaryngological hospital-based departments and private practices in Germany.

PURPOSE: The COVID-19 pandemic has affected healthcare systems worldwide. Data on the impact on otolaryngological clinics and private practices is sparse. This study aimed to present data on healthcare worker (HCW) screening, status of HCW, pre-interventional testing, the use of personal protective equipment (PPE) and the economic impact of the pandemic. METHODS: Otolaryngological private practices and hospital-based departments were surveyed nationwide using an online questionnaire. Participating facilities were recruited via the German Society for Oto-Rhino-Laryngology and the German Association for Otolaryngologists in Bavaria. RESULTS: 365 private practices (2776 employees) and 65 hospitals (2333 employees) were included. Significantly more hospitals (68.7%) than practices (40.5%) performed pre-interventional testing in their outpatients (p < 0.00). Most inpatients were tested in practices and hospitals (100.0% and 95.0%; p = 0.08). HCW screening was performed in 73.7% of practices and in 77.3% of hospitals (p = 0.54). Significantly more HCW infections were reported in private practices (4.7%) than in hospital (3.6%; p = 0.03). The private or home environment was the most frequent source of infection among HCW in hospitals (44%) and practices (63%). The use of PPE increased over the course of the pandemic. The number of procedures and the revenue decreased in 2020. CONCLUSION: The rate of pre-interventional testing among outpatients in otolaryngological practices is low and HCW infections were found to be more frequent in practices than in hospitals. In addition, a high rate of infections in otolaryngological HCW seems to stem from the private or home environment.

A prospective whole-mixture approach to assess risk of the food and chemical exposome.

Many widely used chemicals result in ubiquitous human exposure from multiple sources, including diet. Legislation mainly deals with the toxicological evaluation of single substances owing to a methodological and conceptual lack of alternatives, and does so within defined silos subject to over 40 distinct regulations in the EU alone. Furthermore, much of the research and many of the initiatives concerned with the assessment and evaluation of chemical mixtures and their potential effects on human health rely on retrospective analysis. Here we propose an approach for the prospective identification, assessment and regulation of mixtures relevant to human health. We address two distinct aspects of toxicology-which chemicals actually do occur together, and how potential mixture-related health hazards can be predicted-with an adapted concept of the exposome and large-scale hazard screens. The proactive use of the likelihood of co-exposure, together with the new approach of methods-based testing, may be a timely and feasible way of identifying those substances and mixtures where hazards may have been overlooked and regulatory action is needed. Ideally, we would generate co-exposure patterns for specific consumer groups, depending on lifestyle and dietary habits, to assess the specific risk of identified mixtures.

Minor structural modifications of bisphenol A strongly affect physiological responses of HepG2 cells.

Bisphenols represent a large group of structurally similar compounds. In contrast to bisphenol A (BPA) and bisphenol S (BPS), however, toxicological data are usually scarce, thus making bisphenols an ideal candidate for read-across assessments. BPA, bisphenol C (BPC) and a newly synthesized bisphenol A/C (BPA/C) differ only by one methyl group attached to the phenolic ring. Their EC50 values for cytotoxicity and logPOW values are comparable. However, the estrogenic activities of these bisphenols are not comparable and among this group only BPC leads to a decrease of the mitochondrial membrane potential and ATP concentration in HepG2 cells. Conversely, the cell division rate was decreased by BPS, BPA, BPC and BPA/C at 10% toxicity (EC10). At lower concentrations, only BPC significantly affected proliferation. The pro-inflammatory cytokines TGFB1 and TNF were significantly upregulated by BPC only, while SPP1 was upregulated by BPA, BPA/C and BPS. BPC led to the release of cytochrome c from mitochondria, indicating that this compound is capable of inducing apoptosis. In conclusion, the read-across approach revealed non-applicable in the case of the various structurally and physicochemically comparable bisphenols tested in this study, as the presence of one or two additional methyl group(s) attached at the phenol ring profoundly affected cellular physiology.

Insights into possibilities for grouping and read-across for nanomaterials in EU chemicals legislation.

This paper presents a comprehensive review of European Union (EU) legislation addressing the safety of chemical substances, and possibilities within each piece of legislation for applying grouping and read-across approaches for the assessment of nanomaterials (NMs). Hence, this review considers both the overarching regulation of chemical substances under REACH (Regulation (EC) No 1907/2006 on registration, evaluation, authorization, and restriction of chemicals) and CLP (Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures) and the sector-specific pieces of legislation for cosmetic, plant protection and biocidal products, and legislation addressing food, novel food, and food contact materials. The relevant supporting documents (e.g. guidance documents) regarding each piece of legislation were identified and reviewed, considering the relevant technical and scientific literature. Prospective regulatory needs for implementing grouping in the assessment of NMs were identified, and the question whether each particular piece of legislation permits the use of grouping and read-across to address information gaps was answered.

Zonation of hepatic fatty acid metabolism - The diversity of its regulation and the benefit of modeling.

A pronounced heterogeneity between hepatocytes in subcellular structure and enzyme activities was discovered more than 50years ago and initiated the idea of metabolic zonation. In the last decades zonation patterns of liver metabolism were extensively investigated for carbohydrate, nitrogen and lipid metabolism. The present review focuses on zonation patterns of the latter. We review recent findings regarding the zonation of fatty acid uptake and oxidation, ketogenesis, triglyceride synthesis and secretion, de novo lipogenesis, as well as bile acid and cholesterol metabolism. In doing so, we expose knowledge gaps and discuss contradictory experimental results, for example on the zonation pattern of fatty acid oxidation and de novo lipogenesis. Thus, possible rewarding directions of further research are identified. Furthermore, recent findings about the regulation of metabolic zonation are summarized, especially regarding the role of hormones, nerve innervation, morphogens, gender differences and the influence of the circadian clock. In the last part of the review, a short collection of models considering hepatic lipid metabolism is provided. We conclude that modeling, despite its proven benefit for understanding of hepatic carbohydrate and ammonia metabolisms, has so far been largely disregarded in the study of lipid metabolism; therefore some possible fields of modeling interest are presented.

Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways.

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.

Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes.

Normal human epidermal keratinocytes were isolated and cultivated in serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0.133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.

Transfection of normal human epidermal keratinocytes with lipid/dna complexes in vitro.

Highly proliferative normal human epidermal keratinocytes (NHK) were isolated from human foreskin biopsies, cultivated in serum-free medium and characterized by flow cytometry. The expression of cytokeratin 19, cytokeratin 14 and vimentin indicated that the suspension contained a high percentage of undifferentiated cells of the basal epidermal layer. The NHK were transfected in vitro with lipid/DNA complexes made of Effectene or Lipofectamine and different reporter genes. The transfection efficiency of Effectene/DNA complexes was 20fold higher compared to Lipofectamine. Transfected keratinocytes continued to grow and developed within 2 weeks a cellular multilayer (3-D culture). Areas of transfected cells were detected within this layer.

Quantification of lipids, liposomes, amino acids, and proteins by thermal ultramicrodigestion and an ultramicrocoulometric assay, based on the reaction of hypobromite with ammonia.

The quantification of nitrogen in organic material is described. It is based on a novel thermal ultramicrodigestion in combination with an ultramicrocoulometric quantification. The lower detection limit of the coulometric measurement is 0.5 microg nitrogen, which corresponds to 20 microg lipid, 3 microg glycine, or 4 microg protein. Therefore it is as sensitive as the frequently used Lowry method. In contrast to the Lowry protein determination it is not disturbed by detergents and most other interfering substances.

Reconstructed human skin as model for liposome-skin interaction.

Reconstructed human skin was prepared from human keratinoblasts. After 1 week of cultivation at the air-liquid interface a stratified layer developed, similar to native human epidermis. Liposomes with an average diameter of 50 nm, made of phosphatidylcholine (PC), phosphatidylserine (PS) and human stratum corneum lipids (hSCL) were applied on top of this culture system. The rate of penetration through the reconstructed human epidermis was 1.38, 0.55 and 0.013 ng lipidh-1cm-2 for PC, hSCL and PS liposomes, respectively. Electron microscopy and confocal laser scanning microscopy showed that PS and hSCL liposomes aggregated at the skin surface, while PC liposomes remained homogeneously dispersed. Fluorescence measurements demonstrated that vesicles, made of native human stratum corneum lipids rapidly mixed with PS liposomes, weakly with hSCL liposomes and did not mix with PC liposomes.

Aminopeptidase P--a cell-surface antigen of endothelial and lymphoid cells: catalytic and immuno-histotopical evidences.

The physiological function of the GPI-anchored ectoenzyme aminopeptidase P (APP) is still elusive. Most researchers suppose that this enzyme inactivates biologically active peptides like bradykinin, neuropeptide tyrosine (NPY) and others (Vanhoof et al., 1995). We demonstrate by immunohistology with a specific antibody raised in rabbits and measurement of enzymatic activity in suspensions and of confluent monolayers on microscopic coverslips ('monolayer kinetics') that APP is a cell surface enzyme (ectoenzyme) of endothelial and lymphoid cells.

Physicochemical characterisation of human stratum corneum lipid liposomes.

Liposomes were prepared from an extract of all human stratum corneum lipids (hSCL) and characterised in terms of temperature and the presence of Ca2+ by different physicochemical methods. Vesicle aggregation and lateral phase separation were induced by divalent cations with Ca2+ being more efficient than Mg2+. At 24.1 degrees C, i.e. well below physiological temperatures the suspensions consisted of a lamellar phase and crystalline cholesterol. At and above 37 degrees C, this cholesterol surplus was dissolved in the hSCL membranes. However, melting of the hSCL was not completed up to 60 degrees C. The presence of Ca2+ (> or = 9 mM) induced lateral phase separation and fusion of vesicles into extended multilamellar lipid sheets (MLLS) at and above 32.5 degrees C. Upon a subsequent cooling cycle recrystallisation of cholesterol occurred within the MLLS. Finally, membrane mixing of hSCL liposomes with vesicles made of synthetic lipids was investigated. No mixing was observed between either of DPPE/oleic acid, DPPC/DPPE, DPPC/lyso-PC and hSCL liposomes. Mixtures of DPPC/cholesterol hemisuccinate showed a temperature-dependent membrane mixing behaviour, whilst hSCL liposomes and phosphatidylserine liposomes fused temperature-independently with hSCL liposomes.

Quantitative determination of thiolipids in organic solution, in membranes, and on HPTLC plates.

In the presence of ortho-phthalaldehyde and glucosamine, thiolipids form fluorescent isoindole derivatives. This reaction can be used to quantify single- and double-chain mercaptans in membranes (liposomes) and micellar solutions. The lower detection limit is 100 pmol. In addition, the assay allows the detection of 1.9 nmol thiolipids on HPTLC plates and the fluorescence signal is stable for days. A minor modification of the commonly used DTNB (Ellman's) assay allows the quantification of thiolipids in organic solutions at a concentration down to 3 nmol.

Individual variation of human plantar stratum corneum lipids, determined by automated multiple development of high-performance thin-layer chromatography plates.

The stratum corneum lipids are unique in composition and have been used frequently as a model system of the skin's lipid barrier. Automated multiple development (AMD) of high-performance thin-layer chromatography plates in combination with a 25-step gradient, based on methanol, diethyl ether and n-hexane separated the six major human plantar stratum corneum lipids. Post-chromatographic staining of these lipids with a solution of MnCl2-H2SO4 at 130 degrees C or a solution of CuSO4-H3PO4 at 140 degrees C allowed visualization of the lipids and quantification. The MnCl2-H2SO4 solution stained saturated fatty acids less intensely. Therefore, the CuSO4-H3PO4 solution was used for quantification and we found, on average, 2.06% (w/w) cholesterol 3-sulphate, 20.16% (w/w) free fatty acids, 20.25% (w/w) ceramides, 43.53% (w/w) non-esterified sterols, 4.56% (w/w) triacylglycerols and 9.4% (w/w) sterolesters in the human plantar stratum corneum extracts. The concentration of phospholipids was less than 1% (w/w). In addition, the lipid composition of twenty different human plantar stratum corneum extracts was determined. Statistics revealed a correlation between the ratio of free fatty acids and non-esterified sterols (r=0.832, p<0.01, n=20). Several control experiments proved that this correlation is not due to the extraction method, the post-chromatographic staining procedure or bacterial contamination of the stratum corneum.

Oxidative damage of human skin lipids. Dependence of lipid peroxidation on sterol concentration.

Photoprotection against sunburn and associated irradiation-induced damages of the human skin is mainly attributed to the darkening of the biochrome melanin by its oxidation. Human skin lipids were examined for an additional protection by sterols. Lipid vesicles prepared from extracted human skin lipids as well as from mixtures of typical lipids of the stratum corneum were irradiated by UV light in the presence and absence of oxygen. The oxidative degradation of various lipids was measured by quantitative HPTLC, by the dichlorofluorescein fluorescent assay, by the thiobarbituric acid assay and a novel luminol-based chemiluminescence technique. Electron spin resonance was used to look for certain radical intermediates. The results indicate, that sterols, mainly free cholesterol, with their high concentration in the lipid barrier of the stratum corneum (up to 50 mol%) effectively compete with the peroxidation of other human skin lipids (ceramides and free fatty acids).

Tumor targeting in vivo by means of thermolabile fusogenic liposomes.

Thermolabile fusogenic liposomes were devised based on the stoichiometric 1/2 mixtures of dipalmitoylphosphatidylcholine (DPPC) and elaidic acid (ELA) and from the similar stoichiometric mixtures of DPPC, dipalmitoylphosphatidylglycerol (DPPG) and elaidoyl alcohol (EL-OH) or palmitelaidoyl alcohol (PEL-OH). The resulting vesicle suspensions are fusogenic in the region of hyperthermia (> or = 42 degrees C) and can be targeted selectively to the heated tumor tissue. Incorporation of DPPG or fatty alcohols into the vesicle membranes also leads to a non-specific, temporary vesicle material accumulation in the lung, however, probably due to platelet activation. Vesicle material accumulation in A-431 tumors, xenotransplanted in nude mice, after 30 min of local hyperthermia (42 degrees C) is 4-fold higher for the DPPC/ELA (1/2), 2.8-fold higher for the DPPC/DPPG/EL-OH (0.8/0.2/2) and 3.7-fold higher for the DPPC/ELA/EL-OH (1/1/1) mixtures than for similar vesicles used at the physiological temperature. Extension of hyperthermia to 60 min induces a 7.8-fold relative material accumulation in the tumor tissue when the thermolabile, fusogenic DPPC/ELA/EL-OH (1/1/1) vesicles are used. Simple DPPC vesicles only reach concentrations in the heated tumor or muscle tissue that are 1.85-fold and 1.38-fold higher than in the normothermic control, respectively. This is probably a consequence of simple vasodilatation. In vitro experiments revealed that the adsorption of serum proteins to the vesicle membrane decreases the chain-melting phase transition temperature and the transition enthalpy of vesicle suspension. Adsorption is most prominent at the chain-melting phase transition temperature of the mixed lipid bilayers, which is also the critical temperature for the induction of liposome fusion. This hampers the practical use of the resulting vesicle suspension in vivo. The serum-induced decrease of the chain-melting phase transition temperature, which is likely to change as a function of time in vivo, depends on the lipid composition and on the local surface charge density of vesicles. Incorporation of ELA and DPPG concentrations above 15 mol-%, for example, reduce the extent of protein adsorption onto vesicles. This has to be borne in mind when devising vesicles for practical applications.

Interaction of phosphatidylcholine liposomes with the human stratum corneum.

The interaction of dimyristoylphosphatidylcholine liposomes with the human stratum corneum was investigated by confocal laser scanning microscopy and differential scanning calorimetry. Human skin is characterized by a high autofluorescence. By introducing appropriate optical filters the autofluorescence of the skin was depressed and the penetration profile of fluorescence labelled vesicles was investigated. From optical sectioning it was obvious that neither the vesicles nor the fluorophore N-(lissamine rhodamine B sulfonyl)diacylphophatidylethanolamine (Rho-PE) penetrates in detectable amounts into the human skin. Differential scanning calorimetry of human stratum corneum revealed, that the peak positions of the human stratum corneum specific endothermic transitions at 10 degrees C, 35 degrees C, 50 degrees C, 62 degrees C, 73 degrees C and 81 degrees C did not change significantly after 18 h of non-occlusive vesicle application. However, the enthalpy of the transitions at 35 degrees C, 50 degrees C, 62 degrees C and 73 degrees C, estimated through peak heights increased, relative to the protein related peak at 81 degrees C. A novel transition at 10 degrees C was observed. From these data we conclude that DMPC liposomes do not penetrate intact into the human skin. We deduce, however, that the vesicles disintegrate at the surface of stratum corneum after non-occlusive application. The individual lipid molecules then interact with the lipid barrier of the stratum corneum and penetrate into the latter, which results in an increase of the enthalpy, related to the lipid components of the SC.

Temperature- and pH-controlled fusion between complex lipid membranes. Examples with the diacylphosphatidylcholine/fatty acid mixed liposomes.

The fusion capability of complex lipid bilayers and its pH as well as temperature sensitivity have been studied by optical and spectroscopic means. The aggregation and fusion efficiency of such lipid membranes can be optimized by controlling the phase characteristics of the individual membrane components. For a practically relevant illustration, the stoichiometric 1:2 (mol/mol) mixtures of phosphatidylcholines and fatty acids are used. Perhaps the most interesting liposomes of this kind, which are made of dipalmitoylphosphatidylcholine/elaidic acid (DPPC/ELA-COOH (1:2)), undergo a chain-melting phase transition between 42 degrees C and 48 degrees C, depending on the bulk pH value. The highest chain-melting phase transition temperatures are measured with the fully protonated fatty acids at pH < or = 5.5 and involve a change into the non-bilayer high-temperature state. Upon increasing pH, this transition reverts into an ordinary gel-to-fluid lamellar phase change and occurs at 42 degrees C, by and large. Simultaneously, the rate and the efficacy of fusion between the PC/FA and PC/FA- mixed vesicles decreases. The fusion efficacy of the PC/FA(-) mixed liposomes at pH > or = pK(FA) approximately 7.5 is practically negligible. This is largely due to the increased interbilayer repulsion and to the relatively high water-solubility of the deprotonated fatty acid molecules at high pH. While the pH-variability chiefly affects the efficacy of the intermembrane aggregation, the vesicle fusion itself is more sensitive to temperature variations. It is more likely that the temperature dependence of the intramembrane defect density is chiefly responsible for this. Optimal conditions for the fusion between DPPC/ELA-COOH (1:2) mixed vesicles are thus 3.5 < or = pH < or = 5.5 (6.3) (aggregation maximum) and T > or = 41.5 degrees C = Tm(DPPC) (defect density and fusion maximum). Under such conditions the average size of PC/FA (1:2) mixed vesicles in a 1 mM suspension increases by a factor of 10 over a period of 10 min. Interbilayer fusion can also be catalyzed by the mechanically induced local membrane defects. Freshly made liposomes thus always fuse more avidly than aged vesicles. This permits estimates of the kinetics of membrane defects annihilation based on the measured temporal dependence of the maximum fusion-rate. From such studies, a quasi-exponential decay on the time scale of 1.2 h is found for the thermolabile fusogenic DPPC/ELA-COOH liposomes.

Hemodynamic findings during exercise on a bicycle ergometer following balloon valvuloplasty of pulmonary stenosis in children and adolescents.

Eleven patients (4 female, 7 male), age range 3.3 to 24.8 years (mean 11.10 years) treated for isolated pulmonary stenosis underwent cardiac catheterization and percutaneous transluminal balloon valvuloplasty (PTVP). The right ventricular systolic pressure (RVSP) before valvuloplasty ranged from 31 to 127 mmHg (mean 79 mmHg) decreasing to 28 to 62 mmHg (mean 42 mmHg) immediately after the dilatation. The peak systolic gradient of the pulmonary valve (delta p RV-PA) before valvuloplasty ranged from 22 to 107 mmHg (mean 61 mmHg) and decreased to a range of 14 and 45 mmHg (mean 23 mmHg) immediately after the dilatation. Balloon valvuloplasty was performed using balloons of 13 to 31 mm in diameter. On 11 patients cardiac catheterization and Doppler echocardiography were repeated between 11 months and 5.3 years (mean 3.11 years) after the balloon valvuloplasty showed a further significant fall in the gradient of pressure. The right ventricular systolic pressure ranged from 20 to 51 mmHg (mean 31.7 mmHg) while the transpulmonary gradient varied from 3 to 24 mmHg (mean 11.6 mmHg). At the time of follow-up examination the patients were aged between 7.2 and 25.7 years (mean 15.9 years). On average the second catheterization was performed 3.11 years following the first hemodynamic study. The follow-up examination encompassed clinical examination, electrocardiogram, Doppler echocardiography, and right heart cardiac catheterization. During right heart cardiac catheterization the children exercised on a bicycle ergometer for three min at 50 or 100 W depending on their body surface area. During this exertion, pressures of the right ventricle and the pulmonary artery as well as heart rate and oxygen saturation were recorded.(ABSTRACT TRUNCATED AT 250 WORDS)