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Refers to Clinically Significant Prostate Cancer Diagnosis Without Histological Proof: A Possibility in the Prostate-specific Membrane Antigen Era? European Urology Open Science, Volume 44, October 2022, Pages 30-32. Joris G. Heetman, Lieke Wever, Leonor J. Paulino Pereira, Roderick C.N. van den Bergh https://doi.org/10.1016/j.euros.2022.06.013
Background: Up- and/or downgrading rates in single intermediate-risk positive biopsy core are unknown.
Methods: We identified single intermediate-risk (Gleason grade group (GGG) 2/GGG3) positive biopsy core prostate cancer patients (≤ cT2c and PSA ≤ 20 ng/mL) within the Surveillance, Epidemiology, and End Results (SEER) database (2010–2015). Subsequently, separate uni- and multivariable logistic regression models tested for independent predictors of up- and downgrading.
Results: Of 1,328 assessable patients with single core positive intermediate-risk prostate cancer at biopsy, 972 (73%) harbored GGG2 versus 356 (27%) harbored GGG3. Median PSA (5.5 vs 5.7; p = 0.3), median age (62 vs 63 years; p = 0.07) and cT1-stage (77 vs 75%; p = 0.3) did not differ between GGG2 and GGG3 patients. Of individuals with single GGG2 positive biopsy core, 191 (20%) showed downgrading to GGG1 versus 35 (4%) upgrading to GGG4 or GGG5 at RP. Of individuals with single GGG3 positive biopsy core, 36 (10%) showed downgrading to GGG1 versus 42 (12%) significant upgrading to GGG4 or GGG5 at RP. In multivariable logistic regression models, elevated PSA (10–20 ng/mL) was an independent predictor of upgrading to GGG4/GGG5 in single GGG3 positive biopsy core patients (OR:2.89; 95%-CI: 1.31–6.11; p = 0.007).
Conclusion: In single GGG2 positive biopsy core patients, downgrading was four times more often recorded compared to upgrading. Conversely, in single GGG3 positive biopsy core patients, up- and downgrading rates were comparable and should be expected in one out of ten patients.
1. Electron micrographs of ultra-thin sections of Staphylococcus aureus and Micrococcus lysodeikticus in Vestopal as embedding medium disclose a multiplicity of DNA containing threads with varying interparticular distances.
2. The diameter of these threads is about one tenth of the average optimal section thickness.
3. This section thickness inevitably is implicated in the visualization of the internal distances between the threads as well as in some common trends in the DNA pool, a fact that has to be accounted for in the analysis of the macromolecules.
4. By spreading lysozyme protoplasts of M. lysodeikticus on a water-air interface in a Langmuir trough and by transferring this surface layer to carbon supported Formvar films, two-dimensional systems can be demonstrated which as a thread of constant width comprise the total DNA content of one microorganism each.
5. Such a macromolecular system shows equally shaped, coiled loops in a peripheral zone and many crossings towards the center. Branching of threads never has been observed so far.
From this evidence we conlude:
a) Intracellular DNA in these bacteria seems to exist in one pool as a “woolen ball” which is centered in the cytoplasm as a more or less dense object.
b) This “woolen ball“ embodies the total amount of DNA most probably as one single threadlike unit.
6. Partial destruction of the thread system of protoplasts will result upon changing optimal spreading conditions.
7. The same kind of destruction is shown upon isolation of the DNA from protoplasts, the length of the threads being an inverse function of the number of precipitation steps showing purification.
UV-mikrospektrophotometrische Messungen der Nucleinsäuren- und Eiweißkörper-Konzentration sowie der Kern- und Nukleolengröße nach Virusinfektion und unspezifischer Reizung der Chorion-Allantoismembran zeigen, daß es in beiden Fällen zu einer gleich starken Stimulierung des nucleinsäuren- und eiweißkörperbildenden Systems der Zelle kommt. Bei der Infektion mit Vaccinevirus auf das Ektoderm setzt die Reaktion der Membranzellen in der Eklipse ein, nach Infektion mit Newcastle-Disease-Virus fällt der Titeranstieg mit der Zellreaktion zeitlich zusammen.
Serumkomplement (C′), welches nach dem heutigen Stand der Forschung aus mindestens vier Komponenten besteht, ist an zahlreichen immunologischen und nicht-immunologischen Vorgängen beteiligt. Am besten erforscht ist die Sequenz der C′-Reaktionsstufen bei der Hämolyse sensibilisierter Hammelerythrocyten, jedoch ist noch ungeklärt, welcher Mechanismus letztlich die Zellveränderung, die zum Austritt von Hämoglobin führt, bewirkt.
In der vorliegenden Arbeit wird über das Endprodukt der C′-Reaktion — eine cytolytisch wirkende Substanz — berichtet. Es wird gezeigt, daß sowohl beim Altern von Komplement, wie auch bei C′ fixierenden Antigen/Antikörperreaktionen im Serum eine Substanz entsteht, die zellschädigend und auflösend wirkt.
Seren, in denen durch mehr oder weniger spezifische Vorbehandlung C′ oder einzelne seiner Komponenten ausgeschaltet wurden, sind nicht mehr zur Cytolysinbildung befähigt.
Das cytolytische Prinzip konnte gereinigt und papierchromatographisch als Lysolecithin charakterisiert werden.
Wirkung von Glia-Maturationsfaktor (GMF) und Adhäsionsfaktor (ADF) auf Kulturen leukämischer Zellen
(1987)
GMF was isolated from the glia of different brain parts of calf. Its influence on the input of 3H-TdR was tested in leukaemic and non-malignant lymphocites. GMF isolated from the diencephalon proved to be the most effective one. The used substance of reference was DBcAMP. The cAMP contents of leukaemic cells were lower than those of non-malignant cells, whereas the level of cGMP was higher in the first ones. We found both in non-malignant cultures and in cultures of leukaemic cells that the impact of GMF and AdF elevated the cAMP value and reduced cGMP. The adhesion of cAMP to the nuclei of leukaemic was reduced in comparison with non-malignant cells. The nuclei of leukaemic cells showed increased adhesive capacity after pre-incubation with GMF and AdF. The adhesive capacity of non-malignant nuclei remained unchanged. The adenylcyclase activity (AC) was diminished in leukaemic cells. It could not be stimulated by means of catecholamines. GMF and AdF produced a slight increase of the basal Ac activity in cultures of leukaemic cells only. But isoproterenol led to a distinct increase of the Ac activity in leukaemic cells when incubation with those two factors preceded it.
(±)-Aeroplysinin-1, an optically active 1.2-dihydroarene-1.2-diol. was isolated from the marine sponges Verongia aerophoba (+-isomer) and lanthella ardis (--isomer). For the experiments presented we used the +-isomer from Verongia aerophoba. Here we describe the hitherto unknown biological and pharmacological property of this compound to display pronounced anticancer activity against L5178y mouse lymphoma cells (ED50: 0.5 μm). Friend erythroleukemia cells (ED50: 0.7μm) , human mamma carcinoma cells (ED50: 0.3μm) and human colon carcinoma cells (ED50: 3.0 μm) in vitro. Furthermore, aeroplysinin caused a preferential inhibition of [3H]thymidine (dThd) incorporation rates in L5178y mouse lymphoma cells if compared with murine spleen lymphocytes in vitro. At concentrations between 1.1 and 28.5 μm, the [3H]dThd incorporation rates in L5178y cells were suppressed to 28% -0% but only to 78% -18% in murine spleen lymphocytes. The same differential effect in vitro was found with the following epithelial cells: 14.70 μm of the compound were required to inhibit normal human fibroblasts to 50% , but only 2.9 μm in the assays with human malign keratinocytes or malignant melanoma cells to observe the same inhibitory effect. Moreover, aeroplysinin-1 displayed antileukemic activity in vivo using the L5178y cell/NMRI mouse system; administered at a dose of 50 mg/kg for five consecutive days, the T/C (% ) value was determined to be 338. Preliminary toxicology studies revealed an acute LD50 of 202 mg/kg and a subacute LD50 of 150 mg/kg. Aeroplysinin-1 is neither a direct mutagen nor a premutagen in the umu/Salmonella typhimurium test system.
New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases
(1995)
Reactive coenzyme analogues ω-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of ω-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability at 0 °C. L actate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazonium pentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3α,20β-Hydroxysteroid dehydrogenase reacts at 0 °C with the ethyl homologue and slowly with the propyl compound. The butyl-and pentyl analogues do not inactivate at 0 °C. Tests with 14C -labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2 ′-phospho-adenosine diphosphate, a structural analogue of NADP +, was prepared by condensation of adenosine-2,3-cyclophospho-5′-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid ester with 2 ′:3′-cyclonucleotide-3′-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehyd rogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP +-dependent dehydrogenases the com pound has been show n to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.
By means of differential thermoanalysis, the miscibility of the main polar tetraether lipid of Thermoplasma acidophilum with two ester lipids, dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol, resp., in the presence of excess water was studied. It is shown that with increasing fraction of tetraether lipid in the mixture, the transition range of dipalmitoyl phosphatidylcholine is broadened and the temperature of the maximum heat flow (Tm) is shifted to lower temperatures; furthermore, the enthaply change (ΔH) of the transition declines. Similar results were obtained with mixtures of tetraether lipid with dipalmitoyl phosphatidylglycerol. It is therefore concluded that the main polar tetraether lipid of Thermoplasma acidophilum , which essentially forms monomolecular layers, is able to form stable common phases with bilayer-forming ester lipids. Miscibility of the tetraether lipid with dipalmitoyl phosphatidylglycerol, which are both monovalent anions at neutral pH, is also observed in the presence of high proton or calcium ion concentrations.
pH-titrations with NADH show two ionizable groups in mitochondrial and cytoplasmic malate dehydrogenase, the first with a pKa in the range 6.8 -8.3 for the mitochondrial and 6.4-7.8 for the cytoplasmic enzyme, the second with a lower limit at 10.2 resp. 11. Comparison with bis-(dihydronicotinamide)-dinucleotide and dihydronicotina-mide-ribosyl-P2-ribose-pyrophosphate instead of NADH indicates that the second alkaline ionization is caused by a residue placed near the adenine binding site of the active centre of the two isoenzymes. Binding studies with NADH and NAD+ give evidence for the participation of a group in the mitochondrial enzyme with pKa 6.8, deprotonation of which is necessary for detectable association of NAD+. In contrast the fixation of NAD+ to the cytoplasmic enzyme is independent of pH.
A single procedure for the preparation of lactate dehydrogenase (EC 1.1.1.27), the mitochondrial and cytoplasmic forms of malate dehydrogenase (EC 1.1.1.37), adenylate kinase (EC 2.7.4.3) and pyruvate kinase (EC 2.7.1.40) from pig heart is described. The five enzymes are obtained in preparative amounts in homogenous form with specific activities equal to or higher than those pre viously reported. Some molecular properties of pig heart pyruvate kinase are determined.
Alkylating NAD-Analogs, Glyceraldehyde-3 Phosphate Dehydrogenase, Half-of-the-Sites Reactivity co-(3-Bromoacetylpyridinio)alkyldiphosphoadenosines with alkyl chain lengths of 2 -6 me thylene groups inactivate glyceraldehyde-3 phosphate dehydrogenase from rabbit muscle. Half-of-the-Sites reactivity is observed in each case: The analogs are covalently bound to highly reactive cysteine residues in two of the four subunits. The remaining two subunits still bind N AD and the reactive SH-groups, although modified by SH-reagents of low molecular weight are not labeled by any of the brominated coenzyme models. This behaviour may be explained by the assumption, that the modification of 2 subunits induces structural changes in the neighboured unoccupied subunits which prevent any attack on reactive cysteine residues caused by fixation and orientation of the bromoketo-coenzyme analog when bound to the active center. Structural similarities of the covalently bound coenzyme analogs in the active center and the native ternary GAPDH-NAD-substrate complex suggest that half-of-the-sites reactivity is a natural characteristic of the enzymes catalytic mechanism.
5-Acetyl-4-methyl-1-(β-D-ribofuranosyl) -imidazole-5′-phosphate reacts with diphenylphospho chloridate forming the asymmetrical pyrophosphate ester. This in turn reacts with tri-n-butylammonium phosphate yielding 5-acetyl-4-methyl-imidazole-riboside-5′-diphosphate and with tri-rcbutylammonium pyrophosphate to give the nucleotide triphosphate.
5-Acetyl-4-methyl-imidazole-riboside-5′-pyrophosphate shows in the test with pyruvate kinase a reaction rate three times slower than that of ADP; but the same Km as that of ADP. The ATP analogue is only about 10% as effective as ATP itself in the test with hexokinase, 3-phosphoglycerate kinase and gluconate kinase. Adenylate kinase and NAD+ kinase show no activity when ATP is replaced by the nucleotide-triphosphate-analogue. In presence of ATP the analogue strongly inhibits the reaction of adenylate kinase.
The NAD analogue [3-(3-acetylpyridinio)-propyl] adenosine pyrophosphate forms enzymically inactive complexes with glyceraldehyde-3-phosphate dehydrogenase from yeast and rabbit skeletal muscle. In the latter enzyme four mol of the analogue are bound with equal affinity inhibiting the enzyme in a competitive way: KI = 0.3 mM as compared to the dissociation constant KD=O.6 mм.
The brominated derivative [3- (3-bromoacetylpyridinio) -propyl] adenosine pyrophosphate is covalently bound to both enzymes causing irreversible loss of enzymic activity. Complete inactivation of the enzyme from muscle requires two moles of the analogue per mol of tetramer. The remaining two sites are still able to bind two mol of NAD+ without regain of enzymic activity. In the case of the yeast enzyme four mol of the analogue are bound. Inactivation of the rabbit muscle enzyme is accompanied by the disappearance of two out of four highly reactive sulfhydryl groups; in the yeast enzyme the four active site cysteine residues are still able to react with DTNB1 the reactivity being diminished significantly.
Hybrid formation between the native enzymes from yeast and skeletal muscle is not affected by the modification of the enzyme. Similarly the sedimentation properties of the covalently modified enzyme are indistinguishable from those of the native molecule. This indicates that both the native and the irreversibly inhibited enzyme are identical regarding their quaternary structure.
Die Einführung von Patient Blood Management (PBM) führt zu einem Paradigmenwechsel bezüglich Erkennen und Therapie der Anämie und zeigt Maßnahmen auf um die Entstehung einer Anämie zu verhindern. PBM unterstützt den Arzt im Entscheidungsdilemma zwischen positiver Wirkung und nachteiligen Nebenwirkungen von Bluttransfusionen. Mit PBM wird der Blutverbrauch deutlich reduziert und die Nebenwirkungen gesenkt. Nicht nur die therapeutischen Maßnahmen, sondern auch die diagnostischen PBM Maßnahmen im Labor führen zu einer relevanten Verringerung des Blutvolumens. PBM Studienergebnisse zeigen eine signifikant Reduktion der Morbidität und Mortalität und die Verbesserung des Patienten- Outcome. Ein weiterer positiver Nebeneffekt ist Schonung von Ressourcen in allen beteiligten Bereichen, welches zu einer relevanten Kostenreduktion und Steigerung der Wirtschaftlichkeit führt. Zusätzlich sensibilisiert das PBM bezüglich des Vorliegens, der Entwicklung und der Therapie einer anämischen Situation sowie den Umgang mit der kostbaren Ressource Blut. Die Bedeutung des PBM wird mittlerweile von der Industrie auch für das Labor unterstützt; für den Bereich POCT ist das PBM jedoch bisher noch nicht adäquat technisch realisiert.
Studies on the transport of anions and zwitterions of acidic amino acids in Streptomyces hydrogenans
(1983)
n Streptomyces hydrogenans, acidic amino acfds are taken up either as anions by a specific transport system or as zwitterions via a nonspecific one. Variations in the zwitterion concentration caused by changes in pH influence the uptake and exchange diffusion by the nonspecific system. Differences in pH-optima for ʟ-glutamate and ʟ-aspartate transport are due to the different pK2-values of these amino acids. The anion transport by the specific system is accompanied by a short hyperpolarization of the membrane potential followed by a secondary influx of potassium ions into the cells.
The combination of pamapimod and pioglitazone (KIN001) has a synergetic antiviral, anti-inflammatory, and antifibrotic activity, which may prevent evolution toward COVID-19-associated severe respiratory failure. In a randomized, placebo-controlled, double-blind, phase 2, multicenter trial, 128 non-critically ill hospitalized patients with confirmed COVID-19 were treated with KIN001 or a placebo for 28 days. The proportion of patients alive and free of oxygen or respiratory support at the end of the therapy was lower than anticipated but not different in the two groups (KIN001 n = 19, 29%, placebo n = 21, 33%). 85 participants had at least one adverse event, with no difference in the number and distribution of events between the two groups. The clinical trial was stopped for futility, mainly due to a lower-than-expected incidence of the primary endpoint. KIN001 was safe and well-tolerated but had no significant effect on clinical outcome.
Background: In March 2020, the COVID-19 outbreak led to the declaration of a pandemic. The accompanying restrictions on public life caused a change in the training routines of athletes worldwide. The present study aimed to investigate the effects of a 13-week supervised home training program on physical performance, sleep quality, and health-related quality of life in professional youth soccer players during the first COVID-19 lockdown in Germany.
Methods: Eight professional soccer players (age range 16–19; height: 1.81 ± 0.07 m; body weight: 72.05 ± 6.96 kg) from a Bundesliga team in Germany participated in this study. During the lockdown, they trained 5–6 days per week with home-based training plans and were monitored via tracking apps and video training. To determine the effects of home training, measurements were taken before (March 2020) and after (June 2020) the home training period. Bioelectrical impedance analysis was used to determine body composition, and an isokinetic strength test and a treadmill step test, including lactate measurements, were used to measure physical performance. Two questionnaires were responded to in order to assess health-related quality of life [Short-Form 36 Health Survey (SF-36)] and sleep quality (Pittsburgh Sleep Quality Index).
Results: When comparing measurements before and after the home training period, we observed significant increases in the following variables: body weight (72.05 ± 6.96 kg vs. 73.50 ± 6.68 kg, p = 0.034), fat mass (11.99 ± 3.13 % vs. 13.98 ± 3.92 %, p = 0.030), body mass index (22.04 ± 0.85 kg/m2 vs. 22.49 ± 0.92 kg/m2, p = 0.049), and mental health component summary score (MCS) of the questionnaire SF-36 (53.95 ± 3.47 vs. 58.33 ± 4.50, p = 0.044). Scores on the general health (77.88 ± 14.56 vs. 89.75 ± 13.76, p = 0.025) and mental health (81.50 ± 9.30 vs. 90.00 ± 11.71, p = 0.018) subscales of the SF-36 also increased significantly.
Conclusion: The COVID-19 lockdown led to an increase in body composition parameters and showed an improvement in the MCS and scores on the general and mental health subscales of the SF-36. Physical performance and sleep quality could be maintained during the home training period. These observations may help trainers for future training planning during longer interruptions in soccer training.
The immune response is known to wane after vaccination with BNT162b2, but the role of age, morbidity and body composition is not well understood. We conducted a cross-sectional study in long-term care facilities (LTCFs) for the elderly. All study participants had completed two-dose vaccination with BNT162b2 five to 7 months before sample collection. In 298 residents (median age 86 years, range 75–101), anti-SARS-CoV-2 rector binding IgG antibody (anti-RBD-IgG) concentrations were low and inversely correlated with age (mean 51.60 BAU/ml). We compared the results to Health Care Workers (HCW) aged 18–70 years (n = 114, median age: 53 years), who had a higher mean anti-RBD-IgG concentration of 156.99 BAU/ml. Neutralization against the Delta variant was low in both groups (9.5% in LTCF residents and 31.6% in HCWs). The Charlson Comorbidity Index was inversely correlated with anti-RBD-IgG, but not the body mass index (BMI). A control group of 14 LTCF residents with known breakthrough infection had significant higher antibody concentrations (mean 3,199.65 BAU/ml), and 85.7% had detectable neutralization against the Delta variant. Our results demonstrate low but recoverable markers of immunity in LTCF residents five to 7 months after vaccination.
The recently developed stereospecific sodium salt glycosylation procedure has been successfully applied to the synthesis of the β-ᴅ-2′-deoxyribofuranosides of benzimidazole, 5,6-dihalogeno benzimidazoles, and some 2-substituted analogues in high yield. The 5,6-dibromo analogue was obtained by bromination of the parent nucleoside. These have all been characterized by spectroscopic methods, including 1H NMR, which permitted analyses of their solution conformations and comparison with those of the corresponding ribofuranosides. Some biological aspects, including preliminary results on cytotoxicity and antiviral activity, are briefly considered.
Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1·EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.
Hundreds of genes have been associated with respiratory chain disease (RCD), the most common inborn error of metabolism so far. Elimination of the respiratory electron chain by depleting the entire mitochondrial DNA (mtDNA, ρ0 cells) has therefore one of the most severe impacts on the energy metabolism in eukaryotic cells. In this study, proteomic data sets including the post-translational modifications (PTMs) phosphorylation and ubiquitination were integrated with metabolomic data sets and selected enzyme activities in the osteosarcoma cell line 143B.TK−. A shotgun based SILAC LC-MS proteomics and a targeted metabolomics approach was applied to elucidate the consequences of the ρ0 state. Pathway and protein–protein interaction (PPI) network analyses revealed a nonuniform down-regulation of the respiratory electron chain, the tricarboxylic acid (TCA) cycle, and the pyruvate metabolism in ρ0 cells. Metabolites of the TCA cycle were dysregulated, such as a reduction of citric acid and cis-aconitic acid (six and 2.5-fold), and an increase of lactic acid, oxalacetic acid (both twofold), and succinic acid (fivefold) in ρ0 cells. Signaling pathways such as GPCR, EGFR, G12/13 alpha, and Rho GTPases were up-regulated in ρ0 cells, which could be indicative for the mitochondrial retrograde response, a pathway of communication from mitochondria to the nucleus. This was supported by our phosphoproteome data, which revealed two main processes, GTPase-related signal transduction and cytoskeleton organization. Furthermore, a general de-ubiquitination in ρ0 cells was observed, for example, 80S ribosomal proteins were in average threefold and SLC amino acid transporters fivefold de-ubiquitinated. The latter might cause the observed significant increase of amino acid levels in ρ0 cells. We conclude that an elimination of the respiratory electron chain, e.g. mtDNA depletion, not only leads to an uneven down-regulation of mitochondrial energy pathways, but also triggers the retrograde response.
The yeast Rcf1 protein is a member of the conserved family of proteins termed the hypoxia-induced gene (domain) 1 (Hig1 or HIGD1) family. Rcf1 interacts with components of the mitochondrial oxidative phosphorylation system, in particular the cytochrome bc1 (complex III)-cytochrome c oxidase (complex IV) supercomplex (termed III-IV) and the ADP/ATP carrier proteins. Rcf1 plays a role in the assembly and modulation of the activity of complex IV; however, the molecular basis for how Rcf1 influences the activity of complex IV is currently unknown. Hig1 type 2 isoforms, which include the Rcf1 protein, are characterized in part by the presence of a conserved motif, (Q/I)X3(R/H)XRX3Q, termed here the QRRQ motif. We show that mutation of conserved residues within the Rcf1 QRRQ motif alters the interactions between Rcf1 and partner proteins and results in the destabilization of complex IV and alteration of its enzymatic properties. Our findings indicate that Rcf1 does not serve as a stoichiometric component, i.e. as a subunit of complex IV, to support its activity. Rather, we propose that Rcf1 serves to dynamically interact with complex IV during its assembly process and, in doing so, regulates a late maturation step of complex IV. We speculate that the Rcf1/Hig1 proteins play a role in the incorporation and/or remodeling of lipids, in particular cardiolipin, into complex IV and. possibly, other mitochondrial proteins such as ADP/ATP carrier proteins.
There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were “uncoupling” NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.
Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) are important modulators of endothelial cell homeostasis. We investigated the signaling pathway linking the activation of CYP 2C9 to enhanced endothelial cell proliferation. Overexpression of CYP 2C9 in cultured human endothelial cells markedly increased proliferation. This effect was paralleled by an up-regulation of the G1 phase regulatory protein, cyclin D1. The specific CYP 2C9 inhibitor, sulfaphenazole, prevented both the enhanced cell proliferation and up-regulation of cyclin D1. CYP 2C9 overexpression also decreased the activity of the c-Jun N-terminal kinase (JNK). Coexpression of wild type JNK with CYP 2C9 attenuated the CYP 2C9-induced increase in cyclin D1 expression and abolished the CYP 2C9-induced proliferation response. In contrast, cotransfecting dominant negative JNK with CYP 2C9 restored the CYP 2C9-mediated up-regulation of cyclin D1 and proliferation. The inactivation of JNK is linked to its dephosphorylation by dual specificity mitogen-activated protein (MAP) kinase phosphatases (MKPs). Overexpression of CYP 2C9 significantly increased the expression of MKP-1, as did incubation with 11,12-EET. These data demonstrate that the mitogenic effect of CYP 2C9 is due to the generation of EETs, which promote the MKP-1-mediated dephosphorylation and inactivation of JNK, effects ultimately culminating in the expression of cyclin D1 and endothelial cell proliferation.
Over the last decade, cases of metabolic syndrome and type II diabetes have increased exponentially. Exercise and ω-3 polyunsaturated fatty acid (PUFA)-enriched diets are usually prescribed but no therapy is effectively able to restore the impaired glucose metabolism, hypertension, and atherogenic dyslipidemia encountered by diabetic patients. PUFAs are metabolized by different enzymes into bioactive metabolites with anti- or pro-inflammatory activity. One important class of PUFA metabolizing enzymes are the cytochrome P450 (CYP) enzymes that can generate a series of bioactive products, many of which have been attributed protective/anti-inflammatory and insulin-sensitizing effects in animal models. PUFA epoxides are, however, further metabolized by the soluble epoxide hydrolase (sEH) to fatty acid diols. The biological actions of the latter are less well understood but while low concentrations may be biologically important, higher concentrations of diols derived from linoleic acid and docosahexaenoic acid have been linked with inflammation. One potential application for sEH inhibitors is in the treatment of diabetic retinopathy where sEH expression and activity is elevated as are levels of a diol of docosahexaenoic acid that can induce the destabilization of the retina vasculature.
Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. In this study, we investigated the involvement of the forkhead box, class O (FOXO) family of transcription factors and their downstream target p27Kip1 in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induced a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 was not significantly affected. This effect on p27Kip1 protein was associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulated the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an “Akt-resistant”/constitutively active FOXO3a mutant reversed the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreased p27Kip1 expression independently of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicited endothelial cell proliferation and this effect was inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. Reducing FOXO expression using RNA interference, on the other hand, attenuated p27Kip1 expression and stimulated endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1.
Tetrahydrobiopterin, a critical factor in the production and role of nitric oxide in mast cells
(2003)
Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-γ (IFN-γ) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-γ-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-γ and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-γ induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.
A gene trap strategy has been used to identify genes that are repressed in cells transformed by an activated epidermal growth factor (EGF)/EGF receptor signal transduction pathway. EGF receptor-expressing NIH3T3 cells (HER1 cells) were infected with a retrovirus containing coding sequences for the human CD2 antigen and for secreted alkaline phosphatase in the U3 region. By selecting for and against CD2 expression, we obtained clones in which the gene trap had integrated into genes selectively repressed by EGF. Two of these clones encoded for the secreted extracellular matrix proteins TIMP3 and COL1A2. We show here that both genes are downstream targets of RAS and are specifically repressed by EGF-induced transformation. Moreover, this strategy tags tumor suppressor genes in their normal chromosomal location, thereby improving target-specific screens for antineoplastic drugs.
Objectives: To analyse carbapenemases in Proteus mirabilis and assess the performance of carbapenemase detection assays.
Methods: Eighty-one clinical P. mirabilis isolates with high-level resistance at least to ampicillin (>32 mg/L) or previous detection of carbapenemases were selected and investigated by three susceptibility testing methods (microdilution, automated susceptibility testing, and disk diffusion), six phenotypic carbapenemase assays (CARBA NP, modified carbapenemase inactivation method [CIM], modified zinc-supplemented CIM, simplified CIM, faropenem, and carbapenem-containing agar), two immunochromatographic assays, and whole-genome sequencing.
Results: Carbapenemases were detected in 43 of 81 isolates (OXA-48-like [n = 13]; OXA-23 [n = 12]; OXA-58 [n = 12]; New Delhi metallo-β-lactamase (NDM) [n = 2]; Verona integron–encoded metallo-β-lactamase (VIM) [n = 2]; Imipenemase (IMP) [n = 1]; Klebsiella pneumoniae carbapenemase (KPC) [n = 1]). Carbapenemase-producing Proteus were frequently susceptible to ertapenem (26/43; 60%), meropenem (28/43; 65%), ceftazidime (33/43; 77%), and some even to piperacillin-tazobactam (9/43; 21%). Sensitivity/specificity of phenotypic tests were 30% (CI: 17–46%)/89% (CI: 75–97%) for CARBA NP, 74% (CI: 60–85%)/82% (CI: 67–91%) for faropenem, 91% (CI: 78–97%)/82% (CI: 66–92%) for simplified CIM, and 93% (CI: 81–99%)/100% (CI: 91–100%) for modified zinc-supplemented CIM. An algorithm for improved detection was developed, which demonstrated sensitivity/specificity of 100% (CI: 92–100%)/100% (CI: 91–100%) on the 81 isolates, and 100% (CI: 29–100%)/100% (CI: 96–100%) in a prospective analysis of additional 91 isolates. Interestingly, several OXA-23-producing isolates belonged to the same clonal lineage reported previously from France.
Discussion: Current susceptibility testing methods and phenotypic tests frequently fail to detect carbapenemases in P. mirabilis, which could result in inadequate antibiotic treatment. In addition, the non-inclusion of blaOXA-23/OXA-58 in many molecular carbapenemase assays further impedes their detection. Therefore, the prevalence of carbapenemases in P. mirabilis is likely underestimated. With the herein proposed algorithm, carbapenemase-producing Proteus can be easily identified.
Adhesion of human pathogenic bacteria to endothelial cells is facilitated by fibronectin interaction
(2023)
Human pathogenic bacteria circulating in the bloodstream need to find a way to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, in particular to fibronectin (Fn). Here, we analysed the general role of EC-expressed Fn for bacterial adhesion. For this, we evaluated the expression levels of ECM coding genes in different ECs, revealing that Fn is the highest expressed gene and thereby, it is highly abundant in the ECM environment of ECs. The role of Fn as a mediator in bacterial cell-host adhesion was evaluated in adhesion assays of Acinetobacter baumannii, Bartonella henselae, Borrelia burgdorferi, and Staphylococcus aureus to ECs. The assays demonstrated that bacteria colocalised with Fn fibres, as observed by confocal laser scanning microscopy. Fn removal from the ECM environment (FN1 knockout ECs) diminished bacterial adherence to ECs in both static and dynamic adhesion assays to varying extents, as evaluated via absolute quantification using qPCR. Interactions between adhesins and Fn might represent the crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection.
A 24-year-old patient from Cameroon presented to our hospital because of a foreign structure in her left eye. To our knowledge, for the first time, fluorescent microscopy revealed motile microfilariae, and the diagnosis of loiasis was established. Despite substantial microfilaremia, eosinophilia only unmasked after the initiation of antiparasitic therapy.
Objectives: The aim of this study was to evaluate the development and status quo of the quality of high throughput in vitro diagnostic testing for tetanus and diphtheria antitoxin antibody (ATX) concentrations based on external quality assessment (EQA) data.
Methods: We analyzed manufacturer-specific data of 22 EQA surveys—each for the detection of tetanus and diphtheria ATX—to check the diagnostic strength of the corresponding in vitro diagnostic systems.
Results: While the results were mostly well aligned, individual surveys showed widely dispersed ATX concentrations. The medians of manufacturer collectives deviated from the overall median by up to 8.9-fold in the case of diphtheria ATX and by up to 3.5-fold in the case of tetanus ATX. Such a distribution in the results is particularly critical in the cut-off range for immunity and may lead to an incorrect assessment of vaccination status.
Conclusion: These results were surprising as there are International Standards for both ATX; however, the results may be linked to the high ATX concentration of the reference material, which deviates considerably from clinically significant concentrations. To increase the accuracy and diagnostic strength of both assays, we recommend a recalibration of the test systems and verification of their traceability to the International Standards.
Objectives: To prospectively evaluate lung ultrasound in comparison with radiography and computed tomography (CT) for detecting HIV-related lung diseases.
Methods: Ultrasound examinations in HIV-positive patients were evaluated by three raters; available conventional imaging was evaluated by another rater. Results were compared with each other and the definite diagnosis. Interrater reliability was calculated for each finding.
Results: Eighty HIV-positive patients received lung ultrasound examinations; 74 received conventional imaging. The overall sensitivity was 97.5% for CT, 90.7% for ultrasound and 78.1% for radiography. The most common diagnoses were Pneumocystis jirovecii pneumonia (21 cases) and bacterial pneumonia (17 cases). The most frequent and sensitive ultrasonographic findings were interstitial abnormalities indicated by B-lines, independent of the aetiology. Interrater reliability was high for interstitial abnormalities (ICC=0.82). The interrater reliability for consolidations and effusion increased during the study (r=0.88 and r=0.37, respectively).
Conclusions: Ultrasound is a fast, reliable and sensitive point-of-care tool, particularly in detecting interstitial lung disease, which is common in HIV-associated illness. It does not effectively discriminate between different aetiologies. A longer learning period might be required to reliably identify consolidations and effusions.
Background: International travel poses the risk of importing SARS-CoV-2 infections and introducing new viral variants into the country of destination. Established measures include mandatory quarantine with the opportunity to abbreviate it with a negative rapid antigen test (RAT).
Methods: A total of 1,488 returnees were tested for SARS-CoV-2 with both PCR and RAT no earlier than 5 days after arrival. We assessed the sensitivity and specificity of the RAT. Positive samples were evaluated for infectivity in vitro in a cell culture outgrowth assay. We tracked if participants who tested negative were reported positive within 2 weeks of the initial test.
Results: Potential infectiousness was determined based on symptom onset analysis, resulting in a sensitivity of the antigen test of 89% in terms of infectivity. The specificity was 100%. All positive outgrowth assays were preceded by a positive RAT, indicating that all participants with proven in vitro infectivity were correctly identified. None of the negative participants tested positive during the follow-up.
Conclusions: RAT no earlier than the 5th day after arrival was a reliable method for detecting infectious travellers and can be recommended as an appropriate method for managing SARS-CoV-2 travel restrictions. Compliance to the regulations and a high standard of test quality must be ensured.
Background: Influenza vaccination of healthcare workers (HCWs) is recommended to prevent the transmission of influenza to vulnerable patients. Nevertheless, vaccination coverage rates of HCWs in European countries have been low.
Aim: To investigate the relative and combined strength of sociocognitive variables, from past research, theory and a qualitative study, in explaining the motivation of HCWs to receive the influenza vaccine.
Methods: An anonymous, online questionnaire was distributed among HCWs in hospital settings in Belgium, Germany and the Netherlands between February and April 2013.
Findings: Attitude and past vaccination uptake explained a considerable amount of variance in the intention of HCWs to receive the influenza vaccine. Moreover, low perceived social norms, omission bias, low moral norms, being older, having no patient contact, and being Belgian or Dutch (compared with German) increased the probability of having no intention to receive the influenza vaccine compared with being undecided about vaccination. High intention to receive the influenza vaccine was shown to be more likely than being undecided about vaccination when HCWs had high perceived susceptibility of contracting influenza, low naturalistic views, and lower motivation to receive the vaccine solely for self-protection.
Conclusion: Country-specific interventions and a focus on different sociocognitive variables depending on the intention/lack of intention of HCWs to receive the influenza vaccine may be beneficial to promote vaccination uptake.
The sphingolipid sphingosine-1-phosphate (S1P) promotes tumor development through a variety of mechanisms including promoting proliferation, survival, and migration of cancer cells. Moreover, S1P emerged as an important regulator of tumor microenvironmental cell function by modulating, among other mechanisms, tumor angiogenesis. Therefore, S1P was proposed as a target for anti-tumor therapy. The clinical success of current cancer immunotherapy suggests that future anti-tumor therapy needs to consider its impact on the tumor-associated immune system. Hereby, S1P may have divergent effects. On the one hand, S1P gradients control leukocyte trafficking throughout the body, which is clinically exploited to suppress auto-immune reactions. On the other hand, S1P promotes pro-tumor activation of a diverse range of immune cells. In this review, we summarize the current literature describing the role of S1P in tumor-associated immunity, and we discuss strategies for how to target S1P for anti-tumor therapy without causing immune paralysis.
The clinical breakthrough of bone tissue engineering (BTE) depends on the ability to provide patients routinely with BTE products of consistent pharmacological quality. The bottleneck of this approach is the availability of stem cells. To avoid this, we suggest immobilization of random-donor-derived heterologous osteoinductive MSCs onto osteoconductive matrices. Such BTE products could then be frozen and, after thawing, could be released as ready-to-use products for permanent implantation during surgery. For this purpose, we developed a simple protocol for cryopreservation of BTE constructs and evaluated the effects of this procedure on human MSC (hMSCs) metabolic and osteogenic activity in vitro. Our findings show that hMSCs can be freeze-thawed on a β-TCP scaffold through a technically simple procedure. Treated cells sustained their metabolic activity and showed favorable osteogenic potential. Mechanistically, HIF1α and YBX1 genes were activated after freeze-thawing, and supposed to be linked to enhanced osteogenesis. However, the detailed mechanisms as to how the cryopreservation procedure beneficially affects the osteogenic potential of hMSCs remains to be evaluated. Additionally, we demonstrated that our BTE products could be stored for 3 days on dry ice; this could facilitate the supply chain management of cryopreserved BTE constructs from the site of manufacture to the operating room.
EphrinB2–EphB4 signaling is critical during embryogenesis for cardiovascular formation and neuronal guidance. Intriguingly, critical expression patterns have been discovered in cancer pathologies over the last two decades. Multiple connections to tumor migration, growth, angiogenesis, apoptosis, and metastasis have been identified in vitro and in vivo. However, the molecular signaling pathways are manifold and signaling of the EphB4 receptor or the ephrinB2 ligand is cancer type specific. Here we explore the impact of these signaling pathways in neurooncological disease, including glioma, brain metastasis, and spinal bone metastasis. We identify potential downstream pathways that mediate cancer suppression or progression and seek to understand it´s role in antiangiogenic therapy resistance in glioma. Despite the Janus-faced functions of ephrinB2–EphB4 signaling in cancer Eph signaling remains a promising clinical target.
To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.
Despite intensive research, glioblastoma remains almost invariably fatal. Various promising drugs targeting specific aspects of glioma biology, in addition to or as an alternative to antiproliferative chemotherapy, were not successful in larger clinical trials. Further insights into the biology of glioma and the mechanisms behind the evasive-adaptive response to targeted therapies is needed to help identify new therapeutic targets, prognostics, or predictive biomarkers. As a modulator of the canonically oncogenic Rho-GTPase pathway, Lipid phosphate phosphatase-related protein type 5 (LPPR5) is pivotal in influencing growth, angiogenesis, and therapeutic resistance. We used a GL261 murine orthotopic allograft glioma model to quantify the tumor growth and to obtain tissue for histological and molecular analysis. Epicortical intravital epi-illumination fluorescence video microscopy of the tumor cell spheroids was used to characterize the neovascular architecture and hemodynamics. GL261-glioma growth was delayed and decelerated after LPPR5 overexpression (LPPR5OE). We observed increased tumor cell apoptosis and decreased expression and secretion of vascular endothelial growth factor A in LPPR5OE glioma. Hence, an altered micro-angioarchitecture consisting of dysfunctional small blood vessels was discovered in the LPPR5OE tumors. Sunitinib therapy eliminated these vessels but had no effect on tumor growth or apoptosis. In general, LPPR5 overexpression generated a more benign, proapoptotic glioma phenotype with delayed growth and a dysfunctional vascular architecture.
Membrane-Phloretin Interaction, Infrared Raman, ESR Spectroscopy The transport inhibitor phloretin was bound to human red cell membrane and the concomitant structural changes were observed by spectroscopic methods. By the spin labeling method a decrease in fluidity of the membrane was found at 1 and 10 |iM concentrations of the reagent. This result was obtained with the 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, and the 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl lipid spin labels. Infrared spectroscopy of modified membranes revealed an intensity increase of the POO~ band at about 1250 cm-1. Moreover, a shift of the peak at 1050 cm -1 to 1100 cm-1 was observed in the presence of phloretin. Raman spectroscopy of the membranes did not contradict the results found with infrared and ESR spectroscopy: In the phloretin modified membrane we observed a lack of the band at 1085 cm-1, which leads to suggest that the POO" and/or C-C regions are less fluid. Changes of the extracted red cell membrane lipids were less characteristic, and the results differed from those found in red cell membrane.
1-anilino-naphthalene-8-sulfonate (ANS) fluorescence measurements have revealed that red blood cell membrane of the Rhnull type undergoes a transition at about 16 degrees C. In contrast, viscosity measurements of the extracted membrane lipids showed the usually observed transition at about 18 degrees C. Lower values of titratable sulfhydryl (SH) groups were observed in Rhnull membrane using 5,5'-dithiobis-(2-nitro-benzoic-acid) (Nbs2). In contrast, disulfide bonds in Rhnull membrane were estimated to be about 3 times the value of the controls. Spin labeling experiments using 2-(3-carboxypropyl)-4, 4 dimethyl-2-tridecyl 3-oxazolidinyloxyl were carried out with phospholipase A2 modified membranes. The mobile part of the spectra was significantly increased on the Rhnull membrane. In the presence of D-glucose, infrared spectrometry showed a larger reduction of the intensity of the POO-band in Rhnull membrane. In contrast to controls, binding of the reagent diethylpyrocarbonate resulted in no significant changes of the Rhnull membrane as determined by electron spin resonance (ESR) measurements. D-glucose transport activity was found to be at the upper level of a group of Rh positive and Rh negative persons. It is suggested that the intensity of the polar protein-lipid interaction is reduced in Rhnull membrane.
Background: Pancreatic ductal adenocarcinoma remains one of the most lethal malignancies, with few treatment options. NAPOLI 3 aimed to compare the efficacy and safety of NALIRIFOX versus nab-paclitaxel and gemcitabine as first-line therapy for metastatic pancreatic ductal adenocarcinoma (mPDAC).
Methods: NAPOLI 3 was a randomised, open-label, phase 3 study conducted at 187 community and academic sites in 18 countries worldwide across Europe, North America, South America, Asia, and Australia. Patients with mPDAC and Eastern Cooperative Oncology Group performance status score 0 or 1 were randomly assigned (1:1) to receive NALIRIFOX (liposomal irinotecan 50 mg/m2, oxaliplatin 60 mg/m2, leucovorin 400 mg/m2, and fluorouracil 2400 mg/m2, administered sequentially as a continuous intravenous infusion over 46 h) on days 1 and 15 of a 28-day cycle or nab-paclitaxel 125 mg/m2 and gemcitabine 1000 mg/m2, administered intravenously, on days 1, 8, and 15 of a 28-day cycle. Balanced block randomisation was stratified by geographical region, performance status, and liver metastases, managed through an interactive web response system. The primary endpoint was overall survival in the intention-to-treat population, evaluated when at least 543 events were observed across the two treatment groups. Safety was evaluated in all patients who received at least one dose of study treatment. This completed trial is registered with ClinicalTrials.gov, NCT04083235.
Findings: Between Feb 19, 2020 and Aug 17, 2021, 770 patients were randomly assigned (NALIRIFOX, 383; nab-paclitaxel–gemcitabine, 387; median follow-up 16·1 months [IQR 13·4–19·1]). Median overall survival was 11·1 months (95% CI 10·0–12·1) with NALIRIFOX versus 9·2 months (8·3–10·6) with nab-paclitaxel–gemcitabine (hazard ratio 0·83; 95% CI 0·70–0·99; p=0·036). Grade 3 or higher treatment-emergent adverse events occurred in 322 (87%) of 370 patients receiving NALIRIFOX and 326 (86%) of 379 patients receiving nab-paclitaxel–gemcitabine; treatment-related deaths occurred in six (2%) patients in the NALIRIFOX group and eight (2%) patients in the nab-paclitaxel–gemcitabine group.
Interpretation: Our findings support use of the NALIRIFOX regimen as a possible reference regimen for first-line treatment of mPDAC.
Background: The surgical treatment of giant olfactory groove meningiomas (OGMs) with marked perilesional brain oedema is still a surgical challenge. After tumour resection, increase of brain oedema may occur causing dramatic neurological deterioration and even death of the patient. The objective of this paper is to describe surgical features of a two-step staged resection of these tumours performed to counter increase of postoperative brain oedema.
Methods: This two-step staged resection procedure was carried out in a consecutive series of 19 patients harbouring giant OGMs. As first step, a bifrontal craniectomy was performed followed by a right-sided interhemispherical approach. About 80% of the tumour mass was resected leaving behind a shell-shaped tumour remnant. In the second step, carried out after the patients’ recovery from the first surgery and decline of oedema, the remaining part of the tumour was removed completely followed by duro- and cranioplasty.
Results: Ten patients recovered quickly from first surgery and the second operation was performed after a mean of 12.4 days. In eight patients, the second operation was carried out later between day 25 and 68 due to surgery-related complications, development of a trigeminal zoster, or to a persisting frontal brain oedema. Mean follow-up was 49.3 months and all but one patient had a good outcome regardless of surgery-related complications.
Conclusions: Our results suggest that a two-step staged resection of giant OGMs minimizes the increase of postoperative brain oedema as far as possible and translates into lower morbidity and mortality.
Acute kidney injury (AKI) complicates the clinical course of hospitalized patients by increasing need for intensive care treatment and mortality. There is only little data about its impact on AML patients undergoing intensive induction chemotherapy. In this study, we analyzed the incidence as well as risk factors for AKI development and its impact on the clinical course of AML patients undergoing induction chemotherapy. We retrospectively analyzed data from 401 AML patients undergoing induction chemotherapy between 2007 and 2019. AKI was defined and stratified according to KIDGO criteria by referring to a defined baseline serum creatinine measured on day 1 of induction chemotherapy. Seventy-two of 401 (18%) AML patients suffered from AKI during induction chemotherapy. AML patients with AKI had more days with fever (7 vs. 5, p = 0.028) and were more often treated on intensive care unit (45.8% vs. 10.6%, p < 0.001). AML patients with AKI had a significantly lower complete remission rate after induction chemotherapy and, with 402 days, a significantly shorter median overall survival (OS) (median OS for AML patients without AKI not reached). In this study, we demonstrate that the KIDGO classification allows mortality risk stratification for AML patients undergoing induction chemotherapy. Relatively mild AKI episodes have impact on the clinical course of these patients and can lead to chronic impairment of kidney function. Therefore, we recommend incorporating risk factors for AKI in decision-making considering nutrition, fluid management, as well as the choice of potentially nephrotoxic medication in order to decrease the incidence of AKI.
Purpose: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) replicates predominantly in the upper respiratory tract and is primarily transmitted by droplets and aerosols. Taking the medical history for typical COVID-19 symptoms and PCR-based SARS-CoV-2 testing have become established as screening procedures. The aim of this work was to describe the clinical appearance of SARS-CoV-2-PCR positive patients and to determine the SARS-CoV-2 contact risk for health care workers (HCW).
Methods: The retrospective study included n = 2283 SARS-CoV-2 PCR tests from n = 1725 patients with otorhinolaryngological (ORL) diseases performed from March to November 2020 prior to inpatient treatment. In addition, demographic data and medical history were assessed.
Results: n = 13 PCR tests (0.6%) were positive for SARS-CoV-2 RNA. The positive rate showed a significant increase during the observation period (p < 0.01). None of the patients had clinical symptoms that led to a suspected diagnosis of COVID-19 before PCR testing. The patients were either asymptomatic (n = 4) or had symptoms that were interpreted as symptoms typical of the ORL disease or secondary diagnoses (n = 9).
Conclusion: The identification of SARS-CoV-2-positive patients is a considerable challenge in clinical practice. Our findings illustrate that taking a medical history alone is of limited value and cannot replace molecular SARS-CoV-2 testing, especially for patients with ORL diseases. Our data also demonstrate that there is a high probability of contact with SARS-CoV-2-positive patients in everyday clinical practice, so that the use of personal protective equipment, even in apparently “routine cases”, is highly recommended.
Evaluation of a rapid turn-over, fully-automated ADAMTS13 activity assay: a method comparison study
(2020)
Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy caused by severely reduced activity of the von-Willebrand factor-cleaving protease ADAMTS13, mainly caused by anti-ADAMTS-13 antibodies. Although several test systems for ADAMTS13 measurement exist, long turn-around times hamper the usability in daily practice. We performed a method comparison study for two commercially available ADAMTS13 assays and evaluated the agreement between the fully-automated rapid turn-over HemosIL AcuStar ADAMTS13 Activity assay and the manually performed TECHNOZYM ADAMTS-13 Activity assay. Twenty-four paired test samples derived from 10 consecutively recruited patients (n = 8, acquired TTP; n = 1, atypical hemolytic uremic syndrome; n = 1, control), of which nine test samples were collected in case of clinically apparent TTP and 13 samples were collected from TTP patients in clinical remission were included. Overall correlation between the TECHNOZYM and AcuStar assay was good with a Pearson R of 0.93 (p < 0.001). Agreement between the assays assessed with the Passing–Bablok analysis showed high agreement with an Intercept of − 2.56 (95% confidence interval [CI], − 5.07 to − 0.86) and Slope of 1.04 (95% CI 0.84–1.17). The absolute mean bias was 2.54% (standard difference [SD], 6.38%; 95% CI to 10.0–15.05%). Intra-method reliability was high with an absolute mean bias of − 0.13% (SD 3.21%; 95% CI to 6.42–6.16%). The observer agreement for categorial thresholds (> or < 10% ADAMTS3 activity) was kappa = 0.82 (95% CI 0.59–1.0). Conclusively, overall agreement between the testing methods was sufficient and we support previously published data suggesting the AcuStar assay being a valuable and accurate tool for ADAMTS13 activity testing and TTP diagnostics.
Macrophages acquire anti-inflammatory and proresolving functions to facilitate resolution of inflammation and promote tissue repair. While alternatively activated macrophages (AAMs), also referred to as M2 macrophages, polarized by type 2 (Th2) cytokines IL-4 or IL-13 contribute to the suppression of inflammatory responses and play a pivotal role in wound healing, contemporaneous exposure to apoptotic cells (ACs) potentiates the expression of anti-inflammatory and tissue repair genes. Given that liver X receptors (LXRs), which coordinate sterol metabolism and immune cell function, play an essential role in the clearance of ACs, we investigated whether LXR activation following engulfment of ACs selectively potentiates the expression of Th2 cytokine-dependent genes in primary human AAMs. We show that AC uptake simultaneously upregulates LXR-dependent, but suppresses SREBP-2-dependent gene expression in macrophages, which are both prevented by inhibiting Niemann–Pick C1 (NPC1)-mediated sterol transport from lysosomes. Concurrently, macrophages accumulate sterol biosynthetic intermediates desmosterol, lathosterol, lanosterol, and dihydrolanosterol but not cholesterol-derived oxysterols. Using global transcriptome analysis, we identify anti-inflammatory and proresolving genes including interleukin-1 receptor antagonist (IL1RN) and arachidonate 15-lipoxygenase (ALOX15) whose expression are selectively potentiated in macrophages upon concomitant exposure to ACs or LXR agonist T0901317 (T09) and Th2 cytokines. We show priming macrophages via LXR activation enhances the cellular capacity to synthesize inflammation-suppressing specialized proresolving mediator (SPM) precursors 15-HETE and 17-HDHA as well as resolvin D5. Silencing LXRα and LXRβ in macrophages attenuates the potentiation of ALOX15 expression by concomitant stimulation of ACs or T09 and IL-13. Collectively, we identify a previously unrecognized mechanism of regulation whereby LXR integrates AC uptake to selectively shape Th2-dependent gene expression in AAMs.