SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. ONE SENTENCE SUMMARY: A novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.

[Network for Oncological Advisory Service (NOF) - a Pilot Project for (Long-Term) Follow-Up Care of Pediatric Cancer Patients]. / Netzwerk für onkologische Fachberatung (NOF) ­ Modellprojekt für (Langzeit-) Nachsorge nach einer Krebserkrankung im Kindes- und Jugendalter.

Background: In Germany some 2 000 children and adolescent are diagnosed with cancer every year. Curing rates are increasing and therewith also the number of survivors is growing. Survivors frequently suffer from long-term effects of the disease and its treatment, but long-term follow-up care shows deficits. Method: The Network for oncological advisory service (NOF) started in 11/2013, researching and building up a network of available support in Lower Saxony. A telephone hotline was installed in 01/2014 in order to advice survivors on their problems. At the same time, an interview study on survivors needs was conducted throughout Germany. Results: In the first 2 years, the NOF gave advice to 79 patients. Whilst enquiries of medical or psychological nature were transferred to the cooperation partner, requests on psychosocial and social legal issues are being deled by the NOF due to lack of appropriate partners. The evaluation of 25 interviews shows key issues in long-term after-care: (1) transition from acute therapy to everyday life, (2) problems due to pediatric cancer and therapy, (3) patients perception of own disposition, (4) social reactions towards survivors, (5) structure of long-term follow-up care, (6) information flow. Conclusion: Many survivors suffer from long-term effects of cancer and treatment. The lack of available contact person and being in limbo between cured and simultaneously affected by the cancer treatment and chronic diseases is perceived as being problematic. This translates to various requirements on a patient-oriented long-term care, mainly in the psychosocial field.

Transformation of chlorinated benzenes and toluenes by Ralstonia sp. strain PS12 tecA (tetrachlorobenzene dioxygenase) and tecB (chlorobenzene dihydrodiol dehydrogenase) gene products.

The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5alpha together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3- and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di- and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloro- and 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12.

Genetic and biochemical analyses of the tec operon suggest a route for evolution of chlorobenzene degradation genes.

The TecA broad-spectrum chlorobenzene dioxygenase of Burkholderia sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4, 5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPbeta incompatibility group. PCR analysis of the genetic environment of the tec genes indicated high similarity to the transposon-organized catabolic tcb chlorobenzene degradation genes of Pseudomonas sp. strain P51. Sequence analysis of the regions flanking the tecA genes revealed an upstream open reading frame (ORF) with high similarity to the todF 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene of Pseudomonas putida F1 and a discontinuous downstream ORF showing high similarity to the todE catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing meta-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in Escherichia coli provided data consistent with the proposed relationships.

Identification of chlorobenzene dioxygenase sequence elements involved in dechlorination of 1,2,4,5-tetrachlorobenzene.

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large alpha-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific todC1 alpha-subunit subsequences by equivalent sequences of the tecA1 alpha-subunit. Substrate transformation specificities and rates by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.

Angiotensin-converting enzyme inhibitors in preventing remodeling and development of heart failure after acute myocardial infarction: results of the German multicenter study of the effects of captopril on cardiopulmonary exercise parameters (ECCE).

Early action of angiotensin-converting enzyme (ACE) inhibitors after myocardial infarction (MI) has been shown in large scale clinical trials to reduce mortality over the first weeks. However, the mechanisms involved are yet unclear and several trials showed a tendency toward a small, albeit unexpected, rise in cardiogenic shock or mortality. Since cardiopulmonary exercise testing (CPX) has become a "gold standard" in assessing the severity of heart failure, we studied--after finishing a pilot trial--the effect of captopril versus placebo in 208 patients who were individually titrated (titrated dose, mean 46/69 mg/day after 7 days/4 weeks, respectively) in order to preserve their blood pressure in the acute phase of myocardial infarction; we followed the development of congestive heart failure (CHF) over 4 weeks by measuring oxygen consumption. After 4 weeks, overall oxygen consumption at the anaerobic threshold (VO2-AT; 13.7 vs 13.1), maximal oxygen consumption (VO2max 19.3 vs 18.9 mL/kg per min) and exercise duration (896 vs 839 sec) showed a nonsignificant difference in favor of the captopril group. The predefined, categorized, combined endpoint of severe heart failure or death (heart failure necessitating ACE inhibition, VO2max < 10 mL/kg per min, or death) was significantly reduced in the captopril group (n = 7/104) versus placebo (n = 18/104; p = 0.03). Differences were mainly caused by fewer CHF events (delta n = 10). We conclude that ACE inhibition with individualized dose titration markedly reduces the 4-week incidence of severe heart failure or death; > 10 patients per 100 treated gained major benefits from this therapy.

Genetic and biochemical characterization of the broad spectrum chlorobenzene dioxygenase from Burkholderia sp. strain PS12--dechlorination of 1,2,4,5-tetrachlorobenzene.

The bacterium, Burkholderia (previously Pseudomonas) sp. strain PS12, reported earlier to degrade 1,2,4-trichlorobenzene is shown here to utilize also 1,2,4,5-tetrachlorobenzene (Cl4-benzene) as a growth substrate. To investigate the possibility that this organism attacks Cl4-benzene with a chlorobenzene dioxygenase which concomitantly causes dehalogenation, and to analyze the substrate range of the initial enzyme, a 5503-bp DNA fragment from PS12, exhibiting high similarity to genes coding for class IIB dioxygenases, was cloned and expressed in Escherichia coli. The sequence includes the tec genes coding for the alpha-subunit and beta-subunit of a terminal dioxygenase, a ferredoxin and a reductase. E. coli cells producing these proteins were able to dioxygenolytically attack a range of aromatic compounds including chlorinated benzenes and toluene, and also dinuclear aromatics such as biphenyl and dibenzo-p-dioxin. The enzyme was shown by (18)O2 incorporation experiments to dioxygenolytically attack a chlorosubstituted carbon atom of Cl4-benzene, thereby forming an unstable diol intermediate which spontaneously rearomatizes with concomitant chloride elimination to the corresponding 3,4,6-trichlorocatechol (Cl3-catechol).

Effect of metoprolol and amlodipine on myocardial total ischaemic burden in patients with stable angina pectoris.

OBJECTIVE: A randomized double-blind cross-over study to assess the effect on myocardial total ischaemic burden and the anti-anginal efficacy of the beta-1-blocker metoprolol given as metoprolol CR/Zok versus the calcium channel blocker amlodipine, both given in the recommended and commonly used doses of 100 mg o.d. and 5 mg o.d., respectively. METHOD: Fifty-two patients with a history of stable exercise-induced angina pectoris and at least six episodes of significant ST-segment depression during 24-h ambulatory electrocardiographic monitoring after 9 days of placebo were included in the study. The patients first completed a 9-day placebo run-in phase with additional administration of a long-acting nitrate during the first 7 days. They then received in a randomized sequence metoprolol CR/Zok and amlodipine each for 4 weeks. During placebo treatment and at the end of each phase of active treatment the patients' clinical progress was assessed and a 24-h ECG monitor performed. RESULTS: Forty-seven patients completed the two 4-week treatment periods. Five patients withdrew from the study. The number of ischaemic episodes during 24 h was 30.4 at baseline with placebo, which was reduced significantly by both treatments (P<0.005) to 6.8 episodes after metoprolol and 15.8 episodes after amlodipine treatment (P<0.001 for the difference between the treatment groups). Metoprolol and amlodipine reduced the total duration of ischaemic episodes from 86.0 min to 15-1 and 48.3 min with a mean episode duration of 1.1 and 2.6 min, respectively. Twenty patients on metoprolol (42.6%) and four on amlodipine (8.4%) showed no ST-segment depression at the end of treatment. Baseline heart rate of 80 b.p.m. decreased by 11.1 b.p.m. after metoprolol and increased by 4b.p.m. after amlodipine. Anginal attacks were reduced from 14.8 attacks per week at baseline to 2.4 attacks on metoprolol and 4.4 attacks on amlodipine treatment. For all variables the observed changes from baseline were significant after either treatment (P<0.005) with a significantly more pronounced effect in favour of metoprolol (P<0.0001). Ten patients reported nine different adverse events during metoprolol treatment. On amlodipine, 12 patients were affected by 11 different symptoms leading to three treatment withdrawals. On metoprolol one patient withdrew due to adverse events. CONCLUSION: Both drugs reduce total ischaemic burden by reducing ischaemic episodes and antianginal attacks, with a significantly greater effect from metoprolol 100 mg o.d. as compared to amlodipine 5 mg o.d.

The assimilation of sulfur from multiple sources and its correlation with expression of the sulfate-starvation-induced stimulon in Pseudomonas putida S-313.

Conditions were optimized for the batch growth of Pseudomonas putida S-313 under sulfur-limited conditions. P. putida grew exponentially with sulfate as the sole source of sulfur, and growth was concomitant with the utilization of sulfate until it was exhausted. A further 20% of protein was synthesized after the apparent disappearance of sulfate. A mass balance for the utilized sulfate in cell material was calculated, given the observed molar growth yield of about 3.6 kg protein (mol S)-1 and a sulfur content of 0.41% S in dry matter. Similar data were obtained for growth with cysteine and thiocyanate. The organism also grew exponentially with 4-toluenesulfonate (TS) as sulfur source, essentially as observed with sulfate, except that negligible protein formation after exhaustion of TS was observed. Similar data were also obtained with 4-nitrocatecholsulfate (NCS) and ethanesulfonate. Any substrate pair selected from sulfate, cysteine and thiocyanate was utilized simultaneously, and although one of the pair of substrates was always preferred, growth continued at the same rate when only one substrate remained. Growth after substrate exhaustion was observed. Any substrate pair selected from TS, NCS and ethanesulfonate gave similar data, but with less growth after exhaustion of the sulfur sources. If a mixed substrate pair was chosen from the two groups, the sulfur source from the first-named group was initially used exclusively, and the second source of sulfur was utilized subsequently, after a lag phase. The data are considered to reflect the control of scavenging for sulfur and of distribution of sulfur in the cell exerted by the sulfate-starvation-induced stimulon.

Purification and characterization of the arylsulfatase synthesized by Pseudomonas aeruginosa PAO during growth in sulfate-free medium and cloning of the arylsulfatase gene (atsA).

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PAO1 and purified 2700-fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDS/PAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57 degrees C and pH 8.9, and a Km of 105 microM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in P. aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate-repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5-751 mutant of P. aeruginosa PAO1. Sequencing revealed a 1602-bp reading frame encoding a 534-amino-acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30-40% and 25-30% identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the P. aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15-17' on the P. aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.

Desulfonation of linear alkylbenzenesulfonate surfactants and related compounds by bacteria.

Pseudomonas putida S-313 (= DSM 6884) grew in sulfate-free medium when the sole sulfur source supplied was one of several arylsulfonates involved in the synthesis, application, or biodegradation of linear alkyl-benzenesulfonate (LAS) surfactants. 2-(4-Sulfophenyl)butyric acid, 4-n-butyl-1-methyl-6-sulfotetralin, and 4-toluenesulfonic acid were each completely utilized during growth, as were the model LAS 1-(4-sulfophenyl) octane and the arylsulfonate dyestuff Orange II. The product in each case was the corresponding phenol, which was identified by gas chromatography-mass spectrometry or H nuclear magnetic resonance. Stoichiometric conversion of 4-toluenesulfonic acid to 4-cresol was observed. The molar growth yields observed were 2.4 to 2.8 kg of protein per mol of S, which were comparable to the yield for sulfate. Commercial LAS disappeared from growth medium inoculated with strain S-313, but negligible growth occurred; digestion of cells in alkali led to recovery of the LAS mixture, which seemingly sorbed to the cells. However, mixed culture L6 was readily obtained from batch enrichment cultures containing commercial LAS as a sole sulfur source and an inoculum from domestic sewage. Culture L6 desulfonated components of the LAS surfactant to the corresponding phenols, which were identified by gas chromatography-mass spectrometry. Compounds with shorter alkyl chains were desulfonated preferentially, as were the centrally substituted isomers. In the presence of 200 muM sulfate, culture L6 grew well and LAS disappeared, although this was due purely to sorption, as shown by digestion of the cells in alkali. Thus, under sulfate-limited conditions, LAS can be desulfonated directly.

Experiences with ACE inhibitors early after acute myocardial infarction. Rationale and design of the German Multicenter Study on the Effects of Captopril on Cardiopulmonary Exercise parameters post myocardial infarction (ECCE).

Left ventricular damage by necrosis of myocardial tissue can lead to compromise of left ventricular function, to left ventricular volume increase and ultimately to development of heart failure. This sequence in the pathophysiology has been shown to be blunted by ACE inhibitors. Volume increase, however, can also be helpful in restoring stroke volume and ameliorate elevation of filling pressures. Furthermore, very early institution of ACE inhibition has failed to improve short-term mortality after myocardial infarction in one large trial. The aim of the ECCE trial therefore is, to investigate the early effects of the ACE inhibitor captopril on compromise of exercise capacity, thought to be a first measurable sign of developing heart failure. The ECCE trial is a randomized, seven-center investigation, studying the effects of ACE inhibition on oxygen uptake in a double blind, placebo controlled design in a group of 204 patients. Sample size was calculated on the basis of a pilot trial. The study design and first not unblinded data of 104 patients are presented. The population consists of predominantly male patients with mostly first myocardial infarction. They were admitted to hospital within five hours of onset of chest pain. End-diastolic volumes were normal, but ejection fraction was moderately compromised. ACE inhibition was started after the first day, but within 72 hours of onset of chest pain. After four and after twelve weeks, oxygen uptake was considerably below expected values and one third of the patients had severe compromise of exercise capacity.(ABSTRACT TRUNCATED AT 250 WORDS)