Secretion by Overexpression and Purification of the Water-Soluble Streptomyces K15 Dd-Transpeptidase/Penicillin-Binding Protein

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity

Nanotube Action between Human Mesothelial Cells Reveals Novel Aspects of Inflammatory Responses

A well-known role of human peritoneal mesothelial cells (HPMCs), the resident cells of the peritoneal cavity, is the generation of an immune response during peritonitis by activation of T-cells via antigen presentation. Recent findings have shown that intercellular nanotubes (NTs) mediate functional connectivity between various cell types including immune cells - such as T-cells, natural killer (NK) cells or macrophages - by facilitating a spectrum of long range cell-cell interactions. Although of medical interest, the relevance of NT-related findings for human medical conditions and treatment, e.g. in relation to inflammatory processes, remains elusive, particularly due to a lack of appropriate in vivo data. Here, we show for the first time that primary cultures of patient derived HPMCs are functionally connected via membranous nanotubes. NT formation appears to be actin cytoskeleton dependent, mediated by the action of filopodia. Importantly, significant variances in NT numbers between different donors as a consequence of pathophysiological alterations were observable. Furthermore, we show that TNF-α induces nanotube formation and demonstrate a strong correlation of NT connectivity in accordance with the cellular cholesterol level and distribution, pointing to a complex involvement of NTs in inflammatory processes with potential impact for clinical treatment

Aortic root surgery in septuagenarians: impact of different surgical techniques

<p>Abstract</p> <p>Background</p> <p>To evaluate the impact and safety of different surgical techniques for aortic root replacement (ARR) on early and late morbidity and mortality in septuagenarians undergoing ARR.</p> <p>Methods</p> <p>Ninety-five patients (73.8 ± 3.2 years) were operated and divided into three groups according to the aortic root procedure; MECH-group (n = 51) patients with a mechanical composite graft, BIO-group (n = 22) patients with a customized biological composite graft, and REIMPL-group (n = 22) patients with a valve sparing aortic root reimplantation (David I). In 42.1% (40/95) of these patients the aortic arch was replaced. Follow-up was completed in 95.2% (79/83) of in-hospital survivors.</p> <p>Results</p> <p>Hospital mortality was 12.6% (12/95) in the entire population (MECH. 15.7% (8/51), BIO 19.7% (4/22), REIMPL 0% (0/22); p = 0.004). Two patients died intraoperatively. The most frequent postoperative complications were prolonged mechanical ventilation ((>48 h) in 16.8% (16/93) (MECH. 7% (7/51), BIO 36.4% (8/22), REIMPL 4.5% (1/22); p = 0.013) and rethoracotomy for postoperative bleeding in 12.6% (12/95) (MECH. 12% (6/51), BIO 22.7% (5/22), REIMPL 4.5% (1/22); p = 0.19). Nineteen late deaths (22.9%) (19/83) (MECH 34.8% (15/43), BIO 16.7% (3/18), REIMPL 4.5% (1/22); p = 0.012) occurred during a mean follow-up of 41 ± 42 months (MECH 48 ± 48 months, BIO 25 ± 37 months, REIMPL 40 ± 28 months, p = 0.028). Postoperative NYHA class decreased significantly (p = 0.017) and performance status (p = 0.027) increased for the entire group compared to preoperative values.</p> <p>Conclusion</p> <p>Our data indicate that valve sparing aortic root reimplantation is safe and effective in septuagenarians, and is associated with low early and late morbidity and mortality.</p

Predictors of packed red cell transfusion after isolated primary coronary artery bypass grafting – The experience of a single cardiac center: A prospective observational study

<p>Abstract</p> <p>Background</p> <p>Preoperative patients' characteristics can predict the need for perioperative blood component transfusion in cardiac surgical operations. The aim of this prospective observational study is to identify perioperative patient characteristics predicting the need for allogeneic packed red blood cell (PRBC) transfusion in isolated primary coronary artery bypass grafting (CABG) operations.</p> <p>Patients and Methods</p> <p>105 patients undergoing isolated, first-time CABG were reviewed for their preoperative variables and followed for intraoperative and postoperative data. Patients were 97 males and 8 females, with mean age 58.28 ± 10.97 years. Regression logistic analysis was used for identifying the strongest perioperative predictors of PRBC transfusion.</p> <p>Results</p> <p>PRBC transfusion was used in 71 patients (67.6%); 35 patients (33.3%) needed > 2 units and 14 (13.3%) of these needed > 4 units. Univariate analysis identified female gender, age > 65 years, body weight ≤ 70 Kg, BSA ≤ 1.75 m², BMI ≤ 25, preoperative hemoglobin ≤ 13 gm/dL, preoperative hematocrit ≤ 40%, serum creatinine > 100 μmol/L, Euro SCORE (standard/logistic) > 2, use of CPB, radial artery use, higher number of distal anastomoses, and postoperative chest tube drainage > 1000 mL as significant predictors. The strongest predictors using multivariate analysis were CPB use, hematocrit, body weight, and serum creatinine.</p> <p>Conclusion</p> <p>The predictors of PRBC transfusion after primary isolated CABG are use of CPB, hematocrit ≤ 40%, weight ≤ 70 Kg, and serum creatinine > 100 μmol/L. This leads to better utilization of blood bank resources and cost-efficient targeted use of expensive blood conservation modalities.</p

Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis

BACKGROUND: Acidithiobacillus ferrooxidans is a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences of A. ferrooxidans are available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism. RESULTS: Two candidate mechanisms for sulfate uptake from the environment were detected but both belong to large paralogous families of membrane transporters and their identification remains tentative. Prospective genes, pathways and regulatory mechanisms were identified that are likely to be involved in the assimilation of sulfate into cysteine and in the formation of Fe-S centers. Genes and regulatory networks were also uncovered that may link sulfur assimilation with nitrogen fixation, hydrogen utilization and sulfur reduction. Potential pathways were identified for sulfation of extracellular metabolites that may possibly be involved in cellular attachment to pyrite, sulfur and other solid substrates. CONCLUSIONS: A bioinformatic analysis of the genome sequence of A. ferrooxidans has revealed candidate genes, metabolic process and control mechanisms potentially involved in aspects of sulfur metabolism. Metabolic modeling provides an important preliminary step in understanding the unusual physiology of this extremophile especially given the severe difficulties involved in its genetic manipulation and biochemical analysis

The penicillin receptor in Streptomyces

Kinetics and optical studies of Streptomyces DD-carboxypeptidases-transpeptidases led to the conclusion that the donor, acceptor, and penicillin sites on these enzymes are different but not independent and that penicillin acts as a modifier of the conformation of the protein. In the presence of penicillin, the penicillin-sensitive enzymes would be frozen in a conformation that prevents catalytic activity

The DZHK research platform: maximisation of scientific value by enabling access to health data and biological samples collected in cardiovascular clinical studies

The German Centre for Cardiovascular Research (DZHK) is one of the German Centres for Health Research and aims to conduct early and guideline-relevant studies to develop new therapies and diagnostics that impact the lives of people with cardiovascular disease. Therefore, DZHK members designed a collaboratively organised and integrated research platform connecting all sites and partners. The overarching objectives of the research platform are the standardisation of prospective data and biological sample collections among all studies and the development of a sustainable centrally standardised storage in compliance with general legal regulations and the FAIR principles. The main elements of the DZHK infrastructure are web-based and central units for data management, LIMS, IDMS, and transfer office, embedded in a framework consisting of the DZHK Use and Access Policy, and the Ethics and Data Protection Concept. This framework is characterised by a modular design allowing a high standardisation across all studies. For studies that require even tighter criteria additional quality levels are defined. In addition, the Public Open Data strategy is an important focus of DZHK. The DZHK operates as one legal entity holding all rights of data and biological sample usage, according to the DZHK Use and Access Policy. All DZHK studies collect a basic set of data and biosamples, accompanied by specific clinical and imaging data and biobanking. The DZHK infrastructure was constructed by scientists with the focus on the needs of scientists conducting clinical studies. Through this, the DZHK enables the interdisciplinary and multiple use of data and biological samples by scientists inside and outside the DZHK. So far, 27 DZHK studies recruited well over 11,200 participants suffering from major cardiovascular disorders such as myocardial infarction or heart failure. Currently, data and samples of five DZHK studies of the DZHK Heart Bank can be applied for

A Photoelectron Spectroscopic Investigation of Vinyl Fluoride (C2H3F). The HeI, Threshold and CIS Photoelectron Spectroscopy.

The threshold photoelectron spectrum (TPES) and the constant ion state (CIS) spectra of the individual ionic states of C2H3F have been recorded using synchrotron radiation. For comparison a well resolved HeI photoelectron spectrum (HeI-PES) has also been measured and analyzed in detail. The TPES has been measured between 9.5 eV and 35 eV photon energy. Numerous vibrational structures, reported for the first time, observed in the ground state and the six excited states of the cation are analyzed. Quantum chemical calculations have been performed and provide strong support to the assignments. State selected CIS spectra highlighted the major importance of autoionization for the production of almost all ionized states of C2H3F observed in this work