Looking at the future of manufacturing metrology: roadmap document of the German VDI/VDE Society for Measurement and Automatic Control

"Faster, safer, more accurately and more flexibly'' is the title of the "manufacturing metrology roadmap'' issued by the VDI/VDE Society for Measurement and Automatic Control (<a href="http://www.vdi.de/gma"target="_blank">http://www.vdi.de/gma</a>). The document presents a view of the development of metrology for industrial production over the next ten years and was drawn up by a German group of experts from research and industry. The following paper summarizes the content of the roadmap and explains the individual concepts of "Faster, safer, more accurately and more flexibly'' with the aid of examples

Energy conservation in acetogenic bacteria : caffeate reduction in Acetobacterium woodii

Aus Fruktose-gezogenen Zellen von A. woodii, die in Gegenwart von Caffeat als Elektronenakzeptor gewachsen waren, wurde durch Behandlung mit Lysozym und anschliessendem French-Press-Aufschluß bei niedrigem Druck ein zellfreier Rohextrakt unter strikt anaeroben Bedingungen hergestellt. Dieser katalysierte eine H2-abhängige Caffeatreduktion mit Raten von 8,7 – 18,7 nmol/min x mg Protein. Die Aktivität war strikt ATP-abhängig. Nach einer Auftrennung des Rohextraktes in Cytoplasma- und Membranfraktion war die H2-abhängige Caffeatreduktion ausschliesslich in der cytoplasmatischen Fraktion lokalisiert. Die Zugabe von Membranen führte zu keiner Stimulierung der Aktivität. Die Membranfraktion selbst wies keine Caffeatreduktionsaktivität auf. Verschiedene Verbindungen wurden auf ihre Fähigkeit getestet, als artifizielle Elektronendonatoren für die Caffeatreduktion zu fungieren. TMPD, 1,5-Diphenylcarbazid und reduziertes Methylviologen konnten Caffeat nicht reduzieren. In Gegenwart von Phenylendiamin wurde in zellfreiem Rohextrakt und in der Cytoplasmafraktion Caffeatreduktionsaktivität beobachtet. In der Membranfraktion wurde dagegen keine Reduktion von Caffeat mit Phenylendiamin als Elektronendonor nachgewiesen. NADH + H+ konnte als physiologischer Elektronendonor für die Reduktion von Caffeat fungieren. Die NADH-abhängige Caffeatreduktion war ausschliesslich in der cytoplasmatischen Fraktion lokalisiert und strikt abhängig von ATP. Die Beobachtung, dass NADH + H+ als Elektronendonor für die Caffeareduktion fungieren kann, führte zu der Frage, wie im Zuge H2-abhängiger Caffeatreduktion NADH + H+ gebildet wird und welche Enzyme daran beteiligt sein könnten. NAD+-abhängige Hydrogenaseaktivität wurde an Membranen und im Cytoplasma nachgewiesen. Rund 70% der Aktivität waren in der löslichen Fraktion lokalisiert. Die Gegenwart einer Elektronendonor:NAD+-Oxidoreduktase in A. woodii wurde untersucht. Gewaschene Membranen vermittelten die Oxidation von NADH + H+ mit Kaliumhexacyanoferrat oder Benzylviologen als Elektronenakzeptor. Diese Beobachtung wurde als Hinweis auf eine NAD+-Reduktase gewertet, da solche Enzyme in der Regel reversibel sind. Eine Hydrogenase konnte hierfür ausgeschlossen werden, da die NADH-oxidierende Aktivität Sauerstoff-unempfindlich war. Die Aktivität des NADH-oxidierenden Enzyms konnte durch zugesetztes Na+ nicht stimuliert werden. In Analogie zu Na+-translozierenden NADH:Chinon-Reduktasen wurde die NADH-oxidierende Aktivität an gewaschenen Membranen aber durch Ag+ oder Cu2+ in mikromolaren Konzentrationen vollständig inhibiert. Membranen von A. woodii vermittelten die Reduktion von NAD+ mit reduzierten Ferredoxin als Elektronendonor. Ob diese Aktivität durch das gleiche membranständige Enzyme katalysiert wurde, das auch die NADH-Oxidation bewerkstelligte, konnte nicht geklärt werden. Die Ferredoxin-abhängige NAD+-Reduktion wurde durch micromolare Konzentrationen Ag+ vollständig inhibiert. Die Inihibition war aber wahrscheinlich unspezifischer Natur. Mittels vergleichender 2D-Gelelektrophorese wurden zwei Caffeat-induzierte Proteine identifiziert. Ein Vergleich der Peptidsequenzen, die durch ESI-MS/MS-Analyse der Proteine erhalten wurden, mit in Datenbanken hinterlegten Proteinsequenzen, ergab eine große Übereinstimmung zu der großen alpha- bzw. kleinen beta-Untereinheit von heterodimeren Elektronentransfer-Flavoproteinen (ETFP). Die Proteine wurden mit EtfA und EtfB bezeichnet. Anhand der Peptidsequenzen von EtfA und EtfB wurden degenerierte Oligonukleotide abgeleitet, die zur Amplifikation von Fragmenten der kodierenden Gene herangezogen wurden. Die Analyse der abgeleiteten Aminosäuresequenzen der erhaltenen PCR-Produkte untermauerte die Zuordnung von EtfA und EtfB als Untereinheiten eines ETFP. Die Fähigkeit zur Caffeatreduktion ist in A. woodii nicht konstitutiv vorhanden, sondern wird in erst durch Gegenwart des Phenylacrylats induziert. Die Spezifität dieser Induktion wurde untersucht. Suspensionen ruhender Zellen, die aus Caffeat-induzierten Zellen hergestellt worden waren, reduzierten neben Caffeat auch die Phenylacrylate Ferulat oder p-Cumarsäure mit H2 als Elektronendonor. Analoge Beoabachtungen wurden mit Ferulat-induzierten und p-Cumarsäure-induzierten Zellsuspensionen gemacht. Die Ergebnisse legen den Schluß nahe, dass in A. woodii die Reduktion von Phenylacrylaten durch ein induzierbares enzymatisches System bewerkstelligt wird. EtfA und EtfB wurden als MalE-Fusionsproteine dargestellt und gereinigt. Dagegen wurden Antiseren hergestellt. Immunologische Untersuchungen zeigten, dass die Produktion von EtfA und EtfB durch Gegenwart verschiedener Phenylacrylate induziert wird. Die Induktion war unabhängig vom Wachstumssubstrat. In Gegenwart von zu Phenylacrylaten ähnlichen Verbindungen erfolgte keine Induktion. Für EtfA und EtfB wurde eine Funktion als universeller Elektronenüberträger des Phenylacrylat-Reduktionssystems in A. woodii postuliert. Für die H2-abhängige Reduktion von Caffeat und anderer Phenylacrylate wurde die folgende Reaktionssequenz postuliert: die Oxidation des Elektronendonors H2 durch eine Hydrogenase geht einher mit der Bildung von reduziertem Ferredoxin. Dieses wird durch eine membranständige Ferredoxin-NAD+-Oxidoreduktase oxidiert, die den Transfer der Elektronen auf NAD+ mit der Translokation von Na+ koppelt. Ein aus EtfA und EtfB gebildetes ETFP fungiert dann als Elektronenüberträger zwischen NADH + H+ und einer löslichen Phenylacrylatreduktase.Acetobacterium woodii was grown with fructose in the presence of caffeate as electron acceptor. Cells were treated with lysozyme and broken up applying low preassure by passage through a „French Press“ cell yielding cell-free extract. All manipulations were performed under strictly anaerobic conditions. This cell-free extract mediated H2-dependent caffeate reduction with rates between 8.7 and 18.7 nmol/min x mg protein. The activity was strictly dependent on the presence of ATP. After separating cell-free extract into the cytoplasmic and the membraneous fraction H2-dependent caffeate reduction was exclusively located in the cytoplasm. The addition of membranes lead to no stimulation of the activity. No activity was found in the membraneous fraction. Various compounds were tested to act as artficial electron donors for caffeate reduction. Tetramethyl phenylendiamine, 1,5-diphenylcarbazide and reduced methyl viologen did not mediate caffeate reduction. In the presence of phenylenediamine caffeate reduction was observed in cell-free extract and the cytoplasmic fraction. In the membraneous fraction no reduction of caffeate was observed with phenylenediamine serving as electron donor. NADH acted as a physiological electron donor for the reduction of caffeate. NADH-dependent caffeate reduction was exclusively located in the cytoplasmic fraction and was strictly dependent on the presence of ATP. The observation that NADH acts as an electron donor for caffeate reduction raised the question how NADH is generated during H2-dependent caffeate reduction and which enzymes could be involved. NAD+-dependent hydrogenase activity was identified in the membraneous and the cytoplasmic fraction. About 70% of the activity was located in the soluble fraction. The presence of an electron donor:NAD+-oxidoreductase in A. woodii was investigated. Washed membranes mediated the oxidation of NADH with ferricyanide or benzyl viologen serving as electron acceptors. This observation was taken as a hint for the presence of a NAD+-reductase, since these enzymes are usually reversible. A hydrogenase could be excluded to mediate this activity since the NADH-oxidizing activity was insensitive towards oxygen. The activity of the NADH-oxidizing enzyme could not be stimulated by Na+. In analogy to Na+-translocating NADH:quinone-reductases the NADH-oxidizing activity located at washed membranes was completly inhibited by micro-molar concentrations of Ag+ or Cu2+. Washed membranes of A. woodii mediated the reduction of NAD+ with reduced ferredoxin serving as electron donor. It could not be clarified, whether this activity was mediated by the membrane-bound NADH-oxidizing enzyme. Ferredoxin-dependent NAD+-reduction was completely inhibited by micro-molar concentrations of Ag+. This inihibition was most likely unspecific. Using 2D gel electrophoresis two caffeate-induced proteins were identified that were subsequently subjected to ESI-MS/MS. A comparision of the obtained peptide sequences with protein sequences from data bases revealed great identities to the large alpha-subunit and the small beta-subunit respectively of heterodimeric electron transfer flavorproteins (ETFP). The caffeate-induced proteins were designated EtfA and EtfB respectively. Based on the peptide sequences degenerated oligonucleotides were deduced and gene fragements coding for EtfA and EtfB were amplified. Analysis of the amino acid sequences deduced from the PCR products confirmed that EtfA and EtfB are subunits of an ETFP. The ability to reduce caffeate is not a constitutive feature of A. woodii but is induced in the presence of the phenyl acrylate. The specificity of this induction was investigated. Suspensions of resting cells prepared from caffeate-induced cells reduced besides caffeate other phenyl acrylates like ferulate or p-coumarate with H2 serving as electron donor. This observation implies that the reduction of phenyl acrylates in A. woodii is accomplished by one single induceable enzymatic system. EtfA and EtfB were heterologously overproduced as MalE-fusion proteins and were purified. Antibodies were generated against MalE-EtfA and MalE-EtfB. Western blot analysis revealed that the production of EtfA and EtfB is induced by various phenyl acrylates. This induction was independent of the growth substrate. In the presence of compounds structurally similar to phenyl acrylates no induction was observed. Thus, it is postulated that EtfA/EtfB act as the universal electron mediator of the phenyl acrylate reducing system in A. woodii. The following reaction sequence was postulated for the reduction of caffeate and other phenyl acrylates: the oxidation of the electron donor H2 is accompanied by the generation of reduced ferredoxin. The latter is oxidized by a membrane-bound ferredoxin-NAD+-oxidoreductase that couples electron transfer to NAD+ with the translocation of Na+. An ETFP consisting of EtfA and EtfB mediates the electron transfer between NADH and a soluble phenyl acrylate reductase

Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix^® RT-PCR.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix &lt;sup&gt;®&lt;/sup&gt; multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix &lt;sup&gt;®&lt;/sup&gt; RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix &lt;sup&gt;®&lt;/sup&gt; multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens

Разработка схемы электроснабжения Вахского нефтяного месторождения ОАО «Томскнефть» ВНК

Выпускная квалификационная работа 185 с., 49 рис., 60 табл., 29 источника, 10 приложение. Ключевые слова: расчётная нагрузка, электроснабжение, выбор сечения, защитной аппаратуры. Объектом исследования является нефтяные месторождения ОАО «Томскнефть» ВНК. Цель работы: разработка системы электроснабжения нефтяного месторождения. В процессе исследования произведён поэтапный расчёт нагрузок, выбор сечения линий, оборудования, защитной аппаратуры. В результате исследования спроектирована конкретная модель электроснабжения месторождения. Основные конструктивные, технологические и технико- эксплуатационные характеристики: объект исследования состоит из 5 кустов и прокатно – ремонтного цеха, все относятся II категории по степени надёжности электроснабжения. Область применения: нефтяные месторожAuspuff arbeiten Qualifikation 185 S., 49 Abb., 60 Tab., 29 Quelle, 10 App Stichworte: Gewichtslast, die Energieversorgung, der Schutzausrüstung Gegenstand der Studie ist die ölfelder Publikumsgesellschaft «Tomskneft» Waher ölkonz Ziel der Arbeit: die Entwicklung des Systems der Elektrizitätsversorgung ölfeld Im forschungsprozeß produziert eine schrittweise Berechnung von Lasten, Auswahl der Querschnitt der Leitungen und Geräte, Schutzausrüstung Infolge der Forschung entwickelt ein konkretes Modell der Energieversorgung vorkommen Grundlegende Konstruktive, technologische und betriebstechnische Daten: das Objekt der Forschung besteht aus 5 Sträuchern und rollende – Reparatur-Werkstatt, alle gehören der Kategorie II durch den Grad der Zuverlässigkeit der Stromversorgung Einsatzbereich: ölfe

Pupylation-dependent and -independent proteasomal degradation in mycobacteria

Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway

Pupylation-dependent and -independent proteasomal degradation in mycobacteria

Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway

Antimicrobial susceptibility testing is crucial when treating Finegoldia magna infections

Finegoldia magna is an anaerobic gram-positive bacterium that can cause invasive human infections. Recently, a 52-year-old patient suffering from a periprosthetic joint infection (PJI) due to F. magna was treated with cefepime on hemodialysis; however, treatment failed due to relapse caused by antibiotic-resistant strains. Reports on the antimicrobial susceptibility of F. magna clinical isolates are rare. We collected 57 clinical F. magna isolates from Zurich, Switzerland, between September 2019 and July 2020 and tested their antimicrobial susceptibility to investigate the local resistance pattern. Antimicrobial susceptibility testing (AST) was evaluated for nine antibiotics (benzylpenicillin, amoxicillin/clavulanic acid, cefuroxime, cefepime, levofloxacin, rifampicin, metronidazole, doxycycline, and clindamycin) by E-test according to CLSI guidelines. All F. magna strains were susceptible to benzylpenicillin, amoxicillin/clavulanic acid, and metronidazole, while 75% to clindamycin. F. magna isolates showed MIC values lower than species-unrelated breakpoints for cefuroxime, levofloxacin, and cefepime in 93%, 56%, and 32% of the cases, respectively. MIC values for rifampicin and doxycycline were lower than locally determined ECOFFs in 98% and 72% of the cases, respectively. In summary, we recommend the use of benzylpenicillin, amoxicillin/clavulanic acid, or metronidazole without prior AST as first-line treatment option against F. magna PJI infections. If cefuroxime, cefepime, levofloxacin, rifampicin, doxycycline, or clindamycin are used, AST is mandatory. Keywords: Antimicrobial susceptibility; Cefepime; Finegoldia magna; Periprosthetic joint infectio

Mycoplasma penetrans bacteremia in an immunocompromised patient detected by metagenomic sequencing: a case report.

Background Mycoplasma sp. are well recognized as etiological agents of respiratory and sexually transmitted disease. Mycoplasma penetrans, a species of Mycoplasma sp., has been frequently detected in HIV-positive patients and associated with the progression of HIV-associated disease. To date, there is only a single case report describing M. penetrans as the causative agent of a severe respiratory tract infection in a HIV-negative patient. Case presentation In this report, we describe the case of M. penetrans bacteremia in a HIV-negative, 38-year-old, female, immunocompromised, solid organ transplant patient (combined kidney and pancreas transplantation in 2016), who was admitted to our hospital with anemic uterine bleeding and fever of 38.3 °C. Several hours before her admission at our university hospital, a latex bladder catheter was inserted into her uterus and she complained about fatigue, dizziness and ongoing vaginal bleeding. Laboratory examination showed severe anemia, but microbiological examination was inconspicuous (culture negative vaginal and cervical smears, negative urine culture). Bacterial blood cultures showed a growth signal after 4 h, but microscopic examination with Gram staining and subcultures on different agar media did not identify bacterial pathogens. To identify the bacterial cause of malignancy in the patient, metagenomic sequencing of the blood culture was performed that identified M. penetrans. Conclusion Metagenomic sequencing identified M. penetrans in an immunosuppressed patient with culture-negative bacteremia. Clinicians should be aware of the opportunistic potential of M. penetrans that may cause severe infections in certain vulnerable patient populations and the limitations of culture and Gram staining for confirming the presence of fastidious bacterial pathogens like Mycoplasma spp