Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression

Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins – transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators – all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs. Molecular analysis of a sequence-optimized transgene revealed positive effects at transcriptional, translational, and mRNA stability levels. Since improved expression was consistent in HEK293T, CHO and insect cells, it was not restricted to distinct mammalian cell systems. Additionally, optimized genes represent powerful tools in functional genomics, as demonstrated by the successful rescue of an siRNA-mediated knockdown using a sequence-optimized counterpart. This is the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression

4CaaSt: Comprehensive management of Cloud services through a PaaS

The 4CaaSt project aims at developing a PaaS framework that enables flexible definition, marketing, deployment and management of Cloud-based services and applications. The major innovations proposed by 4CaaSt are the blueprint and its management and lifecycle, a one stop shop for Cloud services and the management of resources in the PaaS level (including elasticity). 4CaaSt also provides a portfolio of ready to use Cloud native services and Cloud- aware immigrant technologies

Nebulization of the acidified sodium nitrite formulation attenuates acute hypoxic pulmonary vasoconstriction

<p>Abstract</p> <p>Background</p> <p>Generalized hypoxic pulmonary vasoconstriction (HPV) occurring during exposure to hypoxia is a detrimental process resulting in an increase in lung vascular resistance. Nebulization of sodium nitrite has been shown to inhibit HPV. The aim of this project was to investigate and compare the effects of nebulization of nitrite and different formulations of acidified sodium nitrite on acute HPV.</p> <p>Methods</p> <p><it>Ex vivo </it>isolated rabbit lungs perfused with erythrocytes in Krebs-Henseleit buffer (adjusted to 10% hematocrit) and <it>in vivo </it>anesthetized catheterized rabbits were challenged with periods of hypoxic ventilation alternating with periods of normoxic ventilation. After baseline hypoxic challenges, vehicle, sodium nitrite or acidified sodium nitrite was delivered via nebulization. In the <it>ex vivo </it>model, pulmonary arterial pressure and nitric oxide concentrations in exhaled gas were monitored. Nitrite and nitrite/nitrate were measured in samples of perfusion buffer. Pulmonary arterial pressure, systemic arterial pressure, cardiac output and blood gases were monitored in the <it>in vivo </it>model.</p> <p>Results</p> <p>In the <it>ex vivo </it>model, nitrite nebulization attenuated HPV and increased nitric oxide concentrations in exhaled gas and nitrite concentrations in the perfusate. The acidified forms of sodium nitrite induced higher levels of nitric oxide in exhaled gas and had longer vasodilating effects compared to nitrite alone. All nitrite formulations increased concentrations of circulating nitrite to the same degree. In the <it>in vivo </it>model, inhaled nitrite inhibited HPV, while pulmonary arterial pressure, cardiac output and blood gases were not affected. All nitrite formulations had similar potency to inhibit HPV. The tested concentration of appeared tolerable.</p> <p>Conclusion</p> <p>Nitrite alone and in acidified forms effectively and similarly attenuates HPV. However, acidified nitrite formulations induce a more pronounced increase in nitric oxide exhalation.</p

Rehabilitation of Watson-Jones proximal tibial avulsion injury in elite academy level football: A report of two separate cases in one season

© 2020 Elsevier Ltd Objectives: Watson-Jones proximal tibial avulsion injuries occur more frequently in athletic and muscular adolescent males. However, they are rare and therefore infrequently described in the medical literature. Two of these injuries occurred in a Category 1 football academy in the same season within a six-month period. We have described the cases with the hope of better informing other clinicians should they encounter this injury. Methods: This case report describes the injury mechanism, surgical management and rehabilitation for the two cases [Players A and B]. Outcomes measures including player speed, agility and power were compared with scores from players of the same age group at the time of injury in the Premier League academies. Risk factors are also discussed. Results: Both players were managed surgically, initially. Player B had the surgical fixation removed during rehabilitation. Player A still has the fixation in situ. Post-surgery, player A returned to full play at thirty-two weeks and thirty-eight weeks for player B. No critical incidents occurred during rehabilitation. Conclusion: Watson-Jones avulsion fractures, although rare, can be managed successfully. Athletes can achieve a successful return to play at their previous level

A study of industry best practices in ethics programming: learning from exemplary ethical organizations to inspire moral courage in the Military

MBA Professional ReportThe objective of this project is to define and identify best practices relating to ethics programming in todayb2ss corporate organizations, and determine if they might be utilized in a military context. In an effort to remain open to all possibilities, we developed a theoretical framework and conducted personal interviews with ethics specialists in industry. We accomplish our objective by commencing with the identification and definition of best practices relating to the promotion of ethical behavior and prevention of unethical behavior in todayb2ss corporate environment. We approach this by examining some of the best practices of active b3sethical businesses.b4s Once the organizations had been selected, we then compare and contrast their methods, looking for similarities and differences. This project will look at processes, practices, and procedures that encourage employees to meet or go beyond the moral minimum in all their daily endeavors. Given this review and analysis we will close with recommendations for feasible application within the United States Navy Supply Corps.http://archive.org/details/astudyofindustry109459977US Navy (USN) authorApproved for public release; distribution is unlimited

a feasibility study in consecutive outpatients

Einleitung: Studien zeigten, dass mit 2-dimensionalem Speckle-Tracking erhobenen Strain und Strain Rate Werten linksventrikuläre systolische Funktionsstörungen früher detektiert werden können, als per Ejektionsfraktion. Diese Studie evaluiert die Eignung von verschiedenen Strain und Strain Rate Werten für Routinenachsorgeuntersuchungen von Anthrazyklin-behandelten Patienten in der täglichen klinischen Anwendung. Methoden: Alle Anthrazyklin- behandelten Patienten wurden eingeschlossen, die während eines Zeitraums von einem Jahr in unserer kinderkardiologischen Ambulanz eine echokardiographische Routinenachsorgeuntersuchung erhielten. Eine alters- und geschlechtsangepasste gesunde Kontrollgruppe wurde etabliert. Im apikalen Vierkammerblick (4CH) wurden der globale longitudinale Strain (GSL) und die globale longitudinale Strain Rate (GSrL) gemessen, in einer kurzen parasternalen Achse (SAX) der globale zirkumferenzielle und gemittelte radiale Strain (GSC, AvSR) und die globale zirkumferenzielle und gemittelte radiale Strain Rate (GSrC, AvSrR). In der Kontrollgruppe wurde der Einfluss von Geschlecht, Alter, Körperoberfläche und Herzfrequenz auf die Messwerte bestimmt. In jeweils 20 Patienten und Kontrollen wurde die Intra- und Inter-Untersucher-Reproduzierbarkeit mittels Bland-Altman-Analyse, Variationskoeffizient (CoV) und Intra-Klassen- Korrelationskoeffizient (ICC) analysiert. Der Einfluss der Breite der Region of Interest (ROI) auf Strain und Strain Rate wurde in 20 gesunden Kontrollen anhand von Messungen mit drei unterschiedlichen ROI-Breiten quantifiziert. Ergebnisse: 131 Patienten und 66 Kontrollen wurden eingeschlossen. Analysen zeigten, dass nur Bilder mit adäquatem Tracking in allen Segmenten verwendet werden sollten, um Messwertabweichungen zu vermeiden. Im 4CH konnten häufiger alle Segmente getrackt werden als in der SAX (76% vs. 62%). Der GSL war in gesunden weiblichen Kontrollen signifikant höher als in männlichen (-21,0 ±1,6% vs. -19,5 ±1,6% (p=0,001)). Die Werte GSL, GSrL, GSC, GSrC und FS waren in Patienten signifikant niedriger, als in Kontrollen (-20,3 ±1,8% vs. -19,2 ±1,9%; -1,11 ±0,12/s vs. -1,02 ±0,16/s; -19,8 ±3,1% vs. -17,6 ±3,0%; -1,27 ±0,22/s vs. -1,13 ±0,20/s; 35,5 ±3,5% vs. 32,7 ±4,1% (p<0,005)). Der AvSR und die AvSrR waren hingegen nicht signifikant unterschiedlich (51,2 ±13,1% vs. 46,2 ±14,1%; 1,94 ±0,33/s vs. 1,85 ±0,38/s (p>0,05)). Nur der GSC und die GSrC zeigten in Subgruppenanalysen (z.B. Diagnose, kumulative Anthrazyklindosis) durchgehend signifikante Ergebnisse. Der GSL zeigte die beste Intra- und Inter-Untersucher-Reproduzierbarkeit (kein Bias, CoV 0,90). Zwischen ROI-Breite und Strain bzw. Strain Rate fand sich eine inverse Korrelation. Schlussfolgerung: Der Einfluss von Geschlecht und ROI-Breite muss beachtet werden. Für die klinische Anwendung haben der GSL und die GSrL die beste Kombination aus Machbarkeit, Sensitivität und Reproduzierbarkeit. Allerdings scheinen der GSC und die GSrC am sensitivsten zu sein. Der AvSR und die AvSrR sind für die klinische Anwendung ungeeignet.Introduction: Studies showed that 2-dimensional speckle-tracking derived strain and strain rate can detect left ventricular systolic dysfunction earlier than ejection fraction or fractional shortening (FS). This study aimed to evaluate the potential value of different strain and strain rate parameters for the follow-up of anthracycline-treated patients in daily clinical practice. Methods: We included all anthracycline-treated patients who attended our outpatient unit in a one-year period for routine echocardiographic follow- up examination. An age and sex-matched healthy control group was established. Global longitudinal strain and strain rate (GSL, GSrL) were derived from the apical four-chamber (4CH) view, and global circumferential and average radial strain and strain rate (GSC, GSrC, AvSR, AvSrR) from a parasternal short axis (SAX) view. The influence of gender, age, body surface area and heart rate on measurements was analyzed in the control group. Intra- and inter-observer variations were measured in 40 randomly selected subjects by the Bland-Altman method, the coefficient of variation (CoV) and intraclass correlation coefficient (ICC). The influence of the Region of Interest (ROI) width on strain and strain rate was assessed in 20 healthy subjects by obtaining measurements from three different ROI-width. Results: 131 patients and 66 control subjects were included. Analyses showed that only loops with adequate tracking in all segments should be analyzed to avoid bias. The feasibility of tracking all segments was better in the 4CH than in the SAX (76% vs. 62%). Certain segments frequently suffered inadequate tracking. GSL was significantly higher in female than male controls (-21.0 ±1.6% vs. -19.5 ±1.6% (p=0.001)). GSL, GSrL, GSC, GSrC and FS were significantly lower in patients than controls (-20.3 ±1.8% vs. -19.2 ±1.9%; -1.11 ±0.12/s vs. -1.02 ±0.16/s; -19.8 ±3.1% vs. -17.6 ±3.0%; -1.27 ±0.22/s vs. -1.13 ±0.20/s; 35.5 ±3.5% vs. 32.7 ±4.1% (p<0.005)). AvSR and AvSrR were not significantly lower in patients (51.2 ±13.1% vs. 46.2 ±14.1%; 1.94 ±0.33/s vs. 1.85 ±0.38/s (p>0.05)). Only GSC and GSrC showed significant results throughout in subgroup-analyses (e.g. diagnosis, cumulative anthracycline dose). GSL showed the best intra- and interobserver reproducibility without bias, CoV 0.90. An inverse correlation between ROI-width and measurements was found. Conclusions: The influence of gender and ROI-width on measurements must be considered. For clinical practice GSL and GSrL provide the best combination of feasibility, sensitivity and reproducibility. However GSC and GSrC seem to be the most sensitive measurements. AvSR and AvSrR failed to detect a difference between patients and controls

VHCF-Verhalten des hochfesten Stahls 100Cr6: Rissinitiierungsmechanismen und Schwellenwerte

Das Ermüdungsverhalten hochfester Stähle wird durch nichtmetallische Einschlüsse im Werkstoff bestimmt, die unter zyklischer Beanspruchung zu Rissinitiierung führen. Bisher noch nicht vollständig verstandene Ermüdungsvorgänge führen auch noch bei sehr hohen Lastspielzahlen über 10^7 Zyklen zu Versagen (Very high cycle fatigue - VHCF) und somit zum Verlust der „klassischen“ Dauerfestigkeit. Im Rahmen dieser Arbeit wurden zur Klärung dieses Phänomens Ermüdungsversuche mit dem hochfesten Stahl 100Cr6 durchgeführt und die VHCF-Rissinitiierung untersucht. Zusätzlich zum natürlichen Versagen durch Einschlüsse wurde die VHCF-Rissinitiierung unter definierten Bedingungen mit künstlichen Defekten nachgestellt und untersucht. Um einen Einblick in die VHCF-Ermüdungsvorgänge zu erhalten, wurde die lokale Mikrostruktur im Bereich der Rissinitiierung mittels REM, FIB, TEM und APT analysiert. Auf Basis der beobachteten Veränderungen der lokalen Mikrostruktur um Defekte und der damit einhergehenden frühen Rissausbreitung im VHCF-Bereich kann der zugrundeliegende Mechanismus, der schlussendlich für die VHCF-Schädigung verantwortlich ist, aufgeklärt werden. Durch bruchmechanische Bewertung der rissinitiierenden Defekte aus Einstufen- und VHCF-Laststeigerungsversuchen konnten zudem Schwellenwerte des Spannungsintensitätsfaktors für VHCF-Versagen in hochfesten Stählen abgeleitet werden, die die Grenzen der VHCF-Schädigung bis zu 10^9 Zyklen festlegen.The fatigue behavior of high-strength steels is determined by non-metallic inclusions, which lead to crack initiation under cyclic loading conditions. Still not fully understood fatigue processes trigger failure at very high numbers of load cycles above 10^7 (Very high cycle fatigue - VHCF) and are responsible for the loss of the “classical” fatigue limit. In the scope of this work fatigue test with the high-strength steel 100Cr6 were executed and the VHCF crack initiation was investigated to clear this phenomenon. Besides the natural failure at inclusions VHCF crack initiation was reproduced with artificial defects under predefined conditions. In order to gain insights into fatigue mechanisms microstructure analyses at the crack initiation site were performed with SEM, FIB, TEM and APT. Based on the observed changes of the local microstructure at defects and the connected early crack propagation in the VHCF-regime the underlying detrimental VHCF crack initiation mechanisms can be cleared. Additionally fracture mechanical evaluations of the crack initiating defects, observed in one-step and VHCF load increase tests, enabled the estimation of a threshold value of the stress intensity factor for VHCF failure in high- strength steels. Below this value no VHCF-damage could be observed up to a fatigue life of 10^9 cycles