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Ectothermic vertebrates experience daily changes in body temperature, and anecdotal observations suggest these changes affect ventricular repolarization such that the T-wave in the ECG changes polarity. Mammals, in contrast, can maintain stable body temperatures, and their ventricular repolarization is strongly modulated by changes in heart rate and by sympathetic nervous system activity. The aim of this study was to assess the role of body temperature, heart rate, and circulating catecholamines on local repolarization gradients in the ectothermic ball python (Python regius). We recorded body-surface electrocardiograms and performed open-chest high-resolution epicardial mapping while increasing body temperature in five pythons, in all of which there was a change in T-wave polarity. However, the vector of repolarization differed between individuals, and only a subset of leads revealed T-wave polarity change. RNA sequencing revealed regional differences related to adrenergic signaling. In one denervated and Ringer's solution-perfused heart, heating and elevated heart rates did not induce change in T-wave polarity, whereas noradrenaline did. Accordingly, electrocardiograms in eight awake pythons receiving intra-arterial infusion of the β-adrenergic receptor agonists adrenaline and isoproterenol revealed T-wave inversion in most individuals. Conversely, blocking the β-adrenergic receptors using propranolol prevented T-wave change during heating. Our findings indicate that changes in ventricular repolarization in ball pythons are caused by increased tone of the sympathetic nervous system, not by changes in temperature. Therefore, ventricular repolarization in both pythons and mammals is modulated by evolutionary conserved mechanisms involving catecholaminergic stimulation., (© 2021 Boukens et al.)

Background: Acute and persistent pain after surgery is well described. However, no large-scale studies on immediate postoperative pain in the operating room (OR) exist, hindering potential areas of research to improve clinical outcomes. Thus, we aimed to describe the occurrence and severity of immediate postoperative pain in a large, unselected cohort., Methods: This was a prospective cohort study, encompassing all procedures in 31 public hospitals in the Danish Realm, during a 5-day period including the weekend. Data on procedures and anesthesia were collected and the main outcome was occurrence of moderate or severe pain in the OR. Secondary outcomes included pain, sedation and nausea in the OR or during the first 15 min in the postanesthesia care unit (PACU) including relevant risk factors. Descriptive and logistic regression statistics were used., Results: A total of 3675 procedures were included for analysis (87% inclusion rate). Moderate or severe pain occurred in 7.4% (95% CI 6.5% to 8.3%) of cases in the OR immediately after awakening, rising to 20.2% in the OR and/or PACU. Large intraprocedure and interprocedure variations occurred (0.0%-37.5%), and in 20% of cases with epidural-general anesthesia patients experienced moderate or severe pain. Independent risk factors were female sex, younger age, preoperative pain, daily opioid use and major surgical procedures., Conclusion: Moderate or severe pain in the immediate postoperative phase occurred in 20% of all cases with procedure and anesthesiological technique variations, suggesting a need for identification of relevant procedure-specific risk factors and development of preventive treatments., Trial Registration Number: RoPR ID 43191., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Vaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants., Results: We describe a holistic approach for the molecular design of recombinant protein antigens-considering both their manufacturability and antigenicity-informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii., Conclusions: This study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.

Komagataella phaffii, also known as Pichia pastoris, is a common host for the production of biologics and enzymes, due to fast growth, high productivity, and advancements in host engineering. Several K. phaffii variants are commonly used as interchangeable base strains, which confounds efforts to improve this host. In this study, genomic and transcriptomic analyses of Y-11430 (CBS7435), GS115, X-33, and eight other variants enabled a comparative assessment of the relative fitness of these hosts for recombinant protein expression. Cell wall integrity explained the majority of the variation among strains, impacting transformation efficiency, growth, methanol metabolism, and secretion of heterologous proteins. Y-11430 exhibited the highest activity of genes involved in methanol utilization, up to two-fold higher transcription of heterologous genes, and robust growth. With a more permeable cell wall, X-33 displayed a six-fold higher transformation efficiency and up to 1.2-fold higher titers than Y-11430. X-33 also shared nearly all mutations, and a defective variant of HIS4, with GS115, precluding robust growth. Transferring two beneficial mutations identified in X-33 into Y-11430 resulted in an optimized base strain that provided up to four-fold higher transformation efficiency and three-fold higher protein titers, while retaining robust growth. The approach employed here to assess unique banked variants in a species and then transfer key beneficial variants into a base strain should also facilitate rational assessment of a broad set of other recombinant hosts., (© 2019 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

The electrocardiogram (ECG) reveals that heart chamber activation and repolarization are much faster in mammals and birds compared to ectothermic vertebrates of similar size. Temperature, however, affects electrophysiology of the heart and most data from ectotherms are determined at body temperatures lower than those of mammals and birds. The present manuscript is a review of the effects of temperature on intervals in the ECG of ectothermic and endothermic vertebrates rather than a hypothesis-testing original research article. However, the conclusions are supported by the inclusion of original data (Iguana iguana, N = 4; Python regius, N = 5; Alligator mississippiensis, N = 4). Most comparisons were of animals of approximately 1 kg. Compared to mammals and birds, the reptiles at 35-37 °C had 4 fold lower heart rates, 2 fold slower atrial and ventricular conduction (longer P- and QRS-wave durations), and 4 fold longer PR intervals (atrioventricular delay) and QT intervals (total ventricular repolarization). We conclude that the faster chamber activation in endotherms cannot be explained by temperature alone. Based on histology, we show that endotherms have a more compact myocardial architecture. In mammals, disorganization of the compact wall by fibrosis associates with conduction slowing and we suggest the compact tissue architecture allows for faster chamber activation. The short cardiac cycle that characterizes mammals and birds, however, is predominantly accommodated by shortening of the atrioventricular delay and the QT interval, which is so long in a 1 kg iguana that it compares to that of an elephant., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Gene expression profiling (GEP) risk models in multiple myeloma are based on 3'-end microarrays. We hypothesized that GEP risk signatures could retain prognostic power despite being translated and applied to whole-transcript microarray data., Methods: We studied CD138-positive bone marrow plasma cells in a prospective cohort of 59 samples from newly diagnosed patients eligible for high-dose therapy (HDT) and 67 samples from previous HDT patients with progressive disease. We used Affymetrix Human Gene 1.1 ST microarrays for GEP. Nine GEP risk signatures were translated by probe set match and applied to our data in multivariate Cox regression analysis for progression-free survival and overall survival in combination with clinical, cytogenetic and biochemical risk markers, including the International Staging System (ISS)., Results: Median follow-up was 66 months (range 42-87). Various translated GEP risk signatures or combinations hereof were significantly correlated with survival: among newly diagnosed patients mainly in combination with cytogenetic high-risk markers and among relapsed patients mainly in combination with ISS stage III., Conclusion: Translated GEP risk signatures maintain significant prognostic power in HDT myeloma patients. We suggest probe set matching for GEP risk signature translation as part of the efforts towards a microarray-independent GEP risk standard. (ClicinalTrials.gov identifier: NCT00639054)., (© 2016 John Wiley & Sons Ltd.)

Background . Distal arm surgery is widely performed under regional anesthesia with brachial plexus block. The preponderance of evidence for the efficacy relies upon injection of local anesthetic in excess of 30 mL. We aimed to compare three different ultrasound-guided brachial plexus block techniques restricting the total volume to 20 mL. Methods . 120 patients were prospectively randomized to ultrasound-guided brachial plexus block with 20 mL ropivacaine 0.75% at either the supraclavicular, infraclavicular, or axillary level. Multiinjection technique was performed with all three approaches. Primary outcome measure was performance time. Results . Performance time and procedural pain were similar between groups. Needle passes and injection numbers were significantly reduced in the infraclavicular group ( P < 0.01). Nerve visibility was significantly reduced in the axillary group ( P = 0.01). Success-rate was significantly increased in the supraclavicular versus the axillary group ( P < 0.025). Total anesthesia-related time was significantly reduced in the supraclavicular compared to the infraclavicular group ( P < 0.01). Block duration was significantly increased in the infraclavicular group ( P < 0.05). No early adverse effects occurred. Conclusion . Supraclavicular and infraclavicular blocks exhibited favorable characteristics compared to the axillary block. Supraclavicular brachial plexus block with the multiinjection intracluster technique exhibited significantly reduced total anesthesia-related time and higher success rate without any early adverse events., Competing Interests: The authors declare that there are no competing interests regarding the publication of this paper.

In many seabird studies, single annual proxies of prey abundance have been used to explain variability in breeding performance, but much more important is probably the timing of prey availability relative to the breeding season when energy demand is at a maximum. Until now, intraseasonal variation in prey availability has been difficult to quantify in seabirds. Using a state-of-the-art ocean drift model of larval cod Gadus morhua, an important constituent of the diet of common guillemots Uria aalge in the southwestern Barents Sea, we were able to show clear, short-term correlations between food availability and measurements of the stress hormone corticosterone (CORT) in parental guillemots over a 3-year period (2009-2011). The model allowed the extraction of abundance and size of cod larvae with very high spatial (4 km) and temporal resolutions (1 day) and showed that cod larvae from adjacent northern spawning grounds in Norway were always available near the guillemot breeding colony while those from more distant southerly spawning grounds were less frequent, but larger. The latter arrived in waves whose magnitude and timing, and thus overlap with the guillemot breeding season, varied between years. CORT levels in adult guillemots were lower in birds caught after a week with high frequencies of southern cod larvae. This pattern was restricted to the two years (2009 and 2010) in which southern larvae arrived before the end of the guillemot breeding season. Any such pattern was masked in 2011 by already exceptionally high numbers of cod larvae in the region throughout chick-rearing period. The findings suggest that CORT levels in breeding birds increase when the arrival of southern sizable larvae does not match the period of peak energy requirements during breeding.

Metabolic markers are measured by microdialysis to detect postoperative ischaemia after reconstructive surgery with myocutaneous flaps. If a haematoma develops around the microdialysis catheter, it can result in misinterpretation of the measurements. The aim of the present study was to investigate whether a haematoma in a flap can be identified and dissociated from ischaemia, or a well-perfused flap, by a characteristic chemical profile. In 7 pigs, the pedicled rectus abdominal muscle flap was mobilised on both sides. A haematoma was made in each flap and two microdialysis catheters were placed, one in the haematoma, and the other in normal tissue. One flap was made ischaemic by ligation of the pedicle. For 6 hours, the metabolism was monitored by measurement every half-an-hour of the concentrations of glucose, lactate, pyruvate, and glycerol from all 4 catheters. After 3 hours of monitoring, intravenous glucose was given as a challenge test to identify ischaemia. The non-ischaemic flap could be differentiated from the ischaemic flap by low glucose, and high lactate, concentrations. It was possible to identify a catheter surrounded by a haematoma in ischaemic as well as non-ischaemic muscle from a low or decreasing concentration of glucose together with a low concentration of lactate. All four sites could be completely dissociated when the concentrations of glucose and lactate were evaluated and combined with the lactate:glucose ratio and a flow chart. The challenge test was useful for differentiating between haematomas in ischaemic and non-ischaemic tissue., (Copyright © 2012 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)