Isobutanol production by combined in vivo and in vitro metabolic engineering.

The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L-1 h-1, and titers of 5.6 and 1.78 g L-1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

One hundred eighteen days on a ventilator: a COVID-19 success story against all odds.

Emerging data suggest that patients with certain comorbidities requiring intensive care unit (ICU) admission for COVID-19 have a poor prognosis. This report describes a case of a patient with multiple comorbidities who contracted COVID-19 pneumonitis but was successfully weaned off invasive mechanical ventilation after 118 days, despite his admission being complicated by recurrent septic episodes and requirement for advanced cardiovascular support and renal replacement therapy. Of note, our patient received three courses of steroids in total during his ICU stay,and current literature strongly supports the use of steroids in critically unwell patients with COVID-19. To the best of our knowledge, this is the longest reported ventilated time and intensive care/hospital stay for a surviving patient with COVID-19 and highlights the importance of allowing sufficient time for clinical interventions to take effect, even when the prognosis appears bleak.

Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity.

The capsid protein purity of adeno-associated virus (AAV) is considered a critical quality attribute of AAV-based gene therapy products. However, the analytical methods currently available to monitor the viral capsid proteins, which are present in extremely low concentrations, have limited sensitivity and robustness, thus limiting their general applicability. As a result, there is an urgent need to develop robust separation methods with highly sensitive detection. In this article, we describe the first denaturation and fluorescence labeling procedure for AAV capsid proteins using the pyrylium dye Chromeo™ P503, enabling the establishment of the first capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method combined with laser-induced fluorescence (LIF) detection for AAV. Upon optimization using a quality-by-design approach, the newly developed method features a simple and robust one-step sample preparation workflow resulting in consistently labeled and denatured viral protein samples, which can subsequently be separated and quantified by CE-LIF. The method has been validated to be accurate and precise with a linear range of 50-150% of the nominal concentration of 2.0 × 1011 vector genomes per mL (vg/mL). The detection limit and quantitation limit were established to be 8.0 × 107 vg/mL (∼0.8 ng/mL) and 4.2 × 108 vg/mL (∼4 ng/mL), respectively, representing the highest sensitivity achieved for AAV capsid protein quantitation reported to date and a linear dynamic range of 8.0 × 107-3.0 × 1011 vg/mL. A comparison of the CE-SDS LIF method with existing methods, such as CE-SDS ultraviolet and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SYPRO Ruby stain, indicated that the new method has superior resolution and a significant increase in signal intensity. Capsid protein purity analysis of multiple AAV serotypes, including AAV5, scAAVrh10, AAV2, and AAV6, has been demonstrated for the first time using the same method, indicating the newly developed AAV labeling procedure and CE-LIF analysis could serve as a Quality Control-friendly platform and best-in-class analytical method for the control of AAV capsid protein purity.

Ligamentum arteriosum calcification on paediatric postmortem computed tomography.

BACKGROUND: Ligamentum arteriosum calcification may be a normal finding in some children, although the frequency has not been well described. OBJECTIVE: To estimate the frequency of ligamentum arteriosum calcification in children at postmortem imaging. MATERIALS AND METHODS: We conducted a single-centre retrospective review of paediatric postmortem CT and chest radiographic imaging over a 6-year period (January 2012 to December 2018). Two independent reviewers assessed the presence of calcification on imaging. We calculated descriptive statistical analysis of ligamentum arteriosum calcification frequency and association with age and gender. RESULTS: During the study period, 220 children underwent whole-body postmortem CT and 182 underwent radiographic imaging. The frequency was higher on postmortem CT than plain radiographs (67/220, 30.5% vs. 3/182, 1.6%) and was highest in children ages 1-7 years (53.6-66.7%), with gradual reduction in frequency in older children, and none in children older than 12 years. There was no gender predilection. CONCLUSION: In the postmortem setting, ligamentum arteriosum calcification is a common finding in children <8 years of age. It can be better identified on postmortem CT than chest radiographs. Radiologists new to reporting postmortem paediatric CT studies should recognise this as a common normal finding to avoid unnecessary further investigations at autopsy.

Canadian wildlife-vehicle collisions: An examination of knowledge and behavior for collision prevention.

OBJECTIVES: This study examines drivers' responses to wildlife on Canadian roads. The objective of this paper is to demonstrate that knowledge of what to do when encountering wildlife on the road does not always translate into the appropriate behavior to avoid a collision. METHODS: Data from the Traffic Injury Research Foundation's (TIRF) 2016 Road Safety Monitor (RSM) and data from TIRF's National Fatality Database from 2000 to 2014 were analyzed to test hypotheses based on the theory of planned behavior. Logistic regression and piecewise linear regression were used. RESULTS: Analyses of the data showed that the prevalence of fatal WVCs has remained relatively consistent, and that the majority of persons killed in WVCs died in crashes that involved large mammals. The majority of fatalities occurred in the summer (182 or 38.4%) and fall (163 or 34.4%). The RSM data revealed that 60.9% [50.5, 70.4] of respondents who previously hit an animal indicated that drivers should slow down and steer straight when confronted with wildlife, while 47.3% [37.1, 57.6] of respondents indicated this was the action they took when they hit wildlife. Comparatively, 59.5% [56.6, 62.4] of respondent who have not hit an animal indicated this was an appropriate response. Additionally, 33.2% [24, 44] of respondents who previously hit an animal indicated that drivers should swerve to avoid a collision with wildlife, while 37.5% [28.2, 47.8] of respondents indicated this was the action they took when they hit wildlife. CONCLUSIONS: Many drivers are unaware of what the safest method of WVC prevention is. Further, while a subgroup of drivers may have the knowledge and intention to slow down and steer straight even if the animal is directly in the path, i.e., the safest possible behavior, they are not necessarily adopting this behavior. Practical applications: Recommendations are formulated to address this discrepancy, as well as practical applications.

Three-Dimensional Imaging-Based Web Application for Predicting Tracheal Tube Depth in Preterm Neonates.

BACKGROUND: Positioning a tracheal tube (TT) to the correct depth in preterm infants is challenging. Currently, there is no reliable single-predictor model for neonates applicable to the whole range of size or age. OBJECTIVE: In this study, we used post-mortem magnetic resonance imaging (PMMRI) of preterm infants to measure tracheal dimensions and to develop a clinical guide for TT positioning. METHODS: We measured tracheal length (TL) and tracheal diameter (TD) in a cohort of normal neonates and foetuses that underwent PMMRI (cause of death unexplained). The distance between the lips and the mid-tracheal point, i.e., the mid-tracheal length (mid-TL), and the TD measurement were obtained. We produced univariate prediction models of mid-TL and TD, using gestational age (GA), foot length (FL), crown-rump length (CRL) and body weight (BW) as potential predictors, as well as multiple prediction models for mid-TL. RESULTS: Tracheal measurements were performed in 117 cases, with a mean GA of 28.8 weeks (range 14-42 weeks). The best linear association was between mid-TL and FL (mid-TL = FL × 0.914 + 1.859; R2 = 0.94), but was improved by multivariate regression models. We developed a prediction tool using only GA and BW (R2 = 0.92), and all four predictors (GA, BW, FL and CRL; R2 = 0.94) which is now available as a web-based application via the Internet. CONCLUSION: Post-mortem imaging data provide estimates of TT insertion depth. Our prediction tool based on age and BW can be used at the bedside and is ready to be tested in clinical practice.

Prefrontal glutamate correlates of methamphetamine sensitization and preference.

Methamphetamine (MA) is a widely misused, highly addictive psychostimulant that elicits pronounced deficits in neurocognitive function related to hypo-functioning of the prefrontal cortex (PFC). Our understanding of how repeated MA impacts excitatory glutamatergic transmission within the PFC is limited, as is information about the relationship between PFC glutamate and addiction vulnerability/resiliency. In vivo microdialysis and immunoblotting studies characterized the effects of MA (ten injections of 2 mg/kg, i.p.) upon extracellular glutamate in C57BL/6J mice and upon glutamate receptor and transporter expression, within the medial PFC. Glutamatergic correlates of both genetic and idiopathic variance in MA preference/intake were determined through studies of high vs. low MA-drinking selectively bred mouse lines (MAHDR vs. MALDR, respectively) and inbred C57BL/6J mice exhibiting spontaneously divergent place-conditioning phenotypes. Repeated MA sensitized drug-induced glutamate release and lowered indices of N-methyl-d-aspartate receptor expression in C57BL/6J mice, but did not alter basal extracellular glutamate content or total protein expression of Homer proteins, or metabotropic or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors. Elevated basal glutamate, blunted MA-induced glutamate release and ERK activation, as well as reduced protein expression of mGlu2/3 and Homer2a/b were all correlated biochemical traits of selection for high vs. low MA drinking, and Homer2a/b levels were inversely correlated with the motivational valence of MA in C57BL/6J mice. These data provide novel evidence that repeated, low-dose MA is sufficient to perturb pre- and post-synaptic aspects of glutamate transmission within the medial PFC and that glutamate anomalies within this region may contribute to both genetic and idiopathic variance in MA addiction vulnerability/resiliency.

The Acute Care Undergraduate TEaching (ACUTE) Initiative: consensus development of core competencies in acute care for undergraduates in the United Kingdom.

BACKGROUND: The care of the acutely ill patient in hospital is often sub-optimal. Poor recognition of critical illness combined with a lack of knowledge, failure to appreciate the clinical urgency of a situation, a lack of supervision, failure to seek advice and poor communication have been identified as contributory factors. At present the training of medical students in these important skills is fragmented. The aim of this study was to use consensus techniques to identify the core competencies in the care of acutely ill or arrested adult patients that medical students should possess at the point of graduation. DESIGN: Healthcare professionals were invited to contribute suggestions for competencies to a website as part of a modified Delphi survey. The competency proposals were grouped into themes and rated by a nominal group comprised of physicians, nurses and students from the UK. The nominal group rated the importance of each competency using a 5-point Likert scale. RESULTS: A total of 359 healthcare professionals contributed 2,629 competency suggestions during the Delphi survey. These were reduced to 88 representative themes covering: airway and oxygenation; breathing and ventilation; circulation; confusion and coma; drugs, therapeutics and protocols; clinical examination; monitoring and investigations; team-working, organisation and communication; patient and societal needs; trauma; equipment; pre-hospital care; infection and inflammation. The nominal group identified 71 essential and 16 optional competencies which students should possess at the point of graduation. CONCLUSIONS: We propose these competencies form a core set for undergraduate training in resuscitation and acute care.