Vaccine process technology-A decade of progress.

In the past decade, new approaches to the discovery and development of vaccines have transformed the field. Advances during the COVID-19 pandemic allowed the production of billions of vaccine doses per year using novel platforms such as messenger RNA and viral vectors. Improvements in the analytical toolbox, equipment, and bioprocess technology have made it possible to achieve both unprecedented speed in vaccine development and scale of vaccine manufacturing. Macromolecular structure-function characterization technologies, combined with improved modeling and data analysis, enable quantitative evaluation of vaccine formulations at single-particle resolution and guided design of vaccine drug substances and drug products. These advances play a major role in precise assessment of critical quality attributes of vaccines delivered by newer platforms. Innovations in label-free and immunoassay technologies aid in the characterization of antigenic sites and the development of robust in vitro potency assays. These methods, along with molecular techniques such as next-generation sequencing, will accelerate characterization and release of vaccines delivered by all platforms. Process analytical technologies for real-time monitoring and optimization of process steps enable the implementation of quality-by-design principles and faster release of vaccine products. In the next decade, the field of vaccine discovery and development will continue to advance, bringing together new technologies, methods, and platforms to improve human health.

Impact of Glycosylation on Protein-Protein Self-Interactions of Monoclonal Antibodies.

Protein self-interactions measured via second osmotic virial coefficients (B22) and dynamic light scattering interaction parameter values (kD) are often used as metrics for assessing the favorability of protein candidates and different formulations during monoclonal antibody (MAb) product development. Model predictions of B22 or kD typically do not account for glycans, though glycosylation can potentially impact experimental MAb self-interactions. To the best of our knowledge, the impact of MAb glycosylation on the experimentally measured B22 and kD values has not yet been reported. B22 and kD values of two fully deglycosylated MAbs and their native (i.e., fully glycosylated) counterparts were measured by light scattering over a range of pH and ionic strength conditions. Significant differences between B22 and kD of the native and deglycosylated forms were observed at a range of low to high ionic strengths used to modulate the effect of electrostatic contributions. Differences were most pronounced at low ionic strength, indicating that electrostatic interactions are a contributing factor. Though B22 and kD values were statistically equivalent at high ionic strengths where electrostatics were fully screened, we observed protein-dependent qualitative differences, which indicate that steric interactions may also play a role in the observed B22 and kD differences. A domain-level coarse-grained molecular model accounting for charge differences was considered to potentially provide additional insight but was not fully predictive of the behavior across all of the solution conditions investigated. This highlights that both the level of modeling and lack of inclusion of glycans may limit existing models in making quantitatively accurate predictions of self-interactions.

Electrostatically Mediated Attractive Self-Interactions and Reversible Self-Association of Fc-Fusion Proteins.

Attractive self-interactions and reversible self-association are implicated in many problematic solution behaviors for therapeutic proteins, such as irreversible aggregation, elevated viscosity, phase separation, and opalescence. Protein self-interactions and reversible oligomerization of two Fc-fusion proteins (monovalent and bivalent) and the corresponding fusion partner protein were characterized experimentally with static and dynamic light scattering as a function of pH (5 and 6.5) and ionic strength (10 mM to at least 300 mM). The fusion partner protein and monovalent Fc-fusion each displayed net attractive electrostatic self-interactions at pH 6.5 and net repulsive electrostatic self-interactions at pH 5. Solutions of the bivalent Fc-fusion contained higher molecular weight species that prevented quantification of typical interaction parameters (B22 and kD). All three of the proteins displayed reversible self-association at pH 6.5, where oligomers dissociated with increased ionic strength. Coarse-grained molecular simulations were used to model the self-interactions measured experimentally, assess net self-interactions for the bivalent Fc-fusion, and probe the specific electrostatic interactions between charged amino acids that were involved in attractive electrostatic self-interactions. Mayer-weighted pairwise electrostatic energies from the simulations suggested that attractive electrostatic self-interactions at pH 6.5 for the two Fc-fusion proteins were due to cross-domain interactions between the fusion partner domain(s) and the Fc domain.

Antibodies Adsorbed to the Air-Water Interface Form Soft Glasses.

When monoclonal antibodies are exposed to an air-water interface, they form aggregates, which negatively impacts their performance. Until now, the detection and characterization of interfacial aggregation have been difficult. Here, we exploit the mechanical response imparted by interfacial adsorption by measuring the interfacial shear rheology of a model antibody, anti-streptavidin immunoglobulin-1 (AS-IgG1), at the air-water interface. Strong viscoelastic layers of AS-IgG1 form when the protein is adsorbed from the bulk solution. Creep experiments correlate the compliance of the interfacial protein layer with the subphase solution pH and bulk concentration. These, along with oscillatory strain amplitude and frequency sweeps, show that the viscoelastic behavior of the adsorbed layers is that of a soft glass with interfacial shear moduli on the order of 10-3 Pa m. Shifting the creep compliance curves under different applied stresses forms master curves consistent with stress-time superposition of soft interfacial glasses. The interfacial rheology results are discussed in the context of the interface-mediated aggregation of AS-IgG1.

Factors Affecting Delayed Gastric Emptying and Long-Term Outcomes of Children With Gastric Emptying.

OBJECTIVES: Our study attempted to identify what factors best predict for delayed gastric emptying (DGE) and whether children respond to treatment. METHODS: Children aged between 0 and 18 were included who had a gastric emptying scintigraphy (GES) study performed between 2009 and 2018. Baseline clinical details were recorded from clinic visit records regarding symptoms, medication, and past medical history. Results were analyzed using multivariate regression analysis and coefficient analysis. Children were followed up at 2 years to assess their symptoms and medication usage. RESULTS: Two hundred and eighty-five children were included in the study of which 174 demonstrated DGE. All children had symptoms prior to GES, the most common symptom being that of vomiting and reflux symptoms which were present in over 90% of patients; other common symptoms like abdominal pain and nausea were seen commonly in around 30%. A genetic disorder and prior surgery were more common in children with DGE but there was no difference in presenting symptoms between normal and DGE groups. Regression analysis showed prior surgery and particularly prior abdominal surgery predicted for DGE and additionally predicted for those with highly DGE. Improvement in symptoms and reduction in medication usage was seen after 2 years. CONCLUSIONS: This study provides one of the largest data sets looking at DGE in children. Prior surgery was found to be a key factor in predicting for highly DGE. Symptoms and medication usage did significantly reduce substantially after 2 years.

Personalised interventions for subgroups of children with conduct problems.

BACKGROUND: Conduct problems are a range of disruptive behaviours in childhood that are associated with long-term adverse outcomes in adolescence and adulthood, including antisocial behaviour, substance misuse, and poor academic achievement. Children with conduct problems can vary according to age of onset, comorbidities, and environmental factors, and it has been suggested that certain groups of children may have different treatment outcomes. Therefore, it is important to assess the extent to which personalised interventions for different groups of children with conduct problems may affect outcomes. To our knowledge, this is the first review to systematically identify and appraise the effectiveness of personalised interventions, adapted, or developed, for prespecified subgroups of children with conduct problems. OBJECTIVES: To assess whether personalised interventions, adapted or developed for subgroups of children with conduct problems are effective in improving outcomes. SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search was 1 February 2022. SELECTION CRITERIA: We included randomised controlled trials (RCTs), in any setting, in children (aged two to 12 years) with conduct problems and within a prespecified subgroup, comparing a personalised intervention with a non-personalised intervention, waitlist control, or treatment as usual. Personalised interventions included adaptations to standard practice, such as parent-training programmes; other recommended interventions for children with conduct problems; or interventions developed specifically to target subgroups of children with conduct problems. We excluded non-personalised and non-psychological interventions (e.g. pharmacological or dietary intervention). Prespecified subgroups of children with conduct problems, however defined, were eligible for inclusion. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods. Our primary outcomes were 1. child conduct problems or disruptive behaviour and 2. ADVERSE EVENTS: Our secondary outcomes were 3. personalised treatment outcomes relevant to each subgroup, 4. parenting skills and knowledge, 5. family functioning, engagement and decreased dropout, and 6. educational outcomes. We used GRADE to assess the certainty of the evidence. MAIN RESULTS: We identified 13 RCTs (858 participants). Seven studies were conducted in the USA, five in Australia, and one in Germany. Eleven studies reported their source of funding, with five studies receiving grants from the National Institute of Mental Health. In total, 15 different funders supported the studies included in the review. We separated subgroups of children with conduct problems into three broad categories: children with co-occurring conditions (e.g. emotional difficulties), parent characteristics (e.g. conflict between parents), or familial/environmental circumstances (e.g. rural families). All studies delivered a personalised intervention that was adapted or developed for a prespecified subgroup of children with conduct problems. We rated all trials at unclear or high risk of bias in most domains. Below, we report the results of improvement in child conduct problems and disruptive behaviour, personalised treatment outcomes, and parenting skills and knowledge for our main comparison: personalised versus non-personalised interventions. Improvement in child conduct problems and disruptive behaviour Compared with a non-personalised intervention, a personalised intervention may result in a slight improvement in child conduct problems or disruptive behaviour measured using the Eyberg Child Behavior Inventory (ECBI) Problem subscale in the short term (mean difference (MD) -3.04, 95% confidence interval (CI) -6.06 to -0.02; 6 studies, 278 participants; P = 0.05), but may have little to no effect on improving child conduct problems or disruptive behaviour measured by the ECBI Intensity subscale (MD -6.25, 95% CI -16.66 to 4.15; 6 studies, 278 participants; P = 0.24), or the Externalising subscale of the Child Behaviour Checklist (CBCL) (MD -2.19, 95% CI -6.97 to 2.59; 3 studies, 189 participants, P = 0.37) in the short term. We graded the certainty of evidence as very low for all three outcomes, meaning any estimate of effect is very uncertain.  Personalised treatment outcomes, relevant to each subgroup Although six studies reported personalised treatment outcomes, relevant to each subgroup, we were unable to pool the data due to differences between the measures used in the studies and the heterogeneity this would produce in analysis. The results for this outcome were inconclusive. Parenting skills and knowledge Although seven studies reported parenting skills and knowledge, we were unable to pool the data due to differences between the measures used in the studies and the heterogeneity this would produce in analysis. The results for this outcome were inconclusive.  Adverse events None of the trials reported monitoring adverse events. Summary of results In summary, there is limited evidence that personalised intervention improves child conduct problems, personalised treatment outcomes, relevant to each subgroup, or parenting skills and knowledge compared with a non-personalised intervention. AUTHORS' CONCLUSIONS: There is limited evidence for the effectiveness of personalised interventions for subgroups of children with conduct problems. The certainty of evidence for all outcomes was very low, meaning that we have very little confidence in the estimated effects and the true effects may be different to our findings, which will limit the relevance of our findings to clinical decisions. To overcome the limitations of the evidence, large-scale RCTs are needed to determine whether personalised interventions, adapted or developed, for subgroups of children with conduct problems are effective in improving outcomes. Consensus on the most appropriate measures to use in these studies is needed in order to facilitate cross-study comparisons. Persistent conduct problems predict a range of adverse long-term outcomes, so future research should investigate the medium- and long-term effects of personalised treatments. Studies are needed in low- and middle-income countries as well as studies recruiting children aged between nine and 12 years, as they were under-represented in the studies.

Preliminary investigation of a significant national Cryptosporidium exceedance in the United Kingdom, August 2023 and ongoing.

Routine laboratory surveillance has identified an unprecedented and ongoing exceedance of Cryptosporidium spp. across the United Kingdom, notably driven by C. hominis transmission, since 14 August 2023. Information from 477 reported cases in England and Wales, followed up with a standardised exposure questionnaire as of 25 September 2023, identified foreign travel in 250 (54%) of 463 respondents and swimming in 234 (66%) of 353 cases. A significant, common exposure has not yet been identified in first analyses.

Preoperative low-molecular weight heparin chemoprophylaxis in head and neck free flap reconstruction.

BACKGROUND: The safety of presurgical thromboprophylaxis using low molecular weight heparin (LMWH) has not been well described in head and neck oncologic surgery with free tissue transfer (HNS-FTT). METHODS: Retrospective chart review of HNS-FTT patients receiving versus not receiving presurgical subcutaneous enoxaparin (Px-LMWH) was performed. Outcomes included estimated blood loss (EBL), hematoma, flap compromise, DVT or pulmonary embolus (PE). Fisher's exact test and Wilcoxon Rank Sum test were performed to compare groups. Odds ratios and associated 95 % confidence intervals were provided as appropriate. RESULTS: 43 of 128 patients (34 %) received Px-LMWH. There was no significant difference in EBL, hematoma, or flap complications between groups. Patients without Px-LMWH had higher rates of DVT and PE, although the difference did not reach statistical significance (p = 1.00, 0.095, respectively). CONCLUSION: Presurgical Px-LMWH can be used in major head and neck reconstructive surgery without increased intraoperative blood loss or postoperative complications. Larger studies will need to be done to determine the impact of Px-LMWH on DVT and PE in this patient population.

Three-dimensional decision support system for treatment of canine impaction.

INTRODUCTION: This study aimed to develop a 3-dimensional (3D) characterization of the severity of maxillary impacted canines and to test the clinical performance of this characterization as a treatment decision support tool. METHODS: Cone-beam computed tomography images obtained from 83 patients with 120 impacted maxillary canines were included. Quantitative information on the canine 3D position and qualitative assessment of root damage of adjacent teeth were evaluated. A severity index was constructed on the basis of the quantitative findings. Clinical applicability was tested by comparing clinical diagnosis and treatment planning for conventional records vs the 3D characterization via a 2-part survey. RESULTS: The average quantitative assessments of impacted maxillary canine position were 6.4 ± 3.6 mm from the midsagittal plane, 11.6 ± 3.1 mm in height relative to the occlusal plane, 31.5° ± 18° of roll, and 48.8° ± 14.3° of pitch. The severity index ranged from 0-13 with a mean score of 4.5 ± 2.2. Overlap with adjacent teeth was the greatest contributor (33%) to the index. Bicortically impacted canines caused the most severe root damage. Cone-beam computed tomography was preferred for assessing root damage and overall severity, whereas conventional imaging was sufficient for height and angulation assessment. The 3D report was very important or important for evaluating root damage, canine position, overall severity, and overlap. The 3D report changed most of the decisions relating to biomechanics, patient education, and treatment time estimate. The decision of exposure and traction vs extraction was changed 22% of the time after the presentation of the 3D report. CONCLUSIONS: The overlap with adjacent teeth frequently contributes the most to the severity index. The 3D report provided relevant clinical information regarding the canine position, damage to adjacent teeth, and the severity index, with a profound impact on the decisions of the clinicians regarding biomechanics, patient education, and treatment time estimate.

Fungal-fungal co-culture: a primer for generating chemical diversity.

Covering: 2002 to 2020In their natural environment, fungi must compete for resources. It has been hypothesized that this competition likely induces the biosynthesis of secondary metabolites for defence. In a quest to discover new chemical diversity from fungal cultures, a growing trend has been to recapitulate this competitive environment in the laboratory, essentially growing fungi in co-culture. This review covers fungal-fungal co-culture studies beginning with the first literature report in 2002. Since then, there has been a growing number of new secondary metabolites reported as a result of fungal co-culture studies. Specifically, this review discusses and provides insights into (1) rationale for pairing fungal strains, (2) ways to grow fungi for co-culture, (3) different approaches to screening fungal co-cultures for chemical diversity, (4) determining the secondary metabolite-producing strain, and (5) final thoughts regarding the fungal-fungal co-culture approach. Our goal is to provide a set of practical strategies for fungal co-culture studies to generate unique chemical diversity that the natural products research community can utilize.

Shifts in predator behaviour following climate induced disturbance on coral reefs.

Coral reefs are increasingly ecologically destabilized across the globe due to climate change. Behavioural plasticity in corallivore behaviour and short-term trophic ecology in response to bleaching events may influence the extent and severity of coral bleaching and subsequent recovery potential, yet our understanding of these interactions in situ remains unclear. Here, we investigated interactions between corallivory and coral bleaching during a severe high thermal event (10.3-degree heating weeks) in Belize. We found that parrotfish changed their grazing behaviour in response to bleaching by selectively avoiding bleached Orbicella spp. colonies regardless of bleaching severity or coral size. For bleached corals, we hypothesize that this short-term respite from corallivory may temporarily buffer coral energy budgets by not redirecting energetic resources to wound healing, and may therefore enable compensatory nutrient acquisition. However, colonies that had previously been heavily grazed were also more susceptible to bleaching, which is likely to increase mortality risk. Thus, short-term respite from corallivory during bleaching may not be sufficient to functionally rescue corals during prolonged bleaching. Such pairwise interactions and behavioural shifts in response to disturbance may appear small scale and short term, but have the potential to fundamentally alter ecological outcomes, especially in already-degraded ecosystems that are vulnerable and sensitive to change.

Biomanufacturing readiness levels [BRL]-A shared vocabulary for biopharmaceutical technology development and commercialization.

The Manufacturing Readiness Levels (MRLs) developed by the Department of Defense are well-established tools for describing the maturity of new technologies resulting from government-sponsored Research and Development programs, from the concept phase to commercial deployment. While MRLs are generally applicable to a wide range of industries and technologies, there is significant value in offering an industry-specific view on how the basic principles may be applied to biomanufacturing. This paper describes Biomanufacturing Readiness Levels (BRLs) developed by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), a public/private partnership that is part of the Manufacturing USA network. NIIMBL brings together private, federal, nonprofit, and academic stakeholders to accelerate the deployment of innovative technologies for biopharmaceutical production and to educate and train a world-leading biomanufacturing workforce. We anticipate that these BRLs will lay the groundwork for a shared vocabulary for assessment of technology maturity and readiness for commercial biomanufacturing that effectively meets the needs of this critical, specialized, and highly regulated industry.

Predicting Experimental B22 Values and the Effects of Histidine Charge States for Monoclonal Antibodies Using Coarse-Grained Molecular Simulations.

Static light scattering (SLS) was used to characterize five monoclonal antibodies (MAbs) as a function of total ionic strength (TIS) at pH values between 5.5 and 7.0. Second osmotic virial coefficient (B22) values were determined experimentally for each MAb as a function of TIS using low protein concentration SLS data. Coarse-grained molecular simulations were performed to predict the B22 values for each MAb at a given pH and TIS. To include the effect of charge fluctuations of titratable residues in the B22 calculations, a statistical approach was introduced in the Monte Carlo algorithm based on the protonation probability based on a given pH value and the Henderson-Hasselbalch equation. The charged residues were allowed to fluctuate individually, based on the sampled microstates and the influence of electrostatic interactions on net protein-protein interactions during the simulations. Compared to static charge simulations, the new approach provided improved results compared to experimental B22 values at pH conditions near the pKa of titratable residues.

Characterizing the Heart Rate Effects From Administration of Sugammadex to Reverse Neuromuscular Blockade: An Observational Study in Patients.

BACKGROUND: Reversal of neuromuscular blockade (NMB) with sugammadex can cause marked bradycardia and asystole. Administration of sugammadex typically occurs in a dynamic period when anesthetic adjuvants and gas concentrations are being titrated to achieve emergence. This evaluation examined the heart rate (HR) responses to sugammadex to reverse moderate to deep NMB during a steady-state period and sought mechanisms for HR changes. METHODS: Patients with normal sinus rhythm, who were undergoing elective surgery that included rocuronium for NMB, were evaluated. After surgery, while at steady-state surgical depth anesthesia with sevoflurane and mechanical ventilation, patients received either placebo or 2 or 4 mg/kg of sugammadex to reverse moderate to deep NMB. Study personnel involved in data analysis were blinded to treatment. Continuous electrocardiogram (ECG) was recorded from the 5 minutes before and 5 minutes after sugammadex/placebo administration. R-R intervals were converted to HR and averaged in 1-minute increments. The maximum prolongation of an R-R interval after sugammadex was converted to an instantaneous HR. RESULTS: A total of 63 patients were evaluated: 8 received placebo, and 38 and 17 received 2 and 4 mg/kg sugammadex. Age, body mass index, and patient factors were similar in groups. Placebo did not elicit HR changes, whereas sugammadex caused maximum instantaneous HR slowing (calculated from the longest R-R interval), ranging from 2 to 19 beats/min. There were 7 patients with maximum HR slowing >10 beats/min. The average HR change and 95% confidence interval (CI) during the 5 minutes after 2 mg/kg sugammadex were 3.1 (CI, 2.3-4.1) beats/min, and this was not different from the 4 mg/kg sugammadex group (4.1 beats/min [CI, 2.5-5.6]). HR variability derived from the standard deviation of consecutive R-R intervals increased after sugammadex. CONCLUSIONS: Sugammadex to reverse moderate and deep NMB resulted in a fast onset and variable magnitude of HR slowing in patients. A difference in HR slowing as a function of dose did not achieve statistical significance. The observational nature of the investigation prevented a full understanding of the mechanism(s) of the HR slowing.

Intravenous dantrolene in hypermetabolic syndromes: a survey of the U.S. Veterans Health Administration database.

BACKGROUND: Intravenous dantrolene is often prescribed for hypermetabolic syndromes other than the approved indication of malignant hyperthermia (MH). To clarify the extent of and indications for dantrolene use in conditions other than MH, we sought to document current practices in the frequency, diagnoses, clinical characteristics and outcomes associated with dantrolene treatment in critical care settings. METHODS: Inpatients receiving intravenous dantrolene from October 1, 2004 to September 30, 2014 were identified retrospectively in the U.S. Veterans Health Administration national database. Extracted data included; diagnoses of hypermetabolic syndromes; triggering drugs; dantrolene dosages; demographics; vital signs; laboratory values; in-hospital mortality; complications; and lengths of stay. Frequency and mortality of patients who did not receive dantrolene were obtained in selected diagnoses for exploratory comparisons. RESULTS: Dantrolene was administered to 304 inpatients. The most frequent diagnoses associated with dantrolene treatment were neuroleptic malignant syndrome (NMS; N = 108, 35.53%) and sepsis (N = 47, 15.46%), with MH accounting for only 13 (4.28%) cases. Over half the patients had psychiatric comorbidities and received psychotropic drugs before dantrolene treatment. Common clinical findings in patients receiving dantrolene included elevated temperature (mean ± SD; 38.7 ± 1.3 °C), pulse (116.33 ± 22.80/bpm), respirations (27.75 ± 9.58/min), creatine kinase levels (2,859.37 ± 6,646.88 IU/L) and low pO2 (74.93 ± 40.16 mmHg). Respiratory, renal or cardiac failure were common complications. Mortality rates in-hospital were 24.01% overall, 7.69% in MH, 20.37% in NMS and 42.55% in sepsis, compared with mortality rates in larger and possibly less severe groups of unmatched patients with MH (5.26%), NMS (6.66%), or sepsis (41.91%) who did not receive dantrolene. CONCLUSIONS: In over 95% of cases, dantrolene administration was associated with diagnoses other than MH in critically-ill patients with hypermetabolic symptoms and medical and psychiatric comorbidities. Exploratory survey data suggested that the efficacy and safety of dantrolene in preventing mortality in hypermetabolic syndromes other than MH remain uncertain. However, randomized and controlled studies using standardized criteria between groups matched for severity are essential to guide practice in using dantrolene.

Risk Assessment of Norovirus Illness from Consumption of Raw Oysters in the United States and in Canada.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Bivalve molluscan shellfish is one commodity commonly identified as being a vector of NoV. Bivalve molluscan shellfish are grown in waters that may be affected by contamination events, tend to bioaccumulate viruses, and are frequently eaten raw. In an effort to better assess the elements that contribute to potential risk of NoV infection and illness from consumption of bivalve molluscan shellfish, the U.S. Department of Health and Human Services/Food and Drug Administration (FDA), Health Canada (HC), the Canadian Food Inspection Agency (CFIA), and Environment and Climate Change Canada (ECCC) collaborated to conduct a quantitative risk assessment for NoV in bivalve molluscan shellfish, notably oysters. This study describes the model and scenarios developed and results obtained to assess the risk of NoV infection and illness from consumption of raw oysters harvested from a quasi-steady-state situation. Among the many factors that influence the risk of NoV illness for raw oyster consumers, the concentrations of NoV in the influent (raw, untreated) and effluent (treated) of wastewater treatment plants (WWTP) were identified to be the most important. Thus, mitigation and control strategies that limit the influence from human waste (WWTP outfalls) in oyster growing areas have a major influence on the risk of illness from consumption of those oysters.

Thyroid and androgen receptor signaling are antagonized by µ-Crystallin in prostate cancer.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3'-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein µ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRß) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3- and androgen-mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth.

In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress.

Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice-liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as -15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near -10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (TCD ∼ -17 °C; calculated via isothermal chemical denaturation and Gibbs-Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near -5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.

Extracorporeal Membrane Oxygenation with Right Ventricular Assist Device for COVID-19 ARDS.

BACKGROUND: Right ventricular failure is an underrecognized consequence of COVID-19 pneumonia. Those with severe disease are treated with extracorporeal membrane oxygenation (ECMO) but with poor outcomes. Concomitant right ventricular assist device (RVAD) may be beneficial. METHODS: A retrospective analysis of intensive care unit patients admitted with COVID-19 ARDS (Acute Respiratory Distress Syndrome) was performed. Nonintubated patients, those with acute kidney injury, and age > 75 were excluded. Patients who underwent RVAD/ECMO support were compared with those managed via invasive mechanical ventilation (IMV) alone. The primary outcome was in-hospital mortality. Secondary outcomes included 30-d mortality, acute kidney injury, length of ICU stay, and duration of mechanical ventilation. RESULTS: A total of 145 patients were admitted to the ICU with COVID-19. Thirty-nine patients met inclusion criteria. Of these, 21 received IMV, and 18 received RVAD/ECMO. In-hospital (52.4 versus 11.1%, P = 0.008) and 30-d mortality (42.9 versus 5.6%, P= 0.011) were significantly lower in patients treated with RVAD/ECMO. Acute kidney injury occurred in 15 (71.4%) patients in the IMV group and zero RVAD/ECMO patients (P< 0.001). ICU (11.5 versus 21 d, P= 0.067) and hospital (14 versus 25.5 d, P = 0.054) length of stay were not significantly different. There were no RVAD/ECMO device complications. The duration of mechanical ventilation was not significantly different (10 versus 5 d, P = 0.44). CONCLUSIONS: RVAD support at the time of ECMO initiation resulted in the no secondary end-organ damage and higher in-hospital and 30-d survival versus IMV in specially selected patients with severe COVID-19 ARDS. Management of severe COVID-19 ARDS should prioritize right ventricular support.

Opportunities and Limitations for Assigning Relative Configurations of Antibacterial Bislactones using GIAO NMR Shift Calculations.

Four new bislactones, dihydroacremonol (1), clonostachyone (2), acremodiol B (3), and acremodiol C (4), along with one known compound, hymeglusin (5), were isolated from cultures of two fungal strains (MSX59876 and MSX59260). Both strains were identified based on phylogenetic analysis of molecular data as Clonostachys spp.; yet, they biosynthesized a suite of related, but different, secondary metabolites. Given the challenges associated with elucidating the structures and configurations of bislactones, GIAO NMR calculations were tested as a complement to traditional NMR and HRESIMS experiments. Fortuitously, the enantiomer of the new natural product (4) was known as a synthetic compound, and the predicted configuration from GIAO NMR calculations (i.e., for the relative configuration) and optical rotation calculations (i.e., for the absolute configuration) matched those of the synthesis product. These results engendered confidence in using similar procedures, particularly the mixture of GIAO NMR shift calculations coupled with an orthogonal technique, to predict the configuration of 1-3; however, there were important limitations, which are discussed for each of these. The metabolites displayed antimicrobial activities, with compounds 1 and 4 being the most potent against Staphylococcus aureus with MICs of 1 and 4 µg/mL, respectively.