Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Noninvasive Ventilation for Preoxygenation during Emergency Intubation.

BACKGROUND: Among critically ill adults undergoing tracheal intubation, hypoxemia increases the risk of cardiac arrest and death. The effect of preoxygenation with noninvasive ventilation, as compared with preoxygenation with an oxygen mask, on the incidence of hypoxemia during tracheal intubation is uncertain. METHODS: In a multicenter, randomized trial conducted at 24 emergency departments and intensive care units in the United States, we randomly assigned critically ill adults (age, ≥18 years) undergoing tracheal intubation to receive preoxygenation with either noninvasive ventilation or an oxygen mask. The primary outcome was hypoxemia during intubation, defined by an oxygen saturation of less than 85% during the interval between induction of anesthesia and 2 minutes after tracheal intubation. RESULTS: Among the 1301 patients enrolled, hypoxemia occurred in 57 of 624 patients (9.1%) in the noninvasive-ventilation group and in 118 of 637 patients (18.5%) in the oxygen-mask group (difference, -9.4 percentage points; 95% confidence interval [CI], -13.2 to -5.6; P<0.001). Cardiac arrest occurred in 1 patient (0.2%) in the noninvasive-ventilation group and in 7 patients (1.1%) in the oxygen-mask group (difference, -0.9 percentage points; 95% CI, -1.8 to -0.1). Aspiration occurred in 6 patients (0.9%) in the noninvasive-ventilation group and in 9 patients (1.4%) in the oxygen-mask group (difference, -0.4 percentage points; 95% CI, -1.6 to 0.7). CONCLUSIONS: Among critically ill adults undergoing tracheal intubation, preoxygenation with noninvasive ventilation resulted in a lower incidence of hypoxemia during intubation than preoxygenation with an oxygen mask. (Funded by the U.S. Department of Defense; PREOXI ClinicalTrials.gov number, NCT05267652.).

Polymicrobial Purulent Pericarditis From a Pancreatico-Pericardial Fistula.

A 54-year-old male with chronic pancreatitis presented with dyspnea. Computed tomography scans demonstrated a subdiaphragmatic fluid collection with pericardial fistulization. Pericardial fluid cultures were polymicrobial in nature. Purulent pericarditis is rare but carries a high mortality rate. We present the first documented case of pancreatico-pericardial fistulization causing purulent pericarditis.

The Neuromechanics of Inspiratory Muscles in Mechanical Ventilation Liberation Success and Failure.

BACKGROUND: Assessing the neuromechanical coupling of inspiratory muscles during mechanical ventilation (MV) could reveal the physiological mechanism of MV failure. This study examined the respiratory neuromechanical characteristics between MV liberation success and failure. METHODS: This is an observational prospective study that included patients during their ventilator liberation process. Assessment of surface electromyography (sEMG) of inspiratory muscles, including the diaphragm and extra-diaphragmatic (scalene, sternocleidomastoid, and parasternal) muscles, was performed 15 minutes after the initiation of spontaneous breathing trials. Neuromechanical efficiency of the diaphragm (NMEDia) and extra-diaphragmatic muscles (NMEExtra) were compared in patients who were successfully liberated from MV with those who failed MV liberation within 72 hours after extubation. RESULTS: A total of 45 patients were enrolled and 28 were female (67%). The sample median age was 63 (IQR 47, 69) years old. One-third of patients failed MV liberation within 72 hours of their spontaneous breathing trials (SBTs). NMEDia was significantly lower in patients who failed MV liberation with a root mean square of (M 0.27), (IQR 0.21, 0.37) compared with (M 0.371), (IQR 0.3, 0.631) for the success group (p=0.0222). The area under the curve for NMEDia was lower in the failure group (M 0.270), (IQR 0.160, 0.370) and (M 0.485), (IQR 0.280, 0.683) for the success group (p=0.024). However, NMEExtra was not statistically different between the two groups. CONCLUSION: Reduced NMEDia is a predictor of MV liberation failure. NMEExtra was not a major contributor to MV liberation outcomes. Further studies should assess the performance of inspiratory muscles NME indices to predict MV liberation outcomes.

Protease-armed, Pathogenic Extracellular Vesicles Link Smoking and Chronic Obstructive Pulmonary Disease.

Rationale: Mounting evidence demonstrates a role for extracellular vesicles (EVs) in driving lung disorders, such as chronic obstructive pulmonary disease (COPD). Although cigarette smoke (CS) is the primary risk factor for COPD, a link between CS and the EVs that could lead to COPD is unknown. Objective: To ascertain whether exposure to CS elicits a proteolytic EV signature capable of driving disease pathogenesis. Methods: Protease expression and enzymatic activity were measured in EVs harvested from the BAL fluid of smoke-exposed mice and otherwise healthy human smokers. Pathogenicity of EVs was examined using pathological tissue scoring after EV transfer into naive recipient mice. Measurements and Main Results: The analyses revealed a unique EV profile defined by neutrophil- and macrophage-derived EVs. These EVs are characterized by abundant surface expression of neutrophil elastase (NE) and matrix metalloproteinase 12 (MMP12), respectively. CS-induced mouse or human-derived airway EVs had a robust capacity to elicit rapid lung damage in naive recipient mice, with an additive effect of NE- and MMP12-expressing EVs. Conclusions: These studies demonstrate the capacity of CS to drive the generation of unique EV populations containing NE and MMP12. The coordinated action of these EVs is completely sufficient to drive emphysematous disease, and their presence could operate as a prognostic indicator for COPD development. Furthermore, given the robust capacity of these EVs to elicit emphysema in naive mice, they provide a novel model to facilitate preclinical COPD research. Indeed, the development of this model has led to the discovery of a previously unrecognized CS-induced protective mechanism against EV-mediated damage.

Prophylactic Administration of Vasopressors Prior to Emergency Intubation in Critically Ill Patients: A Secondary Analysis of Two Multicenter Clinical Trials.

Hypotension affects approximately 40% of critically ill patients undergoing emergency intubation and is associated with an increased risk of death. The objective of this study was to examine the association between prophylactic vasopressor administration and the incidence of peri-intubation hypotension and other clinical outcomes. DESIGN: A secondary analysis of two multicenter randomized clinical trials. The clinical effect of prophylactic vasopressor administration was estimated using a one-to-one propensity-matched cohort of patients with and without prophylactic vasopressors. SETTING: Seven emergency departments and 17 ICUs across the United States. PATIENTS: One thousand seven hundred ninety-eight critically ill patients who underwent emergency intubation at the study sites between February 1, 2019, and May 24, 2021. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was peri-intubation hypotension defined as a systolic blood pressure less than 90 mm Hg occurring between induction and 2 minutes after tracheal intubation. A total of 187 patients (10%) received prophylactic vasopressors prior to intubation. Compared with patients who did not receive prophylactic vasopressors, those who did were older, had higher Acute Physiology and Chronic Health Evaluation II scores, were more likely to have a diagnosis of sepsis, had lower pre-induction systolic blood pressures, and were more likely to be on continuous vasopressor infusions prior to intubation. In our propensity-matched cohort, prophylactic vasopressor administration was not associated with reduced risk of peri-intubation hypotension (41% vs 32%; p = 0.08) or change in systolic blood pressure from baseline (-12 vs -11 mm Hg; p = 0.66). CONCLUSIONS: The administration of prophylactic vasopressors was not associated with a lower incidence of peri-intubation hypotension in our propensity-matched analysis. To address potential residual confounding, randomized clinical trials should examine the effect of prophylactic vasopressor administration on peri-intubation outcomes.

Video versus Direct Laryngoscopy for Tracheal Intubation of Critically Ill Adults.

BACKGROUND: Whether video laryngoscopy as compared with direct laryngoscopy increases the likelihood of successful tracheal intubation on the first attempt among critically ill adults is uncertain. METHODS: In a multicenter, randomized trial conducted at 17 emergency departments and intensive care units (ICUs), we randomly assigned critically ill adults undergoing tracheal intubation to the video-laryngoscope group or the direct-laryngoscope group. The primary outcome was successful intubation on the first attempt. The secondary outcome was the occurrence of severe complications during intubation; severe complications were defined as severe hypoxemia, severe hypotension, new or increased vasopressor use, cardiac arrest, or death. RESULTS: The trial was stopped for efficacy at the time of the single preplanned interim analysis. Among 1417 patients who were included in the final analysis (91.5% of whom underwent intubation that was performed by an emergency medicine resident or a critical care fellow), successful intubation on the first attempt occurred in 600 of the 705 patients (85.1%) in the video-laryngoscope group and in 504 of the 712 patients (70.8%) in the direct-laryngoscope group (absolute risk difference, 14.3 percentage points; 95% confidence interval [CI], 9.9 to 18.7; P<0.001). A total of 151 patients (21.4%) in the video-laryngoscope group and 149 patients (20.9%) in the direct-laryngoscope group had a severe complication during intubation (absolute risk difference, 0.5 percentage points; 95% CI, -3.9 to 4.9). Safety outcomes, including esophageal intubation, injury to the teeth, and aspiration, were similar in the two groups. CONCLUSIONS: Among critically ill adults undergoing tracheal intubation in an emergency department or ICU, the use of a video laryngoscope resulted in a higher incidence of successful intubation on the first attempt than the use of a direct laryngoscope. (Funded by the U.S. Department of Defense; DEVICE ClinicalTrials.gov number, NCT05239195.).

Laryngoscopy and Tracheal Intubation: Does Use of a Video Laryngoscope Facilitate Both Steps of the Procedure?

STUDY OBJECTIVE: To compare the effect of the use of a video laryngoscope versus a direct laryngoscope on each step of emergency intubation: laryngoscopy (step 1) and intubation of the trachea (step 2). METHODS: In a secondary observational analysis of data from 2 multicenter, randomized trials that enrolled critically ill adults undergoing tracheal intubation but did not control for laryngoscope type (video laryngoscope vs direct laryngoscope), we fit mixed-effects logistic regression models examining the 1) the association between laryngoscope type (video laryngoscope vs direct laryngoscope) and the Cormack-Lehane grade of view and 2) the interaction between grade of view, laryngoscope type (video laryngoscope vs direct laryngoscope), and the incidence of successful intubation on the first attempt. RESULTS: We analyzed 1,786 patients: 467 (26.2%) in the direct laryngoscope group and 1,319 (73.9%) in the video laryngoscope group. The use of a video laryngoscope was associated with an improved grade of view as compared with a direct laryngoscope (adjusted odds ratio for increasingly favorable grade of view 3.14, 95% confidence interval [CI] 2.47 to 3.99). Successful intubation on the first attempt occurred in 83.2% of patients in the video laryngoscope group and 72.2% of patients in the direct laryngoscope group (absolute difference 11.1%, 95% CI 6.5% to 15.6%). Video laryngoscope use modified the association between grade of view and successful intubation on the first attempt such that intubation on the first attempt was similar between video laryngoscope and direct laryngoscope at a grade 1 view and higher for video laryngoscope than direct laryngoscope at grade 2 to 4 views (P<.001 for interaction term). CONCLUSIONS: Among critically ill adults undergoing tracheal intubation, the use of a video laryngoscope was associated both with a better view of the vocal cords and with a higher probability of successfully intubating the trachea when the view of the vocal cords was incomplete in this observational analysis. However, a multicenter, randomized trial directly comparing the effect of a video laryngoscope with a direct laryngoscope on the grade of view, success, and complications is needed.

Therapeutic effect of two strategies directed at disruption of pathogenic neutrophil extracellular vesicles in a murine emphysema model.

Chronic obstructive pulmonary disease (COPD) is characterized by lung extracellular matrix (ECM) remodeling that contributes to obstruction. This is driven, in part by extracellular vesicles (EVs) from activated neutrophils (PMNs), which express on their surface an α-1 antitrypsin (AAT) insensitive form of neutrophil elastase (NE). These EVs are predicted to bind to collagen fibers via Mac-1 integrins, during which time NE can enzymatically degrade the collagen. Protamine sulfate (PS), a cationic compound used safely for decades in humans, has been shown, in vitro, to dissociate this NE from the EV surface, rendering it AAT-sensitive. In addition, a nonapeptide inhibitor, MP-9, has been shown to prevent EV association with collagen. We sought to test whether PS, MP-9, or a combination of the two could effectively prevent NE+ EV-driven ECM remodeling in an animal COPD model. EVs were preincubated with PBS, protamine sulfate (25 µM), MP-9 (50 µM), or a combination of PS and MP-9. These were delivered intratracheally to anesthetized female 10- to 12-wk-old A/J mice for a 7-day time period. One group of mice was euthanized and lungs sectioned for morphometry, and the other group was used for live pulmonary function testing. The effect of alveolar destruction by activated neutrophil EVs was abrogated by pretreatment with PS or MP-9. However, in pulmonary function tests, only the PS groups (and combined PS/MP-9 groups) returned pulmonary function to near-control levels. These data presented here offer an insight into the effective use of PS in therapeutic setting for EV-derived alveolar damage.NEW & NOTEWORTHY Protamine sulfate facilitates the removal of neutrophil elastase (NE) from the surface of extracellular vesicles from activated neutrophils. This "free" NE is no longer protected from inhibition by its endogenous anti-protease, α-1-anti-trypsin. This function of protamine sulfate highlights it as a potential therapeutic strategy for COPD, which may attenuate the disease process.

Defining Successful Intubation on the First Attempt Using Both Laryngoscope and Endotracheal Tube Insertions: A Secondary Analysis of Clinical Trial Data.

STUDY OBJECTIVES: Successful intubation on the first attempt has historically been defined as successful placement of an endotracheal tube (ETT) using a single laryngoscope insertion. More recent studies have defined successful placement of an ETT using a single laryngoscope insertion followed by a single ETT insertion. We sought to estimate the prevalence of first-attempt success using these 2 definitions and estimate their associations with the duration of intubation and serious complications. METHODS: We performed a secondary analysis of data from 2 multicenter randomized trials of critically ill adults being intubated in the emergency department or ICU. We calculated the percent difference in successful intubations on the first attempt, median difference in the duration of intubation, and percent difference in the development of serious complications by definition. RESULTS: The study population included 1,863 patients. Successful intubation on the first attempt decreased by 4.9% (95% confidence interval 2.5% to 7.3%) when defined as 1 laryngoscope insertion followed by 1 ETT insertion (81.2%) compared with when defined as only 1 laryngoscope insertion (86.0%). When successful intubation with 1 laryngoscope and 1 ETT insertion was compared with 1 laryngoscope and multiple ETT insertions, the median duration of intubation decreased by 35.0 seconds (95% confidence interval 8.9 to 61.1 seconds). CONCLUSION: Defining successful intubation on the first attempt as placement of an ETT in the trachea using 1 laryngoscope and 1 ETT insertion identifies attempts with the shortest apneic time.

Interferon-dependent signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections, including COVID-19-related acute respiratory distress syndrome, although its contribution to disease pathogenesis remains poorly understood. Blood and airway immune cells from 52 patients with severe COVID-19 were phenotyped by flow cytometry. Samples and clinical data were collected at 2 separate time points to assess changes during ICU stay. Blockade of type I interferon and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) signaling was performed in vitro to determine their contribution to viral clearance in A2 neutrophils. We identified 2 neutrophil subpopulations (A1 and A2) in the airway compartment, where loss of the A2 subset correlated with increased viral burden and reduced 30-day survival. A2 neutrophils exhibited a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation, with consequent reduced viral catabolism, providing the first discrete mechanism to our knowledge of type I interferon signaling in neutrophils. The identification of this neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Type I interferon-dependent IFIT3 signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections including COVID-19-related ARDS, although its contribution to disease pathogenesis remains poorly understood. Here, we identified two neutrophil subpopulations (A1 and A2) in the airway compartment of 52 severe COVID-19 subjects, where loss of the A2 subset correlated with increased viral burden and reduced 30-days survival. A2 neutrophils showcased a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation with consequent reduced viral catabolism, providing the first discrete mechanism of type I interferon signaling in neutrophils. The identification of this novel neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Protocol and statistical analysis plan for the PREOXI trial of preoxygenation with noninvasive ventilation vs oxygen mask.

Background: Hypoxemia is a common and life-threatening complication during emergency tracheal intubation of critically ill adults. The administration of supplemental oxygen prior to the procedure ("preoxygenation") decreases the risk of hypoxemia during intubation. Research Question: Whether preoxygenation with noninvasive ventilation prevents hypoxemia during tracheal intubation of critically ill adults, compared to preoxygenation with oxygen mask, remains uncertain. Study Design and Methods: The PRagmatic trial Examining OXygenation prior to Intubation (PREOXI) is a prospective, multicenter, non-blinded randomized comparative effectiveness trial being conducted in 7 emergency departments and 17 intensive care units across the United States. The trial compares preoxygenation with noninvasive ventilation versus oxygen mask among 1300 critically ill adults undergoing emergency tracheal intubation. Eligible patients are randomized in a 1:1 ratio to receive either noninvasive ventilation or an oxygen mask prior to induction. The primary outcome is the incidence of hypoxemia, defined as a peripheral oxygen saturation <85% between induction and 2 minutes after intubation. The secondary outcome is the lowest oxygen saturation between induction and 2 minutes after intubation. Enrollment began on 10 March 2022 and is expected to conclude in 2023. Interpretation: The PREOXI trial will provide important data on the effectiveness of noninvasive ventilation and oxygen mask preoxygenation for the prevention of hypoxemia during emergency tracheal intubation. Specifying the protocol and statistical analysis plan prior to the conclusion of enrollment increases the rigor, reproducibility, and interpretability of the trial. Clinical trial registration number: NCT05267652.

Effect of Laryngoscope Blade Size on First Pass Success of Tracheal Intubation in Critically Ill Adults.

Tracheal intubation (TI) is a common procedure in critical care, often performed with a Macintosh curved blade used for direct laryngoscopy (DL). Minimal evidence informs the choice between Macintosh blade sizes during TI. We hypothesized that Macintosh 4 blade would have higher first-attempt success than Macintosh 3 blade during DL. DESIGN: Retrospective analysis using a propensity score and inverse probability weighting of data from six prior multicenter randomized trials. SETTING AND PARTICIPANTS: Adult patients who underwent nonelective TI at participating emergency departments and ICUs. We compared the first-pass success of TI with DL in subjects intubated with a size 4 Macintosh blade on the first TI attempt to subjects with a size 3 Macintosh blade on the first TI attempt. MAIN RESULTS: Among 979 subjects, 592 (60.5%) had TI using DL with a Macintosh blade, of whom 362 (37%) were intubated with a size 4 blade and 222 (22.7%) with a size 3 blade. We used inverse probability weighting with a propensity score for analyzing data. We found that patients intubated with a size 4 blade had a worse (higher) Cormack-Lehane grade of glottic view than patients intubated with a size 3 blade (adjusted odds ratio [aOR], 1.458; 95% CI, 1.064-2.003; p = 0.02). Patients intubated with a size 4 blade had a lower first pass success than those with a size 3 blade (71.1% vs 81.2%; aOR, 0.566; 95% CI, 0.372-0.850; p = 0.01). CONCLUSIONS AND RELEVANCE: In critically ill adults undergoing TI using DL with a Macintosh blade, patients intubated using a size 4 blade on first attempt had a worse glottic view and a lower first pass success than patients intubated with a size 3 Macintosh blade. Further prospective studies are needed to examine the optimal approach to selecting laryngoscope blade size during TI of critically ill adults.

DirEct versus VIdeo LaryngosCopE (DEVICE): protocol and statistical analysis plan for a randomised clinical trial in critically ill adults undergoing emergency tracheal intubation.

INTRODUCTION: Among critically ill patients undergoing orotracheal intubation in the emergency department (ED) or intensive care unit (ICU), failure to visualise the vocal cords and intubate the trachea on the first attempt is associated with an increased risk of complications. Two types of laryngoscopes are commonly available: direct laryngoscopes and video laryngoscopes. For critically ill adults undergoing emergency tracheal intubation, it remains uncertain whether the use of a video laryngoscope increases the incidence of successful intubation on the first attempt compared with the use of a direct laryngoscope. METHODS AND ANALYSIS: The DirEct versus VIdeo LaryngosCopE (DEVICE) trial is a prospective, multicentre, non-blinded, randomised trial being conducted in 7 EDs and 10 ICUs in the USA. The trial plans to enrol up to 2000 critically ill adults undergoing orotracheal intubation with a laryngoscope. Eligible patients are randomised 1:1 to the use of a video laryngoscope or a direct laryngoscope for the first intubation attempt. The primary outcome is successful intubation on the first attempt. The secondary outcome is the incidence of severe complications between induction and 2 min after intubation, defined as the occurrence of one or more of the following: severe hypoxaemia (lowest oxygen saturation <80%); severe hypotension (systolic blood pressure <65 mm Hg or new or increased vasopressor administration); cardiac arrest or death. Enrolment began on 19 March 2022 and is expected to be completed in 2023. ETHICS AND DISSEMINATION: The trial protocol was approved with waiver of informed consent by the single institutional review board at Vanderbilt University Medical Center and the Human Research Protection Office of the Department of Defense. The results will be presented at scientific conferences and submitted for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT05239195).

Estimating the bioaccessibility of flocculants in the presence of sediments in model wastewater.

The cationic degradable polymer poly(lactic acid) choline iodide ester methacrylate, poly(PLA4ChMA), can be used to flocculate particles and dewater sediments from tailings ponds and wastewater. A suitable bioaccessibility method is required to characterize the interactions of this novel flocculant in the human gastrointestinal system. To this end, a physiologically based extraction test (PBET) was modified to evaluate the bioaccessibility of flocculants. Bioaccessibility (bioaccessible fraction) is a measure of the solubility of a contaminant in gastrointestinal fluids and that may be available for systemic absorption. The flocculants poly(PLA4ChMA), SNF C3276, and FLOPAM A3338 were tested at a solid-to-liquid ratio of 1:200 in the absence and presence of kaolin clay, which is used as a model sediment compound. Bioaccessible fractions were characterized by proton nuclear magnetic resonance spectroscopy and estimated by gravimetry. The bioaccessibility of poly(PLA4ChMA) in gastric and intestinal PBET solutions decreases from 78% to 100%, respectively, in the absence of kaolin to approximately 0% with kaolin, indicating that poly(PLA4ChMA) remains adsorbed onto the clay surface throughout the PBET, a result confirmed by thermogravimetric analysis. The bioaccessibility of cationic SNF C3276 and anionic FLOPAM A3338 in gastric solution is approximately 76% and 26%, respectively, and is not affected by the presence of kaolin. However, in intestinal solutions, the bioaccessibility of SNF C3276 and FLOPAM A3338 (60-85% in the absence of kaolin) changes to 0% and 100%, respectively, in the presence of kaolin. These results, interpreted in terms of solution pH and surface charge, demonstrate that interactions with kaolin influence the solubility of flocculants and must be considered in the evaluation of bioaccessibility. In future works, such bioaccessibility methods can be applied to assess the human-health safety of using flocculants in wastewater treatments.

I-SPY COVID adaptive platform trial for COVID-19 acute respiratory failure: rationale, design and operations.

INTRODUCTION: The COVID-19 pandemic brought an urgent need to discover novel effective therapeutics for patients hospitalised with severe COVID-19. The Investigation of Serial studies to Predict Your Therapeutic Response with Imaging And moLecular Analysis (ISPY COVID-19 trial) was designed and implemented in early 2020 to evaluate investigational agents rapidly and simultaneously on a phase 2 adaptive platform. This manuscript outlines the design, rationale, implementation and challenges of the ISPY COVID-19 trial during the first phase of trial activity from April 2020 until December 2021. METHODS AND ANALYSIS: The ISPY COVID-19 Trial is a multicentre open-label phase 2 platform trial in the USA designed to evaluate therapeutics that may have a large effect on improving outcomes from severe COVID-19. The ISPY COVID-19 Trial network includes academic and community hospitals with significant geographical diversity across the country. Enrolled patients are randomised to receive one of up to four investigational agents or a control and are evaluated for a family of two primary outcomes-time to recovery and mortality. The statistical design uses a Bayesian model with 'stopping' and 'graduation' criteria designed to efficiently discard ineffective therapies and graduate promising agents for definitive efficacy trials. Each investigational agent arm enrols to a maximum of 125 patients per arm and is compared with concurrent controls. As of December 2021, 11 investigational agent arms had been activated, and 8 arms were complete. Enrolment and adaptation of the trial design are ongoing. ETHICS AND DISSEMINATION: ISPY COVID-19 operates under a central institutional review board via Wake Forest School of Medicine IRB00066805. Data generated from this trial will be reported in peer-reviewed medical journals. TRIAL REGISTRATION NUMBER: NCT04488081.