Advancing surgical instrument safety: A screen of oxidative and alkaline prion decontaminants using real-time quaking-induced conversion with prion-coated steel beads as surgical instrument mimetic.

Iatrogenic transmission of prions, the infectious agents of fatal Creutzfeldt-Jakob disease, through inefficiently decontaminated medical instruments remains a critical issue. Harsh chemical treatments are effective, but not suited for routine reprocessing of reusable surgical instruments in medical cleaning and disinfection processes due to material incompatibilities. The identification of mild detergents with activity against prions is therefore of high interest but laborious due to the low throughput of traditional assays measuring prion infectivity. Here, we report the establishment of TESSA (sTainlESs steel-bead Seed Amplification assay), a modified real-time quaking induced cyclic amplification (RT-QuIC) assay that explores the propagation activity of prions with stainless steel beads. TESSA was applied for the screening of about 70 different commercially available and novel formulations and conditions for their prion inactivation efficacy. One hypochlorite-based formulation, two commercially available alkaline formulations and a manual alkaline pre-cleaner were found to be highly effective in inactivating prions under conditions simulating automated washer-disinfector cleaning processes. The efficacy of these formulations was confirmed in vivo in a murine prion infectivity bioassay, yielding a reduction of the prion titer for bead surface adsorbed prions below detectability. Our data suggest that TESSA represents an effective method for a rapid screening of prion-inactivating detergents, and that alkaline and oxidative formulations are promising in reducing the risk of potential iatrogenic prion transmission through insufficiently decontaminated instrument surfaces.

Characterization of a canine freeze-dried platelet-derived hemostatic agent: A preclinical model for surgical and traumatic hemorrhage.

INTRODUCTION: A freeze-dried, platelet-derived hemostatic agent (FPH) was developed for acute hemorrhage. The canine product (cFPH) was developed for use in preclinical models supporting human product (hFPH) investigations. MATERIALS AND METHODS: A carotid artery bypass graft (CABG) study in dogs compared 3 dosages of cFPH to canine liquid stored platelets (cLSP) and vehicle (VEH) control groups. Histopathological analysis and blood loss assessments were completed. A separate ex-vivo synthetic graft study assessed thrombogenicity via blood from human and canine donors that was combined with species-specific FPH or apheresis platelets. Characterization of cFPH and hFPH included thrombin generation, total thrombus formation, and scanning electron microscopy. RESULTS: Blood loss was reduced in CABG dogs receiving standard of care (cLSP) or cFPH treatment compared to VEH control; a cFPH dose effect signal was observed. Further, cFPH dosing up to 5 × 109 cells/kg was not associated with increased mortality or occlusion of the anastomosis sites, and histopathologic evidence of off-target thrombosis was not detected. When passed through a synthetic graft (ex vivo), whole blood combined with species-specific FPH did not result in thrombosis beyond that of whole blood control. In vitro testing and imaging of cFPH and FPH were comparable. CONCLUSIONS: A single dose of cFPH or cLSP reduced blood loss in a pilot surgical study and was well tolerated with no related adverse events. Further, the hemostatic activity and characteristics of cFPH are comparable to that of hFPH, suggesting that research findings from the canine product are likely to inform the development of the human product.

Mechanisms of action of an investigational new freeze-dried platelet-derived hemostatic product.

BACKGROUND: A safe and efficacious hemostatic product with a long shelf-life is needed to reduce mortality from hemorrhage due to trauma and improve surgical outcomes for persons with platelet deficiency or dysfunction. Thrombosomes, a trehalose-stabilized, leukoreduced, pooled blood group-O freeze-dried platelet-derived hemostatic (FPH) with a 3-year shelf-life, may satisfy this need. OBJECTIVES: To characterize the mechanism of action of FPH. METHODS: FPH's ability to adhere to collagen, aggregate with and without platelets, and form clots was evaluated in vitro. Nonobese diabetic-severe combined immunodeficiency mouse models were used to assess circulation persistence and hemostatic efficacy. RESULTS: FPH displays the morphology and surface proteins of activated platelets. FPH adheres to collagen, aggregates, and promotes clots, producing an insoluble fibrin mesh. FPH is rapidly cleared from circulation, has hemostatic efficacy comparable to apheresis platelets in a murine tail-cut, and acts in a dose-dependent manner. CONCLUSION: FPH is a first-in-class investigational treatment and shows strong potential as a hemostatic agent that is capable of binding exposed collagen, coaggregating with endogenous platelets, and promoting the coagulation cascade. These properties may be exploited to treat active platelet-related or diffuse vascular bleeding. FPH has the potential to fulfill a large unmet patient need as an acute hemostatic treatment in severe bleeding, such as surgery and trauma.

Pilot study of frozen platelet extracellular vesicles as a therapeutic agent in hemorrhagic shock in rats.

BACKGROUND: Hemorrhage accounts for the most preventable deaths after trauma. Resuscitation is guided by studies that demonstrate improved outcomes in patients receiving whole blood or balanced administration of blood products. Platelets present a logistical challenge due to short shelf life and need for refrigeration. Platelet-derived extracellular vesicles (PEVs) are a possible platelet alternative. Platelet-derived extracellular vesicles are secreted from platelets, have hemostatic effects and mitigate inflammation and vascular injury, similar to platelets. This pilot study aimed to elucidate the therapeutic effects of PEVs in a rat model of uncontrolled hemorrhage. METHODS: Male rats were anesthetized and femoral vessels cannulated. Vital signs (MAP, HR, and RR) were monitored. Electrolytes, lactate and ABG were obtained at baseline, 1-hour and 3-hours post injury. Laparotomy was performed, 50% of the middle hepatic lobe excised and the abdomen packed with gauze. Rats received 2 mL PEVs or lactated Ringers (LR) over 6 minutes immediately after injury. Peritoneal blood loss was quantified using preweighed gauze at 5 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes. Laparotomy was closed 1-hour postinjury. Animals were monitored for 3 hours postinjury then euthanized. Generalized Linear Mixed Effects models were performed to assess effects of treatment and time on lactate and MAP. RESULTS: Twenty-one rats were included (11 LR, 10 PEV). Overall blood loss was between 6 mL and 10 mL and not significantly different between groups. There was a 36% mortality rate in the LR group and 0% mortality in the PEV group ( p = 0.03). The LR group had significantly higher lactates at 1 hour ( p = 0.025). At 15 minutes, 45 minutes, 60 minutes, and 180 minutes, the MAP of the PEV group was significantly higher than the LR group. CONCLUSION: Early studies are encouraging regarding the potential use of PEVs in uncontrolled hemorrhagic shock based on improved survival and hemodynamics.

A large-scale forward genetic screen for maize mutants with altered lignocellulosic properties.

The development of efficient pipelines for the bioconversion of grass lignocellulosic feedstocks is challenging due to the limited understanding of the molecular mechanisms controlling the synthesis, deposition, and degradation of the varying polymers unique to grass cell walls. Here, we describe a large-scale forward genetic approach resulting in the identification of a collection of chemically mutagenized maize mutants with diverse alterations in their cell wall attributes such as crystalline cellulose content or hemicellulose composition. Saccharification yield, i.e. the amount of lignocellulosic glucose (Glc) released by means of enzymatic hydrolysis, is increased in two of the mutants and decreased in the remaining six. These mutants, termed candy-leaf (cal), show no obvious plant growth or developmental defects despite associated differences in their lignocellulosic composition. The identified cal mutants are a valuable tool not only to understand recalcitrance of grass lignocellulosics to enzymatic deconstruction but also to decipher grass-specific aspects of cell wall biology once the genetic basis, i.e. the location of the mutation, has been identified.

Telehealth for Pediatric Disorders of Gut-Brain Interaction During the COVID-19 Pandemic.

A retrospective chart review was completed to examine psychological treatment duration and response among pediatric patients with a disorder of gut-brain interaction including functional abdominal pain and irritable bowel syndrome. Cognitive behavioral therapy (CBT) was delivered via telehealth with a licensed psychologist or supervised psychology trainee embedded in a pediatric gastroenterology clinic. Participants were 22 youth (mean age = 14.36 years) who received CBT via telehealth between February and September of 2021, after completing an initial evaluation between February and July of 2021. Patients completed reliable and valid self-report measures of functional disability and pain during treatment. A unique CBT model was employed with an initial focus on psychoeducation and function regardless of level of severity of functional impairment. Consistent with study hypotheses, nonparametric statistical analyses demonstrated statistically significant reductions in functional disability and pain following implementation of the CBT model via telehealth. Contrary to predictions, there was no relation found between severity of functional impairment and duration of treatment.

Utilization of Pediatric Psychology Services in Outpatient Pediatric Gastroenterology Within a Rural Health System.

Increased utilization of pediatric psychology services has been demonstrated following integration into urban pediatric gastroenterology clinics; however, examination within rural health systems is lacking. Utilization of pediatric psychology services was assessed through a retrospective analysis of Electronic Health Record data contrasting referrals occurring six months pre- and post-integration of pediatric psychology in an outpatient pediatric gastroenterology clinic within a rural setting. Significant increases in the number of referrals to pediatric psychology and number of billed initial visits were observed after integration, as was a significant decrease in time to be seen. Patients with public insurance were 3.1 times more likely to complete a billed initial visit compared with patients with nonpublic insurance. The current findings support the integration of pediatric psychology within rural outpatient pediatric gastroenterology clinics to increase utilization and allow more traditionally underserved families to benefit from these services.

Patient reported data integration for management of Eosinophilic Esophagitis.

Eosinophilic Esophagitis (EoE) is an esophageal allergic inflammatory disorder triggered by food proteins. Symptoms of EoE are variable within and between individuals. Presenting symptoms may include dysphagia, food bolus impaction, dyspepsia, or more subtle symptoms such as feeding disorders, regurgitation sensation, or nausea. The development and validation of a pediatric EoE patient self-reported and parent proxy-reported outcome symptom scoring tool was created by Franciosi et al. published in BMJ 2011, titled the Pediatric Eosinophilic Esophagitis Symptom Score (PEESS™ v2.0). To date, its use is largely for research purposes. We propose to evaluate the implementation of the PEESS™ v2.0 in a prospective interventional controlled clinical practice. The study included 620 patients over an 18-month period. Surveys were delivered and administered digitally every month through the MyGeisinger.org Patient Portal. Our analysis demonstrated symptom severity and symptom frequency scores significantly improved over time. However, counter to our hypothesis, patients who completed the PEESS™v2.0 ultimately had higher EoE-related health care utilization of office visits and endoscopies compared with those who did not complete the PEESS™v2.0. This could be related to greater awareness of disease activity and/or increased willingness to seek care. Our study, in the context of mobile health tool and patient-reported outcome trends, represents an opportunity for improved disease monitoring at-home within the field of eosinophilic gastrointestinal diseases.

Defects in Cell Wall Differentiation of the Arabidopsis Mutant rol1-2 Is Dependent on Cyclin-Dependent Kinase CDK8.

Plant cells are encapsulated by cell walls whose properties largely determine cell growth. We have previously identified the rol1-2 mutant, which shows defects in seedling root and shoot development. rol1-2 is affected in the Rhamnose synthase 1 (RHM1) and shows alterations in the structures of Rhamnogalacturonan I (RG I) and RG II, two rhamnose-containing pectins. The data presented here shows that root tissue of the rol1-2 mutant fails to properly differentiate the cell wall in cell corners and accumulates excessive amounts of callose, both of which likely alter the physical properties of cells. A surr (suppressor of the rol1-2 root developmental defect) mutant was identified that alleviates the cell growth defects in rol1-2. The cell wall differentiation defect is re-established in the rol1-2 surr mutant and callose accumulation is reduced compared to rol1-2. The surr mutation is an allele of the cyclin-dependent kinase 8 (CDK8), which encodes a component of the mediator complex that influences processes central to plant growth and development. Together, the identification of the surr mutant suggests that changes in cell wall composition and turnover in the rol1-2 mutant have a significant impact on cell growth and reveals a function of CDK8 in cell wall architecture and composition.

A mixed-linkage (1,3;1,4)-ß-D-glucan specific hydrolase mediates dark-triggered degradation of this plant cell wall polysaccharide.

The presence of mixed-linkage (1,3;1,4)-ß-d-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.