Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme.

The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O2), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O2. The encoded mutations cluster around a putative O2 tunnel, increasing flexibility and accessibility to O2 in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.

Blasting the myth of predictive INR changes related to plasma transfusion: an academic institution's experience

Abstract Introduction Plasma transfusion is a common therapeutic strategy used to lower international normalized ratio (INR) values in the non-emergent setting. However, due to lack of evidence of its efficacy, standardized guidelines for this practice have not been well established. Methods This retrospective observational cohort study analyzed 276 inpatient encounters that involved plasma transfusions focusing on change in INR values from pre- to post-transfusion, with respect to the following predictor variables: vitamin K co-administration, number of plasma units transfused, order indication and body mass index (BMI). Results The overall average change in the INR was 1.35. Patients who received vitamin K showed an average change of 2.51, while patients that did not receive vitamin K demonstrated an average change of 0.70. Increased numbers of plasma units transfused showed benefit up to three-unit orders. Greater decreases in the INR were observed for patients requiring plasma for anticoagulation reversal or active bleeding. There was no significant difference in the change in INR based on the BMI. By multivariate and regression analyses, the stepwise addition of each successive predictor variable demonstrated an increase in the shared variance in the outcome of the post-transfusion INR: the pre-transfusion INR and vitamin K co-administration alone was not significant (p= 0.45); the additional number of plasma units transfused was significant (R² = 0.13, p < 0.001), and; the subsequent additional plasma order indications (R² = 0.19, p < 0.001) and BMI (R² = 0.18, p < 0.001) were increasingly significant. Conclusion Taking into consideration the combination of multiple predictive factors may aid in a more efficient use of plasma products.

Blasting the myth of predictive INR changes related to plasma transfusion: an academic institution's experience.

INTRODUCTION: Plasma transfusion is a common therapeutic strategy used to lower international normalized ratio (INR) values in the non-emergent setting. However, due to lack of evidence of its efficacy, standardized guidelines for this practice have not been well established. METHODS: This retrospective observational cohort study analyzed 276 inpatient encounters that involved plasma transfusions focusing on change in INR values from pre- to post-transfusion, with respect to the following predictor variables: vitamin K co-administration, number of plasma units transfused, order indication and body mass index (BMI). RESULTS: The overall average change in the INR was 1.35. Patients who received vitamin K showed an average change of 2.51, while patients that did not receive vitamin K demonstrated an average change of 0.70. Increased numbers of plasma units transfused showed benefit up to three-unit orders. Greater decreases in the INR were observed for patients requiring plasma for anticoagulation reversal or active bleeding. There was no significant difference in the change in INR based on the BMI. By multivariate and regression analyses, the stepwise addition of each successive predictor variable demonstrated an increase in the shared variance in the outcome of the post-transfusion INR: the pre-transfusion INR and vitamin K co-administration alone was not significant (p = 0.45); the additional number of plasma units transfused was significant (R²â€¯= 0.13, p < 0.001), and; the subsequent additional plasma order indications (R²â€¯= 0.19, p < 0.001) and BMI (R²â€¯= 0.18, p < 0.001) were increasingly significant. CONCLUSION: Taking into consideration the combination of multiple predictive factors may aid in a more efficient use of plasma products.

Binding Interface and Electron Transfer Between Nicotine Oxidoreductase and Its Cytochrome c Electron Acceptor.

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. 2022, 298 (8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein-protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2's surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2's flavin to CycN's heme occurs without the assistance of a protein-derived wire.

Cytologic analysis of vitreous fluids: A retrospective review of our 24 years of experience.

BACKGROUND: Cytologic analysis of vitreous fluid is an important component in diagnosis of vitreitis. No standard reporting guidelines exist for these specimens. This study chronicles our 24 years experience and proposes a tentative diagnostic model. METHODS: Retrospective cytology reports review and database study. Clinical indications, cytologic patterns, ancillary studies performed, and diagnoses were recorded. RESULTS: 176 samples from 160 patients were included and main cytologic patterns are reflected in Table 1. Most fluids were negative for malignancy (88%) and patterns IIB (53%) and IIA (19%) were dominant. The non-diagnostic rate was 7%; atypical and suspicious categories represented <0.5% of fluids tested and only 2% were positive for malignancy (3 intraocular lymphoma and one melanoma). Clinical indications for fluid examination were infection/inflammation (59%), to rule out lymphoma (11%), amyloidosis (3%), melanoma (2%), or to investigate intraocular hemorrhage. Fungal elements were demonstrated in 7 cases. No viral inclusions were appreciated; however, one case was positive for HSV 2 by IHC and 2 were negative by PCR. One case had Gram + cocci. Flow cytometry studies were suboptimal in 6 fluids, negative for an aberrant lymphocyte population in 11, and positive for high grade lymphoma in 3 cases. Atypical, suspicious and positive for melanoma were reported in 3 samples. Amyloid was identified in 1 aspirate. CONCLUSIONS: Cytologic analysis of vitreous fluid is a useful tool. Modern techniques like flow cytometry and PCR testing further expand the diagnostic possibilities. Standardization of diagnostic terminology will aid clinicians caring for patients suffering from ocular disease.

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase.

Nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, is of medical relevance as it shows potential as a therapeutic to aid cessation of smoking due to its ability to oxidize nicotine into a non-psychoactive metabolite. However, the use of NicA2 in this capacity is stymied by its dismal O2-dependent activity. Unlike other enzymes in the amine oxidase family, NicA2 reacts very slowly with O2, severely limiting its nicotine-degrading activity. Instead of using O2 as an oxidant, we discovered that NicA2 donates electrons to a cytochrome c, which means that NicA2 is actually a dehydrogenase. This is surprising, as enzymes of the flavin-containing amine oxidase family were invariably thought to use O2 as an electron acceptor. Our findings establish new perspectives for engineering this potentially useful therapeutic and prompt a reconsideration of the term 'oxidase' in referring to members of the flavin-containing amine 'oxidase' family.

Whole Blood Administration: Comparison of In Vitro Platelet Function of Pressure Bag, Pressure Bag With Fluid Warming Device, and Rapid Infuser Methods.

BACKGROUND: Use of low-titer group O whole blood for emergent transfusion of patients with unknown blood type became AABB approved in January 2018. Since that time, there is increasing use of whole blood in massive transfusion protocols. Whole blood stored at refrigerator temperature (2-4 °C) contains functional platelets that some research proposes may provide better clot dynamics than standard platelets, which are stored at room temperature (20-24 °C). Conventional teaching does not promote infusion of platelet products with pressure or warming, due to concerns of activation and subsequent inactivity of the infused platelets. Although a few reports found no significant changes in platelet function with warming or pressure during infusion of conventional room-temperature-stored platelets, there is limited data to support use of warming or pressure for infusion of whole blood products containing cold-stored platelets. METHODS: This study design is to evaluate and compare three commonly used methods of administering blood products in a massive transfusion setting for their potential effects on platelets contained within whole blood units (pressure bag alone, pressure bag with fluid warming line, and rapid infuser). RESULTS: Platelet function of 10 units tested pre- and post-infusion by thromboelastography (TEG) and platelet aggregation studies found no significant difference in platelet activity pre- and post-infusion with any of the three methods evaluated. CONCLUSIONS: This study supports the use of rapid infuser or pressure bag devices (with or without warming) as acceptable for infusion of whole blood products. Infusion of whole blood with warming is preferable to prevent potential transfusion-associated hypothermia.

Radiation-Induced Sarcoma following Prolonged Coronary Stent Placement.

Radiation exposure for the average coronary stent placement varies based on a number of factors but typically amounts to 6-11 mSv per patient (compared to 3 mSv background). As with all procedures which utilize radiation, there is an inherent risk of genetic mutation and the possible development of malignancy. Here, we present the case of a 75-year-old male who presented with an exophytic mass on his back following prolonged coronary catheterization with a radiation burn seven years prior. Biopsy of the lesion revealed the mass was consistent with an undifferentiated pleomorphic sarcoma emanating from the site of the radiation burn. After staging studies demonstrated no evidence of metastatic disease, radical excision with negative margins was performed. This case demonstrates that despite the rarity of radiation injury, each incidence necessitates strict monitoring of radiation exposure and continual follow-up due to the risk of malignancy.

Acute Hemolytic Transfusion Reaction in Group B Recipient Associated with Group A Apheresis Platelet Donor: Case Report and Literature Review.

Acute hemolytic transfusion reaction is a known but rare potential adverse event related to platelet transfusion. Most reported cases of platelet-related hemolytic transfusion reaction have resulted from transfusion of platelets from group O donor to group A recipient. We identified only one prior case report in the literature of hemolytic transfusion reactions resulting from transfusion of apheresis platelets from group A donor to group B recipient. In that case report, two platelet units were obtained from a single donation and transfused into two separate patients. Both patients exhibited acute hemolytic reactions. The donor is reported to have high anti-B titers, as well as report of probiotic use. We report a case of acute hemolytic reaction in group B recipient following transfusion of apheresis platelets from group A donor with high-titer anti-B but unknown status of probiotic use. This case demonstrates that while low, there still exists potential risk for hemolysis from out-of-group A plasma transfusion.

Activated Prothrombin Complex Concentrate versus Plasma for Reversal of Warfarin-Associated Hemorrhage.

STUDY OBJECTIVE: To evaluate the efficacy and safety of an activated four-factor prothrombin complex concentrate (aPCC) versus plasma for the reversal of warfarin-associated hemorrhage. DESIGN: Single-center, retrospective cohort analysis of adult patients with warfarin-associated hemorrhage treated with either aPCC or plasma. PATIENTS: Patients received either aPCC or plasma as treatment for warfarin-associated hemorrhage between January 1, 2011, and July 1, 2013. Patients with missing data points were excluded from the final analysis. Of the 276 patients included in the final analysis, 128 received aPCC and 148 received plasma. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Those patients who received aPCC achieved a lower posttreatment INR (1.1 [0.1] vs 1.6 [0.5]; p<0.05). In addition, patients who received aPCC had a 4.3 times higher odds of achieving an INR of less than 1.4 (97 [75.8%] vs 65 [43.9%]; p<0.05; odds ratio [OR] = 4.3 [95% confidence interval (CI) 2.6-7.3]). When controlling for vitamin K administration, history of diabetes mellitus, receipt of the recommended reversal agent dose, and pretreatment INR, aPCC administration remained an independent predictor for achieving an international normalized ratio (INR) of less than 1.4 in the first 24 hours after treatment (OR = 3.75 [95% CI 2.11-6.65]; p<0.001). In addition, there was no statistical difference between the groups with regard to occurrences of infusion reaction, pulmonary embolism, deep vein thrombosis, stroke, or myocardial infarction. CONCLUSIONS: Compared with patients who received plasma, patients who received aPCC achieved a lower posttreatment INR, had a larger INR change, and were more likely to achieve an INR less than the prespecified goal. Those patients who received aPCC did not have a higher incidence of thromboembolic events.

How Sensitive Is the Upper Gastrointestinal Tract to 90Y Radioembolization? A Histologic and Dosimetric Analysis in a Porcine Model.

In 90Y radioembolization, nontarget embolization to the stomach or small bowel can result in gastrointestinal injury, a rare but difficult to manage clinical complication. However, dosimetric thresholds for toxicity to these tissues from radioembolization have never been evaluated in a controlled setting. We performed an analysis of the effect of 90Y radioembolization in a porcine model at different absorbed-dose endpoints. METHODS: Six female pigs underwent transfemoral angiography and infusion of 90Y-resin microspheres into arteries supplying part of the gastric wall. Esophagogastroduodenoscopy was performed after 4 wk to assess interim gastrointestinal health. Animals were monitored for side effects for 9 wk after 90Y infusion, after which they were euthanized and their upper gastrointestinal tracts were excised for analysis. Histologic sections were used to map microsphere location, and a microdosimetric evaluation was performed to determine the absorbed-dose profile within the gastrointestinal wall. RESULTS: 90Y radioembolization dosages from 46.3 to 105.1 MBq were infused, resulting in average absorbed doses of between 35.5 and 91.9 Gy to the gastric wall. No animal exhibited any signs of pain or gastrointestinal distress through the duration of the study. Excised tissue showed 1-2 small (<3.0 cm2) healed or healing superficial gastric lesions in 5 of 6 animals. Histologic analysis demonstrated that lesion location was superficial to areas of abnormally high microsphere deposition. An analysis of microsphere deposition patterns within the gastrointestinal wall indicated a high preference for submucosal deposition. Dosimetric evaluation at the luminal mucosa performed on the basis of microscopic microsphere distribution confirmed that 90Y dosimetry techniques conventionally used in hepatic dosimetry provide a first-order estimate of absorbed dose. CONCLUSION: The upper gastrointestinal tract may be less sensitive to 90Y radioembolization than previously thought. Lack of charged-particle equilibrium at the luminal mucosa may contribute to decreased toxicity of 90Y radioembolization compared with external-beam radiation therapy in gastrointestinal tissue. Clinical examples of injury from 90Y nontarget embolization have likely resulted from relatively large 90Y activities being deposited in small tissue volumes, resulting in absorbed doses in excess of 100 Gy.

Effectiveness of Provider Education Followed by Computerized Provider Order Entry Alerts in Reducing Inappropriate Red Blood Cell Transfusion.

To reduce the rate of inappropriate red blood cell transfusion, a provider education program, followed by alerts in the computerized provider order entry system (CPOE), was established to encourage AABB transfusion guidelines. Metrics were established for nonemergent inpatient transfusions. Service lines with high order volume were targeted with formal education regarding AABB 2012 transfusion guidelines. Transfusion orders were reviewed in real time with email communications sent to ordering providers falling outside of AABB recommendations. After 12 months of provider education, alerts were activated in CPOE. With provider education alone, the incidence of pretransfusion hemoglobin levels greater than 8 g/dL decreased from 16.64% to 6.36%, posttransfusion hemoglobin levels greater than 10 g/dL from 14.03% to 3.78%, and number of nonemergent two-unit red blood cell orders from 45.26% to 22.66%. Red blood cell utilization decreased by 13%. No additional significant reduction in nonemergent two-unit orders was observed with CPOE alerts. Provider education, an effective and low-cost method, should be considered as a first-line method for reducing inappropriate red blood cell transfusion rates in stable adult inpatients. Alerts in the computerized order entry system did not significantly lower the percentage of two-unit red blood cells orders but may help to maintain educational efforts.

Critically ill and septic patient: is red blood cell transfusion adding to the Domino Effect?

Sepsis is a major cause of patient morbidity and mortality. Many critically ill patients are septic, and red blood cell transfusion is often part of their treatment plan. Studies have shown that red blood cell transfusion is associated with a dose-dependent increase in patient morbidity and mortality. Although red blood cells are transfused to increase the recipient's oxygen-carrying capacity, there are new and emerging data to support that red blood cell transfusion may potentially decrease perfusion and oxygen delivery to the microcirculation, particularly when older red blood cells are transfused. In addition, there are similar effects in the pathophysiology of sepsis that may overlap with the changes that occur with storage of red blood cells. This article will discuss recent literature addressing red cell transfusion in critically ill and septic patients and discuss general guidelines for red cell transfusion in this patient population. This article will also discuss the epidemiology and pathophysiology of sepsis and relate how storage and transfusion of red cells may potentially contribute to changes observed in a septic patient.

Spirocyclic ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors: from hit to lowering of cerebrospinal fluid (CSF) amyloid ß in a higher species.

A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aß), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aß is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aß in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aß lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aß in rodents and in monkey.

Photoletter to the editor: Diffuse cocaine-related purpura.

Diffuse purpura is an uncommon skin manifestation found in platelet and coagulation disorders, meningococcemia, vasculitides and cocaine use. Reports of cocaine-related purpura predominantly involve adulteration with the anti-helminthic, levamisole. Levamisole enhances the effects of cocaine and is known to cause vasculitis. Recently, the CDC also released an advisory of oxymorphone being used intravenously causing thrombogenic thrombocytopenic purpura (TTP). We report the case of a patient with diffuse purpura ultimately diagnosed with cocaine-related thrombogenic vasculopathy. In the current environment of adulterated cocaine usage and increased prescription narcotic abuse, it is crucial to investigate substance abuse as a cause of diffuse purpura.

Synthesis and evaluation of substrate analogue inhibitors of trypanothione reductase.

Trypanothione reductase (TR) is found in the trypanosomatid parasites, where it catalyses the NADPH-dependent reduction of the glutathione analogue, trypanothione, and is a key player in the parasite's defenses against oxidative stress. TR is a promising target for the development of antitrypanosomal drugs; here, we report our synthesis and evaluation of compounds 3-5 as low micromolar Trypanosoma cruzi TR inhibitors. Although 4 and 5 were designed as potential irreversible inhibitors, these compounds, as well as 3, displayed reversible competitive inhibition. Compound 3 proved to be the most potent inhibitor, with a K(i) = 2 µM.

Recent efforts and available technologies for safety in delivery of blood products.

Over the past few decades, many efforts to improve the safety of blood products have concentrated on the reduction of transfusion-associated infectious diseases, particularly human immunodeficiency virus and hepatitis C. As health care enters a time of significant economic changes and governmental agencies develop guidances to improve patient safety, new efforts are being implemented and new technologies are being developed to ensure safe delivery of blood products. This article outlines the structure of agencies responsible for blood safety in the United States, reviews the fairly recent implementation of a blood product bar-code labeling system (ISBT 128) in the United States, describes safety efforts at the level of the hospital transfusion service, and reviews some of the emerging technologies for safety in blood delivery at the bedside.