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.

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.

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.

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.

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.

Transfusion-related acute lung injury: clinical features and diagnostic dilemmas.

Transfusion-related acute lung injury (TRALI) is recognized as the number-one cause of transfusion-associated death in the United States. Although TRALI was not designated as the number-one cause until 2004, respiratory-related symptoms associated with blood transfusion have been reported for decades. More recently, our understanding of the pathophysiology and clinical significance of TRALI has advanced significantly, and great efforts are being implemented for patient safety. This article reviews the background, history, definitions, clinical features, and pathophysiology of TRALI. It also discusses some of the difficulties in diagnosing TRALI, compares some of the important components in the differential diagnosis, and describes some of the efforts being implemented for patient safety.

Primary adenoid cystic carcinoma of the breast: case report and review of the literature.

Adenoid cystic carcinoma (ACC) of the breast is a rare neoplasm accounting for 0.1% of all breast carcinomas, and presenting most commonly as a painful breast mass. In contrast to the aggressive nature of ACC at other sites, ACC of the breast has a favorable prognosis, lymph node involvement or distant metastases seldom occur. Treatment is basically of simple mastectomy. Chemotherapy, radiation and hormonal treatment have been infrequently used and evaluated. We report a case of ACC of the breast managed with mastectomy and review the literature.

Primary malignant melanoma of the lung: a case report and review of the literature.

Melanomas of the respiratory tract are usually metastatic in origin, and finding a primary melanoma is very rare. We report the case of a 60-year-old man who presented with a 2-month history of cough with associated weight loss. The histopathologic findings were consistent with those of a melanoma. Before considering the diagnosis of primary melanoma of the lung, an extensive diagnostic workup was done to rule out metastasis from an occult primary tumor. In addition, previously reported cases of primary lung melanoma were reviewed, and the established criteria for determining the primary site were applied. Because the precise etiology has not yet been elucidated, the possible mechanism is discussed.