Heme proteins of Giardia intestinalis.

Among the few organisms that cannot make the iron cofactor heme, some nonetheless possess heme proteins. This includes the protozoan parasite Giardia intestinalis, which encodes five known heme proteins: a flavohemoglobin and four members of the cytochrome b5 family. Giardia flavohemoglobin closely resembles those of the Enterobacteriaceae in structure and function, acting as a nitric oxide dioxygenase that is induced when trophozoites are exposed to reactive nitrogen species. The Giardia cytochromes b5 are soluble proteins having relatively low reduction potentials and lack several features that are expected to promote rapid electron transfer with redox partners. Only one potential electron donor, and no electron acceptors, have yet been identified in the Giardia genome, and the roles of these cytochromes are presently unknown. The answer may lie in the sequences that flank the heme-binding core of these proteins which could serve to localize them within the cell through reversible post-translational modifications and to promote specific protein-protein interactions.

Catalytic Differences between Flavohemoglobins of Giardia intestinalis and E. coli.

The sole known heme enzyme of the parasitic protist Giardia intestinalis is a flavohemoglobin (gFlHb) that acts as a nitric oxide dioxygenase (NOD) and protects the organism from the free radical nitric oxide. To learn more about the properties of this enzyme, we measured its nitric oxide dioxygenase, NADH oxidase, and cytochrome c reductase activities and compared these to the activities of the E. coli flavohemoglobin (Hmp). The turnover number for the NOD activity of gFlHb (23 s-1) is about two-thirds of that of Hmp (34 s-1) at pH 6.5 and 37 °C. The two enzymes differ in their sensitivity towards molecules that act as heme ligands. For both gFlHb and Hmp, inhibition with miconazole, a large imidazole ligand, is adequately described by simple competitive inhibition, with KI = 10 µM and 0.27 µM for gFlHb and Hmp, respectively. Inhibition plots with the small ligand imidazole were biphasic, which is consistent with previous experiments with carbon monoxide as a probe that show that the active site of flavohemoglobins exists in two conformations. Interestingly, the largest difference is observed with nitrite, which, like imidazole, also shows a biphasic inhibition plot; however, nitrite inhibits gFlHb at sub-millimolar concentrations while Hmp is not significantly affected. NADH oxidase activity measured under aerobic conditions in the absence of nitric oxide for Hmp was more than twice the activity of gFlHb. The addition of 1 mM hydrogen peroxide in these assays stimulated the NADH oxidase activity of gFlHb but not Hmp. Both enzymes had nearly identical cytochrome c reductase activities but the extent of the contribution of indirect reduction by flavohemoglobin-generated superoxide was much lower with gFlHb (4% SOD-inhibited) than with Hmp (17% SOD-inhibited). Although the active sites of the two enzymes share the same highly conserved residues that are important for catalysis, differences in the distal ligand binding site may account for these differences in activity and sensitivity towards NOD inhibitors. The differences observed in the NADH oxidase and cytochrome c reductase assays suggest that gFlHb may have evolved to protect the protist, which lacks both superoxide dismutase and catalase, from the damaging effects of superoxide by minimizing its production and from peroxide by actively reducing it.

Enhancing Intraoperative Cholangiography Interpretation Skills: A Perceptual Learning Approach for Surgical Residents.

OBJECTIVE: Laparoscopic cholecystectomy is a commonly performed surgery with risk of serious complications. Intraoperative cholangiography (IOC) can mitigate these risks by clarifying the anatomy of the biliary tree and detecting common bile duct injuries. However, mastering IOC interpretation is largely through experience, and studies have shown that even expert surgeons often struggle with this skill. Since no formal curriculum exists for surgical residents to learn IOC interpretation, we developed a perceptual learning (PL)-based training module aimed at improving surgical residents' IOC interpretation skills. DESIGN: Surgical residents were assessed on their ability to identify IOC characteristics and provide clinical recommendations using an online training module based on PL principles. This research had 2 phases. The first phase involved pre/post assessments of residents trained via the online IOC interpretation module, measuring their IOC image recognition and clinical management accuracy (percentage of correct responses), response time and confidence. During the second phase, we explored the impact of combining simulator-based IOC training with the online interpretation module on same measures as used in the first phase (accuracy, response time, and confidence). SETTING: The study was conducted at Rush University Medical College in Chicago. The participants consisted of surgical residents from each postgraduate year (PGY). Residents participated in this study during their scheduled monthly rotation through Rush's surgical simulation center. RESULTS: Total 23 surgical residents participated in the first phase. A majority (95.7%) found the module helpful. Residents significantly increased confidence levels in various aspects of IOC interpretation, such as identifying complete IOCs and detecting abnormal findings. Their accuracy in making clinical management decisions significantly improved from pretraining (mean accuracy 68.1 +/- 17.3%) to post-training (mean accuracy 82.3 +/- 10.4%, p < 0.001). Furthermore, their response time per question decreased significantly from 25 +/- 12 seconds to 17 +/- 12 seconds (p < 0.001). In the second phase, we combined procedural simulator training with the online interpretation module. The 20, first year residents participated and 88% found the training helpful. The training group exhibited significant confidence improvements compared to the control group in various aspects of IOC interpretation with observed nonsignificant accuracy improvements related to clinical management questions. Both groups demonstrated reduced response times, with the training group showing a more substantial, though nonsignificant, reduction. CONCLUSION: This study demonstrated the effectiveness of a PL-based training module for improving aspects of surgical residents' IOC interpretation skills. The module, found helpful by a majority of participants, led to significant enhancements in clinical management accuracy, confidence levels, and decreased response time. Incorporating simulator-based training further reinforced these improvements, highlighting the potential of our approach to address the lack of formal curriculum for IOC interpretation in surgical education.

Creating a Patient-Based Diagnostic Checklist for Functional Tics During the COVID-19 Pandemic.

Background and Objectives: Since the onset of the COVID-19 pandemic, there has been a dramatic change in the presentation of patients with tics. The explosive presentation of atypical tics (TT) has been noted worldwide and thought to be the manifestation of a pandemic-associated functional neurologic disorder following social media exposure to tics. Nevertheless, despite the frequent diagnosis of functional tics (FT), there are no existing formal diagnostic criteria. The primary aim of this study was to create a patient-based diagnostic checklist for making the diagnosis of a functional tic disorder (FTD) during the COVID-19 pandemic. Methods: A retrospective chart review at a single institution during the pandemic was performed. Based on the available literature, diagnostic criteria were created for TT, FT, and patients with dramatically evolving symptoms (i.e., mixed with prior history of mild tics with later fulminant functional worsening). Patient demographics, comorbidities, and tic characteristics of these groups were then compared. Following initial assessments, new diagnostic criteria were established and statistically reanalyzed. Results: One hundred ninety-eight patients underwent investigation. Significant differences in age, sex, psychological comorbidities, tic characteristics, and tic severity were found between patients with TT when compared with either of the 2 the functional groups. Only the presence of rostrocaudal progression and increased obsessive-compulsive behaviors were significantly different between patients with new-onset FT and those with functional worsening of a previous tic disorder. Results also showed that age at tic onset was not a contributing factor for group differentiation. Many patients with FT were not exposed to videos depicting tics on social media. Discussion: This study confirms the presence of a distinct presentation of aTT during the pandemic period. It further establishes the validity of specific criteria useful in dividing patients with tics into 3 formal diagnostic criteria: (1) primary tic disorders (PTDs), (2) a strictly FTD, and (3) a mixed tic disorder consisting of patients with an initial history of a PTD and the later development of FT. Explicit diagnostic criteria should enable clinicians and researchers to make a definitive identification and assist patients and families become more knowledgeable and accepting of the diagnosis of FT.

Mutation of conserved tryptophan residues at the dimer interface of Staphylococcus aureus nitric oxide synthase.

Nitric oxide synthases (NOSs) share two invariant tryptophan residues within a conserved helical lariat that is part of the pterin-binding site and dimer interface. We mutated Staphylococcus aureus NOS Trp-314 (to alanine, phenylalanine, tyrosine and histidine) and Trp-316 (to alanine, phenylalanine and tyrosine) and characterized the effects of mutation on heme environment, quaternary structure, enzymatic activity, and substrate affinity. With arginine present, all saNOS variants bound heme with native thiolate ligation, formed high spin ferric complexes and were dimeric. All variants catalyze the peroxide-dependent oxidation of N-hydroxy-l-arginine, at rates from 10% to 55% of wild type activity. Arginine-free proteins are dimeric with the exception of W314A. Arginine affinity for all variants decreases with increasing temperature between 15 and 42 °C but is precipitous for position-314 variants. Previous structural and biophysical characterization of NOS oxygenase domains demonstrated that the protein can exist in either a tight or loose conformation, with the former corresponding to the active state of the protein. In the position-314 variants it is likely that the loose conformation is favoured, owing to the loss of a hydrogen bond between the indole side chain and the polypeptide backbone of the helical lariat.

Use of Acellular Dermal Matrix Following Fasciectomy for the Treatment of Dupuytren's Disease.

BACKGROUND: Recurrence rates following surgical intervention for Dupuytren's disease (DD) remains high. In this study, we investigate the use of acellular dermal matrix (ADM) to reduce recurrence and improve long-term clinical outcome. METHODS: We examined 132 patients undergoing open fasciectomy for DD from 2007 to 2017. The experimental group had a sheet of ADM (FlexHD) sutured into the surgical bed controls were not closed with ADM. Patient characteristics, range of motion, and complications were examined. RESULTS: Twenty-eight (21.2%) patients were treated with acellular dermal matrix, whereas 104 (78.8%) patients were not. The median age was 67.0 years (range 34-91 years). with no differences between group regarding age, comorbidities, and laterality. The mean preoperative interphalangeal joint flexion contracture in the ADM group of 66.5 ± 29.9 degrees was corrected to 9.7 ± 12.4 degrees, whereas the mean metacarpophalangeal joint preoperative flexion contracture of 51.4 ± 23.9 degrees was corrected to 7.8 ± 14.1 degrees at postoperative examination (P < 0.05). The median follow-up was 18.7 months, during which the recurrence of contracture was observed in 1 of 28 patients in the group receiving ADM compared with 9 of 104 in the control group (P = 0.37). There were no differences in the incidence of minor wound complications observed. CONCLUSION: The adjunct placement of acellular dermal matrix into the wound bed following fasciectomy for DD may be an important surgical strategy to reduce recurrence rates as well as augment coverage of exposed vital structures in cases of severe flexion contracture.