Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress.

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCß domain within the CTD reveals an unusual double-layered ß-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the ß-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

IgG4-related disease causing facial nerve and optic nerve palsies: Case report and literature review.

IgG4-related disease (IgG4-RD) is increasingly being recognized as an entity effecting the head and neck region. Although most commonly seen with salivary gland or paranasal sinus involvement, IgG4-RD may also involve the temporal bone and skull base. We report a rare care of a 61-year-old female with IgG4-RD presenting as synchronous lesions of the middle ear and middle cranial fossa with polyneuropathy of cranial nerves II, VI, and VII. Initial histopathological evaluation of her resected ear mass suggested a benign inflammatory process but no specific diagnosis. Her symptoms progressed over 10months prompting re-evaluation of the specimen and consideration of the IgG4-RD diagnosis. Key pathologic features included prominent lymphoplasmacytic population, storiform fibrosis, obliterative phlebitis, and IgG4 specific staining. The patient was treated with high-dose intravenous and oral steroids but was transitioned to azathioprine secondary to steroid-induced myopathy. Radiographic studies before and after treatment reveal marked improvement of the intracranial and extracranial disease. Correspondingly, her cranial neuropathies resolved. A high degree of clinical suspicion is necessary to diagnosis IgG4-RD. The diagnosis can be supported by elevated serum IgG, elevated IgG index, and pathognomonic histopathological findings. Primary treatment is with corticosteroids. However, immunotherapy using azathioprine or rituximab can be utilized in recurrent disease or patients with steroid intolerance.

Vascularized scapular free bone graft after nonunion of a tarsal navicular stress fracture: a case report.

Nonunion of a tarsal navicular stress fracture, although relatively uncommon, is often amenable to open reduction and internal fixation. Furthermore, avascular necrosis of the navicular whether intact or occurring after fracture is rare, secondary to the adequate blood supply it receives. However, persistent nonunion after primary surgical repair in conjunction with avascular necrosis often results in limited treatment options. Thus, the purpose of the present case report is to describe the surgical approach and complications of a vascularized scapular free bone graft for augmentation of revision talonavicular and naviculocuneiform arthrodesis.

Quantification of Aerosol Concentrations During Endonasal Instrumentation in the Clinic Setting.

OBJECTIVE: Recent anecdotal reports and cadaveric simulations have described aerosol generation during endonasal instrumentation, highlighting a possible risk for transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) during endoscopic endonasal instrumentation. This study aims to provide a greater understanding of particle generation and exposure risk during endoscopic endonasal instrumentation. STUDY DESIGN: Prospective quantification of aerosol generation during office-based nasal endoscopy procedures. METHODS: Using an optical particle sizer, airborne particles concentrations 0.3 to 10 microns in diameter, were measured during 30 nasal endoscopies in the clinic setting. Measurements were taken at time points throughout diagnostic and debridement endoscopies and compared to preprocedure and empty room particle concentrations. RESULTS: No significant change in airborne particle concentrations was measured during diagnostic nasal endoscopies in patients without the need for debridement. However, significant increases in mean particle concentration compared to preprocedure levels were measured during cold instrumentation at 2,462 particles/foot3 (95% CI 837 to 4,088; P = .005) and during suction use at 2,973 particle/foot3 (95% CI 1,419 to 4,529; P = .001). In total, 99.2% of all measured particles were ≤1 µm in diameter. CONCLUSION: When measured with an optical particle sizer, diagnostic nasal endoscopy with a rigid endoscope is not associated with increased particle aerosolization in patient for whom sinonasal debridement is not needed. In patients needing sinonasal debridement, endonasal cold and suction instrumentation were associated with increased particle aerosolization, with a trend observed during endoscope use prior to tissue manipulation. Endonasal debridement may potentially pose a higher risk for aerosolization and SARS-CoV-2 transmission. Appropriate personal protective equipment use and patient screening are recommended for all office-based endonasal procedures. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:E1415-E1421, 2021.

A Comparison of Sphenoid Sinus Osteoneogenesis in Aspirin-Exacerbated Respiratory Disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by excessive leukotriene production, diffuse polyp burden and osteitic bone changes. These bony changes have not been previously characterized. OBJECTIVE: The aim of this radiographic study is to characterize the bony changes noted on computed tomography (CT) scans of the sphenoid sinus in patients with AERD compared to other diseased sinonasal inflammatory states and non-diseased controls. METHODS: A retrospective review of 43 patients with clinically confirmed AERD were included and compared to 22 non-diseased, 9 allergic fungal sinusitis, and 43 chronic rhinosinusitis controls (23 without polyps and 18 with polyps). Comparative measurements were performed using fine-cut CT scans. Sites of comparison were the intersinus septum, the left and right lateral sphenoid wall, the roof, and left and right floor of the sphenoid sinus. Standardized measurements were averaged by two separate rhinologists. RESULTS: Patients with AERD had an average statistically significant increase in bone thickness compared to healthy and diseased controls in nearly every site with the most pronounced changes in the intersinus septum (p < 0.05). CONCLUSION: Patients with AERD have significantly increased thickness of the sphenoid bone compared to control groups with the most pronounced difference in the intersinus septum. These findings may help clinicians increase suspicion for a diagnosis of AERD who clinically have diffuse nasal polyposis.

Prolonged Implantation of Sinus Devices and Implications for Chronic Rhinosinusitis: A Case Report and Review of the Literature.

BACKGROUND: Implantation of sinus stents and spacers can be used as adjuvant management to maintain patency of sinuses after endoscopic sinus surgery for chronic rhinosinusitis. These implants are typically removed several weeks after surgery. We present two cases of different patients who were initially treated by different physicians and were found to have retained sinus spacers in their paranasal sinuses 6-10 years after implantation. CASE PRESENTATION: Case 1: a 40-year old male with chronic rhinosinusitis and history of balloon sinuplasty six years prior presented with worsening symptoms of chronic rhinosinusitis refractory to medical management. He underwent revision functional endoscopic sinus surgery and was found to have retained sinus implants in the left and right frontal sinus recesses. Case 2: a 48-year-old female with long-standing chronic rhinosinusitis refractory to medical management presented after two prior sinus surgeries most recently 10 years ago. She underwent revision functional endoscopic surgery and was found to have a retained sinus implant from prior surgery in the right frontal recess outflow tract embedded within scar tissue and reactive hyperostosis. Foreign bodies from both patients were removed without complication and patients were healing appropriately in the post-operative period. CONCLUSIONS: While sinus stents and spacers can help with post-operative scarring, leaving then unmonitored and in place will eventually result in them becoming a nidus for scarring and infection. It is critical that patients are aware of any foreign bodies we place, if they need scheduled removal or routine observation, and what symptoms may indicate that they are causing a problem.

Understood? Evaluating the readability and understandability of intranasal corticosteroid delivery instructions.

BACKGROUND: Allergic rhinitis is a widespread disease that has significant quality-of-life ramifications. Symptoms include rhinorrhea, nasal obstruction, cough, and postnasal drip. Intranasal corticosteroids are a hallmark of treatment of allergic rhinitis. However, the benefits of treatment are dependent on correct nasal spray technique, of which many patients are not aware. Patient instructions are included with the purchase of these medications. The readability and understandability for these educational materials has been minimally assessed in the medical literature. The aim of this study was to evaluate the readability and understandability of commonly used intranasal steroids. METHODS: Three readability measures (Gunning Fog, Simple Measure of Gobbledygook [SMOG], and FORCAST) and an understandability assessment (Patient Education Materials Assessment Tool for Printable Materials [PEMAT-P]) were used to evaluate the instructions for use of commonly prescribed intranasal steroids. Instructions with 6th grade readability level or lower were considered to meet health literacy experts' recommendations. Higher understandability values correlate to easier understandability. RESULTS: Instructions for 10 intranasal corticosteroid brands were reviewed. Gunning Fog consistently estimated easiest readability, whereas FORCAST estimated most difficult readability. Twenty percent (20%) of analyzed instructions met National Institutes of Health and health literacy experts' recommended reading levels. Understandability of instructions ranged from 33% to 90%, with an average of 66%. CONCLUSION: The benefit of intranasal corticosteroids is contingent on correct use by patients. However, the prepackaged instructions provided are most often above recommended reading levels and are difficult to understand. Future development of intranasal steroid instructions should meet recommended readability levels and be understandable to maximize their utility.

Frostbite of the feet after cryotherapy: a report of two cases.

UNLABELLED: The use of cryotherapy for the reduction of postoperative swelling and pain has become commonplace in orthopedic, podiatric, and cosmetic surgery. Prolonged exposure to extreme cold may induce an injury nearly identical to that of frostbite. The authors report on 2 patients who underwent podiatric orthopedic surgery and were exposed to prolonged cold therapy, which resulted in limb-threatening problems. Both patients had prolonged and essentially uninterrupted application of cryotherapy for 4 to 7 days, resulting in rewarming injuries of tissue necrosis. A team of specialists used limb salvage therapy to successfully treat the patients. Because of the problems encountered with the cold therapy devices, the authors caution against unmonitored and prolonged cold exposure. Newer devices that prevent cold exposure below 65 degrees F for longer than 2 hours could be a better option in cases in which cryotherapy is used. LEVEL OF EVIDENCE: 4.

A molecular basis for water motion detection by the mechanosensory lateral line of zebrafish.

Detection of water motion by the lateral line relies on mechanotransduction complexes at stereocilia tips. This sensory system is comprised of neuromasts, patches of hair cells with stereociliary bundles arranged with morphological mirror symmetry that are mechanically responsive to two opposing directions. Here, we find that transmembrane channel-like 2b (Tmc2b) is differentially required for mechanotransduction in the zebrafish lateral line. Despite similarities in neuromast hair cell morphology, three classes of these cells can be distinguished by their Tmc2b reliance. We map mechanosensitivity along the lateral line using imaging and electrophysiology to determine that a hair cell's Tmc2b dependence is governed by neuromast topological position and hair bundle orientation. Overall, water flow is detected by molecular machinery that can vary between hair cells of different neuromasts. Moreover, hair cells within the same neuromast can break morphologic symmetry of the sensory organ at the stereocilia tips.

E6 and E7 Antibody Levels Are Potential Biomarkers of Recurrence in Patients with Advanced-Stage Human Papillomavirus-Positive Oropharyngeal Squamous Cell Carcinoma.

Purpose: There is a paucity of biomarkers to predict failure in human papillomavirus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) following curative therapy. E6/E7 viral oncoproteins are constitutively expressed in HPV+ tumors and highly immunogenic, resulting in readily detected serum antibodies. The purpose of this study is to determine whether serum E6 and E7 antibody levels can potentially serve as a biomarker of recurrence in patients with HPV+OPSCC.Experimental Design: We evaluated E6/E7 antibody levels in patients with previously untreated, advanced stage (III, IVa-b), HPV+OPSCC receiving definitive chemoradiation under a uniform protocol from 2003 to 2010. Baseline and longitudinal serum samples were obtained from our archived repository. E6/E7 serum levels were measured using a glutathione-S-transferase capture ELISA and quantified by approximating the area under the dilution curve, and were analyzed using ANOVA and linear mixed model for longitudinal analysis.Results: We compared 22 HPV+OPSCC patients who developed recurrence with 30 patients who remained disease-free. There were no differences in T classification, N classification, disease subsite, or smoking status between the groups. In a longitudinal analysis, recurrent patients had significantly higher E6 and E7 serum antibody levels than the nonrecurrent patients over the follow-up period (P = 0.02 and P = 0.002, respectively). Patients who recurred had a lower clearance of E7 antibody than patients who remained disease-free (P = 0.0016).Conclusions: Patients with HPV+OPSCC whose disease recurs have a lower clearance of E6 and E7 antibodies than patients who do not have recurrence. The ratio of E7 antibody at disease recurrence compared with baseline is potentially a clinically significant measurement of disease status in HPV+OPSCC. Clin Cancer Res; 23(11); 2723-9. ©2016 AACR.

Estrogen treatment after ovariectomy protects against fatty liver and may improve pathway-selective insulin resistance.

Pathway-selective insulin resistance where insulin fails to suppress hepatic glucose production but promotes liver fat storage may underlie glucose and lipid abnormalities after menopause. We tested the mechanisms by which estrogen treatment may alter the impact of a high-fat diet (HFD) when given at the time of ovariectomy (OVX) in mice. Female C57BL/6J mice underwent sham operation, OVX, or OVX with estradiol (E2) treatment and were fed an HFD. Hyperinsulinemic-euglycemic clamps were used to assess insulin sensitivity, tracer incorporation into hepatic lipids, and liver triglyceride export. OVX mice had increased adiposity that was prevented with E2 at the time of OVX. E2 treatment increased insulin sensitivity with OVX and HFD. In sham and OVX mice, HFD feeding induced fatty liver, and insulin reduced hepatic apoB100 and liver triglyceride export. E2 treatment reduced liver lipid deposition and prevented the decrease in liver triglyceride export during hyperinsulinemia. In mice lacking the liver estrogen receptor α, E2 after OVX limited adiposity but failed to improve insulin sensitivity, to limit liver lipid deposition, and to prevent insulin suppression of liver triglyceride export. In conclusion, estrogen treatment may reverse aspects of pathway-selective insulin resistance by promoting insulin action on glucose metabolism but limiting hepatic lipid deposition.

Cyanobacterial polyketide synthase docking domains: a tool for engineering natural product biosynthesis.

Modular type I polyketide synthases (PKSs) are versatile biosynthetic systems that initiate, successively elongate, and modify acyl chains. Intermediate transfer between modules is mediated via docking domains, which are attractive targets for PKS pathway engineering to produce natural product analogs. We identified a class 2 docking domain in cyanobacterial PKSs and determined crystal structures for two docking domain pairs, revealing a distinct class 2 docking strategy for promoting intermediate transfer. The selectivity of class 2 docking interactions, demonstrated in binding and biochemical assays, could be altered by mutagenesis. We determined the ideal fusion location for exchanging class 1 and class 2 docking domains and demonstrated effective polyketide chain transfer in heterologous modules. Thus, class 2 docking domains are tools for rational bioengineering of a broad range of PKSs containing either class 1 or 2 docking domains.