MAVS Cys508 palmitoylation promotes its aggregation on the mitochondrial outer membrane and antiviral innate immunity.

Cysteine palmitoylation or S-palmitoylation catalyzed by the ZDHHC family of acyltransferases regulates the biological function of numerous mammalian proteins as well as viral proteins. However, understanding of the role of S-palmitoylation in antiviral immunity against RNA viruses remains very limited. The adaptor protein MAVS forms functionally essential prion-like aggregates upon activation by viral RNA-sensing RIG-I-like receptors. Here, we identify that MAVS, a C-terminal tail-anchored mitochondrial outer membrane protein, is S-palmitoylated by ZDHHC7 at Cys508, a residue adjacent to the tail-anchor transmembrane helix. Using superresolution microscopy and other biochemical techniques, we found that the mitochondrial localization of MAVS at resting state mainly depends on its transmembrane tail-anchor, without regulation by Cys508 S-palmitoylation. However, upon viral infection, MAVS S-palmitoylation stabilizes its aggregation on the mitochondrial outer membrane and thus promotes subsequent propagation of antiviral signaling. We further show that inhibition of MAVS S-palmitoylation increases the host susceptibility to RNA virus infection, highlighting the importance of S-palmitoylation in the antiviral innate immunity. Also, our results indicate ZDHHC7 as a potential therapeutic target for MAVS-related autoimmune diseases.

The Effect of Supine Versus Upright Positioning on Eyelid Height in the Nonsurgical Patient.

PURPOSE: To directly compare margin-reflex distance 1, margin-reflex distance 2, and palpebral fissure in the upright versus supine positions in nonsurgical patients. METHODS: A total of 43 patients (31 female, 12 male, and age range 26-96) were enrolled. Photos were taken in the clinic in the upright and supine position with a ruler placed vertically in the same plane as the eyelid, and the above computer-analyzed measurements were obtained. RESULTS: Among the 86 eyes observed, the average upright margin-reflex distance 1 was 2.97 mm (95% confidence interval [CI], 2.70-3.24), while the average supine margin-reflex distance 1 was 2.38 mm (95% CI, 2.13-2.63). These differences were statistically significant (p < 0.001). Similarly, statistically significant differences were seen with margin-reflex distance 2 (p < 0.001), where upright measurements averaged 5.57 mm (95% CI, 5.33-5.81), and supine measurements averaged 5.01 mm (95% CI, 4.73-5.28). Finally, palpebral fissure showed similar significance (p < 0.001). Upright measurements averaged 8.54 mm (95% CI, 8.19-8.90), while supine measurements averaged 7.38 mm (95% CI, 7.00-7.76). CONCLUSIONS: Supine positioning provides a decrease in margin-reflex distance 1, margin-reflex distance 2, and palpebral fissure when compared with upright positioning in nonsurgical patients. This contradicts a previous study on intraoperative patients when lidocaine with epinephrine was used. This study helps us better understand the normal physiologic response to position changes with regard to eyelid height in the nonsurgical patient. As such, this study may function as a control for future studies comparing anesthetic/surgical parameter effects on intra- and post-operative eyelid heights.

Heterotopic Ossification and Platelet-Rich Plasma in Core Muscle Injuries: A Single-Institution Experience Over 6 Years.

BACKGROUND: A 2015 study of platelet-rich plasma (PRP) for groin injuries in National Football League (NFL) players alerted the authors to the possibility that PRP is associated with heterotopic ossification (HO). The current study of athletes seen between 2014 and 2019 provides a more comprehensive analysis of that observation. PURPOSE/HYPOTHESIS: This report describes the early results of groin surgery for athletes who had experienced failed PRP therapy performed by different practitioners and with an assortment of PRP techniques. The primary goal of this cohort study was to determine short-term clinical outcomes after surgery of PRP-treated patients. It was hypothesized that previous PRP treatment would be associated with the presence of HO among patients with core muscle injuries (CMIs). STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: All athletes seen at 1 institution and identified at their first visit as having received PRP for a CMI were followed and compared with patients with a CMI who had not previously received PRP. Although in many cases HO was observed on clinical examination or imaging, HO was identified intraoperatively in all surgical cases and confirmed pathologically. Successful surgery was defined as return to play at previously high levels of performance or greater as determined by the athletes' own assessments. All patients who had received PRP were followed for ≥2 years. RESULTS: Among 3642 patients with a new CMI seen between 2014 and 2019, 68 (1.9%) patients developed HO within the core muscles and/or adjacent soft tissues. Of the 68 patients, 60 (88.2%) were men, and the mean age was 34.5 years. Of the 68 patients, 62 (91.2%) were athletes and 44 (64.7%) had been treated previously with PRP. HO was observed in 24 (0.7%) patients without previous PRP treatment. Three athletes who received PRP retired early from sports because of HO and scar issues. In total, 22 of 28 (78.6%) NFL players who received PRP developed HO, compared with 0 (0%) of 28 randomly selected, age-, position-, and injury-matched NFL players. After surgical repair, 3-month success rates were 67.9% and 96.4%, respectively, in the PRP and non-PRP groups (P = .006). By 6 months postoperatively, PRP-treated patients were back to similarly high success rates compared with the non-PRP cohort. Scar tissue issues played a prominent role in the relative delay in definitive success. CONCLUSION: The present, more comprehensive study confirms the previous preliminary analysis that treating CMIs with PRP may be associated with HO.

Orbital Venous Malformation Excision after Transcaruncular Embolization with Onyx.

Orbital venous malformations are low-flow lesions resulting from vascular dysgenesis during development. Patients may present with vision loss, proptosis accentuated by Valsalva, and/or painful spontaneous thrombosis. The preferred treatment for symptomatic lesions is embolization combined with excision. A 34-year-old male presented to our institution from an outside emergency department with a diagnosis of presumed idiopathic orbital inflammation. For the prior month, he had been experiencing left orbital pressure, subjective eye bulging, and both diplopia and blurry vision when in peripheral gaze or when bending over. Despite initial improvement with steroids, his symptoms recurred with tapering. Visual acuity was reduced to 20/25, but pupils and motility remained normal. Biopsy demonstrated a vascular lesion characterized by fibroadipose tissue with histologically unremarkable blood vessels, and cerebral arteriography showed no high-flow components. A diagnosis of orbital venous malformation was made. He then underwent intraoperative angiography and Onyx embolization followed by excision via a transcaruncular approach. Two prior reports have described the use of Onyx in venolymphatic malformations. This report highlights a detailed approach to defining flow characteristics pre- and intraoperatively and expands upon our understanding of the use of Onyx for such cases.

Public Perception of Ménière's Disease: A Social Media Analysis.

OBJECTIVE: To analyze tweets associated with Ménière's disease (MD), including type of users who engage, change in usage patterns, and temporal associations, and to compare the perceptions of the general public with healthcare providers. METHODS: An R-program code, academictwitterR API, was used to query Twitter. All tweets mentioning MD from 2007 to 2021 were retrieved and analyzed. Valence Aware Dictionary and Sentiment Reasoning was used as a model to assess sentiment of tweets. Two reviewers assessed 1,007 tweets for qualitative analysis, identifying the source and the topic of the tweet. RESULTS: A total of 37,402 tweets were analyzed. The number of tweets per user ranged from 1 to 563 (M = 33.7, SD = 91.1). Quantitative analysis showed no temporal or seasonal association; however, tweeting increased when celebrities were diagnosed with MD. Of the 1007 representative tweets analyzed, 60.6% of tweets came from the general public and were largely of negative sentiment focusing on quality of life and support, whereas healthcare providers accounted for 23% of all tweets and focused on treatment/prevention. Tweets by news sources accounted for the remaining 13% of all tweets and were primarily positive in sentiment and focused on awareness. CONCLUSIONS: MD is commonly tweeted about by the general public, with limited input regarding the disease from healthcare providers. Healthcare providers must provide accurate information and awareness regarding MD, especially when awareness is highest, such as when celebrities are diagnosed. LEVEL OF EVIDENCE: Level IV.Indicate IRB or IACUCNot applicable.

Golgi stress induces SIRT2 to counteract Shigella infection via defatty-acylation.

Enzymes from pathogens often modulate host protein post-translational modifications (PTMs), facilitating survival and proliferation of pathogens. Shigella virulence factors IpaJ and IcsB induce proteolytic cleavage and lysine fatty acylation on host proteins, which cause Golgi stress and suppress innate immunity, respectively. However, it is unknown whether host enzymes could reverse such modifications introduced by pathogens' virulence factors to suppress pathogenesis. Herein, we report that SIRT2, a potent lysine defatty-acylase, is upregulated by the transcription factor CREB3 under Golgi stress induced by Shigella infection. SIRT2 in turn removes the lysine fatty acylation introduced by Shigella virulence factor IcsB to enhance host innate immunity. SIRT2 knockout mice are more susceptible to Shigella infection than wildtype mice, demonstrating the importance of SIRT2 to counteract Shigella infection.

Nanohybrid Membrane Synthesis with Phosphorene Nanoparticles: A Study of the Addition, Stability and Toxicity.

Phosphorene is a promising candidate as a membrane material additive because of its inherent photocatalytic properties and electrical conductance which can help reduce fouling and improve membrane properties. The main objective of this study was to characterize structural and morphologic changes arising from the addition of phosphorene to polymeric membranes. Here, phosphorene was physically incorporated into a blend of polysulfone (PSf) and sulfonated poly ether ether ketone (SPEEK) doping solution. Protein and dye rejection studies were carried out to determine the permeability and selectivity of the membranes. Since loss of material additives during filtration processes is a challenge, the stability of phosphorene nanoparticles in different environments was also examined. Furthermore, given that phosphorene is a new material, toxicity studies with a model nematode, Caenorhabditis elegans, were carried out to provide insight into the biocompatibility and safety of phosphorene. Results showed that membranes modified with phosphorene displayed a higher protein rejection, but lower flux values. Phosphorene also led to a 70% reduction in dye fouling after filtration. Additionally, data showed that phosphorene loss was negligible within the membrane matrix irrespective of the pH environment. Phosphorene caused toxicity to nematodes in a free form, while no toxicity was observed for membrane permeates.

NMT1 and NMT2 are lysine myristoyltransferases regulating the ARF6 GTPase cycle.

Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD+-dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases.

Correction: Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential.

After publication of this Article the authors noticed errors in several figures. In Fig. 2b the Gapdh panels are incorrect. The lysates are identical to those used in Fig. 1b, therefore the Gapdh panels should be the same in both figures. In Fig. 3b the Gapdh panels for Ad-Fhit-wt and Ad-Fhit-Y114F are incorrect and have been replaced with scans from original films. In Fig. 4A the Gapdh panels are incorrect. The lysates are identical to those used in Fig. 3b, therefore the Gapdh panels should be the same in both figures. In Fig. 4Bb the Gapdh panels for Fhit siRNA were incorrect and have been replaced with scans from original films. All resupplied figures are provided below. In Fig. 5C several panels are incorrect. The Authors were unable to locate the original films for all of these panels so Fig. 5c has been deleted. The scientific conclusions of this paper have not been affected.

Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential.

The Fhit tumor suppressor binds and hydrolyses diadenosine polyphosphates and the Fhit-substrate complex has been proposed as a proapoptotic effector, as determined by infection of susceptible cancer cells with adenoviruses carrying wild-type fragile histidine triad (FHIT) or catalytic site mutants. The highly conserved Fhit tyrosine 114 (Y114), within the unstructured loop C-terminal of the catalytic site, can be phosphorylated by Src family tyrosine kinases, although endogenous phospho-Fhit is rarely detected. To explore the importance of Y114 and identify Fhit-mediated signaling events, wild-type and Y114 mutant FHIT-expressing adenoviruses were introduced into two human lung cancer cell lines. Caspase-dependent apoptosis was effectively induced only by wild-type but not Y114 mutant Fhit proteins. By expression profiling of FHIT versus mutant FHIT-infected cells, we found that survivin, an Inhibitor of Apoptosis Protein (IAP) family member, was significantly decreased by wild-type Fhit. In addition, Fhit inhibited activity of Akt, a key effector in the phosphatidylinositol 3-OH kinase (PI3K) pathway; loss of endogenous Fhit expression caused increased Akt activity in vitro and in vivo, and overexpression of constitutively active Akt inhibited Fhit-induced apoptosis. The results indicate that the Fhit Y114 residue plays a critical role in Fhit-induced apoptosis, occurring through inactivation of the PI3K-Akt-survivin signal pathway.

Effects of histone deacetylase inhibitors on the Ah receptor gene promoter.

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent basic helix-loop-helix-PAS-containing transcription factor which is activated by chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Constitutive expression of the AhR gene occurs in a tissue- and developmentally specific manner and appears to be altered by chemicals which affect histone deacetylase (HDAC) activity in cells in culture. Here we have directly characterized the effects of two HDAC inhibitors, n-butyrate and trichostatin A, on the promoter activity of the murine AhR gene. HDAC inhibitors increased the constitutive activity of the AhR gene promoter in a luciferase reporter construct by five- to sevenfold in a dose- and time-dependent manner in several cell lines and was correlated with an increase in endogenous AhR activity in an AhR-deficient cell line. Deletion analysis of the upstream region of the AhR gene localized the HDAC inhibitor effect to a 167-bp region encompassing -77 to +90 of the AhR gene promoter. Cotransfection of an AhR promoter-luciferase reporter plasmid with a vector expressing the E1A(12s) oncoprotein, a negative regulator of p300, a protein with histone acetylase activity, decreased AhR promoter activity fivefold. Overall, our results support a role for histone acetylation in the transcriptional activity of the AhR gene promoter.

Analysis of the murine AhR gene promoter.

The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates biological and toxicological actions of halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Although much is known about the biochemical and molecular mechanisms of AhR action, little is known about the factors and events that control expression of the AhR gene itself. The 5'-flanking region of the murine AhR gene was characterized and deletion analysis demonstrated that regulatory elements necessary for full constitutive promoter activity are contained within a fragment encompassing -184 to +380 of the AhR gene. The murine AhR gene promoter is a GC-rich, TATA-less promoter that which contains at least five putative Spl-like binding sites. Transient transfection experiments not only identified a region between -1431 and -721 that represses constitutive promoter activity by 2- to 3-fold, but also demonstrate that basal AhR promoter activity occurs in a cell- and species-specific manner. n-Butyrate, a nonspecific histone deacetylase inhibitor, increased AhR promoter activity 8-fold, suggesting a role for histone acetylation in AhR gene promoter activity. Overall, this study defines upstream regulatory regions important for constitutive AhR gene expression and identifies a novel activator of AhR gene expression.

Stereochemical retention of the configuration in the action of Fhit on phosphorus-chiral substrates.

Fhit is the protein product of FHIT, a candidate human tumor suppressor gene. Fhit catalyzes the hydrolysis of diadenosine triphosphate (Ap3A) to AMP and ADP. Fhit is here shown to catalyze the hydrolysis in H218O with production of adenosine 5'-[18O]phosphate and ADP, proving that the substitution of water is at Palpha and not at Pbeta. The chain fold of Fhit is similar to that of galactose-1-phosphate uridylyltransferase, which functions by a double-displacement mechanism through the formation of a covalent nucleotidyl-enzyme intermediate and overall retention of configuration at Palpha. The active site of Fhit contains a histidine motif that is reminiscent of the HPH motif in galactose-1-phosphate uridylyltransferases, in which the first histidine residue serves as the nucleophilic catalyst to which the nucleotidyl group is bonded covalently in the covalent intermediate. In this work, the Fhit-catalyzed cleavage of (RP)- and (SP)-gamma-(m-nitrobenzyl) adenosine 5'-O-1-thiotriphosphate (mNBATPalphaS) in H218O to adenosine 5'-[18O]thiophosphate is shown to proceed with overall retention of configuration at phosphorus. gamma-(m-Nitrobenzyl) adenosine 5'-O-triphosphate (mNBATP) is approximately as good a substrate for Fhit as Ap3A, and both (RP)- and (SP)-mNBATPalphaS are substrates that react at about 0.5% of the rate of Ap3A. The stereochemical evidence indicates that hydrolysis by Fhit proceeds by a double-displacement mechanism, presumably through a covalent AMP-enzyme intermediate.

Genetic, biochemical, and crystallographic characterization of Fhit-substrate complexes as the active signaling form of Fhit.

Alterations in the FHIT gene at 3p14.2 occur as early and frequent events in the development of several common human cancers. The ability of human Fhit-negative cells to form tumors in nude mice is suppressed by stable reexpression of Fhit protein. Fhit protein is a diadenosine P1,P3-triphosphate (ApppA) hydrolase whose fungal and animal homologs form a branch of the histidine triad (HIT) superfamily of nucleotide-binding proteins. Because the His-96 --> Asn substitution of Fhit, which retards ApppA hydrolase activity by seven orders of magnitude, did not block tumor-suppressor activity in vivo, we determined whether this mutation affected ApppA binding or particular steps in the ApppA catalytic cycle. Evidence is presented that His-96 --> Asn protein binds ApppA well and forms an enzyme-AMP intermediate extremely poorly, suggesting that Fhit-substrate complexes are the likely signaling form of the enzyme. The cocrystal structure of Fhit bound to Ado-p-CH2-p-ps-Ado (IB2), a nonhydrolyzable ApppA analog, was refined to 3.1 A, and the structure of His-96 --> Asn Fhit with IB2 was refined to 2.6 A, revealing that two ApppA molecules bind per Fhit dimer; identifying two additional adenosine-binding sites on the dimer surface; and illustrating that His-98 is positioned to donate a hydrogen bond to the scissile bridging oxygen of ApppA substrates. The form of Fhit bound to two ApppA substrates would present to the cell a dramatically phosphorylated surface, prominently displaying six phosphate groups and two adenosine moieties in place of a deep cavity lined with histidines, arginines, and glutamines.

Beta-blocker-induced complications and the patient with glaucoma. Newer treatments to help reduce systemic adverse events.

Primary open-angle glaucoma is a condition associated with an elevated intraocular pressure (IOP) that is defined as optic degeneration with a slowly progressive deterioration of the visual field that may lead to blindness. More than 1 million Americans are being treated for glaucoma, and 80,000 are legally blind as a result of the disease. Glaucoma has its highest prevalence among the elderly population, with an incidence of approximately 1% in those older than 60 years, 3% in those between the ages of 70 and 80 years, and more than 9% in those older than 80 years. Treatment is directed at lowering high ocular pressures. The initial treatment, in most cases topical therapy with a beta-adrenergic blocking agent, reduces the IOP to help preserve sight. But such topical agents may also have adverse systemic effects on cardiac, pulmonary, central nervous system (CNS), and endocrine functions.

The role of the FHIT/FRA3B locus in cancer.

Common fragile sites form gaps at characteristic chromosome bands in metaphases from normal cells after aphidicolin induction. The distribution of common fragile sites parallels the positions of neoplasia-associated chromosomal rearrangements, prompting the proposal that fragility disposes to chromosomal rearrangements. Implicit in this hypothesis is that genes at fragile sites are altered by chromosome rearrangement and thus contribute to neoplastic growth. Chromosome band 3p14.2, encompassing the most inducible common fragile region, FRA3B, has been cloned and the FHIT gene, straddling FRA3B, characterized. The gene is inactivated by deletions in cancer-derived cell lines and primary tumors and Fhit protein is absent or reduced in lung, stomach, kidney, and cervical carcinomas, consistent with function as a tumor suppressor. FRA3B thus fulfills the prophecy that fragile site alterations contribute to the neoplastic process through inactivation of a tumor suppressor gene.

Validation of cold chain procedures suitable for distribution of vaccines by public health programs in the USA.

To enhance quality assurance of vaccine distribution by public health programs in the US, various methods for packing vaccines were validated. Validation involved both tests in an environmental chamber and actual shipping of packages by commercial overnight delivery service. Dry ice was used with vaccines needing to be kept at temperatures lower than -14 degrees C, and water-based cold packs with other vaccines. The latter could be used in two ways. When frozen, and placed over two or three faces of well-insulated boxes, assortments of vaccines were kept cold but not frozen for 2 days or more. However, packages with -15 degrees C cold packs may reach < 0 degree C. When cold packs at refrigerator temperature cover four to six faces of well insulated boxes, vaccine freezing in winter conditions or warming in temperate conditions was slowed considerably. These approaches, which require materials costing less than approximately 1% of the cost of the vaccines they protect, provide examples of packaging suitable for overnight delivery of vaccines in the US in different seasons.