Membranome-based identification of amino acid substitution in Haemophilus influenzae multidrug efflux pump HmrM for reduced chloramphenicol susceptibility.

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Amino acid substitutions in the region between RpoB clusters II and III on rifampin susceptibility in Haemophilus influenzae.

BACKGROUND: Rifampin is a potent chemoprophylactic antibiotic for Haemophilus influenzae infection, and the resistance rate in H. influenzae is low. In this study, we assessed rifampin resistance-related genetic variations in H. influenzae. METHODS: Rifampin susceptibility testing and whole-genome sequencing were performed in 51 H. influenzae isolates. Variations associated with rifampin resistance were identified using Fisher's exact tests. Functional assays were performed to evaluate the effect of RpoB substitutions on rifampin susceptibility. RESULTS: Using the genome of the Rd KW20 H. influenzae strain as the reference, we detected 40 genetic variations in rpoB, which resulted in 39 deduced amino acid substitutions among the isolates. Isolate A0586 was resistant to rifampin, with a minimum inhibitory concentration (MIC) = 8 µg/mL. Phylogenetic analyses revealed that the RpoB sequence of isolate A0586 was distinct from other isolates. Five substitutions, including H526N located in cluster I and L623F, R628C, L645F, and L672F in the region between clusters II and III, were unique to isolate A0586. In two rifampin-susceptible H. influenzae isolates, RpoB-H526N alone and in combination with RpoB-L672F increased the MICs of rifampin to 4 and 8 µg/mL, respectively. RpoB-L672F did not affect cell growth and transcription in H. influenzae isolates. No amino acid substitutions in the AcrAB-TolC efflux pump or outer membrane proteins were found to be associated with rifampin resistance in H. influenzae. CONCLUSIONS: Our findings indicate that L672F substitution in the region between RpoB clusters II and III has an aggravating effect on rifampin resistance in H. influenzae.

Molecular characterization of multidrug-resistant non-typeable Haemophilus influenzae with high-level resistance to cefuroxime, levofloxacin, and trimethoprim-sulfamethoxazole.

BACKGROUND: Non-typeable Haemophilus influenzae (NTHi) has become the major cause of invasive H. influenzae diseases in the post-H. influenzae type b vaccine era. The emergence of multidrug-resistant (MDR) NTHi is a growing public health problem. Herein, we investigated the molecular basis of MDR in NTHi. The isolated NTHi were subjected to antimicrobial susceptibility testing for 12 agents. Whole genome and plasmid sequencing were conducted and analyzed to identify significant genetic variations and plasmid-encoded genes conferred antibiotic resistance. RESULTS: Thirteen (50%) MDR NTHi isolates were obtained; of these, 92.3% were non-susceptible to ampicillin, 30.8% to amoxicillin-clavulanate, 61.5% to cefuroxime, 61.5% to ciprofloxacin/levofloxacin, 92.3% to trimethoprim-sulfamethoxazole, 30.8% to tetracycline, and 7.7% to azithromycin. Eight ampicillin-resistant isolates were ß-lactamase positive; of these, 6 carried blaTEM-1 and 2 carried blaROB-1, whereas 4 were ß-lactamase negative. Genetic variations in mrdA, mepA, and pbpG were correlated with amoxicillin-clavulanate non-susceptibility, whereas variations in ftsI and lpoA conferred cefuroxime resistance. Five variations in gyrA, 2 in gyrB, 3 in parC, 1 in parE, and 1 in the parC-parE intergenic region were associated with levofloxacin/ciprofloxacin non-susceptibility. Among these genes, 8 variations were linked to high-level levofloxacin resistance. Six variations in folA were associated with trimethoprim-sulfamethoxazole resistance. Plasmid-bearing tet(B) and mef(A) genes were responsible for tetracycline and azithromycin resistance in 4 and 1 MDR isolates, respectively. CONCLUSIONS: This study clarified the molecular epidemiology of MDR in NTHi. This can benefit the monitoring of drug resistance trends in NTHi and the adequate medical management of patients with NTHi infection.

Ambulatory Orbital Fracture Repair: An Analysis of ER Visits After Surgery From a Multistate Study.

OBJECTIVE: To determine the 30-day postoperative emergency room (ER) visit rate following ambulatory orbital fracture repair with same-day discharge, and the causes and risk factors associated with ER visit. STUDY DESIGN: Database study. SETTING: State Ambulatory Surgery and Services Database (SASD) and State Emergency Department Database (SEDD) for California, New York, and Florida for 2011. METHODS: We identified orbital fracture repair procedures among adults from the SASD, which was linked to the SEDD to identify the incidence and causes of ER visits within 30 days. Univariate and multivariable logistic regression models were used to determine the factors associated with ER visit. RESULTS: Among 762 patients, the 30-day postoperative ER visit rate was 4.5%. Most ER visits (58.9%) occurred during the first week after surgery. The most common reasons for ER visits were related to pain, swelling, headache, dizziness, and fatigue (29.4%), followed by ophthalmologic etiologies including visual disturbances and infection of the eye (14.7%). There was no case of retrobulbar hematoma. In the multivariate analysis, patients living in Florida were at a significantly higher risk for ER visit compared to those in California (odds ratio: 4.48 [1.43-14.10], p = .010). CONCLUSION: Ambulatory orbital fracture repair appears to be safe. Common reasons for ER visit included pain, swelling, and ophthalmic symptoms. An increased risk for ER visit was seen with certain geographic regions but not with medical comorbidities or concurrent facial fractures or procedures.

Molecular characterization of cefepime and aztreonam nonsusceptibility in Haemophilus influenzae.

BACKGROUND: Cefepime and aztreonam are highly efficacious against H. influenzae, and resistant strains are rare. In this study, we isolated cefepime- and aztreonam-nonsusceptible H. influenzae strains and addressed the molecular basis of their resistance to cefepime and aztreonam. METHODS: Two hundred and 28 specimens containing H. influenzae were screened, of which 32 isolates were enrolled and applied to antimicrobial susceptibility testing and whole-genome sequencing. Genetic variations that were detected in all nonsusceptible isolates with statistical significance by Fisher's exact tests were identified as cefepime or aztreonam nonsusceptibility related. Functional complementation assays were conducted to assess the in vitro effects of proteins with sequence substitutions on drug susceptibility. RESULTS: Three H. influenzae isolates were nonsusceptible to cefepime, one of which was also nonsusceptible to aztreonam. Genes encoding TEM, SHV and CTX-M extended-spectrum ß-lactamases were not detected in the cefepime- and aztreonam-nonsusceptible isolates. Five genetic variations in four genes and 10 genetic variations in five genes were associated with cefepime and aztreonam nonsusceptibility, respectively. Phylogenetic analyses revealed that changes in FtsI were correlated strongly with the MIC of cefepime and moderately with aztreonam. FtsI Thr532Ser-Tyr557His cosubstitution linked to cefepime nonsusceptibility and Asn305Lys-Ser385Asn-Glu416Asp cosubstitution to aztreonam nonsusceptibility. Functional complementation assays revealed that these cosubstitutions increased MICs of cefepime and aztreonam in susceptible H. influenzae isolates, respectively. CONCLUSIONS: Genetic variations relevant to resistant phenotypes of cefepime and aztreonam nonsusceptibility in H. influenzae were identified. Moreover, the effects of FtsI cosubstitutions on increasing MICs of cefepime and aztreonam in H. influenzae were demonstrated.

Hepatitis B virus virion secretion is a CRM1-spike-mediated late event.

BACKGROUND: Hepatitis B virus (HBV) is a major human pathogen worldwide. To date, there is no curative treatment for chronic hepatitis B. The mechanism of virion secretion remains to be investigated. Previously, we found that nuclear export of HBc particles can be facilitated via two CRM1-specific nuclear export signals (NES) at the spike tip. METHODS: In this study, we used site-directed mutagenesis at the CRM1 NES, as well as treatment with CRM1 inhibitors at a low concentration, or CRM1-specific shRNA knockdown, in HBV-producing cell culture, and measured the secretion of various HBV viral and subviral particles via a native agarose gel electrophoresis assay. Separated HBV particles were characterized by Western blot analysis, and their genomic DNA contents were measured by Southern blot analysis. Secreted extracellular particles were compared with intracellular HBc capsids for DNA synthesis and capsid formation. Virion secretion and the in vivo interactions among HBc capsids, CRM1 and microtubules, were examined by proximity ligation assay, immunofluorescence microscopy, and nocodazole treatment. RESULTS: We report here that the tip of spike of HBV core (HBc) particles (capsids) contains a complex sensor for secretion of both HBV virions and naked capsids. HBV virion secretion is closely associated with HBc nuclear export in a CRM1-dependent manner. At the conformationally flexible spike tips of HBc particles, NES motifs overlap extensively with motifs important for secretion of HBV virions and naked capsids. CONCLUSIONS: We provided experimental evidence that virions and naked capsids can egress via two distinct, yet overlapping, pathways. Unlike the secretion of naked capsids, HBV virion secretion is highly CRM1- and microtubule-dependent. CRM1 is well known for its involvement in nuclear transport in literature. To our knowledge, this is the first report that CRM1 is required for virion secretion. CRM1 inhibitors could be a promising therapeutic candidate for chronic HBV patients in clinical medicine.

Tracheostomy Outcomes in COVID-19 Patients in a Low Resource Setting.

OBJECTIVES: COVID-19 predominately affects safety net hospitals. Tracheostomies improve outcomes and decrease length of stay for COVID-19 patients. Our objectives are to determine if (1) COVID-19 tracheostomies have similar complication and mortality rates as non-COVID-19 tracheostomies and (2) to determine the effectiveness of our tracheostomy protocol at a safety net hospital. METHODS: Patients who underwent tracheostomy at Los Angeles County Hospital between August 2009 and August 2020 were included. Demographics, SARS-CoV-2 status, body mass index (BMI), Charlson Co-morbidity Index (CCI), length of intubation, complication rates, decannulation rates, and 30-day all-cause mortality versus tracheostomy related mortality rates were all collected. RESULTS: Thirty-eight patients with COVID-19 and 130 non-COVID-19 patients underwent tracheostomies. Both groups were predominately male with similar BMI and CCI, though the COVID-19 patients were more likely to be Hispanic and intubated for a longer time (P = .034 and P < .0001, respectively). Both groups also had similar, low intraoperative complications at 2% to 3% and comparable long-term post-operative complications. However, COVID-19 patients had more perioperative complications within 7 days of surgery (P < .01). Specifically, they were more likely to have perioperative bleeding at their tracheostomy sites (P = .03) and long-term post-operative mucus plugging (P < .01). However, both groups had similar 30-day mortality rates. There were no incidences of COVID-19 transmission to healthcare workers. CONCLUSIONS: COVID-19 tracheostomies are safe for patients and healthcare workers. Careful attention should be paid to suctioning to prevent mucus plugging. LEVEL OF EVIDENCE: 3.

Rapid, reliable, and reproducible cell fusion assay to quantify SARS-Cov-2 spike interaction with hACE2.

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.

Thermal Conductance Through the Skull Base From Endoscopic Surgery Cauterization Instruments: Cadaver and Goat Model.

OBJECTIVE: Cauterization prevents hemorrhage and optimizes the surgical field during endoscopic sinus surgery but may cause injury to nearby structures. The objective of this study is to examine thermal conductance from cauterization equipment across the skull base. STUDY DESIGN: Cadaver and animal model. SETTING: Surgical skills laboratory of an academic tertiary medical institution. METHODS: A pilot study was conducted with a deidentified cadaver head and expanded to a goat head animal model. Endoscopic dissection was performed to expose the lamina papyracea, ethmoid roof, sphenoid roof, and frontal sinus. Cautery was applied to the frontal sinus of goat heads, and temperatures were measured via thermocouple sensors placed along the intracranial skull base. Surgical instruments studied included monopolar, bipolar, and endoscopic bipolar devices at various power settings. RESULTS: Temperature increase, as averaged across all cautery powers and measurement positions, was highest for the monopolar cautery (17.55 °C) when compared with the bipolar and endoscopic bipolar devices (<2 °C for bipolar, Endo-Pen, Stammberger, and Wormald; P < .001). Monopolar cautery reached 30.86 °C at high power when averaged over all positions (P < .001) as compared with <3 °C for the other instruments. Temperatures rose as power of cautery was increased from low to medium and high. Temperatures decreased as the distance of the thermocouple sensor probe from the cautery origin increased. CONCLUSION: Thermal conductance across the skull base varies depending on equipment and power of cautery, with monopolar resulting in the largest temperature increase. Choice and implementation of cauterization instruments have implications on inadvertent transmission of thermal energy during endoscopic sinus surgery.

Trends in Operative Complex Middle and Upper Maxillofacial Trauma: A 17-Year Study.

OBJECTIVES/HYPOTHESIS: Over 3 million incidents of facial trauma occur each year in the United States. This study aims to determine trends in operative middle and upper maxillofacial trauma in one of the largest US cities. STUDY DESIGN: Retrospective case-control study. METHODS: Retrospective chart review of all operative middle and upper maxillofacial trauma from July 1993 to July 2010 presenting to Los Angeles County Hospital, a Level I Trauma Center. Data included demographics, mechanism of injury, and fracture characteristics. RESULTS: Analysis was performed for a total of 4,299 patients and 5,549 facial fractures. Mean patient age was 34.6, and most patients were male (88%). Between the two time periods (1993-2001 and 2002-2010), there was a 42% reduction in operative maxillofacial trauma (3,510 to 2,039). Orbital floor and zygomaticomaxillary complex fractures were the most prevalent types of fractures. Panfacial fractures demonstrated the largest reduction in number of fractures (325 to 5, P<0.01). Assault and motor vehicle accidents (MVA) were the two most common mechanisms of injury. Operative fractures due to MVAs decreased (390 to 214, P = .74), whereas fractures due to assault increased (749 to 800, P<0.01). Compared to adults, pediatric facial trauma (age < 18) were caused by a higher percentage of MVAs (27% vs. 13%), auto versus pedestrian (9% vs. 5%), and gunshot wounds (8% vs. 4%) (P<0.01). CONCLUSIONS: Operative middle and upper maxillofacial trauma decreased over a 17-year period. Assault was the most significant mechanism of trauma overall. These trends suggest that focusing future prevention strategies on curtailing interpersonal violence may more effectively address the burden of facial trauma. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:1985-1989, 2021.

CSF Leaks due to Electric Scooter Injury.

BACKGROUND: There has been a rapid increase in electric motorized scooter (e-scooter) usage after the introduction of dockless, shareable devices. METHODS: Case series from three tertiary hospitals in Los Angeles between May-September 2019. RESULTS: Five patients had skull base fractures and CSF leaks or pneumocephalus after e-scooter accident, none wore helmets. Two patients were treated with observation alone, two patients were treated with lumbar drain or external ventriculostomy placement, and one patient died of their injuries prior to definitive management. CONCLUSION: Without appropriate safety policies in place, the number of such injuries may increase as the use of e-scooters increase. Laryngoscope, 131:E1035-E1037, 2021.

Advanced Practice Providers Utilization Trends in Otolaryngology From 2012 to 2017 in the Medicare Population.

OBJECTIVE: Nurse practitioners and physician assistants form a growing advanced practice provider (APP) group. We aim to analyze the trends and types of services provided by APPs in otolaryngology. STUDY DESIGN: Cross-sectional study. SETTING: Medicare Provider Utilization and Payment Data: Physician and Other Supplier Public Use Files, 2012-2017. METHODS: The Medicare database was searched for 13 commonly used otolaryngology-specific Current Procedural Terminology (CPT) codes, and 10 evaluation and management (E/M) codes were evaluated by provider type. Changes in code utilization were compared between physicians and APPs over time. RESULTS: From 2012 to 2017, there was a 51% increase in the number of otolaryngology APPs, compared to a 2.2% increase in physician providers. APPs increased their share of new and established patient visits from 4% to 7%d 11% to 15%, respectively. There was not a significant difference over time in number of patient visits performed annually per provider according to provider type. The increase in number of APP vs physician providers was significantly greater for every procedure except for balloon sinus dilation and tympanostomy tube placement. CONCLUSION: Due to increasing numbers, APPs are accounting for more patient visits and procedures over time. The physician workforce and the numbers of procedures performed per physician have remained relatively stable from 2012 to 2017. Increasing complexity of patients seen and a broader range of procedures offered by work-experienced or postgraduate-trained APPs may further improve access to health care in the face of possible physician shortages.

Extensively drug-resistant Haemophilus influenzae - emergence, epidemiology, risk factors, and regimen.

BACKGROUND: Concern about Haemophilus influenzae infection has been increasing over recent decades. Given the emergence of H. influenzae with severe drug resistance, we assessed the prevalence of as well as risk factors and potential therapies for extensively drug-resistant (XDR) H. influenzae infection in Taiwan. RESULTS: In total, 2091 H. influenzae isolates with disk diffusion-based antibiotic susceptibility testing from 2007 to 2018 were enrolled. H. influenzae strains resistant to ampicillin, chloramphenicol, levofloxacin, and trimethoprim-sulfamethoxazole tended to be isolated from patient wards (≧41%), whereas those resistant to amoxicillin-clavulanate, cefotaxime, and cefuroxime were more likely to be isolated from intensive care units (approximately 50%). XDR H. influenzae was first identified in 2007, and its incidence did not significantly change thereafter. Overall prevalence of single, multiple, and extensively drug-resistant H. influenzae over 2007-2018 was 21.5% (n = 450), 26.6% (n = 557), and 2.5% (n = 52), respectively. A stepwise logistic regression analysis revealed that blood culture (odds ratio: 4.069, 95% confidence intervals: 1.339-12.365, P = 0.013) was an independent risk factor for XDR H. influenzae infection. No nosocomial transmission of XDR H. influenzae observed. Antibiotic susceptibility testing results demonstrated that cefotaxime was effective against 78.8% (n = 41) of the XDR strains. CONCLUSIONS: The presence of XDR H. influenzae strains was identified in Taiwan, and cefotaxime was efficacious against most of these strains.

Adding a C-terminal Cysteine (CTC) Can Enhance the Bactericidal Activity of Three Different Antimicrobial Peptides.

The emergence of antibiotic-resistant bacteria has threatened our health worldwide. There is an urgent need for novel antibiotics. Previously, we identified a novel 37-mer antimicrobial peptide (AMP), HBcARD, with broad spectrum antimicrobial activity. Here, we improved the efficacy of HBcARD, by re-engineering the peptide, including the addition of a new cysteine to its C-terminus (CTC). The new 28-mer derivative, D-150-177C, contains all D-form arginines, in addition to a C-terminal cycteine. This peptide can kill antibiotic-resistant clinical isolates of Gram-negative bacteria, and is more potent than the parental HBcARD peptide in a mouse sepsis model. In another lung infection mouse model, D-150-177C showed protection efficacy against colistin-resistant Acinetobacter baumannii. Unlike colistin, we observed no acute toxicity of D-150-177C in vivo. Interestingly, we found that CTC modification could enhance the antibacterial activity of several other AMPs, such as buforinII and lysin. The potential application and mechanism of this CTC method as a general approach to improving drug efficacy, warrants further investigation in the future.

HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

Societal Identification of Facial Paralysis and Paralysis Location.

IMPORTANCE: When able to identify facial paralysis, members of society regard individuals with facial paralysis differently. They perceive a decrease in attractiveness, more negative affect, and lower quality of life. However, the ability of lay people in society to accurately identify the presence of facial paralysis has not yet been defined. OBJECTIVE: To determine societal members' ability to (1) identify paralysis in varying degrees of paralysis severity and (2) localize the defect on the face. DESIGN, SETTING, AND PARTICIPANTS: A prospective observational study conducted in an academic tertiary referral center using a group of 380 casual observers was carried out. MAIN OUTCOMES AND MEASURES: Surveys were designed containing smiling and repose images of normal faces and faces with unilateral facial paralysis of 3 severity levels (low, medium, and high) as categorized by House-Brackmann (HB) grade. The photographs were then shown to casual observers in a web-based survey. After reviewing both normal faces and faces with varying degrees of paralysis, they then indicated (1) whether paralysis was present and (2) if so, where the paralysis was on the face. RESULTS: A total of 380 participants (267 [70.3%] women and 113 [29.7%] men with a mean [SD] age of 29 [12] years) successfully completed the survey, viewing 2860 facial photographs in aggregate. The accuracy rate of identifying paralysis increased from low-grade through high-grade paralysis. Facial paralysis was identified in 249 (34.6%) of 719 facial photographs with low-grade paralysis, 448 (63.2%) of 709 with medium-grade paralysis, and 696 (96.7%) of 720 with high-grade paralysis (χ2 = 912.6, P < .001); 6.2% (44/731) of normal faces were incorrectly identified as having paralysis (χ2 = 912.6, P < .001). Participants correctly localized paralysis in 157 (63.0%) of 249 low-grade photographs, 307 (68.5%) of 448 medium-grade photographs, and 554 (79.6%) of 696 high-grade photographs (χ2 = 32.5, P < .001). In general, participants tended to identify facial paralysis more accurately in smiling vs repose faces (48.6% vs 20.6%, 92.4% vs 33.7%, and 96.7% vs 96.6% in low-, medium-, and high-grade paralysis, respectively) (χ2 = 62.2, P < .001; χ2 = 262.6, P < .001; χ2 = 0.0, P = .96, respectively). CONCLUSIONS AND RELEVANCE: The ability of individuals to identify the presence of facial paralysis increased as paralysis severity increased. Further, smiling increased accurate identification. However, even when individuals can identify paralysis, they are not necessarily able to accurately localize the paralysis on a face. This may speak to a phenomenon in which perception of a facial defect comes from a holistic interpretation of a face, rather than a clinically accurate specification of the defect location. These findings are important in the future counseling of patients. LEVEL OF EVIDENCE: NA.

Medical student healthcare consulting groups: A novel way to train the next generation of physician-executives.

Meeting the challenges of the evolving healthcare environment requires leadership of physicians well-trained in clinical medicine and healthcare management. However, many physicians lack training in business and leadership. While some residency programs have management tracks, training at the medical school level is currently lacking. We developed the Hopkins Health Management Advisory Group, an extracurricular program at Johns Hopkins University School of Medicine that exposes medical students to healthcare management and fosters development of leadership skills. Teams of students work directly with health system executives on 3-6 month-long projects using management consulting principles to address problems spanning health system domains, including strategy, operations, and quality improvement. Since the program's inception, 23 students have completed seven projects, with 13 additional students currently working on three more projects. Sponsors leading six out of seven completed projects have implemented recommendations. Qualitative survey respondents have found the program beneficial, with students frequently describing how the program has helped to develop professional skills and foster knowledge about healthcare management. These early assessments show positive impact for both students and the institution, and suggest that such programs can train students in management early and concurrently in their medication education by immersing them in team-based health system projects.

Prevalence of Body Dysmorphic Disorder and Surgeon Diagnostic Accuracy in Facial Plastic and Oculoplastic Surgery Clinics.

IMPORTANCE: Body dysmorphic disorder (BDD) is a relative contraindication for facial plastic surgery, but formal screening is not common in practice. The prevalence of BDD in patients seeking facial plastic surgery is not well documented. OBJECTIVE: To establish the prevalence of BDD across facial plastic and oculoplastic surgery practice settings, and estimate the ability of surgeons to screen for BDD. DESIGN, SETTING, AND PARTICIPANTS: This multicenter prospective study recruited a cohort of 597 patients who presented to academic and private facial plastic and oculoplastic surgery practices from March 2015 to February 2016. METHODS: All patients were screened for BDD using the Body Dysmorphic Disorder Questionnaire (BDDQ). After each clinical encounter, surgeons independently evaluated the likelihood that a participating patient had BDD. Validated instruments were used to assess satisfaction with facial appearance including the FACE-Q, Blepharoplasty Outcomes Evaluation (BOE), Facelift Outcomes Evaluation (FOE), Rhinoplasty Outcomes Evaluation (ROE), and Skin Rejuvenation Outcomes Evaluation (SROE). RESULTS: Across participating practices (9 surgeons, 3 sites), a total of 597 patients were screened for BDD: 342 patients from site 1 (mean [SD] age, 44.2 [16.5] years); 158 patients, site 2 (mean [SD] age, 46.0 [16.2] years), site 3, 97 patients (mean [SD] age, 56.3 [15.5] years). Overall, 58 patients [9.7%] screened positive for BDD by the BDDQ instrument, while only 16 of 402 patients [4.0%] were clinically suspected of BDD by surgeons. A higher percentage of patients presenting for cosmetic surgery (37 of 283 patients [13.1%]) compared with those presenting for reconstructive surgery (21 of 314 patients [6.7%]) screened positive on the BDDQ (odds ratio, 2.10; 95% CI, 1.20-3.68; P = .01). Surgeons were only able to correctly identify 2 of 43 patients (4.7%) who screened positive for BDD on the BDDQ, and the positive likelihood ratio was only 1.19 (95% CI, 0.28-5.07). Patients screening positive for BDD by the BDDQ had lower satisfaction with their appearance as measured by the FACE-Q, ROE, BOE, SROE, and FOE. CONCLUSIONS AND RELEVANCE: Body dysmorphic disorder is a relatively common condition across facial plastic and oculoplastic surgery practice settings. Patients who screen positive on the BDDQ have lower satisfaction with their facial appearance at baseline. Surgeons have a poor ability to screen for patients with BDD when compared with validated screening instruments such as the BDDQ. Routine implementation of validated BDD screening instruments may improve patient care. LEVEL OF EVIDENCE: NA.

Societal Value of Surgery for Facial Reanimation.

IMPORTANCE: Patients with facial paralysis are perceived negatively by society in a number of domains. Society's perception of the health utility of varying degrees of facial paralysis and the value society places on reconstructive surgery for facial reanimation need to be quantified. OBJECTIVE: To measure health state utility of varying degrees of facial paralysis, willingness to pay (WTP) for a repair, and the subsequent value of facial reanimation surgery as perceived by society. DESIGN, SETTING, AND PARTICIPANTS: This prospective observational study conducted in an academic tertiary referral center evaluated a group of 348 casual observers who viewed images of faces with unilateral facial paralysis of 3 severity levels (low, medium, and high) categorized by House-Brackmann grade. Structural equation modeling was performed to understand associations among health utility metrics, WTP, and facial perception domains. Data were collected from July 16 to September 26, 2015. MAIN OUTCOMES AND MEASURES: Observer-rated (1) quality of life (QOL) using established health utility metrics (standard gamble, time trade-off, and a visual analog scale) and (2) their WTP for surgical repair. RESULTS: Among the 348 observers (248 women [71.3%]; 100 men [28.7%]; mean [SD] age, 29.3 [11.6] years), mixed-effects linear regression showed that WTP increased nonlinearly with increasing severity of paralysis. Participants were willing to pay $3487 (95% CI, $2362-$4961) to repair low-grade paralysis, $8571 (95% CI, $6401-$11 234) for medium-grade paralysis, and $20 431 (95% CI, $16 273-$25 317) for high-grade paralysis. The dominant factor affecting the participants' WTP was perceived QOL. Modeling showed that perceived QOL decreased with paralysis severity (regression coefficient, -0.004; 95% CI, -0.005 to -0.004; P < .001) and increased with attractiveness (regression coefficient, 0.002; 95% CI, 0.002 to 0.003; P < .001). Mean (SD) health utility scores calculated by the standard gamble metric for low- and high-grade paralysis were 0.98 (0.09) and 0.77 (0.25), respectively. Time trade-off and visual analog scale measures were highly correlated. We calculated mean (SD) WTP per quality-adjusted life-year, which ranged from $10 167 ($14 565) to $17 008 ($38 288) for low- to high-grade paralysis, respectively. CONCLUSIONS AND RELEVANCE: Society perceives the repair of facial paralysis to be a high-value intervention. Societal WTP increases and perceived health state utility decreases with increasing House-Brackmann grade. This study demonstrates the usefulness of WTP as an objective measure to inform dimensions of disease severity and signal the value society places on proper facial function. LEVEL OF EVIDENCE: NA.