Schizophrenia-Linked Protein tSNARE1 Regulates Endosomal Trafficking in Cortical Neurons.

TSNARE1, which encodes the protein tSNARE1, is a high-confidence gene candidate for schizophrenia risk, but nothing is known about its cellular or physiological function. We identified the major gene products of TSNARE1 and their cytoplasmic localization and function in endosomal trafficking in cortical neurons. We validated three primary isoforms of TSNARE1 expressed in human brain, all of which encode a syntaxin-like Qa SNARE domain. RNA-sequencing data from adult and fetal human brain suggested that the majority of tSNARE1 lacks a transmembrane domain that is thought to be necessary for membrane fusion. Biochemical data demonstrate that tSNARE1 can compete with Stx12 for incorporation into an endosomal SNARE complex, supporting its possible role as an inhibitory SNARE. Live-cell imaging in cortical neurons from mice of both sexes demonstrated that brain tSNARE1 isoforms localized to the endosomal network. The most abundant brain isoform, tSNARE1c, localized most frequently to Rab7+ late endosomes, and endogenous tSNARE1 displayed a similar localization in human neural progenitor cells and neuroblastoma cells. In mature rat neurons from both sexes, tSNARE1 localized to the dendritic shaft and dendritic spines, supporting a role for tSNARE1 at the postsynapse. Expression of either tSNARE1b or tSNARE1c, which differ only in their inclusion or exclusion of an Myb-like domain, delayed the trafficking of the dendritic endosomal cargo Nsg1 into late endosomal and lysosomal compartments. These data suggest that tSNARE1 regulates endosomal trafficking in cortical neurons, likely by negatively regulating early endosomal to late endosomal trafficking.SIGNIFICANCE STATEMENT Schizophrenia is a severe and polygenic neuropsychiatric disorder. Understanding the functions of high-confidence candidate genes is critical toward understanding how their dysfunction contributes to schizophrenia pathogenesis. TSNARE1 is one of the high-confidence candidate genes for schizophrenia risk, yet nothing was known about its cellular or physiological function. Here we describe the major isoforms of TSNARE1 and their cytoplasmic localization and function in the endosomal network in cortical neurons. Our results are consistent with the hypothesis that the majority of brain tSNARE1 acts as a negative regulator to endolysosomal trafficking.

Aerosol and droplet generation from orbital repair: Surgical risk in the pandemic era.

INTRODUCTION: The highly contagious COVID-19 has resulted in millions of deaths worldwide. Physicians performing orbital procedures may be at increased risk of occupational exposure to the virus due to exposure to secretions. The goal of this study is to measure the droplet and aerosol production during repair of the inferior orbital rim and trial a smoke-evacuating electrocautery handpiece as a mitigation device. MATERIAL AND METHODS: The inferior rim of 6 cadaveric orbits was approached transconjunctivally using either standard or smoke-evacuator electrocautery and plated using a high-speed drill. Following fluorescein inoculation, droplet generation was measured by counting under ultraviolet-A (UV-A) light against a blue background. Aerosol generation from 0.300-10.000 µm was measured using an optical particle sizer. Droplet and aerosol generation was compared against retraction of the orbital soft tissue as a negative control. RESULTS: No droplets were observed following the orbital approach using electrocautery. Visible droplets were observed after plating with a high-speed drill for 3 of 6 orbits. Total aerosol generation was significantly higher than negative control following the use of standard electrocautery. Use of smoke-evacuator electrocautery was associated with significantly lower aerosol generation in 2 of 3 size groups and in total. There was no significant increase in total aerosols associated with high-speed drilling. DISCUSSION AND CONCLUSIONS: Droplet generation for orbital repair was present only following plating with high-speed drill. Aerosol generation during standard electrocautery was significantly reduced using a smoke-evacuating electrocautery handpiece. Aerosols were not significantly increased by high-speed drilling.

Aerosol and droplet generation from mandible and midface fixation: Surgical risk in the pandemic era.

PURPOSE: The COVID-19 pandemic has led to concerns over transmission risk from healthcare procedures, especially when operating in the head and neck such as during surgical repair of facial fractures. This study aims to quantify aerosol and droplet generation from mandibular and midface open fixation and measure mitigation of airborne particles by a smoke evacuating electrocautery hand piece. MATERIALS AND METHODS: The soft tissue of the bilateral mandible and midface of two fresh frozen cadaveric specimens was infiltrated using a 0.1% fluorescein solution. Surgical fixation via oral vestibular approach was performed on each of these sites. Droplet splatter on the surgeon's chest, facemask, and up to 198.12 cm (6.5 ft) away from each surgical site was measured against a blue background under ultraviolet-A (UV-A) light. Aerosol generation was measured using an optical particle sizer. RESULTS: No visible droplet contamination was observed for any trials of mandible or midface fixation. Total aerosolized particle counts from 0.300-10.000 µm were increased compared to baseline following each use of standard electrocautery (n = 4, p < 0.001) but not with use of a suction evacuating electrocautery hand piece (n = 4, p = 0.103). Total particle counts were also increased during use of the powered drill (n = 8, p < 0.001). CONCLUSIONS: Risk from visible droplets during mandible and midface fixation is low. However, significant increases in aerosolized particles were measured after electrocautery use and during powered drilling. Aerosol dispersion is significantly decreased with the use of a smoke evacuating electrocautery hand piece.

Epigenetic regulation of Kcna3-encoding Kv1.3 potassium channel by cereblon contributes to regulation of CD4+ T-cell activation.

The role of cereblon (CRBN) in T cells is not well understood. We generated mice with a deletion in Crbn and found cereblon to be an important antagonist of T-cell activation. In mice lacking CRBN, CD4(+) T cells show increased activation and IL-2 production on T-cell receptor stimulation, ultimately resulting in increased potassium flux and calcium-mediated signaling. CRBN restricts T-cell activation via epigenetic modification of Kcna3, which encodes the Kv1.3 potassium channel required for robust calcium influx in T cells. CRBN binds directly to conserved DNA elements adjacent to Kcna3 via a previously uncharacterized DNA-binding motif. Consequently, in the absence of CRBN, the expression of Kv1.3 is derepressed, resulting in increased Kv1.3 expression, potassium flux, and CD4(+) T-cell hyperactivation. In addition, experimental autoimmune encephalomyelitis in T-cell-specific Crbn-deficient mice was exacerbated by increased T-cell activation via Kv1.3. Thus, CRBN limits CD4(+) T-cell activation via epigenetic regulation of Kv1.3 expression.

Prevalence of Invasive Infections Due to Carbapenem-Resistant Enterobacteriaceae among Adult Patients in U.S. Hospitals.

This large-scale retrospective analysis (n = 60,551) of the Premier inpatient database (1 January 2011 to 31 December 2014) found an overall prevalence of carbapenem-resistant Enterobacteriaceae strains of 2.3% (range, 0.9% to 5.8% by geographic region) among patients with infections due to Enterobacteriaceae Ongoing monitoring and development of decision support tools/algorithms are needed for identification of high-risk patients.

Arhgef16, a novel Elmo1 binding partner, promotes clearance of apoptotic cells via RhoG-dependent Rac1 activation.

Elmo is an evolutionarily conserved mammalian ortholog of Caenorhabditis elegans CED-12 with proposed roles during the removal of apoptotic cells, cell migration, neurite outgrowth, and myoblast fusion (Katoh and Negishi (2003) [1], Park and Tosello (2007) [2], Grimsley et al. (2004) [3], Hamoud et al. (2014) [4]). Elmo mediates these cellular processes by interacting with various proteins located in the plasma membrane, cytoplasm and nucleus, and by modulating their activities although it has no intrinsic catalytic activity (Park and Tosello (2007) [2], Hamoud et al. (2014) [4], Li et al. (2013) [5], Margaron, Fradet and Cote (2013) [6], and Mauldin et al. (2013)[7]). Because there are a limited number of proteins known to interact with Elmo, we performed a yeast two-hybrid screen using Elmo1 as bait to identify Elmo1-interacting proteins and to evaluate their mode of regulation. Arhgef16 was one of the proteins identified through the screen and subsequent analyses revealed that Arhgef16 interacted with Elmo1 in mammalian cells as well. Expression of Arhgef16 in phagocytes promoted engulfment of apoptotic cells, and engulfment mediated by Arhgef16 increased synergistically in the presence of Elmo1 but was abrogated in the absence of Elmo1. In addition, Arhgef16-mediated removal of apoptotic cells was dependent on RhoG, but independent of Dock1. Taken together, this study suggests that the newly identified Elmo1-interacting protein, Arhgef16, functions synergistically with Elmo1 to promote clearance of apoptotic cells in a RhoG-dependent and Dock1-independent manner.

Developing forebrain synapses are uniquely vulnerable to sleep loss.

Sleep is an essential behavior that supports lifelong brain health and cognition. Neuronal synapses are a major target for restorative sleep function and a locus of dysfunction in response to sleep deprivation (SD). Synapse density is highly dynamic during development, becoming stabilized with maturation to adulthood, suggesting sleep exerts distinct synaptic functions between development and adulthood. Importantly, problems with sleep are common in neurodevelopmental disorders including autism spectrum disorder (ASD). Moreover, early life sleep disruption in animal models causes long lasting changes in adult behavior. Different plasticity engaged during sleep necessarily implies that developing and adult synapses will show differential vulnerability to SD. To investigate distinct sleep functions and mechanisms of vulnerability to SD across development, we systematically examined the behavioral and molecular responses to acute SD between juvenile (P21-28), adolescent (P42-49) and adult (P70-100) mice of both sexes. Compared to adults, juveniles lack robust adaptations to SD, precipitating cognitive deficits in the novel object recognition test. Subcellular fractionation, combined with proteome and phosphoproteome analysis revealed the developing synapse is profoundly vulnerable to SD, whereas adults exhibit comparative resilience. SD in juveniles, and not older mice, aberrantly drives induction of synapse potentiation, synaptogenesis, and expression of peri-neuronal nets. Our analysis further reveals the developing synapse as a convergent node between vulnerability to SD and ASD genetic risk. Together, our systematic analysis supports a distinct developmental function of sleep and reveals how sleep disruption impacts key aspects of brain development, providing mechanistic insights for ASD susceptibility.

Design and synthesis of highly selective, orally active Polo-like kinase-2 (Plk-2) inhibitors.

Polo-like kinase-2 (Plk-2) is a potential therapeutic target for Parkinson's disease and this Letter describes the SAR of a series of dihydropteridinone based Plk-2 inhibitors. By optimizing both the N-8 substituent and the biaryl region of the inhibitors we obtained single digit nanomolar compounds such as 37 with excellent selectivity for Plk-2 over Plk-1. When dosed orally in rats, compound 37 demonstrated a 41-45% reduction of pS129-α-synuclein levels in the cerebral cortex.

Treatment of post-tonsillectomy hemorrhage with nebulized tranexamic acid: A retrospective study.

OBJECTIVE: To evaluate the association of treatment with nebulized tranexamic acid (TXA) with rates of operative intervention in post-tonsillectomy hemorrhage (PTH). METHODS: Single tertiary-referral center and satellite hospitals, retrospective cohort of adult and pediatric patients who were diagnosed with PTH in 2015-2022 and treated with nebulized TXA and standard care, compared with an age- and gender-matched control cohort treated with standard care. Patients were typically treated in the emergency department with a single dose of 500mg/5 mL TXA delivered via nebulizer. RESULTS: 1110 total cases of PTH were observed, and 83 were treated with nebulized TXA. Compared to 249 age- and gender-matched PTH controls, TXA-treated patients had a rate of operating room (OR) intervention of 36.1% versus 60.2% (p < 0.0001) and a rate of repeat bleeding of 4.9% versus 14.2% (p < 0.02). The odds ratio for OR intervention with TXA treatment was 0.37 (95% CI 0.22, 0.63). There were no adverse effects identified with an average follow-up time of 586 days. CONCLUSION: Treatment of PTH with nebulized TXA is associated with lower rates of operative intervention and lower rates of repeat bleeding events. Prospective studies are needed to further characterize efficacy and optimal treatment protocols.

Development of an in vitro antigen-detection test as an alternative method to the in vivo plaque reduction neutralization test for the quality control of Japanese encephalitis virus vaccine.

Japanese encephalitis virus (JEV) causes diseases that attack the human central nervous system. Traditionally, the quality control for JEV vaccines, in which the plaque reduction neutralization (PRN) titer is measured by the national control laboratories before the vaccine batches are marketed, has required laboratory animal testing. However, classical animal tests have inherent problems, including the very fact that animals are used, ethical issues, and the possibility of error. In this study, JEV antigen was measured in an in vitro assay to assess the feasibility of replacing in vivo assays that measure the PRN titers of JEV vaccines. We constructed a double-sandwich enzyme-linked immunosorbent assay (DS-ELISA) that could detect JEV envelope (E). Initially, monoclonal antibodies (mAbs) directed against the JEV E protein were generated and characterized. We isolated 18 mAbs against JEV E protein, and most were the IgG1 or IgG2a isotype. The mAbs (5F15 and 7D71) were selected as the most suitable mAb pair to detect JEV E protein. DS-ELISA with this pair detected as little as approximately 3 µg/mL JEV E protein and demonstrated a relationship between the amount of JEV E protein and the PRN titer. From these results, we surmise that this DS-ELISA may be useful, not only in terms of measuring the amount of JEV E protein, but also as a substitute for the PRN test for JEV vaccine evaluation.

Effect of long-term administration of cordycepin from Cordyceps militaris on testicular function in middle-aged rats.

This study was carried out to examine the potential beneficial effect of cordycepin on the decline of testicular function induced with age. A total of 30 male Sprague-Dawley rats (twenty-four 12-month-olds and six 2-month-olds) were divided into five groups. The young control (YC) and middle-aged control (MC) groups received vehicle only. Cordycepin-treated groups were administered daily doses of oral cordycepin at 5, 10, and 20 mg/kg body weight for 4 months. As a result, the MC group exhibited epididymal weight loss, decreased sperm motility, and reduced spermatogenesis compared to the young control group. Interestingly, the epididymal weights of middle-aged rats were dose-dependently increased by treatment with cordycepin. Cordycepin also improved calcium levels and decreased urea and nitrogen, uric acid, and creatinine in the blood of middle-aged rats. In addition, cordycepin significantly increased sperm motility and the progressiveness of sperm movement. All cordycepin-treated groups showed well-arranged spermatogonia, densely packed cellular material, and increased numbers of mature spermatozoa in the seminiferous lumen compared to the middle-aged control group. These results indicate that long-term administration of cordycepin can counteract the decline of testicular function in middle-aged rats.

A peptide vaccine based on a B-cell epitope on the VP1 protein of enterovirus 70 induces a strong antibody response.

Enterovirus 70 (EV70) is the causative agent of acute hemorrhagic conjunctivitis (AHC), for which no effective vaccine is available. This study revealed a high reactivity of the N-terminal region of EV70 VP1 (VP1-1) with an anti-EV70 mouse serum. The analysis of overlapping synthetic peptides of VP1-1 identified a B-cell epitope in this region. The E-peptide (14-ANTVESEIKAELGVI-28) showing the highest reactivity with the anti-EV70 serum induced neutralizing antibodies in mice and reduced the virus titer in the eyes, suggesting that it is a candidate vaccine against AHC caused by EV70.

Syndesmotic Screw Breakage May Be More Problematic Than Previously Reported: Increased Rates of Hardware Removal Secondary to Pain With Intraosseous Screw Breakage.

BACKGROUND: The majority of retained syndesmotic screws will either loosen or break once the patient resumes weight-bearing. While evidence is limited, anecdotal experience suggests that intraosseous screw breakage may be problematic for some patients due to painful bony erosion. This study seeks to identify the incidence of intraosseous screw breakage, variables that may predict intraosseous screw breakage, and whether intraosseous screw breakage is associated with higher rates of implant removal secondary to pain. METHODS: Five hundred thirty-one patients undergoing syndesmotic stabilization were screened, of which 43 patients (with 58 screws) experiencing postoperative screw breakage met inclusion criteria. Patient charts were retrospectively reviewed for demographic data, comorbidities, time to screw breakage, location of screw breakage, and implant removal. Several radiographic parameters were evaluated for their potential to influence the site of screw breakage. RESULTS: Intraosseous screw breakage occurred in 32 patients (74.4%). Screw breakage occurred exclusively in the tibiofibular clear space in the remaining 11 instances (25.6%). Intraosseous screw breakage was significantly associated with eventual implant removal after breakage (P = .034). Screws placed further from the tibiotalar joint were at less risk for intraosseous breakage (odds ratio 0.818, P = .002). Screws placed at a threshold height of 20 mm or greater were more likely to break in the clear space (odds ratio 12.1, P = .002). CONCLUSION: Syndesmotic screw breakage may be more problematic than previously described. Intraosseous breakage was associated with higher rates of implant removal secondary to pain in this study. Placement of screws 20 mm or higher from the tibiotalar joint may decrease risk of intraosseous breakage.Levels of Evidence: Level III: Retrospective study.

Boosting the enantioresolution of zwitterionic-teicoplanin chiral stationary phases by moving to wide-pore core-shell particles.

A novel zwitterionic-teicoplanin chiral stationary phase (CSP), based on superficially porous particles (SPPs) of 2.7 µm particle diameter and 160 Å pore size, has been prepared and evaluated towards the enantioseparation of important classes of compounds, including chiral drugs, pesticides, and N-derivatized amino acids. The comparison with two analogous CSPs prepared on SPPs with 2.7 and 2.0 µm particle diameter and 90 Å pore size has revealed that the use of large-pore particles allows to dramatically improve both the enantioselectivity and the resolution-per-analysis-time, at the point that the column prepared with the new CSP outperformed the one packed with the finest particles. On the novel wide-pore CSP, the separation of fifteen racemates of pratical importance was significantly improved in terms of both enantioselectivity and resolution-per-analysis time-compared to the CSPs based on SPPs with smaller pores (90 Å). Such a CSP would be suitable for very fast enantioseparations allowing the saving of solvent for greener high-efficiency/high-throughput applications.

Coordinated Regulation of CB1 Cannabinoid Receptors and Anandamide Metabolism Stabilizes Network Activity during Homeostatic Downscaling.

Neurons express overlapping homeostatic mechanisms to regulate synaptic function and network properties in response to perturbations of neuronal activity. Endocannabinoids (eCBs) are bioactive lipids synthesized in the postsynaptic compartments to regulate synaptic transmission, plasticity, and neuronal excitability primarily through retrograde activation of presynaptic cannabinoid receptor type 1 (CB1). The eCB system is well situated to regulate neuronal network properties and coordinate presynaptic and postsynaptic activity. However, the role of the eCB system in homeostatic adaptations to neuronal hyperactivity is unknown. To address this issue, we used Western blotting and targeted lipidomics to measure adaptations in eCB system to bicuculline (BCC)-induced chronic hyperexcitation in mature cultured rat cortical neurons, and used multielectrode array (MEA) recording and live-cell imaging of glutamate dynamics to test the effects of pharmacological manipulations of eCB on network activities. We show that BCC-induced chronic hyperexcitation triggers homeostatic downscaling and a coordinated adaptation to enhance tonic eCB signaling. Hyperexcitation triggers first the downregulation of fatty acid amide hydrolase (FAAH), the lipase that degrades the eCB anandamide, then an accumulation of anandamide and related metabolites, and finally a delayed upregulation of surface and total CB1. Additionally, we show that BCC-induced downregulation of surface AMPA-type glutamate receptors (AMPARs) and upregulation of CB1 occur through independent mechanisms. Finally, we show that endocannabinoids support baseline network activities before and after downscaling and is engaged to suppress network activity during adaptation to hyperexcitation. We discuss the implications of our findings in the context of downscaling and homeostatic regulation of in vitro oscillatory network activities.

Pediatric intraosseous cranial myxoma: A case report and review of literature.

While intraosseous cranial myxoma is a rare pathology, it is important for providers to be aware of it, as early diagnosis and treatment is imperative for prognosis. Long-term follow-up is needed as high rates of recurrence have been documented.

Aerosol generation during cadaveric simulation of otologic surgery and live cochlear implantation.

OBJECTIVE: The risk of SARS-CoV-2 transmission to healthcare workers through airborne aerosolization during otologic surgery has not been characterized. The objective of this study was to describe and quantify the aerosol generation during common otologic procedures in both cadaveric surgical simulation and live patient surgery. METHODS: The number concentrations of generated aerosols in the particle size range of 0.30 to 10.0 µm were quantified using an optical particle sizer during both a cadaveric simulation of routine otologic procedures as well as cochlear implant surgery on live patients in the operating room. RESULTS: In the cadaveric simulation, temporalis fascia graft harvest using cold techniques (without electrocautery) (n = 4) did not generate aerosols above baseline concentrations. Tympanoplasty (n = 3) and mastoidectomy (n = 3) both produced statistically significant increases in concentrations of aerosols (P < 0.05), predominantly submicron particles (< 1.0 µm). High-speed, powered drilling of the temporal bone during mastoidectomy with a Multi Flute cutting burr resulted in higher peak concentrations and greater number of spikes in aerosols than with a diamond burr. In the operating room, spikes in aerosols occurred during both cochlear implant surgeries. CONCLUSION: In the cadaveric simulation, temporalis fascia graft harvest without electrocautery did not generate aerosol levels above baseline, while significant aerosol levels were generated during mastoidectomy and to a much less degree during tympanoplasty. Aerosol spikes were appreciated during cochlear implantation surgery in live patients. LEVEL OF EVIDENCE: 2.

Aerosol Generation During Myringotomy With Tympanostomy Tube Insertion: Implications for Otolaryngology in the COVID-19 Era.

The risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission to health care workers during myringotomy and tympanostomy tube (MT) insertion is unknown. To determine the need for enhanced precautions to prevent potential spread via aerosolized particles, we used an optical particle sizer to measure aerosol generation intraoperatively during a case series of MT insertion. We also discuss our institutional experience with safe pandemic-era perioperative practices. There was no measured increase in aerosol particle number during the procedure at a distance of 30 cm from the external auditory canal. These initial data are reassuring regarding the risk of SARS-CoV-2 transmission to the operating room team due to aerosol generation, but further study is necessary before making definitive recommendations.

Droplet Exposure Risk to Providers From In-Office Flexible Laryngoscopy: A COVID-19 Simulation.

To provide data on risk of respiratory droplets from common otolaryngologic procedures during the COVID-19 pandemic, a novel simulation of droplet exposure from flexible laryngoscopy was performed. After completion of a nasal symptom questionnaire, topical fluorescein spray was administered into the nasal and oropharynx of 10 healthy volunteers, who then underwent flexible laryngoscopy under 2 conditions: routine without provoked response and with prompted sneeze/cough. After each, droplets on the proceduralist and participant were counted under ultraviolet A light. Droplets were observed on 1 of 10 volunteers after routine laryngoscopy and 4 of 10 during laryngoscopy with sneeze/cough. A nasal symptom score based on congestion and rhinorrhea was significantly elevated among droplet producers after sneeze/cough (P = .0164). No droplets were observed on the provider. Overall, with adequate personal protective equipment, flexible laryngoscopy poses minimal droplet risk to providers. Nasal symptoms can identify patients more likely to produce droplets after sneeze/cough.