The Prostaglandin D2 Receptor CRTH2 Promotes IL-33-Induced ILC2 Accumulation in the Lung.

Group 2 innate lymphoid cells (ILC2s) are rare innate immune cells that accumulate in tissues during allergy and helminth infection, performing critical effector functions that drive type 2 inflammation. ILC2s express ST2, the receptor for the cytokine IL-33, and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for the bioactive lipid prostaglandin D2 (PGD2). The IL-33-ST2 and the PGD2-CRTH2 pathways have both been implicated in promoting ILC2 accumulation during type 2 inflammation. However, whether these two pathways coordinate to regulate ILC2 population size in the tissue in vivo remains undefined. In this study, we show that ILC2 accumulation in the murine lung in response to systemic IL-33 treatment was partially dependent on CRTH2. This effect was not a result of reduced ILC2 proliferation, increased apoptosis or cell death, or differences in expression of the ST2 receptor in the absence of CRTH2. Rather, data from adoptive transfer studies suggested that defective accumulation of CRTH2-deficient ILC2s in response to IL-33 was due to altered ILC2 migration patterns. Whereas donor wild-type ILC2s preferentially accumulated in the lungs compared with CRTH2-deficient ILC2s following transfer into IL-33-treated recipients, wild-type and CRTH2-deficient ILC2s accumulated equally in the recipient mediastinal lymph node. These data suggest that CRTH2-dependent effects lie downstream of IL-33, directly affecting the migration of ILC2s into inflamed lung tissues. A better understanding of the complex interactions between the IL-33 and PGD2-CRTH2 pathways that regulate ILC2 population size will be useful in understanding how these pathways could be targeted to treat diseases associated with type 2 inflammation.

Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy.

Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance.

Association of erosion with timing of detection and selected risk factors in primary dentition: a longitudinal study.

OBJECTIVE: The aims of this study were to determine the prevalence of erosion in a birth cohort at 24, 36, and 48 months and to investigate risk factors for erosion. METHODS: One hundred and fifty-four children from a birth cohort were followed at 24, 36, and 48 months of age. RESULTS: Of the 154 children examined, 0% (0/154), 7% (11/154), and 28% (40/154) had erosion detected for the first time at 24, 36, and 48 months, respectively (P < 0.001). A cumulative total of 51 (33%) children and 256 (8%) teeth had erosion by the age of 48 months. There were no significant associations between erosive lesions first detected at 36 months and oral hygiene behaviour, medical conditions, or dietary habits reported at the 24- or 36-month examinations (all P > 0.05). In contrast, erosive lesion first detected at 48 months was positively associated with the use of a feeding bottle reported at the 36-month examination (P = 0.026). CONCLUSIONS: The prevalence of dental erosion in young children increased with age, with clinically detectable lesions forming between 24 and 36 months of age. Erosive lesions first detected at 48 months were positively associated with the use of a feeding bottle reported at 36 months.

Memantine for the prevention of primary headache disorders.

OBJECTIVE: To describe the current data evaluating the efficacy and safety of memantine for the prevention of primary headache disorders. DATA SOURCES: A literature search using MEDLINE (1966-July 2014) and EMBASE (1973-July 2014) was conducted using the search terms memantine, headache, migraine, glutamate, and NMDA. References of identified articles were reviewed for additional, relevant citations. STUDY SELECTION AND DATA EXTRACTION: All English-language articles dealing with the use of memantine for prevention of primary headache disorders were included. DATA SYNTHESIS: Data from several retrospective reports and 2 prospective clinical trials suggest that memantine may be a useful treatment option for the prevention of primary headache disorders. The majority of available literature focuses specifically on chronic migraine prevention in refractory patients who had failed multiple previous prophylactic therapies. In these patients, 10 to 20 mg of memantine daily reduced the frequency and intensity of migraine headaches and was generally well tolerated, with few adverse events. Data regarding the efficacy of memantine for other primary headache disorders such as chronic tension type and cluster headaches are limited. CONCLUSION: Although further studies evaluating the efficacy of memantine for prevention of primary headache disorders are warranted, memantine may be a reasonable option, used either as monotherapy or adjunctive therapy, in the refractory chronic migraine prophylaxis setting.

Meiotic Cytokinesis in Saccharomyces cerevisiae: Spores That Just Need Closure.

In the budding yeast Saccharomyces cerevisiae, sporulation occurs during starvation of a diploid cell and results in the formation of four haploid spores forming within the mother cell ascus. Meiosis divides the genetic material that is encapsulated by the prospore membrane that grows to surround the haploid nuclei; this membrane will eventually become the plasma membrane of the haploid spore. Cellularization of the spores occurs when the prospore membrane closes to capture the haploid nucleus along with some cytoplasmic material from the mother cell, and thus, closure of the prospore membrane is the meiotic cytokinetic event. This cytokinetic event involves the removal of the leading-edge protein complex, a complex of proteins that localizes to the leading edge of the growing prospore membrane. The development and closure of the prospore membrane must be coordinated with other meiotic exit events such as spindle disassembly. Timing of the closure of the prospore membrane depends on the meiotic exit pathway, which utilizes Cdc15, a Hippo-like kinase, and Sps1, an STE20 family GCKIII kinase, acting in parallel to the E3 ligase Ama1-APC/C. This review describes the sporulation process and focuses on the development of the prospore membrane and the regulation of prospore membrane closure.

A Cross-Sectional Survey Study of Cannabis Use for Fibromyalgia Symptom Management.

OBJECTIVE: To assess the use of cannabis as a symptom management strategy for patients with fibromyalgia. PATIENTS AND METHODS: An electronic, cross-sectional survey was conducted among patients diagnosed with fibromyalgia and treated in Integrative Medicine & Health at Mayo Clinic, Rochester, Minnesota. The survey was constructed with the Symptom Management Theory tool and was sent anonymously via web-based software to patients with a diagnosis of fibromyalgia. RESULTS: Of 5234 patients with fibromyalgia sent the online survey, 1336 (25.5%) responded and met the inclusion criteria. Survey respondents had a median age of 48 (Q1-Q3: 37.5-58.0) years, and most identified as female. Nearly half of respondents (49.5%, n=661) reported cannabis use since their fibromyalgia diagnosis. The most common symptoms for which respondents reported using cannabis were pain (98.9%, n=654); fatigue (96.2%; n=636); stress, anxiety, or depression (93.9%; n=621); and insomnia (93.6%; n=619). Improvement in pain symptoms with cannabis use was reported by 82.0% (n=536). Most cannabis-using respondents reported that cannabis also improved symptoms of stress, anxiety, and depression and of insomnia. CONCLUSION: Considering that cannabis is a popular choice among patients for managing fibromyalgia symptoms, clinicians should have adequate knowledge of cannabis when discussing therapeutic options for fibromyalgia with their patients.

Overcoming patient reluctance to statin intolerance.

Statin therapy is a cornerstone in the management of dyslipidemia, both in primary and secondary prevention of cardiovascular events. Despite strong guidelines supporting statin use, concerns regarding side effects, particularly musculoskeletal symptoms, contribute to statin intolerance and patient reluctance. While statin intolerance is reported in 5% to 30% of patients, its true prevalence may be overestimated due to the influence of the nocebo effect. Factors associated with higher incidence of statin intolerance include older age, female sex, comorbidities such as diabetes and chronic kidney disease, and concurrent use of medications such as antiarrhythmic agents or calcium channel blockers. Clinical characterization of statin intolerance requires thorough evaluation and exclusion of alternative causes of musculoskeletal symptoms. Strategies to address statin intolerance include reassessing cardiovascular risk, engaging in shared decision-making, statin rechallenge after appropriate washout periods, dosage titration for tolerability, and consideration of alternative therapies when low-density lipoprotein goals cannot be achieved with statins. This review provides an overview of the spectrum of statin intolerance, its clinical assessment, and a systematic approach to caring for a patient with statin intolerance.

SPO71 mediates prospore membrane size and maturation in Saccharomyces cerevisiae.

The mechanisms that control the size and shape of membranes are not well understood, despite the importance of these structures in determining organelle and cell morphology. The prospore membrane, a double lipid bilayer that is synthesized de novo during sporulation in S. cerevisiae, grows to surround the four meiotic products. This membrane determines the shape of the newly formed spores and serves as the template for spore wall deposition. Ultimately, the inner leaflet of the prospore membrane will become the new plasma membrane of the cell upon germination. Here we show that Spo71, a pleckstrin homology domain protein whose expression is induced during sporulation, is critical for the appropriate growth of the prospore membrane. Without SPO71, prospore membranes surround the nuclei but are abnormally small, and spore wall deposition is disrupted. Sporulating spo71Δ cells have prospore membranes that properly localize components to their growing leading edges yet cannot properly localize septin structures. We also found that SPO71 genetically interacts with SPO1, a gene with homology to the phospholipase B gene that has been previously implicated in determining the shape of the prospore membrane. Together, these results show that SPO71 plays a critical role in prospore membrane development.

Meiosis II spindle disassembly requires two distinct pathways.

During exit from meiosis II, cells undergo several structural rearrangements, including disassembly of the meiosis II spindles and cytokinesis. Each of these changes is regulated to ensure that they occur at the proper time. Previous studies have demonstrated that both SPS1, which encodes a STE20-family GCKIII kinase, and AMA1, which encodes a meiosis-specific activator of the Anaphase Promoting Complex, are required for both meiosis II spindle disassembly and cytokinesis in the budding yeast Saccharomyces cerevisiae. We examine the relationship between meiosis II spindle disassembly and cytokinesis and find that the meiosis II spindle disassembly failure in sps1Δ and ama1∆ cells is not the cause of the cytokinesis defect. We also see that the spindle disassembly defects in sps1Δ and ama1∆ cells are phenotypically distinct. We examined known microtubule-associated proteins Ase1, Cin8, and Bim1, and found that AMA1 is required for the proper loss of Ase1 and Cin8 on meiosis II spindles while SPS1 is required for Bim1 loss in meiosis II. Taken together, these data indicate that SPS1 and AMA1 promote distinct aspects of meiosis II spindle disassembly, and that both pathways are required for the successful completion of meiosis.

Gabapentinoid Prescribing Practices at a Large Academic Medical Center.

Objective: To evaluate indications for gabapentinoid prescription at an academic medical center. Patients and Methods: We retrospectively reviewed patients aged 18 years or older who were prescribed gabapentinoids (gabapentin or pregabalin) during the 2019 calendar year at an academic medical center in the US Midwest. Patient demographic characteristics, indications for gabapentinoid prescription, and prescribing clinician specialities were abstracted from a random sample, and the findings were extrapolated to the overall cohort. Results: A total of 6205 prescriptions for gabapentinoids were initially identified. In the random sample of prescriptions (n=721), 89.5% were for gabapentin and 10.5% were for pregabalin. More women than men were prescribed gabapentinoids, and the mean ± SD patient age was 58.6±16.9 years. The top 5 indications for gabapentinoid prescriptions were neuropathic pain, musculoskeletal pain, restless legs syndrome, anxiety, and headache. A majority (66.7%) of prescriptions had substantial-to-modest evidence, but 29.0% of prescriptions had conflicting or insufficient evidence. Conclusion: To our knowledge, this study is one of the first to manually review clinical notes from multiple clinical specialities to ascertain indications for gabapentinoid prescriptions. Although most prescriptions had modest evidence to support their use, a high percentage of gabapentinoid prescriptions were issued for indications not supported by robust evidence. This suggests that prescribers are gravitating toward gabapentinoid use for reasons that are currently not fully understood. Clinician intent for off-label gabapentinoid prescriptions at the point of care should be further studied to understand the factors that lead to these clinical decisions.

Association between frailty status and complications in patients undergoing surgical excision of malignant esophageal neoplasms.

Background: Research within the last decade highlights the patients' frailty status as an important predictor of esophageal cancer outcomes, but the literature evaluating frailty's role in these patients remains limited. We evaluated the role of frailty in patients undergoing resection of malignant esophageal neoplasms. Methods: We used the Nationwide Readmissions Database from 2016 and 2017 to identify patients who underwent excision of a malignant esophageal neoplasm. Patient frailty was queried using the Johns Hopkins Adjusted Clinical Groups frailty-defining diagnosis indicator. Propensity score matching identified 289 frail patients and 281 non-frail patients. Mann-Whitney U testing was performed and receiver operating characteristic (ROC) curves were created, following the creation of logistic regression models for predicting discharge disposition. The area under the curve (AUC) served as a proxy for model performance. Results: Frail patients had significantly more nonroutine discharges, longer inpatient lengths of stay, higher costs, more acute infections, posthemorrhagic anemia and deep vein thrombosis, and greater mortality (P<0.05). No significant differences were found between the 2 cohorts with respect to readmission rates, pulmonary embolism or dysphagia. Predictive models for patient discharge disposition demonstrated that frailty status in combination with age resulted in better ROC curves (AUC: 0.652) compared to models using age alone (AUC: 0.601). Conclusions: Frailty was found to be significantly correlated with higher rates of inpatient medical complications following esophagectomy. The inclusion of patient frailty status in predictive models for discharge disposition resulted in a better predictive capacity compared to those using age alone.

The Smk1 MAPK and Its Activator, Ssp2, Are Required for Late Prospore Membrane Development in Sporulating Saccharomyces cerevisiae.

During sporulation in the budding yeast Saccharomyces cerevisiae, proper development of the prospore membrane is necessary for the formation of viable spores. The prospore membrane will eventually become the plasma membrane of the newly formed haploid spore and also serves as the template for the deposition of the spore wall. The prospore membrane is generated de novo during meiosis II and the growing edge of the prospore membrane is associated with the Leading Edge Protein (LEP) complex. We find that the Smk1 MAP kinase, along with its activator Ssp2, transiently localizes with the LEP during late meiosis II. SSP2 is required for the leading edge localization of Smk1; this localization is independent of the activation state of Smk1. Like other LEP components, the localization of Smk1 at the leading edge also depends on Ady3. Although prospore membrane development begins normally in smk1 and ssp2 mutants, late prospore membrane formation is disrupted, with the formation of ectopic membrane compartments. Thus, MAP kinase signaling plays an important role in the formation of the prospore membrane.

Gaboxadol in Fragile X Syndrome: A 12-Week Randomized, Double-Blind, Parallel-Group, Phase 2a Study.

Background: Fragile X syndrome (FXS), the most common single-gene cause of intellectual disability and autism spectrum disorder (ASD), is caused by a >200-trinucleotide repeat expansion in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. Individuals with FXS can present with a range of neurobehavioral impairments including, but not limited to: cognitive, language, and adaptive deficits; ASD; anxiety; social withdrawal and avoidance; and aggression. Decreased expression of the γ-aminobutyric acid type A (GABAA) receptor δ subunit and deficient GABAergic tonic inhibition could be associated with symptoms of FXS. Gaboxadol (OV101) is a δ-subunit-selective, extrasynaptic GABAA receptor agonist that enhances GABAergic tonic inhibition, providing the rationale for assessment of OV101 as a potential targeted treatment of FXS. No drug is approved in the United States for the treatment of FXS. Methods: This 12-weeks, randomized (1:1:1), double-blind, parallel-group, phase 2a study was designed to assess the safety, tolerability, efficacy, and optimal daily dose of OV101 5 mg [once (QD), twice (BID), or three-times daily (TID)] when administered for 12 weeks to adolescent and adult men with FXS. Safety was the primary study objective, with key assessments including treatment-emergent adverse events (TEAEs), treatment-related adverse events leading to study discontinuation, and serious adverse events (SAEs). The secondary study objective was to evaluate the effect of OV101 on a variety of problem behaviors. Results: A total of 23 participants with FXS (13 adolescents, 10 adults) with moderate-to-severe neurobehavioral phenotypes (Full Scale Intelligence Quotient, 41.5 ± 3.29; ASD, 82.6%) were randomized to OV101 5 mg QD (n = 8), 5 mg BID (n = 8), or 5 mg TID (n = 7) for 12 weeks. OV101 was well tolerated across all 3 treatment regimens. The most common TEAEs were upper respiratory tract infection (n = 4), headache (n = 3), diarrhea (n = 2), and irritability (n = 2). No SAEs were reported. Improvements from baseline to end-of-treatment were observed on several efficacy endpoints, and 60% of participants were identified as treatment responders based on Clinical Global Impressions-Improvement. Conclusions: Overall, OV101 was safe and well tolerated. Efficacy results demonstrate an initial signal for OV101 in individuals with FXS. These results need to be confirmed in a larger, randomized, placebo-controlled study with optimal outcomes and in the most appropriate age group. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT03697161.

A Noncanonical Hippo Pathway Regulates Spindle Disassembly and Cytokinesis During Meiosis in Saccharomyces cerevisiae.

Meiosis in the budding yeast Saccharomyces cerevisiae is used to create haploid yeast spores from a diploid mother cell. During meiosis II, cytokinesis occurs by closure of the prospore membrane, a membrane that initiates at the spindle pole body and grows to surround each of the haploid meiotic products. Timely prospore membrane closure requires SPS1, which encodes an STE20 family GCKIII kinase. To identify genes that may activate SPS1, we utilized a histone phosphorylation defect of sps1 mutants to screen for genes with a similar phenotype and found that cdc15 shared this phenotype. CDC15 encodes a Hippo-like kinase that is part of the mitotic exit network. We find that Sps1 complexes with Cdc15, that Sps1 phosphorylation requires Cdc15, and that CDC15 is also required for timely prospore membrane closure. We also find that SPS1, like CDC15, is required for meiosis II spindle disassembly and sustained anaphase II release of Cdc14 in meiosis. However, the NDR-kinase complex encoded by DBF2/DBF20MOB1 which functions downstream of CDC15 in mitotic cells, does not appear to play a role in spindle disassembly, timely prospore membrane closure, or sustained anaphase II Cdc14 release. Taken together, our results suggest that the mitotic exit network is rewired for exit from meiosis II, such that SPS1 replaces the NDR-kinase complex downstream of CDC15.

Student Perceptions of Classic and Game-Based Online Student Response Systems.

BACKGROUND: Online student response systems (OSRSs), driven by the Internet and cell phone technology, provide a free, easily accessible method to increase student engagement, facilitate active learning, and provide learners and teachers with instant feedback about learning progress. PURPOSE: This article describes undergraduate nursing students' use of 2 OSRSs and their perceptions of the impact of the tools on class participation and engagement. METHODS: Students used their own mobile phones or computers to access 2 types of OSRSs: a classic and a game-based OSRS. RESULTS: Students indicated that both systems increased participation and engagement. The game-based OSRS was favored over the classic OSRS. The potential for use of the game-based OSRS for assessing rapid-answer fact-based knowledge and the classic OSRS for assessing more complex learning tasks is discussed. CONCLUSION: Nurse educators are encouraged to consider integrating online response system technology into their classroom teaching.

A Bayesian machine learning approach for drug target identification using diverse data types.

Drug target identification is a crucial step in development, yet is also among the most complex. To address this, we develop BANDIT, a Bayesian machine-learning approach that integrates multiple data types to predict drug binding targets. Integrating public data, BANDIT benchmarked a ~90% accuracy on 2000+ small molecules. Applied to 14,000+ compounds without known targets, BANDIT generated ~4,000 previously unknown molecule-target predictions. From this set we validate 14 novel microtubule inhibitors, including 3 with activity on resistant cancer cells. We applied BANDIT to ONC201-an anti-cancer compound in clinical development whose target had remained elusive. We identified and validated DRD2 as ONC201's target, and this information is now being used for precise clinical trial design. Finally, BANDIT identifies connections between different drug classes, elucidating previously unexplained clinical observations and suggesting new drug repositioning opportunities. Overall, BANDIT represents an efficient and accurate platform to accelerate drug discovery and direct clinical application.

Forskolin induction of late-LTP and up-regulation of 5' TOP mRNAs translation via mTOR, ERK, and PI3K in hippocampal pyramidal cells.

The late phase of long-term potentiation (LTP) requires activation of the mammalian target of rapamycin (mTOR) pathway and synthesis of new proteins. mTOR regulates protein synthesis via phosphorylation of 4E-binding proteins (4E-BPs) and S6K, and via selective up-regulation of 5' terminal oligopyrimidine (5' TOP) mRNAs that encode components of the translational machinery. In this study, we explored the regulation of 5' TOP mRNAs during late-LTP (L-LTP). Synaptic plasticity was studied at Schaffer collateral--CA1 pyramidal cell synapses in rat organotypic hippocampal slices. Forskolin, an adenylate cyclase activator, induced L-LTP in organotypic slices that was mTOR-dependent. To determine if 5' TOP mRNAs are specifically up-regulated during L-LTP, we generated a 5' TOP-myr-dYFP reporter to selectively monitor 5' TOP translation. Confocal imaging experiments in cultured slices revealed an increase in somatic and dendritic fluorescence after forskolin treatment. This up-regulation was dependent on an intact TOP sequence and was mTOR, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-dependent. Our findings indicate that forskolin induces L-LTP in hippocampal neurons and up-regulates 5' TOP mRNAs translation via mTOR, suggesting that up-regulation of the translational machinery is a candidate mechanism for the stabilization of LTP.

Ultrawide angle fluorescein angiographic imaging: a comparison to conventional digital acquisition systems.

BACKGROUND AND OBJECTIVE: A prototype ultrawide field angiographic system and conventional technology were compared. PATIENTS AND METHODS: Thirty subjects with diabetic retinopathy were imaged in three centers by conventional (Topcon Inc., Paramus, NJ, or Carl Zeiss Meditec, Dublin, CA) and ultrawide field (Optos P200A; Optos Inc., Dunfermline, Scotland) digital fluorescein angiography units. A calibrated grid was digitally placed on the images to facilitate measurements. Main outcome measures were field of view and the number of grid sectors displaying retinal ischemia and neovascularization. Secondary measures were image quality and patient satisfaction scores. RESULTS: The P200A provided a significantly greater visual field extent of 8.7 +/- 1.6 disc diameters (DD) compared to the conventional systems (3.4 +/- 0.76 DD) (P < .001). Retinal ischemia was better revealed (16.9 +/- 15 vs 3.4 +/- 4.26 sectors) with the prototype, but image quality was superior with the conventional system. Patient satisfaction scores did not differ between systems. CONCLUSION: A prototype ultrawide angle imaging system displayed significantly more diabetic retinal pathology in a single image than conventional systems.

The conserved ATPase Get3/Arr4 modulates the activity of membrane-associated proteins in Saccharomyces cerevisiae.

The regulation of cellular membrane dynamics is crucial for maintaining proper cell growth and division. The Cdc48-Npl4-Ufd1 complex is required for several regulated membrane-associated processes as part of the ubiquitin-proteasome system, including ER-associated degradation and the control of lipid composition in yeast. In this study we report the results of a genetic screen in Saccharomyces cerevisiae for extragenic suppressors of a temperature-sensitive npl4 allele and the subsequent analysis of one suppressor, GET3/ARR4. The GET3 gene encodes an ATPase with homology to the regulatory component of the bacterial arsenic pump. Mutants of GET3 rescue several phenotypes of the npl4 mutant and transcription of GET3 is coregulated with the proteasome, illustrating a functional relationship between GET3 and NPL4 in the ubiquitin-proteasome system. We have further found that Get3 biochemically interacts with the trans-membrane domain proteins Get1/Mdm39 and Get2/Rmd7 and that Deltaget3 is able to suppress phenotypes of get1 and get2 mutants, including sporulation defects. In combination, our characterization of GET3 genetic and biochemical interactions with NPL4, GET1, and GET2 implicates Get3 in multiple membrane-dependent pathways.