Bile acid-induced IRF3 phosphorylation mediates cell death, inflammatory responses, and fibrosis in cholestasis-induced liver and kidney injury via regulation of ZBP1.

BACKGROUND AND AIMS: Cell death and inflammation play critical roles in chronic tissue damage caused by cholestatic liver injury leading to fibrosis and cirrhosis. Liver cirrhosis is often associated with kidney damage, which is a severe complication with poor prognosis. Interferon regulatory factor 3 (IRF3) is known to regulate apoptosis and inflammation, but its role in cholestasis remains obscure. In this study. APPROACH AND RESULTS: We discovered increased IRF3 phosphorylation in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. In the bile duct ligation model of obstructive cholestasis in mice, we found that tissue damage was associated with increased phosphorylated IRF3 (p-IRF3) in the liver and kidney. IRF3 knockout ( Irf3-/- ) mice showed significantly attenuated liver and kidney damage and fibrosis compared to wide-type mice after bile duct ligation. Cell-death pathways, including apoptosis, necroptosis, and pyroptosis, inflammasome activation, and inflammatory responses were significantly attenuated in Irf3-/- mice. Mechanistically, we show that bile acids induced p-IRF3 in vitro in hepatocytes. In vivo , activated IRF3 positively correlated with increased expression of its target gene, Z-DNA-Binding Protein-1 (ZBP1), in the liver and kidney. Importantly, we also found increased ZBP1 in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. We discovered that ZBP1 interacted with receptor interacting protein 1 (RIP1), RIP3, and NLRP3, thereby revealing its potential role in the regulation of cell-death and inflammation pathways. In conclusion. CONCLUSIONS: Our data indicate that bile acid-induced p-IRF3 and the IRF3-ZBP1 axis play a central role in the pathogenesis of cholestatic liver and kidney injury.

Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis.

OBJECTIVE: Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges exacerbate liver injury in a high fat-cholesterol-sugar diet (MASH diet)-induced model of MASH. DESIGN: C57BL/6 mice received either chow or the MASH diet for 3 months with or without weekly alcohol binges. Neutrophil infiltration, neutrophil extracellular traps (NETs) and fibrosis were evaluated. RESULTS: We found that alcohol binges in MASH increase liver injury and fibrosis. Liver transcriptomic profiling revealed differential expression of genes involved in extracellular matrix reorganisation, neutrophil activation and inflammation compared with alcohol or the MASH diet alone. Alcohol binges specifically increased NET formation in MASH livers in mice, and NETs were also increased in human livers with MASH plus alcohol use. We discovered that cell-free NETs are sensed via Nod-like receptor protein 3 (NLRP3). Furthermore, we show that cell-free NETs in vitro induce a profibrotic phenotype in hepatic stellate cells (HSCs) and proinflammatory monocytes. In vivo, neutrophil depletion using anti-Ly6G antibody or NET disruption with deoxyribonuclease treatment abrogated monocyte and HSC activation and ameliorated liver damage and fibrosis. In vivo, inhibition of NLRP3 using MCC950 or NLRP3 deficiency attenuated NET formation, liver injury and fibrosis in MASH plus alcohol diet-fed mice (graphical abstract). CONCLUSION: Alcohol binges promote liver fibrosis via NET-induced activation of HSCs and monocytes in MASH. Our study highlights the potential of inhibition of NETs and/or NLRP3, as novel therapeutic strategies to combat the profibrotic effects of alcohol in MASH.

Molecular Dynamics in Rydberg Tweezer Arrays: Spin-Phonon Entanglement and Jahn-Teller Effect.

Atoms confined in optical tweezer arrays constitute a platform for the implementation of quantum computers and simulators. State-dependent operations are realized by exploiting electrostatic dipolar interactions that emerge, when two atoms are simultaneously excited to high-lying electronic states, so-called Rydberg states. These interactions also lead to state-dependent mechanical forces, which couple the electronic dynamics of the atoms to their vibrational motion. We explore these vibronic couplings within an artificial molecular system in which Rydberg states are excited under so-called facilitation conditions. This system, which is not necessarily self-bound, undergoes a structural transition between an equilateral triangle and an equal-weighted superposition of distorted triangular states (Jahn-Teller regime) exhibiting spin-phonon entanglement on a micrometer distance. This highlights the potential of Rydberg tweezer arrays for the study of molecular phenomena at exaggerated length scales.

Changes of microbiome in response to supplements with silver nanoparticles in cotton rhizosphere.

The current study focuses on analyzing the effects of supplements containing silver nanoparticles (AgNPs) on plant growth and rhizospheric bacterial communities. Specifically, the impact of AgNP supplements was assessed on both plant growth promoting traits and bacterial communities in the soil. To do this, a screening process was conducted to select bacteria capable of synthesizing AgNPs through extracellular biosynthesis. UV-Visible spectrophotometer, Fourier transform infrared, X-ray diffraction, scanning electron microscope, and field emission scanning electron microscopy all confirmed, produced AgNPs is in agglomerates form. The resulting AgNPs were introduced into soil along with various supplements and their effects were evaluated after 10 days using next generation sequencing (Illumina-16S rDNA V3-V4 region dependent) to analyze changes in bacterial communities. Seed germination, root-shoot biomass and chlorophyll content were used to assess the growth of the cotton plant, whereas the bacterial ability to promote growth was evaluated by measuring its culturable diversity including traits like phosphate solubilization and indole acetic acid production. The variance in Bray-Curtis ß diversity among six selected combinations including control depends largely on the type of added supplements contributing to 95%-97% of it. Moreover, seed germination improves greatly between 63% and 100% at a concentration range of 1.4 to 2.8 mg/L with different types of supplements. Based on the results obtained through this study, it is evident that using AgNPs along with fructose could be an effective tool for promoting Gossypium hirsutum growth and enhancing plant growth traits like profiling rhizospheric bacteria. The results that have been obtained endorse the idea of boosting the growth of rhizospheric bacteria in a natural way when AgNPs are present. Using these supplements in fields that have been contaminated will lead to a better understanding of how ecological succession occurs among rhizospheric bacteria, and what effect it has on the growth of plants.

ß 2-Adrenoceptor Agonists Promote Extracellular Signal-Regulated Kinase 1/2 Dephosphorylation in Human Airway Epithelial Cells by Canonical, cAMP-Driven Signaling Independently of ß-Arrestin 2.

Chronic use of ß 2-adrenoceptor agonists as a monotherapy in asthma is associated with a loss of disease control and an increased risk of mortality. Herein, we tested the hypothesis that ß 2-adrenoceptor agonists, including formoterol, promote biased, ß-arrestin (Arr) 2-dependent activation of the mitogen-activated protein kinases, ERK1/2, in human airway epithelial cells and, thereby, effect changes in gene expression that could contribute to their adverse clinical outcomes. Three airway epithelial cell models were used: the BEAS-2B cell line, human primary bronchial epithelial cells (HBEC) grown in submersion culture, and HBEC that were highly differentiated at an air-liquid interface. Unexpectedly, treatment of all epithelial cell models with formoterol decreased basal ERK1/2 phosphorylation. This was mediated by cAMP-dependent protein kinase and involved the inactivation of C-rapidly-activated fibrosarcoma, which attenuated downstream ERK1/2 activity, and the induction of dual-specificity phosphatase 1. Formoterol also inhibited the basal expression of early growth response-1, an ERK1/2-regulated gene that controls cell growth and repair in the airways. Neither carvedilol, a ß 2-adrenoceptor agonist biased toward ßArr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells, although ß 2-adrenoceptor desensitization was compromised in ARRB2-deficient cells. Collectively, these results contest the hypothesis that formoterol activates ERK1/2 in airway epithelia by nucleating a ßArr2 signaling complex; instead, they indicate that ß 2-adrenoceptor agonists inhibit constitutive ERK1/2 activity in a cAMP-dependent manner. These findings are the antithesis of results obtained using acutely challenged native and engineered HEK293 cells, which have been used extensively to study mechanisms of ERK1/2 activation, and highlight the cell type dependence of ß 2-adrenoceptor-mediated signaling. SIGNIFICANCE STATEMENT: It has been proposed that the adverse effects of ß 2-adrenoceptor agonist monotherapy in asthma are mediated by genomic mechanisms that occur principally in airway epithelial cells and are the result of ß-arrestin 2-dependent activation of ERK1/2. This study shows that ß 2-adrenoceptor agonists, paradoxically, reduced ERK1/2 phosphorylation in airway epithelia by disrupting upstream rat sarcoma-C-rapidly accelerated fibrosarcoma complex formation and inducing dual-specificity phosphatase 1. Moreover, these effects were cAMP-dependent protein kinase-dependent, suggesting that ß 2-adrenoceptor agonists were not biased toward ß-arrestin 2 and acted via canonical, cAMP-dependent signaling.

Prostanoid Receptors of the EP4-Subtype Mediate Gene Expression Changes in Human Airway Epithelial Cells with Potential Anti-Inflammatory Activity.

There is a clear, unmet clinical need to identify new drugs to treat individuals with asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) in whom current medications are either inactive or suboptimal. In preclinical models, EP4-receptor agonists display efficacy, but their mechanism of action is unclear. In this study, using human bronchial epithelial cells as a therapeutically relevant drug target, we hypothesized that changes in gene expression may play an important role. Several prostanoid receptor mRNAs were detected in BEAS-2B cells, human primary bronchial epithelial cells (HBECs) grown in submersion culture and HBECs grown at an air-liquid interface with PTGER4 predominating. By using the activation of a cAMP response element reporter in BEAS-2B cells as a surrogate of gene expression, Schild analysis determined that PTGER4 mRNAs encoded functional EP4-receptors. Moreover, inhibitors of phosphodiesterase 4 (roflumilast N-oxide [RNO]) and cAMP-dependent protein kinase augmented and attenuated, respectively, reporter activation induced by 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxo-cyclopentyl]sulphanylpropylsulphanyl] acetic acid (ONO-AE1-329), a selective EP4-receptor agonist. ONO-AE1-329 also enhanced dexamethasone-induced activation of a glucocorticoid response element reporter in BEAS-2B cells, which was similarly potentiated by RNO. In each airway epithelial cell variant, numerous genes that may impart therapeutic benefit in asthma, COPD, and/or IPF were differentially expressed by ONO-AE1-329, and those changes were often augmented by RNO and/or dexamethasone. We submit that an EP4-receptor agonist, either alone or as a combination therapy, may be beneficial in individuals with chronic lung diseases in whom current treatment options are inadequate. SIGNIFICANCE STATEMENT: Using human bronchial epithelial cells as a therapeutically relevant drug target, we report that EP4-receptor activation promoted gene expression changes that could provide therapeutic benefit in individuals with asthma, COPD, and IPF in whom current treatment options are ineffective or suboptimal.

Impact of Phosphodiesterase 4 Inhibition on the Operational Efficacy, Response Maxima, and Kinetics of Indacaterol-Induced Gene Expression Changes in BEAS-2B Airway Epithelial Cells: A Global Transcriptomic Analysis.

The effects of phosphodiesterase (PDE) 4 inhibitors on gene expression changes in BEAS-2B human airway epithelial cells are reported and discussed in relation to the mechanism(s) of action of roflumilast in chronic obstructive pulmonary disease (COPD). Microarray-based gene expression profiling failed to identify mRNA transcripts that were differentially regulated by the PDE4 inhibitor 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) after 1, 2, 6, or 18 hours of exposure. However, real-time polymerase chain reaction analysis revealed that GSK 256066 was a weak stimulus, and the negative microarray results reflected low statistical power due to small sample sizes. Furthermore, GSK 256066, roflumilast, and its biologically active metabolite roflumilast N-oxide generally potentiated gene expression changes produced by the long-acting ß 2-adrenoceptor agonists (LABAs) salmeterol, indacaterol, and formoterol. Many of these genes encode proteins with antiviral, anti-inflammatory, and antibacterial activities that could contribute to the clinical efficacy of roflumilast in COPD. RNA-sequencing experiments established that the sensitivity of genes to salmeterol varied by ∼7.5-fold. Consequently, the degree to which a PDE4 inhibitor potentiated the effect of a given concentration of LABA was gene-dependent. Operational model fitting of concentration-response curve data from cells subjected to fractional, ß 2-adrenoceptor inactivation determined that PDE4 inhibition increased the potency and doubled the efficacy of LABAs. Thus, adding roflumilast to standard triple therapy, as COPD guidelines recommend, may have clinical relevance, especially in target tissues where LABAs behave as partial agonists. Collectively, these results suggest that the genomic impact of roflumilast, including its ability to augment LABA-induced gene expression changes, may contribute to its therapeutic activity in COPD.

5-HT2A deletion protects against Clozapine-induced hyperglycemia.

Clozapine is an antipsychotic known for its superior efficacy in treating drug-resistant Schizophrenia. However, Clozapine induces various side effects such as hyperglycemia, agranulocytosis, weight gain etc. The mechanisms of these Clozapine-induced side effects have remained largely elusive though an important role is ascribed to 5-HT2A (Serotonin receptor subtype-2A). In this pilot study, we report for the first time that the 5-HT2A 'global' knockout mice (Htr2a-/-) are resistant to the Clozapine-induced hyperglycemia. Importantly though, the Htr2a-/- mice exhibit near normal basal glucose metabolism in the glucose tolerance tests. Collectively, the Htr2a-/- mice provide an important tool to study the Clozapine-induced hyperglycemia.

Analysis of the Indacaterol-Regulated Transcriptome in Human Airway Epithelial Cells Implicates Gene Expression Changes in the Adverse and Therapeutic Effects of ß2-Adrenoceptor Agonists.

The contribution of gene expression changes to the adverse and therapeutic effects of ß2-adrenoceptor agonists in asthma was investigated using human airway epithelial cells as a therapeutically relevant target. Operational model-fitting established that the long-acting ß2-adrenoceptor agonists (LABA) indacaterol, salmeterol, formoterol, and picumeterol were full agonists on BEAS-2B cells transfected with a cAMP-response element reporter but differed in efficacy (indacaterol ≥ formoterol > salmeterol ≥ picumeterol). The transcriptomic signature of indacaterol in BEAS-2B cells identified 180, 368, 252, and 10 genes that were differentially expressed (>1.5- to <0.67-fold) after 1-, 2-, 6-, and 18-hour of exposure, respectively. Many upregulated genes (e.g., AREG, BDNF, CCL20, CXCL2, EDN1, IL6, IL15, IL20) encode proteins with proinflammatory activity and are annotated by several, enriched gene ontology (GO) terms, including cellular response to interleukin-1, cytokine activity, and positive regulation of neutrophil chemotaxis The general enriched GO term extracellular space was also associated with indacaterol-induced genes, and many of those, including CRISPLD2, DMBT1, GAS1, and SOCS3, have putative anti-inflammatory, antibacterial, and/or antiviral activity. Numerous indacaterol-regulated genes were also induced or repressed in BEAS-2B cells and human primary bronchial epithelial cells by the low efficacy LABA salmeterol, indicating that this genomic effect was neither unique to indacaterol nor restricted to the BEAS-2B airway epithelial cell line. Collectively, these data suggest that the consequences of inhaling a ß2-adrenoceptor agonist may be complex and involve widespread changes in gene expression. We propose that this genomic effect represents a generally unappreciated mechanism that may contribute to the adverse and therapeutic actions of ß2-adrenoceptor agonists in asthma.

The effects of type I collagenase on the degelification of chimpanzee (Pan troglodytes) semen plug and sperm quality.

BACKGROUND: Semen from the chimpanzee species becomes a colloidal solid after ejaculation. The formation of this copulatory plug is believed to prevent additional spermatozoa of subsequent mating events from accessing the ova. However, this naturally preserved strategy hampers the processes for sperm preparation. In this study, we investigated whether collagenase can be used to degelify the semen plug and accelerate the semen liquefaction process in zoo captive chimpanzee species (Pan troglodytes). RESULTS: We showed that incubation of chimpanzee ejaculates with 0.1% type I collagenase efficiently and significantly (p < 0.05) releases 2.7-fold more spermatozoa from the coagulated ejaculates, and this degelification process did not alter sperm morphology or viability; nor did it stimulate spontaneous capacitation or an acrosome reaction as assessed by tyrosine phosphorylation and peanut agglutinin stains; moreover, based on computer assisted sperm analysis assay, motility-related parameters remained similar to those of untreated spermatozoa. When collagenase effects were evaluated on cryopreserved sperm samples, we observed post collagenase treatment in which 2.5% glycerol, as a cryoprotectant, preserved sperm acrosome integrity better than 7.8%; however, 7.8% glycerol, as a cryoprotectant, maintained sperm motility better than that of 2.5% glycerol. CONCLUSIONS: Our results demonstrated for the first time that type I collagenase can be used to obtain a significantly higher number of spermatozoa from colloid chimpanzee semen ejaculate without affecting the physiological properties of spermatozoa, and these results are critical for the subsequent gamete development. Our results would benefit sperm preparation processes and cryopreservation efficiency per ejaculate, as more unaffected spermatozoa can be released from the semen plug within a shorter period of time. These results would also benefit the genetic diversity of the chimpanzee species, using sperm cells from less dominant individuals, and for achieving better pregnancy success in primates with significantly higher amounts of sperm for artificial insemination.

Effects of ß-blockers on house dust mite-driven murine models pre- and post-development of an asthma phenotype.

BACKGROUND: Our previous studies suggested certain ß-adrenoceptor blockers (ß-blockers) attenuate the asthma phenotype in ovalbumin driven murine models of asthma. However, the ovalbumin model has been criticized for lack of clinical relevance. METHODS: We tested the non-selective ß-blockers, carvedilol and nadolol, in house dust mite (HDM) driven murine asthma models where drugs were administered both pre- and post-development of the asthma phenotype. We measured inflammation, mucous metaplasia, and airway hyper-responsiveness (AHR). We also measured the effects of the ß-blockers on extracellular-signal regulated kinase (ERK 1/2) phosphorylation in lung homogenates. RESULTS: We show that nadolol, but not carvedilol, attenuated inflammation and mucous metaplasia, and had a moderate effect attenuating AHR. Following HDM exposure, ERK1/2 phosphorylation was elevated, but the level of phosphorylation was unaffected by ß-blockers, suggesting ERK1/2 phosphorylation becomes dissociated from the asthma phenotype. CONCLUSION: Our findings in HDM models administering drugs both pre- and post-development of the asthma phenotype are consistent with previous results using ovalbumin models and show differential effects for nadolol and carvedilol on the asthma phenotype. Lastly, our data suggest that ERK1/2 phosphorylation may be involved in development of the asthma phenotype, but may have a limited role in maintaining the phenotype.

Identification, characterization and purification of porcine Quiescin Q6-Sulfydryl Oxidase 2 protein.

BACKGROUND: Post-spermiogenesis membrane surface modifications rely on molecules present in the reproductive tracts. Two isoforms (isoform 1 and 2) from Quiescin Q6-Sulfydryl Oxidase protein family have been identified in the male reproductive tract of rodent species. However, unlike isoform 1, scarce information is available for isoform 2, likely due to its lower expression level and lack of proper purification methods to obtain sufficient protein quantity for further assays. RESULTS: This study demonstrated the presence of short and long forms of Quiescin Q6-Sulfydryl Oxidase 2 in boar, likely representing the secretory (short form) and transmembrane (long form) forms of Quiescin Q6-Sulfydryl Oxidase 2. Immunohistochemistry studies revealed the presence of Quiescin Q6-Sulfydryl Oxidase 2 in a broad range of porcine tissues; the pronounced vesicle-contained Quiescin Q6-Sulfydryl Oxidase 2 at the apical region of epididymis and seminal vesicles epithelium suggested its involvement in sperm physiology and its participation in semen formation. The majority of porcine Quiescin Q6-Sulfydryl Oxidase 2 could be purified via either antibody affinity column or be salted out using 10%-40% ammonium sulfate. Higher amount of low molecular weight Quiescin Q6-Sulfydryl Oxidase 2 observed in the seminal vesicle likely represents the secretory form of Quiescin Q6-Sulfydryl Oxidase 2 and reflects an exuberant secretory activity in this organ. CONCLUSIONS: We demonstrated for the first time, the presence of Quiescin Q6-Sulfydryl Oxidase 2 in porcine species; moreover, two forms of Quiescin Q6-Sulfydryl Oxidase 2 were identified and exhibited distinct molecular weights and properties during protein purification processes. This study also provided feasible Quiescin Q6-Sulfydryl Oxidase 2 purification methods from slaughterhouse materials that could potentially allow obtaining sufficient amount of Quiescin Q6-Sulfydryl Oxidase 2 for future functional investigations.

Phosphodiesterase 4 Inhibitors Attenuate the Asthma Phenotype Produced by ß2-Adrenoceptor Agonists in Phenylethanolamine N-Methyltransferase-Knockout Mice.

Mice lacking the endogenous ß2-adrenoceptor (ß2AR) agonist epinephrine (phenylethanolamine N-methyltransferase [PNMT]-knockout mice) are resistant to developing an "asthma-like" phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, and chronic administration of ß2AR agonists to PNMT-KO mice restores the phenotype. Based on these and other studies showing differential effects of various ß2AR ligands on the asthma phenotype, we have speculated that the permissive effect of endogenous epinephrine and exogenous ß2AR agonists on allergic lung inflammation can be explained by qualitative ß2AR signaling. The ß2AR can signal through at least two pathways: the canonical Gαs-cAMP pathway and a ß-arrestin-dependent pathway. Previous studies suggest that ß-arrestin-2 is required for allergic lung inflammation. On the other hand, cell-based assays suggest antiinflammatory effects of Gαs-cAMP signaling. This study was designed to test whether the in vitro antiinflammatory effects of phosphodiesterase 4 inhibitors, known to increase intracellular cAMP in multiple airway cell types, attenuate the asthma-like phenotype produced by the ß2AR agonists formoterol and salmeterol in vivo in PNMT-KO mice, based on the hypothesis that skewing ß2AR signaling toward Gαs-cAMP pathway is beneficial. Airway inflammatory cells, epithelial mucus production, and airway hyperresponsiveness were quantified. In Ova S/C PNMT-KO mice, formoterol and salmeterol restored the asthma-like phenotype comparable to Ova S/C wild-type mice. However, coadministration of either roflumilast or rolipram attenuated this formoterol- or salmeterol-driven phenotype in Ova S/C PNMT-KO. These findings suggest that amplification of ß2AR-mediated cAMP by phosphodiesterase 4 inhibitors attenuates the asthma-like phenotype promoted by ß-agonists.

Baroreflex dysfunction in sick newborns makes heart rate an unreliable surrogate for blood pressure changes.

BACKGROUND: Cerebral pressure passivity (CPP) in sick newborns can be detected by evaluating coupling between mean arterial pressure (MAP) and cerebral blood flow measured by near infra-red spectroscopy hemoglobin difference (HbD). However, continuous MAP monitoring requires invasive catheterization with its inherent risks. We tested whether heart rate (HR) could serve as a reliable surrogate for MAP in the detection of CPP in sick newborns. METHODS: Continuous measurements of MAP, HR, and HbD were made and partitioned into 10-min epochs. Spectral coherence (COH) was computed between MAP and HbD (COHMAP-HbD) to detect CPP, between HR and HbD (COHHR-HbD) for comparison, and between MAP and HR (COHMAP-HR) to quantify baroreflex function (BRF). The agreement between COHMAP-HbD and COHHR-HbD was assessed using ROC analysis. RESULTS: We found poor agreement between COHMAP-HbD and COHHR-HbD in left hemisphere (area under the ROC curve (AUC) 0.68) and right hemisphere (AUC 0.71). Baroreflex failure (COHMAP-HR not significant) was present in 79% of epochs. Confining comparison to epochs with intact BRF showed an AUC of 0.85 for both hemispheres. CONCLUSIONS: In these sick newborns, HR was an unreliable surrogate for MAP required for the detection of CPP. This is likely due to the prevalence of BRF failure in these infants.

No-Show Rates in an Academic Otolaryngology Practice Before and During the COVID-19 Pandemic.

OBJECTIVE: Our objectives were to determine the no-show and nonattendance rate for an outpatient academic otolaryngology practice, to identify patient and systemic factors associated with nonattendance, and to evaluate the impact that the COVID-19 pandemic had on the rate of nonattendance. METHODS: This is a retrospective review of the Epic practice management and billing reports from all scheduled outpatient visits at a multi-physician, academic, general, and sub-specialty otolaryngology practice from January 2019 to December 2021. RESULTS: Over three years, 121,347 clinic visits were scheduled in the otolaryngology practice. The overall nonattendance rate was 18.3%. A statistically significant increase in nonattendance was noted during the COVID-19 pandemic (16.8% vs. 19.8%, p < 0.001). The rate of nonattendance in patients of younger age (under 18 years) (p <0.001), female gender (p=0.03), afternoon appointments (p=0.04), and extended time between the day of scheduling and the day of appointment (p <0.001) increased. Head and neck clinics were found to have the lowest nonattendance rates, while pediatric otolaryngology clinics had the highest (12.6% vs. 21.3%). On multivariate regression, younger age (p < 0.001), female gender (p=0.01), afternoon appointments (p< 0.001), and online self-scheduling (p< 0.001) were significantly associated with nonattendance. CONCLUSIONS: Both patient and appointment-related factors were found to impact rates of nonattendance in this academic otolaryngology practice. In this study, young age, female gender, afternoon appointments, and online self-scheduling were associated with increased nonattendance. In addition, the COVID-19 pandemic significantly impacted no-show rates across all otolaryngologic subspecialties.

G-CSF increases calprotectin expression, liver damage and neuroinflammation in a murine model of alcohol-induced ACLF.

Background and aims: Granulocyte colony-stimulating factor (G-CSF) has been proposed as a therapeutic option for patients with ACLF, however clinical outcomes are controversial. We aimed at dissecting the role of G-CSF in an alcohol-induced murine model of ACLF. Methods: ACLF was triggered by a single alcohol binge (5 g/kg) in a bile duct ligation (BDL) liver fibrosis model. A subgroup of mice received two G-CSF (200 µg/kg) or vehicle injections prior to acute decompensation with alcohol. Liver, blood and brain tissues were assessed. Results: Alcohol binge administered to BDL-fibrotic mice resulted in features of ACLF indicated by a significant increase in liver damage and systemic inflammation compared to BDL alone. G-CSF treatment in ACLF mice induced an increase in liver regeneration and neutrophil infiltration in the liver compared to vehicle-treated ACLF mice. Moreover, liver-infiltrating neutrophils in G-CSF-treated mice exhibited an activated phenotype indicated by increased expression of CXC motif chemokine receptor 2, leukotriene B4 receptor 1, and calprotectin. In the liver, G-CSF triggered increased oxidative stress, type I interferon response, extracellular matrix remodeling and inflammasome activation. Circulating IL-1ß was also increased after G-CSF treatment. In the cerebellum, G-CSF increased neutrophil infiltration and S100a8/9 expression, induced microglia proliferation and reactive astrocytes, which was accompanied by oxidative stress, and inflammasome activation compared to vehicle-treated ACLF mice. Conclusion: In our novel ACLF model triggered by alcohol binge that mimics ACLF pathophysiology, neutrophil infiltration and S100a8/9 expression in the liver and brain indicate increased tissue damage, accompanied by oxidative stress and inflammasome activation after G-CSF treatment.

IRF3 regulates neuroinflammatory responses and the expression of genes associated with Alzheimer's disease.

The pathological role of interferon signaling is emerging in neuroinflammatory disorders, yet, the specific role of Interferon Regulatory Factor 3 (IRF3) in neuroinflammation remains poorly understood. Here, we show that global IRF3 deficiency delays TLR4-mediated signaling in microglia and attenuates the hallmark features of LPS-induced inflammation such as cytokine release, microglial reactivity, astrocyte activation, myeloid cell infiltration, and inflammasome activation. Moreover, expression of a constitutively active IRF3 (S388D/S390D:IRF3-2D) in microglia induces a transcriptional program reminiscent of the Activated Response Microglia and the expression of genes associated with Alzheimer's Disease, notably apolipoprotein-e. Lastly, using bulk-RNAseq of IRF3-2D brain myeloid cells, we identified Z-DNA binding protein-1 as a target of IRF3 that is relevant across various neuroinflammatory disorders. Together, our results identify IRF3 as an important regulator of LPS-mediated neuroinflammatory responses and highlight IRF3 as a central regulator of disease-specific gene activation in different neuroinflammatory diseases.

Regional and socioeconomic inequalities in access to pre-primary education in India: evidence from a recent household survey.

In India, the National Education Policy 2020 recommends ensuring universal access to high-quality early childhood care and education for children aged 3-6 years by 2030. Using the 75th round of National Statistical Office data (2017-2018), this paper analyses the regional and socioeconomic inequalities in access to pre-primary education. Also, we investigate the specific role of households' economic status and educational attainment in explaining these inequalities. We find considerable regional (rural/urban) and socioeconomic inequalities in access to pre-primary education in India, with girls and children belonging to historically disadvantaged social groups (scheduled castes and scheduled tribes) less likely to attend early childhood education, particularly in rural areas. We find that a substantial portion of the rural-urban gap in access to pre-primary education can be removed by controls for households' economic condition and household head's educational status. In addition, we find gender and socioeconomic inequalities in the household investment in early years education. These findings highlight the need to put policy efforts and commitments to reducing barriers to accessing pre-primary education for children in disadvantaged conditions in India.

Innate immune activation: Parallels in alcohol use disorder and Alzheimer's disease.

Alcohol use disorder is associated with systemic inflammation and organ dysfunction especially in the liver and the brain. For more than a decade, studies have highlighted alcohol abuse-mediated impairment of brain function and acceleration of neurodegeneration through inflammatory mechanisms that directly involve innate immune cells. Furthermore, recent studies indicate overlapping genetic risk factors between alcohol use and neurodegenerative disorders, specifically regarding the role of innate immunity in the pathomechanisms of both areas. Considering the pressing need for a better understanding of the relevance of alcohol abuse in dementia progression, here we summarize the molecular mechanisms of neuroinflammation observed in alcohol abuse and Alzheimer's disease, the most common cause of dementia. In addition, we highlight mechanisms that are already established in the field of Alzheimer's disease that may be relevant to explore in alcoholism to better understand alcohol mediated neurodegeneration and dementia, including the relevance of the liver-brain axis.

α and ß catalytic subunits of cAMP-dependent protein kinase regulate formoterol-induced inflammatory gene expression changes in human bronchial epithelial cells.

BACKGROUND AND PURPOSE: It has been proposed that genomic mechanisms contribute to adverse effects often experienced by asthmatic subjects who take regular, inhaled ß2 -adrenoceptor agonists as a monotherapy. Moreover, data from preclinical models of asthma suggest that these gene expression changes are mediated by ß-arrestin-2 rather than PKA. Herein, we tested this hypothesis by comparing the genomic effects of formoterol, a ß2 -adrenoceptor agonist, with forskolin in human primary bronchial epithelial cells (HBEC). EXPERIMENTAL APPROACH: Gene expression changes were determined by RNA-sequencing. Gene silencing and genome editing were employed to explore the roles of ß-arrestin-2 and PKA. KEY RESULTS: The formoterol-regulated transcriptome in HBEC treated concurrently with TNFα was defined by 1480 unique gene expression changes. TNFα-induced transcripts modulated by formoterol were annotated with enriched gene ontology terms related to inflammation and proliferation, notably "GO:0070374~positive regulation of ERK1 and ERK2 cascade," which is an apparent ß-arrestin-2 target. However, expression of the formoterol- and forskolin-regulated transcriptomes were highly rank-order correlated and the effects of formoterol on TNFα-induced inflammatory genes were abolished by an inhibitor of PKA. Furthermore, formoterol-induced gene expression changes in BEAS-2B bronchial epithelial cell clones deficient in ß-arrestin-2 were comparable with those expressed by their parental counterparts. Contrariwise, gene expression was partially inhibited in clones lacking the α-catalytic subunit (Cα) of PKA and abolished following the additional knockdown of the ß-catalytic subunit (Cß) paralogue. CONCLUSIONS: The effects of formoterol on inflammatory gene expression in airway epithelia are mediated by PKA and involve the cooperation of PKA-Cα and PKA-Cß.