Single cell-resolved study of advanced murine MASH reveals a homeostatic pericyte signaling module.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is linked to insulin resistance and type 2 diabetes and marked by hepatic inflammation, microvascular dysfunction, and fibrosis, impairing liver function and aggravating metabolic derangements. The liver homeostatic interactions disrupted in MASH are still poorly understood. We aimed to elucidate the plasticity and changing interactions of non-parenchymal cells associated with advanced MASH. METHODS: We characterized a diet-induced mouse model of advanced MASH at single-cell resolution and validated findings by assaying chromatin accessibility, bioimaging murine and human livers, and via functional experiments in vivo and in vitro. RESULTS: The fibrogenic activation of hepatic stellate cells (HSCs) led to deterioration of a signaling module consisting of the bile acid receptor NR1H4/FXR and HSC-specific GS-protein-coupled receptors (GSPCRs) capable of preserving stellate cell quiescence. Accompanying HSC activation, we further observed the attenuation of HSC Gdf2 expression, and a MASH-associated expansion of a CD207-positive macrophage population likely derived from both incoming monocytes and Kupffer cells. CONCLUSION: We conclude that HSC-expressed NR1H4 and GSPCRs of the healthy liver integrate postprandial cues, which sustain HSC quiescence and, through paracrine signals, overall sinusoidal health. Hence HSC activation in MASH not only drives fibrogenesis but may desensitize the hepatic sinusoid to liver homeostatic signals. IMPACT AND IMPLICATIONS: Homeostatic interactions between hepatic cell types and their deterioration in metabolic dysfunction-associated steatohepatitis are poorly characterized. In our current single cell-resolved study of advanced murine metabolic dysfunction-associated steatohepatitis, we identified a quiescence-associated hepatic stellate cell-signaling module with potential to preserve normal sinusoid function. As expression levels of its constituents are conserved in the human liver, stimulation of the identified signaling module is a promising therapeutic strategy to restore sinusoid function in chronic liver disease.

Multipartite Fluorogenic Sensors for Monitoring Tyrosine Phosphatase Activity.

Fluorogenic substrates are essential tools for studying the activity of many enzymes including the protein tyrosine phosphatases (PTPs). Here, we have taken the first step toward the development of genetically encodable sensors for PTP activity using fluorescent and fluorogen-activating proteins. The Fluorescence-Activating and absorption Shifting Tag (FAST) is a small protein that becomes fluorescent upon binding to a small molecule dye. We demonstrate that FAST protein can be used as a sensor for PTP-mediated dephosphorylation of phosphorylated dye molecules. Phosphorylated 4-hydroxybenzylidene rhodanine (pHBR) is not able to bind to the FAST protein and induce fluorescence, but provides a sensitive assay for PTP activity, readily detecting 100 pM concentrations of PTP1B in the presence of FAST with a kcat value of 19 ± 1 s-1 and a KM value of 93 ± 3 µM. In addition, while phosphorylation of the C-terminal peptide of split GFP does not result in appreciable change in fluorescence of the reconstituted protein, phosphorylation of the C-terminal peptide of the split FAST protein abrogates fluorescence. Upon PTP-mediated dephosphorylation of the C-terminal peptide, the ability of the N- and C-terminal components to form a fluorescent complex with the small molecule dye is restored, leading to fluorescence.

A multi-center comparison of surgical techniques for corpus Callosotomy in pediatric drug-resistant epilepsy.

OBJECTIVES: Corpus callosotomy (CC) is used to reduce seizures, primarily in patients with generalized drug-resistant epilepsy (DRE). The invasive nature of the procedure contributes to underutilization despite its potential superiority to other palliative procedures. The goal of this study was to use a multi-institutional epilepsy surgery database to characterize the use of CC across participating centers. METHODS: Data were acquired from the Pediatric Epilepsy Research Consortium (PERC) Surgery Database, a prospective observational study collecting data on children 0-18 years referred for surgical evaluation of DRE across 22 U.S. pediatric epilepsy centers. Patient, epilepsy, and surgical characteristics were collected across multiple CC modalities. Outcomes and complications were recorded and analyzed statistically. RESULTS: Eighty-three patients undergoing 85 CC procedures at 14 participating epilepsy centers met inclusion criteria. Mean age at seizure onset was 2.3 years (0-9.4); mean age for Phase I evaluation and surgical intervention were 9.45 (.1-20) and 10.46 (.2-20.6) years, respectively. Generalized seizure types were the most common (59%). Complete CC was performed in 88%. The majority of CC procedures (57%) were via open craniotomy, followed by laser interstitial thermal therapy (LiTT) (20%) and mini-craniotomy/endoscopic (mc/e) (22%). Mean operative times were significantly longer for LiTT, whereas mean estimated blood loss was greater in open cases. Complications occurred in 11 cases (13%) and differed significantly between surgical techniques (p < .001). There was no statistically significant difference in length of postoperative stay across approaches. Mean follow-up was 12.8 months (range 1-39). Favorable Engel outcomes were experienced by 37 (78.7%) of the patients who underwent craniotomy, 10 (58.8%) with LiTT, and 12 (63.2%) with mc/e; these differences were not statistically significant. SIGNIFICANCE: CC is an effective surgical modality for children with DRE. Regardless of surgical modality, complication rates are acceptable and seizure outcomes generally favorable. Newer, less-invasive, surgical approaches may lead to increased adoption of this efficacious therapeutic option for pediatric DRE.

The mechanism of covalent inhibition of LAR phosphatase by illudalic acid.

Leukocyte antigen-related (LAR) phosphatase is a receptor-type protein tyrosine phosphatase involved in cellular signaling and associated with human disease including cancer and metabolic disorders. Selective inhibition of LAR phosphatase activity by well characterized and well validated small molecules would provide key insights into the roles of LAR phosphatase in health and disease, but identifying selective inhibitors of LAR phosphatase activity has been challenging. Recently, we described potent and selective inhibition of LAR phosphatase activity by the fungal natural product illudalic acid. Here we provide a detailed biochemical characterization of the adduct formed between LAR phosphatase and illudalic acid. A mass spectrometric analysis indicates that two cysteine residues are covalently labeled by illudalic acid and a related analog. Mutational analysis supports the hypothesis that inhibition of LAR phosphatase activity is due primarily to the adduct with the catalytic cysteine residue. A computational study suggests potential interactions between the illudalic acid moiety and the enzyme active site. Taken together, these data offer novel insights into the mechanism of inhibition of LAR phosphatase activity by illudalic acid.

Association between thrombus composition and stroke etiology in the MR CLEAN Registry biobank.

PURPOSE: The composition of thrombi retrieved during endovascular thrombectomy (EVT) in acute ischemic stroke (AIS) due to large vessel occlusion (LVO) may differ depending on their origin. In this study, we investigated the association between thrombus composition and stroke etiology in a large population of patients from the Dutch MR CLEAN Registry treated with EVT in daily clinical practice. METHODS: The thrombi of 332 patients with AIS were histologically analyzed for red blood cells (RBC), fibrin/platelets (F/P), and white blood cells (leukocytes) using a machine learning algorithm. Stroke etiology was assessed using the Trial of Org 10,172 in acute stroke treatment (TOAST) classification. RESULTS: The thrombi of cardioembolic origin contained less RBC and more F/P than those of non-cardioembolic origin (25.8% vs 41.2% RBC [p = 0.003] and 67.1% vs 54.5% F/P [p = 0.004]). The likelihood of a non-cardioembolic source of stroke increased with increasing thrombus RBC content (OR 1.02; [95% CI 1.00-1.06] for each percent increase) and decreased with a higher F/P content (OR 1.02; [95% CI 1.00-1.06]). Thrombus composition in patients with a cardioembolic origin and undetermined origin was similar. CONCLUSION: Thrombus composition is significantly associated with stroke etiology, with an increase in RBC and a decrease in F/P raising the odds for a non-cardioembolic cause. No difference between composition of cardioembolic thrombi and of undetermined origin was seen. This emphasizes the need for more extensive monitoring for arrhythmias and/or extended cardiac analysis in case of an undetermined origin.

Scalable-resolution structured illumination microscopy.

Structured illumination microscopy suffers from the need of sophisticated instrumentation and precise calibration. This makes structured illumination microscopes costly and skill-dependent. We present a novel approach to realize super-resolution structured illumination microscopy using an alignment non-critical illumination system and a reconstruction algorithm that does not need illumination information. The optical system is designed to encode higher order frequency components of the specimen by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in structured illumination microscopy. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. This algorithm eliminates the need of clean peaks in illumination and the knowledge of illumination patterns, which makes instrumentation simple and flexible for use with a variety of microscope objective lenses. We present a variety of experimental results on beads and cell samples to demonstrate resolution enhancement by a factor of 2.6 to 3.4 times, which is better than the enhancement supported by the conventional linear structure illumination microscopy where the same objective lens is used for structured illumination as well as collection of light. We show that the same system can be used in SIM configuration with different collection objective lenses without any careful re-calibration or realignment, thereby supporting a range of resolutions with the same system.

Observer Variability as a Determinant of Measurement Error of Ultrasonographic Measurements of the Optic Nerve Sheath Diameter: A Systematic Review.

BACKGROUND: Ultrasonographic measurements of the diameter of the sheath of the optic nerve can be used to assess intracranial pressure indirectly. These measurements come with measurement error. OBJECTIVE: Our aim was to estimate observer's measurement error as a determinant of ultrasonographic measurement variability of the optic nerve sheath diameter. METHODS: A systematic search of the literature was conducted in Embase, Medline, Web of Science, the Cochrane Central Register of Trials, and the first 200 articles of Google Scholar up to April 19, 2021. Inclusion criteria were the following: healthy adults, B-mode ultrasonography, and measurements 3 mm behind the retina. Studies were excluded if standard error of measurement could not be calculated. Nine studies featuring 389 participants (median 40; range 15-100) and 22 observers (median 2; range 1-4) were included. Standard error of measurement and minimal detectable differences were calculated to quantify observer variability. Quality and risk of bias were assessed with the Guidelines for Reporting Reliability and Agreement Studies. RESULTS: The standard error of measurement of the intra- and interobserver variability had a range of 0.10-0.41 mm and 0.14-0.42 mm, respectively. Minimal detectable difference of a single observer was 0.28-1.1 mm. Minimal detectable difference of multiple observers (range 2-4) was 0.40-1.1 mm. Quality assessment showed room for methodological improvement of included studies. CONCLUSIONS: The standard errors of measurement and minimal detectable differences of ultrasonographic measurements of the optic nerve sheath diameter found in this review with healthy participants indicate caution should be urged when interpreting results acquired with this measurement method in clinical context.

Transcriptional Dynamics of Hepatic Sinusoid-Associated Cells After Liver Injury.

BACKGROUND AND AIMS: Hepatic sinusoidal cells are known actors in the fibrogenic response to injury. Activated hepatic stellate cells (HSCs), liver sinusoidal endothelial cells, and Kupffer cells are responsible for sinusoidal capillarization and perisinusoidal matrix deposition, impairing vascular exchange and heightening the risk of advanced fibrosis. While the overall pathogenesis is well understood, functional relations between cellular transitions during fibrogenesis are only beginning to be resolved. At single-cell resolution, we here explored the heterogeneity of individual cell types and dissected their transitions and crosstalk during fibrogenesis. APPROACH AND RESULTS: We applied single-cell transcriptomics to map the heterogeneity of sinusoid-associated cells in healthy and injured livers and reconstructed the single-lineage HSC trajectory from pericyte to myofibroblast. Stratifying each sinusoidal cell population by activation state, we projected shifts in sinusoidal communication upon injury. Weighted gene correlation network analysis of the HSC trajectory led to the identification of core genes whose expression proved highly predictive of advanced fibrosis in patients with nonalcoholic steatohepatitis (NASH). Among the core members of the injury-repressed gene module, we identified plasmalemma vesicle-associated protein (PLVAP) as a protein amply expressed by mouse and human HSCs. PLVAP expression was suppressed in activated HSCs upon injury and may hence define hitherto unknown roles for HSCs in the regulation of microcirculatory exchange and its breakdown in chronic liver disease. CONCLUSIONS: Our study offers a single-cell resolved account of drug-induced injury of the mammalian liver and identifies key genes that may serve important roles in sinusoidal integrity and as markers of advanced fibrosis in human NASH.

Fluorescence fluctuation-based super-resolution microscopy using multimodal waveguided illumination.

Photonic chip-based total internal reflection fluorescence microscopy (c-TIRFM) is an emerging technology enabling a large TIRF excitation area decoupled from the detection objective. Additionally, due to the inherent multimodal nature of wide waveguides, it is a convenient platform for introducing temporal fluctuations in the illumination pattern. The fluorescence fluctuation-based nanoscopy technique multiple signal classification algorithm (MUSICAL) does not assume stochastic independence of the emitter emission and can therefore exploit fluctuations arising from other sources, as such multimodal illumination patterns. In this work, we demonstrate and verify the utilization of fluctuations in the illumination for super-resolution imaging using MUSICAL on actin in salmon keratocytes. The resolution improvement was measured to be 2.2-3.6-fold compared to the corresponding conventional images.

Water transport in polymer composites through swelling-induced networks of hydrogel particles.

Water diffusion in polymer composites is not only affected by the chemical nature of the materials but also by their internal structures. To enable the design of polymer composites with controlled diffusion kinetics, we investigate the effect of hydrogel particle networks on the water transport. The composites in this study comprise hydrogel particles based on sodium poly(acrylic acid), which are incorporated at different concentrations into a soft and sticky polymer matrix. Through the use of X-ray micro computed tomography, the internal structure of the polymer composites is examined and the interparticle distances are calculated. The structure of the composites is then related to the water diffusion kinetics upon exposure to saline solution as well as humid air. Even though the hydrogel particles are isolated and the interparticle distances are in the order of several micrometers, a sudden increase in the water diffusion kinetics is observed above a critical concentration. Due to the low water permeability of the matrix, such a change in the water diffusion kinetics is indicative of network formation. During hydration, swelling enables the hydrogels to overcome the interparticle distances and form a network for water transport.

Evaluation of pediatric glioma outcomes using intraoperative MRI: a multicenter cohort study.

BACKGROUND: The use of intraoperative MRI (iMRI) during treatment of gliomas may increase extent of resection (EOR), decrease need for early reoperation, and increase progression-free and overall survival, but has not been fully validated, particularly in the pediatric population. OBJECTIVE: To assess the accuracy of iMRI to identify residual tumor in pediatric patients with glioma and determine the effect of iMRI on decisions for resection, complication rates, and other outcomes. METHODS: We retrospectively analyzed a multicenter database of pediatric patients (age ≤ 18 years) who underwent resection of pathologically confirmed gliomas. RESULTS: We identified 314 patients (mean age 9.7 ± 4.6 years) with mean follow-up of 48.3 ± 33.6 months (range 0.03-182.07 months) who underwent surgery with iMRI. There were 201 (64.0%) WHO grade I tumors, 57 (18.2%) grade II, 24 (7.6%) grade III, 9 (2.9%) grade IV, and 23 (7.3%) not classified. Among 280 patients who underwent resection using iMRI, 131 (46.8%) had some residual tumor and underwent additional resection after the first iMRI. Of the 33 tissue specimens sent for pathological analysis after iMRI, 29 (87.9%) showed positive tumor pathology. Gross total resection was identified in 156 patients (55.7%), but this was limited by 69 (24.6%) patients with unknown EOR. CONCLUSIONS: Analysis of the largest multicenter database of pediatric gliomas resected using iMRI demonstrated additional tumor resection in a substantial portion of cases. However, determining the impact of iMRI on EOR and outcomes remains challenging because iMRI use varies among providers nationally. Continued refinement of iMRI techniques for use in pediatric patients with glioma may improve outcomes.

Development and Validation of the Foot Union Scoring Evaluation Tool for Arthrodesis of Foot Structures.

Reliable evaluation of osseous consolidation after pedal arthrodesis can be difficult, and the presence or absence of radiographic healing often dictates care. Plain radiographs remain the mainstay imaging tool owing to their cost, efficiency, and low radiation exposure. Applying radiographic parameters that can reliably determine osseous healing is essential. However, currently, no reliable or validated measures are available to determine osseous union of any joint in the foot or ankle. The purpose of the present study was to develop a radiographic healing scoring system that would enhance the diagnostic healing assessment after joint arthrodesis of the foot or ankle. We adapted several existing scales previously validated for fracture healing in the leg, because no study has attempted to apply this to a joint fusion model. A total of 150 cases were evaluated by 6 blinded assessors to test the interrater reliability of the subjective healing assessment compared with the proposed scoring system. The radiographs were classified by the postoperative period: ≤4 weeks, 5 to 12 weeks, and >12 weeks. The initial proposed scale was found to have high interrater reliability but was burdensome. Using a priori item reduction protocols, a limited 5-item scale further improved the internal consistency and reduced the burden. The result was excellent interrater reliability (α = 0.978, standard deviation 0.02, 95% confidence interval 0.96 to 0.99) among all assessors compared with the reduced reliability (α = 0.752) for subjective arthrodesis healing. Intrarater reliability was also found to be superior using a test-retest method. The reliability of this system appeared superior to the subjective assessment of arthrodesis healing, even in the absence of clinical correlates, after foot arthrodesis.

Long-Term Outcomes of Corrective Osteotomies Using Porous Titanium Wedges for Flexible Flatfoot Deformity Correction.

Common corrective osteotomies used in flexible flatfoot deformity reconstruction include Cotton and Evans osteotomies, which require structural graft to maintain correction. Auto-, allo-, and xenografts are associated with a number of limitations, including disease transmission, rejection, donor site morbidity, technical challenges related to graft fashioning, and graft resorption. Porous titanium is a synthetic substance designed to address these flaws; however, few studies have been reported on the efficacy, safety, and long-term outcomes. A multicenter retrospective cohort of 63 consecutive preconfigured porous titanium wedges (PTWs) used in flexible flatfoot reconstructions from June 1, 2009 to June 30, 2015 was evaluated. The primary outcome measure was the pre- to postdeformity correction efficacy. The secondary outcomes included maintenance of correction at a minimum follow-up point of 12 months, complications, graft incorporation, and graft safety profile. Multivariate linear regression found a statistically significant improvement in all radiographic parameters from preoperatively to the final weightbearing radiographs (calcaneocuboid 18.850 ± 4.020 SE, p < .0001; Kite's, 7.810 ± 3.660 SE, p = .04; Meary's 13.910 ± 3.100 SE, p = .0001; calcaneal inclination, 5.550 ± 2.140 SE, p = .015). When restricted to patients with >4 years of follow-up data, maintenance of correction appeared robust in all 4 measurements, demonstrating a lack of bone or graft resorption. No patients were lost to follow-up, no major complications or implant explantation or migration occurred, and all implants were incorporated. Minor complications included hardware pain from plates over grafts (8%), 1 case of scar neuritis, and a 5% table incidence of transfer pain associated with the PTWs. These results support the use of PTWs for safety and degree and maintenance of correction in flatfoot reconstruction.

Connexin Hemichannels in Astrocytes: An Assessment of Controversies Regarding Their Functional Characteristics.

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a 'docked' counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.

Perioperative antibiotic use in vagus nerve stimulator implantation: a clinical series.

PURPOSE: Preoperative antibiosis contributes up to one third of total antibiotic use in major hospitals. Choice of antibiotic is not uniformly standardized, and polypharmacy regimens may be used without knowing the effect on rates of surgical site infection, nonsurgical infections, or antibiotic resistance. Careful examination of trends in surgical prophylaxis is warranted. In this study, we aimed to examine our institution's experience with vagus nerve stimulator (VNS) implantation, focusing on association between perioperative antibiotic practices and postoperative infectious outcomes. METHODS: We conducted a single-center case-control study using a retrospective chart review of 50 consecutively operated patients undergoing VNS implantation over 24 months by two experienced surgeons at our institution from April 2014 to March 2016. In each surgery, the technical procedure, operating room, and surgical team were the same, while surgeon's preference in antibiotic prophylaxis differed. Group 1 received a single dose of intravenous (IV) cefazolin (n = 26), and Group 2 received IV cefazolin, paired with one or both of gentamicin/vancomycin, in addition to a 10-day outpatient oral course of clindamycin (n = 24). Patient demographics, perioperative details, and minimum 3-month follow-up for infection and healthcare utilization were recorded. Student t tests were computed for significance. RESULTS: Group 1 patients on average were older than group 2 patients (10.2, 7.1 years, p = 0.01), and length of surgery was longer (115.5, 91.9 min, p = 0.007). There were no differences in number of surgeons gowned (p = 0.11), presence of tracheostomy (p = 0.43) or gastrostomy (p = 0.20) tube, nonsurgical infections (p = 0.32), and number of postoperative emergency department (ED) visits (p = 0.22) or readmissions (p = 0.23). Neither group had VNS infections in the follow-up period. CONCLUSION: Single preoperative dosing of one antibiotic appropriately chosen to cover typical skin flora conferred equal benefit to perioperative prophylactic polypharmacy in this study. There were no differences in postoperative infection events or ED visits/readmissions. Restraint with preoperative antibiosis shows equipoise in postoperative infection and overall resource utilization.

Transfrontal and Transsphenoidal Approaches to Pediatric Craniopharyngioma: A National Perspective.

INTRODUCTION: This study compared transsphenoidal (TS) and transfrontal (TF) approaches to craniopharyngioma utilizing a national database. METHODS: The Kids' Inpatient Database (2003, 2006, and 2009) was surveyed for patients with a diagnosis of craniopharyngioma who underwent a subset of surgical interventions to compare TS and TF surgery. Demographics, hospital variables, and complications/comorbidities were analyzed with multivariate regression. RESULTS: 314 admissions (TS = 104, TF = 210) were identified. The mean age was 14.8 (TS) versus 9.8 (TF) years (p < 0.001). The mean number of diagnoses was 4.6 (TS) versus 6.2 (TF) (p < 0.001). Diabetes insipidus was associated with 38% (TS) and 69% (TF). Cerebrospinal fluid (CSF) leak affected 19% TS and 4% TF resections. Other complications and comorbidities included postoperative stroke (2% TS vs. 5% TF), panhypopituitarism (5 vs. 8%), death (0 vs. 1%), cranial nerve deficits (1 vs. 6%), thrombotic events (7 vs. 17%), and seizures (0 vs. 12%). 98% of patients were discharged home after a mean 6-day length of stay (LOS) after TS, whereas 90% of TF patients had a LOS of 12 days. TS cases were more likely to be privately insured (68%) and from higher income brackets (61%) than TF ones (56 and 2%, respectively) (p < 0.05). In multivariate regression models adjusting for age, sex, race, number of diagnoses, surgical approach, hospital volume, and insurance type, the TS approach was associated with an increased incidence of CSF leak (OR 10, p < 0.001). More documented diagnoses (OR 16-60, p < 0.01) and TF approach (OR 2.6, p < 0.01) were associated with an increased incidence of other complications and comorbidities. Age younger than 10 (ß-coefficient 2.3, p = 0.01), more diagnoses (ß-coefficient 1.2, p < 0.001), and TF approach (ß- coefficient 3.0, p < 0.01) were associated with increased LOS. A higher number of diagnoses were associated with nonhome discharge destinations (ß-coefficient 1.29, p < 0.001). CONCLUSION: TS surgery was associated with an increased incidence of CSF leak but shorter LOS; TF surgery was associated with an increased incidence of other complications. Patients undergoing TS surgery were more likely to have private insurance and a higher family income bracket.

HCO3(-)-independent pH regulation in astrocytes in situ is dominated by V-ATPase.

The mechanisms of HCO3(-)-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H(+) pump by Cl(-) removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3-6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3(-)-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.

Activation, permeability, and inhibition of astrocytic and neuronal large pore (hemi)channels.

Astrocytes and neurons express several large pore (hemi)channels that may open in response to various stimuli, allowing fluorescent dyes, ions, and cytoplasmic molecules such as ATP and glutamate to permeate. Several of these large pore (hemi)channels have similar characteristics with regard to activation, permeability, and inhibitor sensitivity. Consequently, their behaviors and roles in astrocytic and neuronal (patho)physiology remain undefined. We took advantage of the Xenopus laevis expression system to determine the individual characteristics of several large pore channels in isolation. Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X7 yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca(2+) and their permeability to dyes and small atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X7, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized the divalent cation-sensitive permeation pathway in primary cultured astrocytes. We observed no activation of membrane conductance or Cx43-mediated dye uptake in astrocytes nor in Cx43-expressing C6 cells. Our data underscore that although Cx43-mediated transport is observed in overexpressing cell systems, such transport may not be detectable in native cells under comparable experimental conditions.

Sensory-motor networks involved in speech production and motor control: an fMRI study.

Speaking is one of the most complex motor behaviors developed to facilitate human communication. The underlying neural mechanisms of speech involve sensory-motor interactions that incorporate feedback information for online monitoring and control of produced speech sounds. In the present study, we adopted an auditory feedback pitch perturbation paradigm and combined it with functional magnetic resonance imaging (fMRI) recordings in order to identify brain areas involved in speech production and motor control. Subjects underwent fMRI scanning while they produced a steady vowel sound /a/ (speaking) or listened to the playback of their own vowel production (playback). During each condition, the auditory feedback from vowel production was either normal (no perturbation) or perturbed by an upward (+600 cents) pitch-shift stimulus randomly. Analysis of BOLD responses during speaking (with and without shift) vs. rest revealed activation of a complex network including bilateral superior temporal gyrus (STG), Heschl's gyrus, precentral gyrus, supplementary motor area (SMA), Rolandic operculum, postcentral gyrus and right inferior frontal gyrus (IFG). Performance correlation analysis showed that the subjects produced compensatory vocal responses that significantly correlated with BOLD response increases in bilateral STG and left precentral gyrus. However, during playback, the activation network was limited to cortical auditory areas including bilateral STG and Heschl's gyrus. Moreover, the contrast between speaking vs. playback highlighted a distinct functional network that included bilateral precentral gyrus, SMA, IFG, postcentral gyrus and insula. These findings suggest that speech motor control involves feedback error detection in sensory (e.g. auditory) cortices that subsequently activate motor-related areas for the adjustment of speech parameters during speaking.

Isoform-specific phosphorylation-dependent regulation of connexin hemichannels.

Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC.