Functional Characterization of a Novel SLC4A4 Variant and Uniparental Isodisomy in Proximal Renal Tubular Acidosis Patient.

SLC4A4 variants are the etiologies of inherited proximal renal tubular acidosis (pRTA), which results in metabolic acidosis, hypokalemia, glaucoma, band keratopathy, and cataract. This study aims to characterize SLC4A4 variant and uniparental isodisomy of chromosome 4 in a patient, and analyse the functional characterization of SLC4A4 variants. This study analyzed renal tubular acidosis disease genes by whole exome sequencing (WES). H3M2 algorithm was used to analyze the run of homozygosity region in chromosomal regions in trio-WES data. The pathogenicity analysis of variants was performed using bioinformatics tools. Additionally, protein stability was analyzed by cycloheximide chase assay. Whole-cell patch clamping was used to examine the electrophysiological properties of NBCe1-A. A novel homozygous SLC4A4 variant was identified in the patient: a missense variant c.496C > T, p. Arg166Trp (NM_003759.4). But the father was heterozygous variant carrier, and the mother did not detect the variant. The H3M2 and UPDio algorithm revealed paternal uniparental isodisomy on chromosome 4 in the patient. SIFT, Poly Phen-2, FATHMM and Mutant Taster showed that the variant might be pathogenic. The tertiary structure analysis showed that the variant could cause structural damage to NBCe1 protein. Foldx results showed that the protein stability of the variant was slightly reduced. Cycloheximide chase assay demonstrated that the variant affects protein stability. The result of electrophysiological studies showed that the variant altered Na+/HCO3- cotransport activity of protein. In conclusion, the study is the first to report a pRTA patient with Arg166Trp variant with UPiD (4) pat and analyze the function of Arg166Trp variant.

Simultaneous calcium recordings of hippocampal CA1 and primary motor cortex M1 and their relations to behavioral activities in freely moving epileptic mice.

Epilepsy is a common neurological disorder characterized by recurrent epileptic seizures. The cause of most cases of epilepsy is unknown. Although changes of calcium events in a single brain region during seizures have been reported before, there have been few studies on relations between calcium events of two different brain regions and epileptic behaviors in freely moving mice. To analyze calcium events simultaneously recorded in hippocampal CA1 (CA1) and primary motor cortex M1 (M1), and to explore their relations to various epileptic behaviors in freely moving epileptic models. Epileptic models were induced by Kainic acid (KA), a direct agonist of glutamatergic receptor, on adult male C57/BL6J mice. Calcium events of neurons and glia in CA1 and M1 labeled by a calcium indicator dye were recorded simultaneously with a multi-channel fiber photometry system. Three typical types of calcium events associated with KA-induced seizures were observed, including calcium baseline-rising, cortical spreading depression (CSD) and calcium flashing with a steady rate. Our results showed that the calcium baseline-rising occurred in CA1 was synchronized with that in M1, but the CSD waves were not. However, synchronization of calcium flashing in the two areas was uncertain, because it was only detected in CA1. We also observed that different calcium events happened with different epileptic behaviors. Baseline-rising events were accompanied by clonus of forelimbs or trembling, CSD waves were closely related to head movements (15 out of 18, 6 mice). Calcium flashing occurred definitely with drastic convulsive motor seizures (CMS, 6 mice). The results prove that the synchronization of calcium event exists in CA1 and M1, and different calcium events are related with different seizure behaviors. Our results suggest that calcium events involve in the synchronization of neural network and behaviors in epilepsy.

Preoperative recovery sleep ameliorates postoperative cognitive dysfunction aggravated by sleep fragmentation in aged mice by enhancing EEG delta-wave activity and LFP theta oscillation in hippocampal CA1.

Sleep fragmentation (SF) is a common sleep problem experienced during the perioperative period by older adults, and is associated with postoperative cognitive dysfunction (POCD). Increasing evidence indicates that delta-wave activity during non-rapid eye movement (NREM) sleep is involved in sleep-dependent memory consolidation and that hippocampal theta oscillations are related to spatial exploratory memory. Recovery sleep (RS), a self-regulated state of sleep homeostasis, enhances delta-wave power and memory performance in sleep-deprived older mice. However, it remains unclear whether RS therapy has a positive effect on cognitive changes following SF in older mouse models. Therefore, this study aimed to explore whether preoperative RS can alleviate cognitive deficits in aged mice with SF. A model of preoperative 24-h SF combined with exploratory laparotomy-induced POCD was established in 18-month-old mice. Aged mice were treated with preoperative 6-h RS following SF and postoperative 6-h RS following surgery, respectively. The changes in hippocampus-dependent cognitive function were investigated using behavioral tests, electroencephalography (EEG), local field potential (LFP), magnetic resonance imaging, and neuromorphology. Mice that underwent 24-h SF combined with surgery exhibited severe spatial memory impairment; impaired cognitive performance could be alleviated by preoperative RS treatment. In addition, preoperative RS increased NREM sleep; enhanced EEG delta-wave activity and LFP theta oscillation in the hippocampal CA1; and improved hippocampal perfusion, microstructural integrity, and neuronal damage. Taken together, these results provide evidence that preoperative RS may ameliorate the severity of POCD aggravated by SF by enhancing delta slow-wave activity and hippocampal theta oscillation, and by ameliorating the reduction in regional cerebral blood flow and white matter microstructure integrity in the hippocampus.

Prognostic role of serum ammonia in patients with sepsis-associated encephalopathy without hepatic failure.

Objectives: Our previous study shows that serum ammonia in sepsis patients without hepatic failure is associated with a poor prognosis. The relationship between serum ammonia level and the prognosis of sepsis-associated encephalopathy (SAE) patients without hepatic failure remains unclear. We aimed to explore the relationship between serum ammonia levels and the prognosis of patients with SAE. Materials and methods: This study is a retrospective cohort study. We collected 465 patients with SAE admitted to the intensive care unit (ICU) from Medical Information Mart for Intensive Care IV (MIMIC IV) from 2008 to 2019. Patients with SAE were divided into a survival group (369 patients) and a non-survival group (96 patients). We used the Wilcoxon signed-rank test and the multivariate logistic regression analysis to analyze the relationship between serum ammonia levels and the prognosis of patients with SAE. R software was used to analyze the dataset. Results: The primary outcome was the relationship between serum ammonia level and hospital mortality of SAE. The secondary outcomes were the relationship between serum ammonia level and hospital stays, simplified acute physiology score (SAPS II), Charlson, Glasgow coma scale (GCS), sequential organ failure assessment (SOFA), and lactate level of SAE. The mortality of patients with SAE was 20.6%. The serum ammonia level was not significantly associated with hospital mortality, longer hospital stays, higher SAPS II and Charlson scores, and lower GCS of patients with SAE. The serum ammonia level was associated with higher SOFA scores and lactate levels in patients with SAE. The SAPS II and Charlson scores were independent risk factors for death in patients with SAE. Conclusion: Serum ammonia level was associated with higher SOFA scores and lactate levels in patients with SAE. In addition, the SAPS II and Charlson scores can be used to assess the prognosis of patients with SAE. Therefore, we should closely monitor serum ammonia, SAPS II, and Charlson levels in patients with SAE.

Two-photon calcium imaging of neuronal and astrocytic responses: the influence of electrical stimulus parameters and calcium signaling mechanisms.

Objective. Electrical brain stimulation has been used to ameliorate symptoms associated with neurologic and psychiatric disorders. The astrocytic activation and its interaction with neurons may contribute to the therapeutic effects of electrical stimulation. However, how the astrocytic activity is affected by electrical stimulation and its calcium signaling mechanisms remain largely unknown. This study is to explore the influence of electrical stimulus parameters on cellular calcium responses and corresponding calcium signaling mechanisms, with a focus on the heretofore largely overlooked astrocytes.Approach. Usingin vivotwo-photon microscopy in mouse somatosensory cortex, the calcium activity in neurons and astrocytes were recorded.Main results. The cathodal stimulation evoked larger responses in both neurons and astrocytes than anodal stimulation. Both neuronal and astrocytic response profiles exhibited the unimodal frequency dependency, the astrocytes prefer higher frequency stimulation than neurons. Astrocytes need longer pulse width and higher current intensity than neurons to activate. Compared to neurons, the astrocytes were not capable of keeping sustained calcium elevation during prolonged electrical stimulation. The neuronal Ca2+influx involves postsynaptic effects and direct depolarization. The Ca2+surge of astrocytes has a neuronal origin, the noradrenergic and glutamatergic signaling act synergistically to induce astrocytic activity.Significance. The astrocytic activity can be regulated by manipulating stimulus parameters and its calcium activation should be fully considered when interpreting the mechanisms of action of electrical neuromodulation. This study brings considerable benefits in the application of electrical stimulation and provides useful insights into cortical signal transduction, which contributes to the understanding of mechanisms underlying the therapeutic efficacy of electrical stimulation for neurorehabilitation applications.

MicroRNA-153-3p sensitizes melanoma cells to dacarbazine by suppressing ATG5-mediated autophagy and apoptosis.

BACKGROUND: Dacarbazine is one of the most commonly used chemotherapeutic agents for the treatment of melanoma; however, only 5-10% of patients benefit from this treatment. MicroRNA-153-3p (miR-153-3p) has a tumor-suppressive effect in melanoma. In the present study, we found that miR-153-3p was downregulated in melanoma cell lines (A357 and M14). METHODS: The target relationship between miR-153-3p and Autophagy-related gene 5 (ATG5) was confirmed by Dual-Luciferase Reporter Assay. Cell Counting Kit-8, flow cytometry, immunofluorescence, and Western blot were used to examine cell viability, apoptosis, and autophagy, respectively. RESULTS: miR-153-3p overexpression decreased the half-maximal inhibitory concentration value of dacarbazine, while increasing the apoptotic rate in both A357 and M14 cells. Moreover, miR-153-3p enhanced dacarbazine-induced autophagy in melanoma cells. Our bioinformatics study revealed that ATG5 is one of the potential targets of miR-153-3p. The overexpression of ATG5 decreased dacarbazine sensitivity and promoted proliferation, as well as inhibited apoptosis and autophagy in melanoma cells. miR-153-3p exhibited suppressive effects via directly binding and downregulating ATG5 expression, which subsequently increased sensitivity to dacarbazine and inhibited proliferation, and enhanced apoptosis and autophagy in melanoma cells. CONCLUSIONS: The results of the present study showed that miR-153-3p sensitizes melanoma cells to dacarbazine by suppressing ATG5-mediated autophagy and apoptosis, and provided a basis to explore the functions of miRNAs on drug resistance in the treatment of melanoma.

Transient ischemia-reperfusion induces cortical hyperactivity and AMPAR trafficking in the somatosensory cortex.

Brain ischemia results from cardiac arrest, stroke or head trauma. The structural basis of rescuing the synaptic impairment and cortical dysfunctions induced in the stage of ischemic-reperfusion can occur if therapeutic interventions are applied in time, but the functional basis for this resilience remains elusive. Here, we explore the changes in cortical activity and a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) GluA1 subunit in spine (sGluA1) after transient ischemia-reperfusion in vivo for 28 days. Using in vivo two-photon microscopy in the mouse somatosensory cortex, we found that the average frequency of Ca2+ transients in the spine (there was an unusual synchrony) was higher after 15 min of ischemia-reperfusion. In addition, the transient ischemia-reperfusion caused a reflective enhancement of AMPARs, which eventually restored to normal. The cortical hyperactivity (Ca2+ transients) and the increase in AMPARs were successfully blocked by an NMDA receptor antagonist. Thus, the increase of AMPARs, cortical hyperactivity and the unusual synchrony might be the reason for reperfusion injury after short-term transient ischemia.

Stereotypical patterns of epileptiform calcium signal in hippocampal CA1, CA3, dentate gyrus and entorhinal cortex in freely moving mice.

Epilepsy is a multi-etiological brain dysfunction syndrome. Hippocampal neuronal damage induced by seizures may be one of the causes leading to cognitive impairment, but the underlying mechanism remains to be further elucidated. The kainic acid (KA) model of temporal lobe epilepsy is widely used in understanding of the epileptogenesis. Fiber photometry is a signal detection technology suitable for recording calcium activity of neurons in the deep brain of freely moving animal. Here, we used the optical fiber-based method to monitor the real-time neuronal population activities of freely moving mice after subcutaneous injection of KA. We observed that KA administration led to one to three kinds of stereotypical patterns of epileptiform calcium activity in CA1, CA3, and dentate gyrus (DG) of the hippocampus, as well as the entorhinal cortex (EC). There were three kinds of waves in the hippocampal CA1, which we named wave 1, wave 2 and slow flash. Wave 1 and wave 2 appeared in both the CA3 and DG regions, but the EC only showed wave 1. In these epileptiform calcium signals, we observed a high amplitude and long duration calcium wave as a part of wave 2, which resembled cortical spreading depression (CSD) and always appeared at or after the end of seizure. Because the same characteristic of epileptiform calcium signal appeared in different brain regions, calcium signal may not exist with region specificity, but may exhibit a cell type specific manner. Thus, our work provides a support for the pathogenesis of epilepsy and epileptiform signal transmission research.

Cortical Plasticity Induced by Anodal Transcranial Pulsed Current Stimulation Investigated by Combining Two-Photon Imaging and Electrophysiological Recording.

Anodal-transcranial pulsed current stimulation (a-tPCS) has been used in human studies to modulate cortical excitability or improve behavioral performance in recent years. Multiple studies show crucial roles of astrocytes in cortical plasticity. The calcium activity in astrocytes could regulate synaptic transmission and synaptic plasticity. Whether the astrocytic activity is involved in a-tPCS-induced cortical plasticity is presently unknown. The purpose of this study is to investigate the calcium responses in neurons and astrocytes evoked by a-tPCS with different current intensities, and thereby provides some indication of the mechanisms underlying a-tPCS-induced cortical plasticity. Two-photon calcium imaging was used to record the calcium responses of neurons and astrocytes in mouse somatosensory cortex. Local field potential (LFP) evoked by sensory stimulation was used to assess the effects of a-tPCS on plasticity. We found that long-duration a-tPCS with high-intensity current could evoke large-amplitude calcium responses in both neurons and astrocytes, whereas long-duration a-tPCS with low-intensity current evoked large-amplitude calcium responses only in astrocytes. The astrocytic Ca2+ elevations are driven by noradrenergic-dependent activation of the alpha-1 adrenergic receptors (A1ARs), while the intense Ca2+ responses of neurons are driven by action potentials. LFP recordings demonstrated that low-intensity a-tPCS led to enhancement of cortical excitability while high-intensity a-tPCS resulted in diminution of cortical excitability. The results provide some evidence that the enhancement of a-tPCS-induced cortical excitability might be partly associated with calcium elevation in astrocytes, whereas the diminution of a-tPCS-induced cortical excitability might be caused by excessive calcium activity in neurons. These findings indicate that the appropriate current intensity should be used in the application of a-tPCS.

Status, sources, and risk assessment of polycyclic aromatic hydrocarbons in urban soils of Xi'an, China.

To identify status, source of polycyclic aromatic hydrocarbons (PAHs) in urban soils and to assess soil environmental quality in Xi'an City, China, total 45 soil samples were collected from surface layer (0-10 cm) in different functional areas. Total concentrations of 16 US EPA priority PAHs ranged from 149.9 to 5770 µg kg-1, with a mean of 1246 µg kg-1. High molecular weight (HMW) PAHs accounted for the majority (42.4-72.2%) of the total PAHs in the urban soils, and phenanthrene (Phe), fluorene (Flo), pyrene (Pyr), benzo(b)fluoranthene (BbF), and chrysene (Chr) were the major compounds. Concentrations of PAHs varied among different functional areas. High level of PAHs was particularly apparent in industrial zones and city road overpass, while low level was recorded in scenic spots and campus. The integration of isomer ratios, principal component analysis (PCA), and positive matrix factor (PMF) indicated that the sources of PAHs in Xi'an urban soils were mainly derived from vehicle emissions and coal combustion. Based on incremental lifetime cancer risks (ILCR) model, the urban soils from the three functional areas (industrial zone, urban road, and city road overpass) posed potential cancer risk, and the cancer risks of direct ingestion for children were apparently higher than that for adolescence and for adult, respectively. Therefore, attention should be paid to the health risk for children exposed to PAHs in the urban soils.

[Effects of lipoic acid on cytokines and chemokines in astrocytes stimulated with lipopolysaccharide].

OBJECTIVE: To investigate the effects of lipoic acid (LA) on the release of TNF-α, IL-1ß, IL-6, IL-10 and the expressions of chemokines in astrocytes stimulated with lipopolysaccharide (LPS). METHODS: Astrocytes were separated from the cerebral cortex of newly-born C57BL/6 mice (within 48 h after birth). After identification and purification, the second-generation astrocytes were stimulated with LPS (1 µg/mL), and then treated with LA (100 µg/mL). The production of nitric oxide (NO) was assayed by Griess assay. The levels of TNF-α, IL-1ß, IL-6 and IL-10 in supernatants were quantified by ELISA. The expressions of CC chemokine ligand-20 (CCL20), monocyte chemoattractive protein 1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) mRNAs were detected using reverse transcription-PCR. RESULTS: Compared with PBS control, LPS significantly increased the production of TNF-α, IL-1ß and IL-6, but decreased the level of IL-10 in cultured astrocytes (P<0.05). LA treatment inhibited LPS-induced NO, TNF-α, IL-1ß and IL-6 production, and enhanced IL-10 secretion, and compared with LPS stimulation alone, the differences were statistically significant (P<0.05). In addition, LA treatment also suppressed the expressions of CCL20, MCP-1 and MIP-1α mRNA in astrocytes stimulated with LPS. CONCLUSION: LA inhibits neuroinflammatory response in LPS-activated astrocytes. The neuroprotection of LA is partly due to the inhibition of pro-inflammatory cytokines and chemokines derived from astrocytes.

Targeting the shift from M1 to M2 macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil.

We observed the therapeutic effect of Fasudil and explored its mechanisms in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Fasudil, a selective Rho kinase (ROCK) inhibitor, was injected intraperitoneally at 40 mg/kg/d in early and late stages of EAE induction. Fasudil ameliorated the clinical severity of EAE at different stages, and decreased the expression of ROCK-II in spleen, accompanied by an improvement in demyelination and inhibition of inflammatory cells. Fasudil mainly inhibited CD4(+)IL-17(+) T cells in early treatment, but also elevated CD4(+)IL-10(+) regulatory T cells and IL-10 production in late treatment. The treatment of Fasudil shifted inflammatory M1 to anti-inflammatory M2 macrophages in both early and late treatment, being shown by inhibiting CD16/32, iNOS, IL-12, TLR4 and CD40 and increasing CD206, Arg-1, IL-10 and CD14 in spleen. By using Western blot and immunohistochemistry, iNOS and Arg-1, as two most specific markers for M1 and M2, was inhibited or induced in splenic macrophages and spinal cords of EAE mice treated with Fasudil. In vitro experiments also indicate that Fasudil shifts M1 to M2 phenotype, which does not require the participation or auxiliary of other cells. The polarization of M2 macrophages was associated with the decrease of inflammatory cytokine IL-1ß, TNF-α and MCP-1. These results demonstrate that Fasudil has therapeutic potential in EAE possibly through inducing the polarization of M2 macrophages and inhibiting inflammatory responses.

[Effect of Fasudil on the phenotype conversion of LPS-stimulated BV-2 microglia].

AIM: To explore the effect of Fasudil on LPS-stimulated BV-2 microglia in inflammatory reaction and phenotype conversion. METHODS: The routinely cultured BV-2 microglia in vitro were divided into PBS control group, PBS plus Fasudil treatment group, LPS stimulation group and LPS plus Fasudil group. We determined the production of NO by Griess reaction, the level of TNF-α by ELISA, and analyzed the M1 and M2 phenotypes of microglia by flow cytometry. RESULTS: The treatment of LPS lead to the characteristics of M1 phenotype in BV-2 microglia. Fasudil inhibited the production of NO and the release of TNF-α in LPS-stimulated BV-2 microglia. Interestingly, Fasudil transformed inflammatory M1 cells to anti-inflammatory M2 cells. CONCLUSION: Fasudil shows an anti-inflammatory effect, which may be associated with the conversion of inflammatory M1 microglia to anti-inflammatory M2 cells.

Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis, acting possibly through antiinflammatory effect.

AIM: The purpose of this investigation was to further explore the mechanism(s) underlying the amelioration in EAE caused by Fasudil, particularly focusing on anti-inflammatory effect. METHODS: We induced a chronic-progressive experimental autoimmune encephalomyelitis (EAE) in B6 mice immunized with myelin oligodendrocyte glycoprotein(35-55) and performed Fasudil intervention in early and late stages of the disease. RESULTS: The administration of Fasudil (40 mg/kg, i.p) had a therapeutic effect in delaying the onset and ameliorating the severity of EAE, accompanied by the improvement in myelination and the decrease in inflammatory cells in spinal cords. Fasudil inhibited TLR-4, p-NF-kB/p65, and inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and enhanced IL-10 production in spinal cords. The ratio of arginase/iNOS was enhanced mainly in the spinal cords of EAE mice treated with Fasudil, reflecting a shift toward the M2 (antiinflammation) macrophage/microglia phenotype. The administration of Fasudil also induced the upregulation of CB2 receptor in spinal cords, but did not significantly trigger CB1 receptor. Levels of neurotrophic factors NGF, BDNF, and GDNF in the CNS were not altered by Fasudil. CONCLUSION: Fasudil ameliorates disease progression in EAE, acting possibly through antiinflammatory pathway.