Yes-associated protein regulates glutamate homeostasis through promoting the expression of excitatory amino acid transporter-2 in astrocytes via ß-catenin signaling.

The homeostasis of glutamate is mainly regulated by the excitatory amino acid transporters (EAATs), especially by EAAT2 in astrocytes. Excessive glutamate in the synaptic cleft caused by dysfunction or dysregulation of EAAT2 can lead to excitotoxicity, neuronal death and cognitive dysfunction. However, it remains unclear about the detailed regulation mechanism of expression and function of astrocytic EAAT2. In this study, first, we found increased neuronal death and impairment of cognitive function in YAPGFAP -CKO mice (conditionally knock out Yes-associated protein [YAP] in astrocytes), and identified EAAT2 as a downstream target of YAP through RNA sequencing. Second, the expression of EAAT2 was decreased in cultured YAP-/- astrocytes and the hippocampus of YAPGFAP -CKO mice, and glutamate uptake was reduced in YAP-/- astrocytes, but increased in YAP-upregulated astrocytes. Third, further investigation of the mechanism showed that the mRNA and protein levels of ß-catenin were decreased in YAP-/- astrocytes and increased in YAP-upregulated astrocytes. Wnt3a activated YAP signaling and up-regulated EAAT2 through ß-catenin. Furthermore, over-expression or activation of ß-catenin partially restored the downregulation of EAAT2, the impairment of glutamate uptake, neuronal death and cognitive decline that caused by YAP deletion. Finally, activation of EAAT2 also rescued neuronal death and cognitive decline in YAPGFAP -CKO mice. Taken together, our study identifies an unrecognized role of YAP signaling in the regulation of glutamate homeostasis through the ß-catenin/EAAT2 pathway in astrocytes, which may provide novel insights into the pathogenesis of brain diseases that closely related to the dysfunction or dysregulation of EAAT2, and promote the development of clinical strategy.

Enamel matrix derivative expedites osteogenic differentiation of BMSCs via Wnt/ß-catenin pathway in high glucose microenvironment.

INTRODUCTION: The influence of enamel matrix derivative (EMD) on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was explored in high glucose (HG) microenvironment with interaction of Wnt/ß-catenin pathway. MATERIALS AND METHODS: Extraction of BMSCs from Sprague-Dawley rats, culture, and identification were manifested. The cells were treated with different concentration of EMD in HG to figure out the most available concentration for proliferation and osteogenic differentiation. Then, observation of cell growth curve and cell cycle changes, and detection of Osterix, runt-related transcription factor 2 (Runx2), COL-I, early osteogenic indexes, Calcium salt deposition, and ß-catenin protein in Wnt/ß-catenin pathway were assured. After adding Wnt/ß-catenin pathway inhibitor (XAV-939) in the cells with osteogenesis induction, detection of binding of ß-catenin to Osterix was clarified. RESULTS: Via identification BMSCs cultured in vitro was qualified. Different concentrations of EMD could accelerate cell proliferation in HG and osteogenesis induction, and 75 µg/mL EMD had the best effect. The HG augmented BMSCs proliferation and the propidium iodide index of flow cytometry cycle was elevated in HG, which were strengthened via the EMD. After BMSCs' osteogenesis induction, Osterix, Runx2, CoL-1, early osteogenic indexes, and calcium salt deposition were reduced, but elevated via EMD. ß-Catenin was the lowest in the HG, but elevated after EMD. After addition of XAV-939, reduction of ß-catenin and the downstream (Osterix and Runx2) were manifested. Detection of binding protein bands was in ß-catenin and Osterix of the HG after EMD treatment. CONCLUSION: EMD may facilitate the osteogenic differentiation of BMSCs via activating the Wnt/ß-catenin pathway in HG.

Astrocytic YAP Promotes the Formation of Glia Scars and Neural Regeneration after Spinal Cord Injury.

Yes-associated protein (YAP) transcriptional coactivator is negatively regulated by the Hippo pathway and functions in controlling the size of multiple organs, such as liver during development. However, it is not clear whether YAP signaling participates in the process of the formation of glia scars after spinal cord injury (SCI). In this study, we found that YAP was upregulated and activated in astrocytes of C57BL/6 male mice after SCI in a Hippo pathway-dependent manner. Conditional knockout (KO) of yap in astrocytes significantly inhibited astrocytic proliferation, impaired the formation of glial scars, inhibited the axonal regeneration, and impaired the behavioral recovery of C57BL/6 male mice after SCI. Mechanistically, the bFGF was upregulated after SCI and induced the activation of YAP through RhoA pathways, thereby promoting the formation of glial scars. Additionally, YAP promoted bFGF-induced proliferation by negatively controlling nuclear distribution of p27Kip1 mediated by CRM1. Finally, bFGF or XMU-MP-1 (an inhibitor of Hippo kinase MST1/2 to activate YAP) injection indeed activated YAP signaling and promoted the formation of glial scars and the functional recovery of mice after SCI. These findings suggest that YAP promotes the formation of glial scars and neural regeneration of mice after SCI, and that the bFGF-RhoA-YAP-p27Kip1 pathway positively regulates astrocytic proliferation after SCI.SIGNIFICANCE STATEMENT Glial scars play critical roles in neuronal regeneration of CNS injury diseases, such as spinal cord injury (SCI). Here, we provide evidence for the function of Yes-associated protein (YAP) in the formation of glial scars after SCI through regulation of astrocyte proliferation. As a downstream of bFGF (which is upregulated after SCI), YAP promotes the proliferation of astrocytes through negatively controlling nuclear distribution of p27Kip1 mediated by CRM1. Activation of YAP by bFGF or XMU-MP-1 injection promotes the formation of glial scar and the functional recovery of mice after SCI. These results suggest that the bFGF-RhoA-YAP-p27Kip1 axis for the formation of glial scars may be a potential therapeutic strategy for SCI patients.

Asymmetric Markovnikov Hydroaminocarbonylation of Alkenes Enabled by Palladium-Monodentate Phosphoramidite Catalysis.

A palladium-catalyzed asymmetric Markovnikov hydroaminocarbonylation of alkenes with anilines has been developed for the atom-economical synthesis of 2-substituted propanamides bearing an α-stereocenter. A novel phosphoramidite ligand L16 was discovered which exhibited very high reactivity and selectivity in the reaction. This asymmetric Markovnikov hydroaminocarbonylation employs readily available starting materials and tolerates a wide range of functional groups, thus providing a facile and straightforward method for the regio- and enantioselective synthesis of 2-substituted propanamides under ambient conditions. Mechanistic studies revealed that the reaction proceeds through a palladium hydride pathway.

Sphingosine 1-phosphate promotes the proliferation of olfactory ensheathing cells through YAP signaling and participates in the formation of olfactory nerve layer.

Olfactory ensheathing cells (OECs) are unique glial cells with axonal growth-promoting properties in the olfactory epithelium and olfactory bulb, covering the entire length of the olfactory nerve. The proliferation of OECs is necessary for the formation of the presumptive olfactory nerve layer (ONL) during development and OECs transplantation. However, the molecular mechanism underlying the regulation of OEC proliferation in the ONL still remains unknown. In the present study, we examined the role of sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) on OEC proliferation. Initially, reverse transcription-PCR (RT-PCR), western blot and immunostaining revealed that S1PRs were highly expressed in the OECs in vitro and in vivo. Furthermore, we found that S1P treatment promoted the proliferation of primary cultured OECs mediated by S1PR1. Mechanistically, yes-associated protein (YAP) was required for S1P-induced OEC proliferation through RhoA signaling. Finally, conditional knockout of YAP in OECs reduced OEC proliferation in ONL, which impaired the axonal projection and growth of olfactory sensory neurons, and olfactory functions. Taken together, these results reveal a previously unrecognized function of S1P/RhoA/YAP pathway in the proliferation of OECs, contributing to the formation of ONL and the projection, growth, and function of olfactory sensory neurons during development.

Voltammetric sensing of recombinant viral dengue virus 2 NS1 based on Au nanoparticle-decorated multiwalled carbon nanotube composites.

A homemade gold electrode is modified with a carbon nanotubes/gold nanoparticles nanocomposite to perform selective and sensitive electrochemical detection of dengue toxin. This nanostructured composite offers a large specific surface and a reactive interface allowing the immobilization of biological material. Dengue antibodies are immobilized on gold nanoparticles via covalent bonding for dengue toxin detection. The porous tridimensional network of carbon nanotubes and gold nanoparticles enhances the electrochemical signal and the overall performance of the sensor. After optimization, the system exhibits a high sensitivity of - 0.44 ± 0.01 µA per decade with wide linear range between 1 × 10-12 and 1 × 10-6 g/mL at a working potential of 0.22 V vs Ag/AgCl. The extremely low detection limit (3 × 10-13 g/mL) ranks this immunosensor as one of the most efficient reported in the literature for the detection of recombinant viral dengue virus 2 NS1. This biosensor also offers good selectivity, characterized by a low response to various non-specific targets and assays in human serum. The outstanding performances and the reproducibility of the system place the biosensor developed among the best candidates for future medical applications and for early diagnosis of dengue fever. Graphical abstract.

Structural Changes of Mercaptohexanol Self-Assembled Monolayers on Gold and Their Influence on Impedimetric Aptamer Sensors.

Despite a large number of publications describing biosensors based on electrochemical impedance spectroscopy (EIS), little attention has been paid to the stability and reproducibility issues of the sensor interfaces. In this work, the stability and reproducibility of faradaic EIS analyses on the aptamer/mercaptohexanol (MCH) self-assembled monolayer (SAM)-functionalized gold surfaces in ferri- and ferrocyanide solution were systematically evaluated prior to and after the aptamer-probe DNA hybridization. It is shown that the EIS data exhibited significant drift, and this significantly affected the reproducibility of the EIS signal of the hybridization. As a result, no significant difference between the charge transfer resistance (RCT) changes induced by the aptamer-target DNA hybridization and that caused by the drift could be identified. A conditioning of the electrode in the measurement solution for more than 12 h was required to reach a stable RCT baseline prior to the aptamer-probe DNA hybridization. The monitored drift in RCT and double layer capacitance during the conditioning suggests that the MCH SAM on the gold surface reorganized to a thinner but more closely packed layer. We also observed that the hot binding buffer used in the following aptamer-probe DNA hybridization process could induce additional MCH and aptamer reorganization, and thus further drift in RCT. As a result, the RCT change caused by the aptamer-probe DNA hybridization was less than that caused by the hot binding buffer (blank control experiment). Therefore, it is suggested that the use of high temperature in the EIS measurement should be carefully evaluated or avoided. This work provides practical guidelines for the EIS measurements. Moreover, because SAM-functionalized gold electrodes are widely used in biosensors, for example, DNA sensors, an improved understanding of the origin of the observed drift is very important for the development of well-functioning and reproducible biosensors.

NMDA receptors inhibit axonal outgrowth by inactivating Akt and activating GSK-3ß via calcineurin in cultured immature hippocampal neurons.

Neurons are highly polarized cells with an axon and dendritic arbors. It is still not well studied that how formation and elaboration of axon and dendrites is controlled by diffusible signaling factors such as glutamate via specific receptors. We found that N-methyl-D-aspartate (NMDA) receptors were enriched (stage 2-3) but decreased expression (stage 4-5) at tip of axon of cultured hippocampal neurons during distinct development stages. Inhibition of NMDA receptor activity by competitive antagonist DL-2-amino-5-phosphonovalerate (APV) or channel blocker MK801 promoted axonal outgrowth at the early stages, whereas inhibited dendritic development in later stages. Meanwhile, knockdown of NMDA receptors also promoted axonal outgrowth and branch in immature neurons. Furthermore, GluN2B but not GluN2A subunit inhibited axonal outgrowth in immature hippocampal neurons. Finally, we found that NMDA receptors inhibited axonal outgrowth by inactivating Akt and activating GSK-3ß signaling in a calcineurin-dependent manner. Taken together, our results demonstrate that stabilization GSK-3ß activation in the axon growth cone by Ca2+ influx through NMDA receptors may be involved in regulation of axon formation in immature neurons at early stages.

[Advances in the roles and mechanisms of nonsense-mediated mRNA decay in embryonic development].

Nonsense-mediated mRNA decay (NMD) is originally identified as a widespread mRNA surveillance machinery in degrading 'aberrant' mRNA species with premature termination codons (PTCs) rapidly, which protects the cells from the accumulation of truncated proteins. Recent studies show that NMD can also regulate the degradation of normal gene transcripts, which execute important cellular and physiological functions. Therefore, NMD is considered as a highly conserved post-transcriptional regulatory mechanism in eukaryotes. NMD modulates 3% to 20% of the transcriptome from yeast to human directly or indirectly, which is essential for various physiological processes, such as cell homeostasis, stress response, proliferation, and differentiation. NMD can regulate the level of transcripts that involves in development, and single knockout of most NMD factors has an embryonic lethal effect. NMD plays an important role in the self-renewal, differentiation of embryonic stem cells and is critical during embryonic development. In this review, we summarized the latest advances in the roles and mechanisms of NMD in embryonic development, in order to provide new ideas for the research on embryonic development and the treatment of embryonic development related diseases.

Nanocellulose Modified Polyethylene Separators for Lithium Metal Batteries.

Poor cycling stability and safety concerns regarding lithium (Li) metal anodes are two major issues preventing the commercialization of high-energy density Li metal-based batteries. Herein, a novel tri-layer separator design that significantly enhances the cycling stability and safety of Li metal-based batteries is presented. A thin, thermally stable, flexible, and hydrophilic cellulose nanofiber layer, produced using a straightforward paper-making process, is directly laminated on each side of a plasma-treated polyethylene (PE) separator. The 2.5 µm thick, mesoporous (≈20 nm average pore size) cellulose nanofiber layer stabilizes the Li metal anodes by generating a uniform Li+ flux toward the electrode through its homogenous nanochannels, leading to improved cycling stability. As the tri-layer separator maintains its dimensional stability even at 200 °C when the internal PE layer is melted and blocks the ion transport through the separator, the separator also provides an effective thermal shutdown function. The present nanocellulose-based tri-layer separator design thus significantly facilitates the realization of high-energy density Li metal-based batteries.

[Study on the Relationship Between Normalization of Tumor Microvessels and CA9 for Rh-Endostatin to Inhibit Lewis Lung Cancer].

OBJECTIVES: To explore the relationship between normalization of tumor microvessels and CA9 for rh-Endostatin to inhibit Lewis lung cancer (LLC) and the expression level of CA9 in LLC. METHODS: Lewis cells of logarithmic growth phase were collected and made into 1×106 mL-1 cell suspensions were prepared. The transplanted tumor model of LLC was established on C57/BL6 mice by injected 0.2 mL cell suspensions/mice into 40 C57/BL6 mice. 40 LLC mice were randomly divided into control group and rh-ES group (20 mice per group). Control group experienced treatment of intraperitoneal injection (ip) for 0.2 mL NS/d, while rh-ES group was treated for 5 mg rh-ES/(kg·d) from the first to the ninth day. The samples of 5 mice were obtained from day 2, day 4, day 6 and day 9 after treatment in control group or rh-ES group, respectively. CA9 was tested by IHC in LLC and paracancerous tissues and estimated by RT-PCR and ELISA in the each time point of both rh-ES group and control group,respectively. RESULTS: The transplanted tumor model of LLC on C57/BL6 mice was established successfully. The expression of CA9 decreased on day 4 and day 6 in rh-ES group estimated by RT-PCR and ELISA, which indicated some great significance when compared with day 2, day 9 in rh-ES group and day 4, day 6 in control group (P<0.05), and the expression of CA9 in day 2, day 4, day 6, day 9 tested by IHC was higher in LLC than in paracancerous tissues in control group (P<0.05). CONCLUSIONS: The expression of CA9 was higher in LLC. Rh-ES could have positive effect on LLC model of C57/BL6 mice, in day 4-6 (a brief normalized time course) decreased the expression of CA9 and reversed the tumor hypoxia.

Iron Carbide Nanoparticles Encapsulated in Mesoporous Fe-N-Doped Carbon Nanofibers for Efficient Electrocatalysis.

Exploring low-cost and high-performance nonprecious metal catalysts (NPMCs) for oxygen reduction reaction (ORR) in fuel cells and metal-air batteries is crucial for the commercialization of these energy conversion and storage devices. Here we report a novel NPMC consisting of Fe3 C nanoparticles encapsulated in mesoporous Fe-N-doped carbon nanofibers, which is synthesized by a cost-effective method using carbonaceous nanofibers, pyrrole, and FeCl3 as precursors. The electrocatalyst exhibits outstanding ORR activity (onset potential of -0.02 V and half-wave potential of -0.140 V) closely comparable to the state-of-the-art Pt/C catalyst in alkaline media, and good ORR activity in acidic media, which is among the highest reported activities of NPMCs.

[Hyoid epiglottis combined with sternocleidomastoid muscle clavicle flap for repairing laryngeal defect after laryngeal trauma infection: a case report].

The initial treatment of open laryngeal trauma must be implemented immediately, with the primary focus on saving lives. However, in the later stages, various factors may cause changes in the structure and function of the larynx, which requires special attention. This article reports on the treatment process of a patient with depression who suffered from laryngeal trauma. Due to the late stage of laryngeal infection causing laryngeal defects, a hyoid epiglottis combined with sternocleidomastoid muscle clavicular flap repair was performed. Additionally, personalized functional exercise was performed, ultimately resulting in recovery.

Polycarboxyl ionic liquid functionalized Yb-MOFs nanoballs based dual-wavelength responsive photoelectrochemical aptasensor for the simultaneous determination of AFB1 and OTA.

Developing an accurate and precise approach for the simultaneous detection of ochratoxin A (OTA) and aflatoxin B1 (AFB1) is significant for food safety surveillance. Herein, a photoelectrochemical sensing platform was constructed based on polycarboxylic ionic liquid functionalized metal-organic framework integrated with gold nanoparticles (Yb-MOFs@AuNPs). Sulfhydryl functionalized hairpin DNA (hDNA) was immobilized on a Yb-MOFs@AuNPs modified glassy carbon electrode (GCE) surface through Au-S bond. After blocking residual active binding sites with BSA, gold nanoparticles-labeled AFB1 aptamer (AuNPs-Apt 1) and gold nanorods-labeled OTA aptamer (AuNRs-Apt 2) were introduced to construct a photoelectrochemical aptasensor for the simultaneous determination of AFB1 and OTA. Due to the surface plasmon resonance effect and the nanometer size effect of gold nanomaterials, the photoelectrochemical aptasensor can output photocurrent responses as being excited with different wavelengths at 520 nm and 808 nm, respectively. When the AFB1 and OTA concentration in the range of 0.001-50.0 ng mL-1, a good linear relationship between the photocurrent difference (ΔI) before and after recognizing targets and the logarithm of AFB1 or OTA concentration was obtained. The detection limits for AFB1 and OTA were 0.40 pg mL-1 and 0.19 pg mL-1, respectively. AFB1 and OTA in corn samples were detected simultaneously by the photoelectrochemical aptasensor.

Erratum: ß-elemene promotes the senescence of glioma cells through regulating YAP-CDK6 signaling.

[This corrects the article on p. 370 in vol. 11, PMID: 33575077.].

Bufei Yishen Formula Inhibits the Cell Senescence in COPD by Up-Regulating the ZNF263 and Klotho Expression.

Background: Bufei Yishen formula (BYF) is an effective prescription for the clinical treatment of chronic obstructive pulmonary disease (COPD). However, the molecular mechanism by which it exerts its pharmacological effects remains to be explored. Methods: The human bronchial cell line BEAS-2B was treated with cigarette smoke extract (CSE). Cellular senescence markers were detected by Western blot and ELISA. Potential transcription factor of klotho was predicted using JASPAR and USCS databases. Results: CSE induced cellular senescence with intracellular accumulation of cellular senescence biomarkers (p16, p21 and p27) and increased secretion of senescence-related secretory phenotypic (SASP) factors (IL-6, IL-8, and CCL3). In contrast, BYF treatment inhibited CSE-induced cellular senescence. CSE suppressed the transcription, expression and secretion of klotho, whereas BYF treatment rescued its transcription, expression and secretion. CSE downregulated the protein level of ZNF263, whereas BYF treatment rescued the expression of ZNF263. Furthermore, ZNF263-overexpressing BEAS-2B cells could inhibit CSE-induced cellular senescence and SASP factor secretion by upregulating the expression of klotho. Conclusion: This study revealed a novel pharmacological mechanism by which BYF alleviates clinical symptoms of COPD patients, and regulating ZNF263 and klotho expression may be beneficial to the treatment and prevention of COPD.

Advanced prodrug strategies in nucleoside analogues targeting the treatment of gastrointestinal malignancies.

Gastrointestinal malignancies are common digestive system tumor worldwide. Nucleoside analogues have been widely used as anticancer drugs for the treatment of a variety of conditions, including gastrointestinal malignancies. However, low permeability, enzymatic deamination, inefficiently phosphorylation, the emergence of chemoresistance and some other issues have limited its efficacy. The prodrug strategies have been widely applied in drug design to improve pharmacokinetic properties and address safety and drug-resistance issues. This review will provide an overview of the recent developments of prodrug strategies in nucleoside analogues for the treatment of gastrointestinal malignancies.

A near-infrared photoelectrochemical aptasensing system based on Bi2O2S nanoflowers and gold nanoparticles for high-performance determination of MCF-7 cells.

Circulating tumor cells (CTCs) are commonly considered as the major cause of tumor metastasis and can eventually lead to death. Therefore, developing a high-performance method for the determination of CTCs is very significant for promoting the cancer survival rate. Photoelectrochemical biosensing systems have been extensively investigated and applied for bioassays. Herein, Bi2O2S nanoflowers were successfully prepared through a simple one-step hydrothermal method. After being integrated with gold nanoparticles with a diameter of ∼45 nm, AuNPs/Bi2O2S nanocomposites were coated onto an ITO electrode surface to build a photoelectrochemical sensing platform which can be excited with near-infrared light to produce photocurrent response. Subsequently, mercapto-group functionalized aptamer (SH-Apt) was fixed onto the AuNPs/Bi2O2S/ITO surface. Due to the overexpress of MUC1 protein in the cell membrane, MCF-7 cells were specifically trapped on the SH-Apt/AuNPs/Bi2O2S/ITO surface. The introduce of MCF-7 cells lead to an obvious decrease on the photocurrent response. The photocurrent variation shows a satisfied linear relationship to the logarithm of MCF-7 cells concentration ranged from 50 to 6 × 105 cell mL-1. The detection limit obtained is 17 cell mL-1. The PEC biosensor shows excellent sensitivity, selectivity and stability for sensing MCF-7 cells, even for determining MCF-7 cells in clinical serum samples.

SARM1 promotes the neuroinflammation and demyelination through IGFBP2/NF-κB pathway in experimental autoimmune encephalomyelitis mice.

AIM: Multiple sclerosis (MS) is an autoimmune disease, and its typical characteristics are neuroinflammation and the demyelination of neurons in the central nervous system (CNS). Sterile alpha and TIR motif containing 1 (SARM1) is an essential factor mediating axonal degeneration and SARM1 deletion reduces the neuroinflammation in spinal cord injury. This study aimed to explore the roles of SARM1 and its underlying mechanisms in MS. METHODS: Experimental autoimmune encephalomyelitis (EAE, a model of MS) model was established. Immunostaining, western blot, electron microscope, and HE staining were used to examine the pathological manifestations such as inflammation, demyelination, and neuronal death in SARM1f/f EAE mice and SARM1Nestin -CKO EAE mice. In addition, RNA-seq, real-time PCR and double-immunostaining were used to examine the underlying mechanism of SARM1 in EAE mice. RESULTS: SARM1 was upregulated in neurons of the spinal cords of EAE mice. SARM1 knockout in CNS ameliorated EAE with less neuroinflammation, demyelination, and dead neurons. Mechanically, SARM1 knockout resulted in the reduction of insulin-like growth factor (IGF)-binding protein 2 (IGFBP2) in neurons of EAE mice, which might inhibit the neuroinflammation through inhibiting NF-κB signaling. Finally, activation of NF-κB partially aggravated the neuroinflammation and demyelination deficits of SARM1Nestin -CKO EAE mice. CONCLUSIONS: These results identified the unknown role of SARM1 in the promotion of neuroinflammation and demyelination and revealed a novel drug target pathway of SARM1/IGFBP2/NF-κB for MS.