The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy.

BACKGROUND: Clinically, Charcot-Marie-Tooth disease (CMT)-associated muscle atrophy still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT type 4F (CMT4F) by destroying the myelin sheath form, which may be related to the inhibitory role of Ezrin in the self-association of L-periaxin. However, it is still unknown whether L-periaxin and Ezrin are independently or interactively involved in the process of muscle atrophy by affecting the function of muscle satellite cells. METHOD: A gastrocnemius muscle atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells were treated with adenovirus-mediated overexpression or knockdown of Ezrin. Then, overexpression of L-periaxin and NFATc1/c2 or knockdown of L-periaxin and NFATc3/c4 mediated by adenovirus vectors were used to confirm their role in Ezrin-mediated myoblast differentiation, myotube formation and gastrocnemius muscle repair in a peroneal nerve injury model. RNA-seq, real-time PCR, immunofluorescence staining and Western blot were used in the above observation. RESULTS: For the first time, instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/fusion in vitro. In vivo transduction of adenovirus vectors carrying Ezrin, but not Periaxin, into the gastrocnemius muscle in a peroneal nerve injury model increased the numbers of muscle myosin heavy chain (MyHC) I and II type myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of overexpressed Ezrin combined with incubation of knockdown L-periaxin within the injured peroneal nerve or injection of knockdown L-periaxin into peroneal nerve-injured gastrocnemius muscle not only increased the number of muscle fibers but also recovered their size to a relatively normal level in vivo. Overexpression of Ezrin promoted myoblast differentiation/fusion, inducing increased MyHC-I+ and MyHC-II + muscle fiber specialization, and the specific effects could be enhanced by the addition of adenovirus vectors for knockdown of L-periaxin by shRNA. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by knockdown of Ezrin by shRNA in vitro but decreased myotube length and size. Mechanistically, overexpressing Ezrin did not alter protein kinase A gamma catalytic subunit (PKA-γ cat), protein kinase A I alpha regulatory subunit (PKA reg Iα) or PKA reg Iß levels but increased PKA-α cat and PKA reg II α levels, leading to a decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the effects of overexpressing-Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by an increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, overexpressing Ezrin enhanced type I muscle fiber specialization, accompanied by an increase in NFATc2/c3 levels and a decrease in NFATc1 levels. Furthermore, overexpressing NFATc2 or knocking down NFATc3 reversed the inhibitory effects of Ezrin knockdown on myoblast differentiation/fusion. CONCLUSIONS: The spatiotemporal pattern of Ezrin/Periaxin expression was involved in the control of myoblast differentiation/fusion, myotube length and size, and myofiber specialization, which was related to the activated PKA-NFAT-MEF2C signaling pathway, providing a novel L-Periaxin/Ezrin joint strategy for the treatment of muscle atrophy induced by nerve injury, especially in CMT4F.

Continuous exposure to isoprenaline reduced myotube size by delaying myoblast differentiation and fusion through the NFAT-MEF2C signaling pathway.

We aimed to explore whether superfluous sympathetic activity affects myoblast differentiation, fusion, and myofiber types using a continuous single-dose isoprenaline exposure model in vitro and to further confirm the role of distinct NFATs in ISO-mediated effects. Compared with delivery of single and interval single, continuous single-dose ISO most obviously diminished myotube size while postponing myoblast differentiation/fusion in a time- and dose-dependent pattern, accompanied by an apparent decrease in nuclear NFATc1/c2 levels and a slight increase in nuclear NFATc3/c4 levels. Overexpression of NFATc1 or NFATc2, particularly NFATc1, markedly abolished the inhibitory effects of ISO on myoblast differentiation/fusion, myotube size and Myh7 expression, which was attributed to a remarkable increase in the nuclear NFATc1/c2 levels and a reduction in the nuclear NFATc4 levels and the associated increase in the numbers of MyoG and MEF2C positive nuclei within more than 3 nuclei myotubes, especially in MEF2C. Moreover, knockdown of NFATc3 by shRNA did not alter the inhibitory effect of ISO on myoblast differentiation/fusion or myotube size but partially recovered the expression of Myh7, which was related to the slightly increased nuclear levels of NFATc1/c2, MyoG and MEF2C. Knockdown of NFATc4 by shRNA prominently increased the number of MyHC +, MyoG or MEF2C + myoblast cells with 1 ~ 2 nuclei, causing fewer numbers and smaller myotube sizes. However, NFATc4 knockdown further deteriorated the effects of ISO on myoblast fusion and myotube size, with more than 5 nuclei and Myh1/2/4 expression, which was associated with a decrease in nuclear NFATc2/c3 levels. Therefore, ISO inhibited myoblast differentiation/fusion and myotube size through the NFAT-MyoG-MEF2C signaling pathway.

Electrothermal Desolvation-Enhanced Dielectric Barrier Discharge Plasma-Induced Vapor Generation for Sensitive Determination of Antimony by Atomic Fluorescence Spectrometry.

A novel simple electrothermal desolvation-enhanced dielectric barrier discharge plasma-induced vapor generation (ETD-DBD-PIVG) method has been developed for sensitive Sb determination by atomic fluorescence spectrometry (AFS). In our proposed ETD-DBD-PIVG, 20 µL sample solution was dried first; then, the resulting solution residue was directly converted into molecular volatile species efficiently through the interactions with hydrogen-doped DBD plasma; and finally, it was transported to AFS for detection. It was found that the desolvation process could greatly enhance Sb vapor generation, and the Sb fluorescence signal intensity is almost independent of its speciation, where comparable sensitivity is achieved for Sb(III) and Sb(V), enabling efficient total Sb detection without pre-reduction. Influencing parameters were evaluated in detail, including heating time, discharge gap, solution pH, and flow rates of argon and hydrogen, as well as coexisting ion interference. Under optimized conditions, the limit of detection was calculated as 0.86 µg L-1 (17.2 pg) for Sb. The accuracy of the proposed method was validated by the analysis of certified reference materials of simulated natural water samples and several river water samples. Compared with conventional hydride generation, the new ETD-DBD-PIVG offers an alternative green vapor generation technique with several advantages: (1) it eliminates the use of a sample flow system (e.g., no use of any syringe or peristaltic pump); instead, 20 µL of a sample is directly pipetted onto the glass plate for analysis; (2) it greatly simplifies the sample pretreatment steps as no pre-reduction process is needed; (3) it is sensitive and suitable for volume-limited sample analysis: efficient Sb vapor generation without chemical reducing reagents in ETD-DBD-PIVG enables Sb detection with an absolute limit at the picogram level. All the results demonstrate that the proposed method provides a simple, green, and sensitive method for Sb determination and it can also be extended to other elements such as Cd and As.

Direct and Sensitive Determination of Antimony in Water by Hydrogen-Doped Solution Anode Glow Discharge-Optical Emission Spectrometry Without Hydride Generation.

In the present work, a novel, simple, and sensitive method for the direct determination of trace Sb in water samples was developed based on hydrogen-doped solution anode glow discharge-optical emission spectrometry (SAGD-OES). It was found that the vapor generation and excitation of Sb occurred simultaneously in the SAGD, contributing to the significant improvement in the sensitivity of Sb as compared with normal pure He-operated SAGD or solution cathode glow discharge. Besides, the proposed hydrogen-doped SAGD-OES could be operated even at pH = 14, which could reduce the interference of coexisting ions as many metal ions could be precipitated and removed. Our results demonstrated that the proposed method offered good tolerance to the interferences of Li, Na, Ca, Mg, Fe, Ni, Mn, and Zn ions even at a concentration of 50 mg L-1. Under optimized conditions, the limit of detection of Sb was 0.85 µg L-1, which was comparable to that of microplasma sources coupled with conventional hydride generation. The linearity of the Sb calibration curve reached R2 > 0.999 in the 5-5000 µg L-1 range. Finally, the accuracy of the proposed method was validated by the determination of certified reference materials [GSB 07-1376-2001 (1) and (2))] and real water samples. The proposed low-power (6 W), green, sensitive, rapid, and robust method provides a promising approach for on-site trace Sb analysis and may also be extended to other elements.

Development of a Portable Method for Serum Lithium Measurement Based on Low-Cost Miniaturized Ultrasonic Nebulization Coupled with Atmospheric-Pressure Air-Sustained Discharge.

An accurate, rapid but cheap, and portable method for monitoring of serum lithium (Li) is highly desirable for mental patients who take Li medicine for treatment. Conventional techniques are usually bulky, costly, and cannot provide on-site real-time measurements. Herein, a miniaturized, reliable, cost-effective, and portable optical emission method for rapid and sensitive determination of serum Li was developed based on a combination of miniaturized ultrasonic nebulization (MUN) and a low-power (≈22 W) atmospheric-pressure air-sustained discharge (APAD) excitation source. The proposed method eliminates the use of any compressed gas or pump and can achieve serum Li detection within 40 s with low sample consumption (less than 20 µL serum). Except for dilution with water, no extra treatment is needed for serum Li analysis by MUN-APAD-OES. In addition, it offers a significant advantage of good tolerance to the coexisting high concentration of Na, K, Ca, and Mg, which is in contrast with the obvious matrix effect encountered in conventional inductively coupled plasma optical emission spectrometry (ICP-OES). Different operating parameters affecting the performance of MUN-APAD-OES were evaluated. Under optimized conditions, the detection limit of Li (670.8 nm) was calculated to be 0.6 µg L-1 (6 µg L-1 in serum). Finally, the accuracy of the proposed method was validated by the analysis of two certified reference materials (Seronorm serum L-1 and L-2 RUO), six real human serum samples, and eight real animal serum samples. All of the results indicate that the low-cost and low-power MUN-APAD-OES provides a promising reliable method for on-site serum Li measurement and may also be extended to other elements.

Development of an Automatic Column Chromatography Separation Device for Metal Isotope Analysis Based on Droplet Counting.

A novel, simple, cost-effective, reliable, and practical automatic column chromatography separation device capable of simultaneously purifying samples for radiogenic and non-traditional stable isotope analysis has been developed. The device avoids the use of any pump and features eluent driving by the siphon effect (gravity) and quantitative control by infrared droplet counting. Several factors affecting the control of droplets were investigated, including types and concentrations of eluents and the height of the liquid level. Results showed that accurate dripping of the eluent could be readily achieved by controlling the number of droplets under selected conditions. The separation performance of the device was first demonstrated by the elution of Sr and Cd in synthetic matrix solutions. The recoveries of Sr and Cd samples were better than 87.6 and 95.0%, respectively, and the whole procedure blank was about 0.3 ng for Sr and 0.1 ng for Cd. Finally, the reliability of the device was further validated by the purification of Sr and Cd from different geological reference materials (NIST 2711a, Nod-A-1, BCR-2, and BHVO-2). The determined Cd and Sr isotope values agree well with their reference values within the uncertainty range. All these results clearly demonstrate the reliability and practicability of the proposed device, which provides a promising method for the automated purification of isotope samples.

The detrimental effects of stress-induced glucocorticoid exposure on mouse uterine receptivity and decidualization.

Glucocorticoids (GCs), stress-induced steroid hormones, are released by adrenal cortex and essential for stress adaptation. Recently, there has been renewed interest in the relationship between GCs and pregnancy following the discovery that glucocorticoid receptor is necessary for implantation. It has been widely recognized that stress is detrimental to pregnancy. However, effects of stress-induced GC exposure on uterine receptivity and decidualization are still poorly understood. This study aims to explore the effects of GCs exposure on uterine receptivity, decidualization, and their underlying mechanisms in mice. Single prolonged stress (SPS) and corticosterone (Cort) injection models were used to analyze effects of GC exposure on early pregnancy, respectively. SPS or Cort injection inhibits embryo implantation by interfering Lif signaling and stimulating the uterine deposition of collagen types I, III, and IV on day 4 of pregnancy. Uterine decidualization is also attenuated by SPS or Cort injection through suppressing Cox-2 expression. Cort-induced collagen disorder also suppresses decidualization through regulating mesenchymal-epithelial transition. Our data should shed lights for a better understanding for the effects of GCs on embryo implantation for clinical research.

Progesterone-regulated Hsd11b2 as a barrier to balance mouse uterine corticosterone.

Glucocorticoids (GCs) are essential for mouse embryo implantation and decidualization. Excess GCs are harmful for mouse embryo implantation and decidualization. 11ß-Hydroxysteroid dehydrogenases type I and II (Hsd11b1/Hsd11b2) are main enzymes for regulating local level of GCs. Hsd11b2 acts as the placental glucocorticoid barrier to protect the fetus from excessive exposure. Although effects of GCs on the fetus and placenta in late pregnancy have been extensively studied, the effects of these adrenal corticosteroids in early pregnancy are far less well defined. Therefore, we examined the expression, regulation and function of Hsd11b1/Hsd11b2 in mouse uterus during early pregnancy. We found that Hsd11b2 is highly expressed in endometrial stromal cells on days 3 and 4 of pregnancy and mainly upregulated by progesterone (P4). In both ovariectomized mice and cultured stromal cells, P4 significantly stimulates Hsd11b2 expression. P4 stimulation of Hsd11b2 is mainly mediated by the Ihh pathway. The uterine level of corticosterone (Cort) is regulated by Hsd11b2 during preimplantation. Embryo development and the number of inner cell mass cells are suppressed by Cort treatment. These results indicate that P4 should provide a low Cort environment for the development of preimplantation mouse embryos by promoting the expression of uterine Hsd11b2.

Oncostatin M expression in the mouse uterus during early pregnancy promotes embryo implantation and decidualization.

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family, which functions in embryo implantation and decidualization. The expression, function and regulation of Osm in mouse uteri during early pregnancy remain unknown. We show that Osm is mainly expressed in the uterine epithelium from days 1 to 4 of pregnancy and in decidual cells on day 5 of pregnancy. Osm promotes the attachment of Osm-soaked blue beads, which mimic blastocysts, to a pseudopregnant mouse uterus. Prostaglandin E2 (PGE2 )-induced Osm in mouse uterine epithelial cells upregulates the levels of Il-33 expression and phosphorylates Stat3. In vitro decidualization is significantly promoted by Osm. Our results indicate that PGE2 -induced Osm may mediate embryo implantation through Il-33 and participate in decidualization via the Stat3-Egr1 pathway.