RESUMEN
Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.
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Cinetocoros , Huso Acromático , Ratones , Animales , Cinetocoros/metabolismo , Espastina/genética , Espastina/metabolismo , Huso Acromático/fisiología , Microtúbulos/metabolismo , Meiosis , Oocitos/fisiologíaRESUMEN
A modified, sensitive and reversible method for protein staining on nitrocellulose (NC) and polyvinylidine fluoride (PVDF) membranes was developed in Western blotting. The method employed Congo red staining to visualize proteins on different blot membranes. Staining of proteins with Congo red dye is more faster procedures. According to the experimental results, approximate 20 ng proteins could be detected in 3 min in room temperature. The staining on the proteins is easily reversible with Congo red destaining solution for NC and PVDF membranes, so that the blot membranes can be reused for Western blotting. In addition, we confirmed that the staining method is fully compatible with Western blot detection. NC and PVDF membranes treatment with Congo red staining does not interfere with conventional chemiluminescent substrates of peroxidase. As compared to MemCode reversible protein stain kits from Pirece Biotechnology, the staining technique is more sensitive, lower of cost, convenient and not adversely affecting subsequent Western blotting results. On the other hand, the stain is more sensitive than the Ponceau S staining. Therefore, Congo red staining is a promising and ideal alternative for current protein stain. Besides, the binding modes of Congo red or Ponceau S stain were investigated using various 2D and 3D molecular docking and demonstrated potential molecular basis for sensitivity of Congo red staining are higher than Ponceau S.
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Compuestos Azo/química , Colorantes/química , Rojo Congo/química , Proteínas/química , Coloración y Etiquetado/métodos , Western Blotting/métodos , Polivinilos/químicaRESUMEN
This paper analyzes how emigration impacts fiscal gap of population-exporting region in the long term. We construct a general equilibrium model of emigration and fiscal gap and make empirical verification using two-step system GMM model. Among the major lessons from this work, five general and striking results are worth highlighting: (1) the economic losses of emigration are the immediate cause of widening the fiscal gap. (2) in the short and long term, emigration can expand the fiscal revenue gap through the superimposed effect of tax rate and tax base. (3) the gap in fiscal expenditure is widened by the outflow of people in the short term. However, local governments would change the strategy to keep the spending gap from widening in the long run. (4) a positive impact of emigration on the fiscal gap. the more severe population emigration, the larger the fiscal gap. (5) when the trend of emigration becomes irreversible, the subsequent efforts of local governments to expand fiscal expenditure for attraction population would not only fail to revive the regional economy, but aggravate the expansion of fiscal gap. The contribution of research is twofold. On the one hand, it fills the theoretical gap between emigration and fiscal gap because previous studies have paid little attention to the fiscal problems of local government of population outflow. On the other hand, the selection of Northeast China that has been subject to long-term out-of-population migration is good evidence to verify this theory, which is tested very well using the 2S-GMM model. The comprehensive discussion on the relationship between emigration and fiscal gap is helpful to guide those continuous population-exporting regions that are facing a huge fiscal gap how to solve the fiscal gap and unsustainability from the perspective of fiscal revenue and expenditure.
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Emigración e Inmigración , Humanos , China , Dinámica Poblacional , Impuestos/economíaRESUMEN
Objective: We aimed to examine whether heparin-binding epidermal growth factor-like growth factor (HB-EGF) affects the lung fibrosis process through the activation of p38 protein in mitogen-activated protein kinases (MAPK) signaling pathway, as well as the expression of downstream inflammatory factors. Methods: The expression levels of HB-EGF, collagen type I (COL-I), and hexokinase 2 (HK2) in peripheral blood mononuclear cells (PBMCs) of patients with connective tissue disease-related interstitial lung disease (CTD-ILD) were examined by qPCR, Western blotting and ELISA. Results: In vitro experiments showed that HB-EGF was increased in almost all subtypes [rheumatoid arthritis (RA), systemic sclerosis (SSc) and idiopathic inflammatory myopathies (IIMs)] as well as in all groups (P < 0.05). For embryonic lung fibroblast (A549) cells, the expression levels of HK2 and α-smooth muscle actin (α-SMA) genes were elevated during 0-4 h and then plateaued. Transforming growth factor-ß1 (TGF-ß1) induced fibrosis in human embryonic lung fibroblasts (MRC-5) cells and A549 for a certain period of time, but the degree of induction varied, which may be related to the redifferentiability of cells at different spatial locations. Moreover, HB-EGF at concentrations above 1 ng/ml stimulation increased COL-I expression (P < 0.05), and for α-SMA gene, even 1 ng/ml concentration of HB-EGF had a stimulatory effect, and different concentrations of HB-EGF did activate the expression of p38 in a concentration-dependent manner within a certain concentration range, and by The qPCR results showed that for interleukin 6 (IL-6), an inflammatory factor regulated downstream of p38, the expression was significantly increased in A549 cells compared to control (P < 0.05), but tumor necrosis factor-α (TNF-α) expression was downregulated (P < 0.05), but for interleukin-1ß (IL-1ß) gene, there was no significant difference in A549 cells, and expression was downregulated in MRC-5 cells. Therefore, it is suggested that HB-EGF regulates the expression of inflammatory factors through p38 will be differential across cells. Conclusion: Our study shows that HB-EGF can suppress pulmonary fibrosis through downstream activation of p38/MAPK pathway activity, as well as the expression of various inflammatory factors downstream of it.
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High-altitude polycythemia (HAPC) affects individuals living at high altitudes, characterized by increased red blood cells (RBCs) production in response to hypoxic conditions. The exact mechanisms behind HAPC are not fully understood. We utilized a mouse model exposed to hypobaric hypoxia (HH), replicating the environmental conditions experienced at 6000 m above sea level, coupled with in vitro analysis of primary splenic macrophages under 1% O2 to investigate these mechanisms. Our findings indicate that HH significantly boosts erythropoiesis, leading to erythrocytosis and splenic changes, including initial contraction to splenomegaly over 14 days. A notable decrease in red pulp macrophages (RPMs) in the spleen, essential for RBCs processing, was observed, correlating with increased iron release and signs of ferroptosis. Prolonged exposure to hypoxia further exacerbated these effects, mirrored in human peripheral blood mononuclear cells. Single-cell sequencing showed a marked reduction in macrophage populations, affecting the spleen's ability to clear RBCs and contributing to splenomegaly. Our findings suggest splenic ferroptosis contributes to decreased RPMs, affecting erythrophagocytosis and potentially fostering continuous RBCs production in HAPC. These insights could guide the development of targeted therapies for HAPC, emphasizing the importance of splenic macrophages in disease pathology.
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Mal de Altura , Ferroptosis , Animales , Ratones , Humanos , Bazo , Esplenomegalia , Leucocitos Mononucleares , Macrófagos , HipoxiaRESUMEN
This experiment investigated the underlying mechanism of ultrasonic-assisted stewing to enhance the aroma intensity of chicken broth by measuring fat content, oil droplet sizes, zeta potential, viscosity, surface protein loading, lipid oxidation, and aroma compound concentrations. As the thermo-ultrasound time increased, the fat content increased from 0.3 % to 1.2 %, resulting in a milky white appearance. After 1 h of thermo-ultrasound, the broth had the smallest particle size and the highest surface protein load, viscosity, and emulsion stability, as well as the highest total amount of aroma-active compounds of 314.70 ng/mg. With the further extension of thermo-ultrasound time, lipid oxidation increased, but the stability of chicken broth decreased, lowering the content of aroma-active compounds. These outcomes suggested that thermo-ultrasound could enhance the aroma intensity of chicken broth by increasing the fat content and the emulsion stability of the broth.
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Pollos , Odorantes , Animales , Emulsiones/química , Lípidos , Proteínas de la Membrana , Odorantes/análisis , UltrasonidoRESUMEN
For western blot analysis, a housekeeping protein, such as ß-actin or glyceraldehyde-3-phosphate dehydrogenase, is used as loading control with the assumption that these proteins are stable. In practice, these internal loading control proteins vary with different cell states and tissue types. These internal standards are not appropriate for use with serum, extracellular secretion, cerebrospinal fluid analysis or for protein purification. We investigated total protein measurement using Congo red staining and found it to be a superior alternative to routine loading controls. Advantages include lower cost, technical simplicity and improved linear regression. We propose using Congo red staining for total protein immunoblotting to evaluate protein loading in western blots.
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Rojo Congo , Gliceraldehído-3-Fosfato Deshidrogenasas , Actinas/metabolismo , Western Blotting , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Coloración y EtiquetadoRESUMEN
High-fat diet (HFD) has been associated with neuroinflammation and apoptosis in distinct brain regions. To explore the effect of short-term (7, 14 and 21 days) high-fat overfeeding on apoptosis, inflammatory signaling proteins, APP changes and glial cell activities in cerebral cortex and cerebellum. Mice were fed with HFD for different lengths (up to 21 days) and after each time body weights of mice was tested, then the apoptotic proteins, IL-1ß, APP, BACE1and MAPKs, Akt and NF-κB signaling activity were evaluated by western blots. Results demonstrate that short period of high-fat overnutrition significantly promotes apoptosis, APP expression at day 21 of cerebral cortex and at day 7 of cerebellum compared to chow diet. In addition, increased GFAP+astrocytes, Iba-1+microglia and IL-1ß 30 were observed in cerebral cortex after 21 days HFD, but no changes for 7 days overfeeding of cerebellum. Serendipitously, ERK1/2 pathway was activated both in cerebral cortex and cerebellum for different time course of HFD. Furthermore, increased phospho-p38 MAPK level was observed in cerebellum only. In consistent with in vivo results, SH-SY5Y cells treatment with cholesterol (50 µM, 100 µM) for 48 h culture in vitro demonstrated that pro-apoptotic proteins were enhanced as well. In brief, short-term HFD consumption increases sensitivity to apoptosis, APP and IL-1ß production as well as gliosis in cerebral cortex and cerebellum, which may be related to enhancement of ERK1/2 and p38 MAPK activation.
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Apoptosis/genética , Corteza Cerebral/metabolismo , Dieta Alta en Grasa/efectos adversos , Gliosis/genética , Sistema de Señalización de MAP Quinasas/genética , Animales , Línea Celular Tumoral , Cerebelo/metabolismo , Gliosis/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
A shortage of freshwater resources has become a fundamental and chronic problem for sustainable agriculture development in arid regions. Use of saline water irrigation has become an important means for alleviating freshwater scarcity. However, long-term irrigation with saline water may cause salt accumulation in the soil, and further affect nitrogen transformation and N2O emission. To investigate this, we conducted a ten-year field experiment to evaluate the effect of irrigation water salinity and N amount on N2O emission and denitrifying bacterial communities. The experimental design was a 2×2 factorial with two irrigation water salinity levels (salinity levels are expressed as electrical conductivity), 0.35 dS·m-1 and 8.04 dS·m-1, and two N amounts, 0 kg·hm-2 and 360 kg·hm-2, representing SFN0, SHN0, SFN360, and SHN360, respectively. The results indicated that long-term saline water irrigation significantly increased soil salinity, moisture, and NH4+-N content, whereas it decreased soil pH, NO3--N, organic matter, and total nitrogen content. Irrigation with saline water significantly inhibited N2O emission, being associated with a decreased in level of 45.19% (unfertilized plots) and 43.50% (fertilized plots) compared with irrigation with fresh water. N2O emission increased as the N amount increased; the N2O emission was 161% higher in the fertilized plots than in the unfertilized plots. In the unfertilized plots, saline water irrigation significantly reduced the activity of denitrifying enzymes, the abundance of nirK, nirS, and nosZ, and the diversity of denitrifying bacterial communities. In the fertilized plots, saline water irrigation did not significantly affect the abundance of nosZ, whereas it significantly reduced the abundance of nirK and nirS. Saline water irrigation and nitrogen application altered the community structures of denitrifying bacteria with nirK, nirS, and nosZ; the irrigation water salinity seemed to have a greater impact on the denitrifying bacterial community in comparison with fertilization. Linear discriminant analysis (LDA) effect size (LEfSe) analysis demonstrated that denitrifying bacterial potential biomarkers increased as the water salinity increased, meaning that saline water irrigation could alter the community structures of denitrifying bacteria, and promote the growth of dominant species. Our findings indicate that increased abundance of nosZ, nirK, and nirS promoted N2O emission, and although long-term saline water reduced soil N2O emission, it resulted in a continuous increase of soil salinity. The emission of N2O had extremely positive correlation with soil NO3--N, organic matter, total nitrogen, denitrifying bacteria abundance, and denitrifying enzyme activities, and was negatively correlated with soil moisture. The soil physiochemical properties and the community structure of denitrifying bacteria had a significant influence on soil N2O emission in cotton fields, and nirS bacteria showed the highest association with N2O emission, thus it might be a dominant microflora in the process of denitrification. This information will aid in reducing atmospheric N2O emissions in agriculturally productive alluvial grey desert soils.
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Óxido Nitroso/análisis , Microbiología del Suelo , Bacterias , Desnitrificación , Aguas Salinas , SueloRESUMEN
We have recently demonstrated that tau hyperphosphorylation causes diabetic synaptic neurodegeneration of retinal ganglion cells (RGCs), which might be the earliest affair during the pathogenesis of diabetic retinopathy (DR). Thus, there is a pressing need to seek therapeutic agents possessing neuroprotective effects against tau hyperphosphorylation in RGCs for arresting the progression of DR. Here, using a well-characterized diabetes model of db/db mouse, we discovered that topical ocular application of 10 mg/kg/day of ginsenoside Rg1 (GRg1), one of the major active ingredients extracted from Panax ginseng and Panax notoginseng, ameliorated hyperphosphorylated tau-triggered RGCs synaptic neurodegeneration in diabetic mice. The neuroprotective effects of GRg1 on diabetic retinae were abrogated when retinal IRS-1 or Akt was suppressed by intravitreal injection with si-IRS-1 or topically coadministered with a specific inhibitor of Akt, respectively. However, selective repression of retinal GSK3ß by intravitreal administration of si-GSK3ß rescued the neuroprotective properties of GRg1 when Akt was inactivated. Therefore, the present study showed for the first time that GRg1 can prevent hyperphosphorylated tau-induced synaptic neurodegeneration of RGCs via activation of IRS-1/Akt/GSK3ß signaling in the early phase of DR. Moreover, our data clarify the potential therapeutic significance of GRg1 for neuroprotective intervention strategies of DR.