AbstractSubarachnoid hemorrhage (SAH) can lead to significant acute neuroinflammation, with treatment outcomes often being inadequate. Olfactory mucosa mesenchymal stem cells (OM‐MSCs) have promising therapeutic potential in nerve regeneration and functional recovery. This investigation sought to elucidate the functional mechanisms through which exosomes derived from OM‐MSCs provide protection against neuroinflammation following SAH. Mouse OM‐MSCs and their exosomes were isolated and characterized using various techniques, including transmission electron microscopy, immunofluorescence staining, Western blotting, flow cytometry, and nanoparticle tracking analysis. Hemin‐induced HT22 cells were subsequently utilized to assess the impact of OM‐MSC‐derived exosomes on the inflammatory response, apoptosis, and mitophagy through ELISAs, Western blotting, qPCR, flow cytometry, and immunofluorescence staining. The impacts of exosomes on neuroinflammation and neuronal damage in SAH model mice were assessed using qPCR, ELISAs, Western blotting, immunofluorescence staining, and TUNEL staining. Exosomes derived from OM‐MSCs had the capacity to reduce the levels of proinflammatory factors (IL‐6, IL‐1β, and TNF‐α) and promote apoptosis in hemin‐induced HT22 cells. Exosomes alleviated neuroinflammation and neuronal injury post‐SAH, as evidenced by the increase in modified Garcia scores, reduction in the brain water content, decrease in blood–brain barrier permeability, decreases in inflammatory marker levels, and reduction in apoptosis rates. Notably, the protective effects of exosomes derived from OM‐MSCs on neuroinflammation and apoptosis, both in vitro and in vivo, were mediated via the activation of mitophagy. These findings provide a fresh perspective for subsequent clinical research in the domain of prevention and treatment strategies.

Actin and actin polymerization factors regulate the immune system in a complex manner. The function in the cytoplasm has been well-established, where they are important components of the cytoskeleton, controlling cell migration, function, and vesicular transport. However, it remains poorly understood how they enter the nucleus to regulate immunological functions in B cells. Here, our study, through constructing a mouse model with specific WASH deletion in B cells, has shown that a deficiency of WASH leads to a decrease in BCR signaling and B cell metabolism, abnormal B cell differentiation, and a reduction of humoral response. Mechanistically, WASH interacts with pSTAT1 to promote the phosphorylation of STAT1, facilitating its translocation into the nucleus and regulating biological functions. Our study has unveiled the potential molecular mechanisms by which WASH influences B cell signaling, metabolism, and function through STAT1. These findings will offer potential avenues for therapeutic strategies targeting autoimmune diseases.

Breast cancer became the most prevalent malignancy among women, and HER2 expression status is critical for treatment decisions. With the emergence of ADC drugs, HER2 low-expressing patients who previously did not respond well to traditional anti-HER2 therapies may now benefit. In this study, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were applied to assess HER2 expression in 349 patients with HER2-non-positive breast cancer. Our analysis revealed that HER2-low tumors exhibited fewer grade III tumors (39.74

Background: Neuronal cell ferroptosis following intracerebral hemorrhage (ICH) is a crucial factor contributing to the poor prognosis of ICH patients. The objective of this investigation was to investigate the molecular mechanism of IL-1(3 (3- induced mesenchymal stem cell-derived exosomes (IL-1(3-Exo) (3- Exo) in mitigating ICH injury. Methods: Exo and IL-1(3-Exo (3- Exo were obtained and identified. Hemin was used to induce an ICH model, and an ICH mouse model was established using Collagenase. Exo and IL-1(3-Exo (3- Exo interventions were conducted to study their impact and molecular mechanisms on neuronal ferroptosis in ICH. Results: Vesicular structure Exo and IL-1(3- (3- Exo, with an average particle size of 141.7 +/- 38.8 nm and 138.8 +/- 37.5 nm, respectively, showed high expression of CD63, CD9 and CD81 could be taken up by SH-SY5Y cells. These Exos reversed Hemin-induced abnormalities in neuronal cells, including elevated iron, Fe2+, 2 + , ROS, MDA, 4-HNE, and decreased SOD, GSHPx, GSH, FTH1 levels, and cell vitality. The RNA content of IL-1(3-Exo (3- Exo was linked to its ability to reduce iron accumulation. There was an interaction between HSPA5 and GPX4. Exo and IL-1(3-Exo (3- Exo reversed Hemin-induced downregulation of HSPA5 and GPX4 expression. Overexpression and knockdown of HSPA5 respectively potentiate or counteract the impacts of Exo and IL-1(3-Exo. (3- Exo. IL-1(3-Exo (3- Exo was more effective than Exo. These findings were further validated in ICH mice. Moreover, both Exo and IL-1(3-Exo (3- Exo reduced the modified neurological severity score and brain water content, as well as alleviated pathological damage in ICH mice. Conclusion: IL-1(3-Exo (3- Exo inhibited neuronal ferroptosis in ICH through the HSPA5/GPX4 axis.

Store-operated Ca2+ entry (SOCE) is a universal Ca2+ influx pathway that is important for the function of many cell types. SOCE is controlled by the interaction of the ER Ca2+ sensor STIM1 with the plasma membrane Ca2+ channel Orai1. S417 is located in the third coiled-coil (CC3) domain of the C-terminus of STIM1. We found that single-point mutation of this residue (S417G) abolished STIM1 C-terminus interactions with Orai1. Mutation of S417 also abolished CAD-Orai1 binding and Orai1 channel activation, eliminated STIM1 puncta formation, and co-localization with Orai1 and SOCE. 2-APB was found to restore the binding of the STIM1 C-terminus mutant (S417G) to Orai1 and dose-dependently activate Orai1 channel. Both CBD and NBD of Orai1 are required for 2-APB-induced coupling between the Orai1 and STIM1 C-terminus mutant (S417G) and CRAC channel activation. We also demonstrated that 2-APB led to delayed activation of Orai1-K85E channel, although Orai1-K85E obviously impairs 2-APB-induced STIM1 C-terminus mutant (S417G)-Orai1 coupling. Our results suggest S417 in the CC3 domain of STIM1 is essential for STIM1-Orai1 binding and CRAC channel activation.

Background and purpose: Store-operated Ca2+ entry (SOCE) is important for the function of many cell types. It is controlled by the interaction between ER Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel Orai1. CAD of STIM1 is required for SOCE. It contains two putative coiled-coil regions (CC2 and CC3). The role of CC3 remains to be elucidated. Experimental approach: various plasmids carrying different fluorescent protein genes were constructed for better understanding the influence of S417G mutation in CC3 on SOCE activation; Confocal imaging system, calcium imaging technique and FRET technique were employed to examine the actions of 2-APB on the interaction between STIM1 C terminus and Orai1. Key results: Single-point mutation of the residue (S417G) abolishes STIM1 C-terminus interactions with Orai1. Mutation of S417 also abolished CAD-Orai1 binding and Orai1 channel activation, eliminated STIM1 puncta formation and co-localization with Orai1 and SOCE. 2-APB were found to restore the binding of STIM1 C-terminus mutant(S417G) to Orai1 and dose-dependently activated Orai1 channel. Both CBD and NBD of Orai1 is required for 2-APB-induced coupling between Orai1 and STIM1 C terminus mutant(S417G) and CRAC channel activation. We also demonstrated 2-APB lead to delayed activation of Orai1-K85E channel, although Orai1-K85E obviously impair 2-APB-induced STIM1 C-terminus mutant(S417G)-Orai1 coupling. Conclusions and implications: Our data suggest that S417 in the CC3 domain of STIM1 is critical for STIM1-Orai1 binding and CRAC channel activation. We also proposed experimental models of combined STM1 or Orai1 mutants with 2-APB to better understand the activation mechanism of CRAC.

To explore the mechanismof Rab5/RAB-5 activation during endocytic recycling, we performa genome-wide RNAi screen and identify a recycling regulator, LET-502/ROCK. LET-502 preferentially interacts with RAB5-(GDP) and activates RABX-5 GEF activity toward RAB-5, presumably by disrupting the self-inhibiting conformation of RABX-5. Furthermore, we find that the concomitant loss of LET-502 and another CED-10 effector, TBC-2/RAB-5-GAP, results in an endosomal buildup of RAB-5, indicating that CED-10 directs TBC-2-mediated RAB-5 inactivation and re-activates RAB-5 via LET-502 afterward. Then, we compare the functional position of LET-502 with that of RME-6/RAB-5-GEF. Loss of LET-502-RABX-5 module or RME-6 leads to diminished RAB-5 presence in spatially distinct endosome groups. We conclude that in the intestine of C. elegans, RAB-5 resides in discrete endosome subpopulations. Under the oversight of CED-10, LET-502 synergizes with RABX-5 to revitalize RAB-5 on a subset of endosomes in the deep cytosol, ensuring the progress of basolateral recycling.

The traditional Petri dish, which has not been changed in almost 60 years, has clear limitations when it is applied to cell cultures in a modern biological laboratory. In this work, by integrating the advantages of both the semidirect breath figure (sDBF) method and the traditional breath figure (BF) method, we proposed for the first time a novel hybrid BF method for the fabrication of honeycomb-patterned Petri dishes with mPEG self-assembly in the pores. Due to the amphiphilic structure of mPEG, the active OH groups of mPEG were located inside the pores of the dishes, which could covalently couple with other functional materials. For instance, in this work, an antibacterial agent was immobilized onto the dish surface via a typical coupling reaction. Because of the size difference between the bacteria and cells, the prepared dishes had selective antibacterial activity but noncytotoxicity against mammalian cells. The present hybrid BF method provides a new insight for endowing commercial PS Petri dishes or other membranes with special topographical structures and functions, which could solve the long-term challenges of cell cultures in the future.

Acute pancreatitis (AP) is an acute inflammatory process of the pancreas that is characterized by inflammation, edema, vacuolization and necrosis, which has significant morbidity and lethality. The pathogenesis of AP has not been established completely. An early and critical feature of AP is the aberrant signaling of Calcium (Ca 2+ ) within the pancreatic acinar cell, termed Ca 2+ overload. Store-operated Ca 2+ (SOC) channels are the principal Ca 2+ influx channels that contribute to Ca 2+ overload in pancreatic acinar cells. Store-operated Ca 2+ entry (SOCE) has been proved to be a key pathogenic step in AP development that leads to trypsin activation, inflammation and vacuolization. However, the molecular mechanisms are still poorly understood. By establishing Ca 2+ overload model and mouse AP model using caerulein, we found that caerulein triggered SOCE via inducing interaction between STIM1 and Orai1, which activated calcineurin (CaN); CaN activated the nuclear factor of activated T cells (NFAT) and transcription factor EB (TFEB), thus promoting the transcriptional activation of multiple chemokines genes and autophagy-associated genes respectively. To the best of our knowledge, this is the first evidence showing that SOCE activates TFEB via CaN activation, which may have noticeable longer-term effects on autophagy and vacuolization in AP development. Our findings reveal the role for SOCE/CaN in AP development and provide potential targets for AP treatment.

RAB-10/Rab10 is a master regulator of endocytic recycling in epithelial cells. To better understand the regulation of RAB-10 activity, we sought to identify RAB-10(GDP)–interacting proteins. One novel RAB-10(GDP)–binding partner that we identified, LET-413, is the Caenorhabditis elegans homologue of Scrib/Erbin. Here, we focus on the mechanistic role of LET-413 in the regulation of RAB-10 within the C. elegans intestine. We show that LET-413 is a RAB-5 effector and colocalizes with RAB-10 on endosomes, and the overlap of LET-413 with RAB-10 is RAB-5 dependent. Notably, LET-413 enhances the interaction of DENN-4 with RAB-10(GDP) and promotes DENN-4 guanine nucleotide exchange factor activity toward RAB-10. Loss of LET-413 leads to cytosolic dispersion of the RAB-10 effectors TBC-2 and CNT-1. Finally, we demonstrate that the loss of RAB-10 or LET-413 results in abnormal overextensions of lateral membrane. Hence, our studies indicate that LET-413 is required for DENN-4–mediated RAB-10 activation, and the LET-413–assisted RAB-5 to RAB-10 cascade contributes to the integrity of C. elegans intestinal epithelia.

2-Aminoethyldiphenyl borate (2-APB) is the most commonly used pharmacological agent in the study of calcium release-activated channels (CRACs); however, its inhibitory mechanism to CRACs remains unclear. To address this issue, we systematically employed confocal imaging, dual-wavelength excitation photometry and FRET to examine the effects of 2-APB on the dynamic activities and function of STIM1 and Orai1, two key components of CRACs. Imaging results support that there are two signaling pathways (Orai1-independent and Orai1-dependent) for the formation of STIM1 puncta. 2-APB could dose dependently block Orai1- independent but not Orai1-dependent STIM1 puncta formation, despite its obvious inhibition effect on store-operated Ca2+ entry (SOCE). In addition, we found that although 2-APB could not visibly alter near plasma membrane CAD-eYFP localization, it could completely block CAD-YFP-induced constitutive Ca2+ entry and promote the interaction between Orai1 and CAD by FRET measurements. Therefore, we proposed that inhibitory action of 2-APB on SOCE might attribute to its direct inhibitory effects on Orai1 channel itself, but not the interference on puncta formation between STIM1 and Orai1.

CRISPR/Cas9技术是近年发展起来的快速基因编辑技术.通过该技术已对多种生物的基因组进行了编辑.由此产生的基因编辑动物的建系与鉴定是随之而来较为繁琐的工作.单导向RNA(single-guide RNA,sgRNA)靶序列的设计和确定不仅影响后续靶向基因组的效率,还可作为优化鉴定、筛选方法的参考.本研究在选取sgRNA靶序列时,不仅依据软件的评分,还分析了sgRNA靶序列是否含有酶切位点,以便对后续纯合子/杂合子进行鉴定.结果显示,以特异引物扩增的野生型小鼠Chrm3基因片段可被限制性内切酶BanⅡ切为两个片段;而纯合子小鼠“丢失”该酶切位点,其PCR产物不能被切开;杂合子小鼠PCR产物被不完全切开,凝胶电泳结果可见三条带.本研究结果提示该策略可有效简化基因编辑动物建系鉴定工作,提高鉴定效率及改善阳性动物辨识效果.

Huntingtin-associated protein 1 (Hap1) was originally identified as a protein that binds to the Huntington disease protein, huntingtin. Growing evidence has shown that Hap1 participates in intracellular trafficking via its association with various microtubule-dependent transporters and organelles. Recent studies also revealed that Hap1 is involved in exocytosis such as insulin release from pancreatic β-cells. However, the mechanism underlying the action of Hap1 on insulin release remains to be investigated. We found that Hap1 knock-out mice had a lower plasma basal insulin level than control mice. Using cultured pancreatic β-cell lines, INS-1 cells, we confirmed that decreasing Hap1 reduces the number of secreted vesicles and inhibits vesicle exocytosis. Electrophysiology and imaging of intracellular Ca2+ measurements demonstrated that Hap1 depletion significantly reduces the influx of Ca2+ mediated by L-type Ca2+ channels (Cav). This decrease is not due to reduced expression of Cav1.2 channel mRNA but results from the decreased distribution of Cav1.2 on the plasma membrane of INS-1 cells. Fluorescence recovery after photobleaching showed a defective movement of Cav1.2 in Hap1 silencing INS-1 cells. Our findings suggest that Hap1 is important for insulin secretion of pancreatic β-cells via regulating the intracellular trafficking and plasma membrane localization of Cav1.2, providing new insight into the mechanisms that regulate insulin release from pancreatic β-cells.

Hydrogen peroxide (H2O2) and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene (C60) is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione (GSH) is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative (C60-GSH) was successfully synthesized, and its beneficial roles in protecting against H2O2-induced oxidative stress and apoptosis in cultured HEK 293T cells were investigated. Fourier Transform infrared spectroscopy and 1H nuclear magnetic resonance were used to confirm the chemical structure of C60-GSH. Our results demonstrated that C60-GSH prevented the reactive oxygen species (ROS)-mediated cell damage. Additionally, C60-GSH pretreatment significantly attenuated H2O2-induced superoxide dismutase (SOD) consumption and malondialdehyde (MDA) elevation. Furthermore, C60-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H2O2 stimulation in HEK 293T cells. Importantly, these protective effects of C60-GSH were superior to those of GSH. In conclusion, these results suggested that C60-GSH has potential to protect against H2O2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity.

Huntingtin-associated protein 1 (HAP1) is enriched in neurons and binds to polyglutamine-expanded huntingtin. It consists of two alternatively spliced isoforms, HAP1A and HAP1B, which differ only in their short C-terminal sequences. Both HAP1A and HAP1B have been also detected in pancreatic β cells, where the loss of HAP1 impairs glucose-stimulated insulin secretion. Here, we use time-lapse laser scanning confocal microscopy to provide direct evidence that HAP1A, but not HAP1B, co-localizes and co-migrates with insulin-containing vesicles and actin-based myosin Va motor protein in the INS-1 pancreatic β cell line. Knocking down HAP1 expression using small interfering RNA significantly inhibited actin-based transport of insulin vesicles following glucose stimulation. Co-immunoprecipitation experiments demonstrated interaction between HAP1A, myosin Va, and phogrin, a transmembrane protein in insulin-containing vesicles. Stimulating INS-1 cells with glucose increased the association of HAP1A with myosin Va, while silencing HAP1 expression reduced the association of myosin Va with phogrin after glucose stimulation, without affecting levels of myosin Va or actin. Our results provide real-time evidence in living cells that HAP1 may help regulate transport of insulin-containing secretory granules along cortical actin filaments. This also raises the possibility that HAP1 may play an important role in actin-based secretory vesicle trafficking in neurons.

The aberrant expression of microRNA-183 (miRNA/miR-183) has been found to be involved in numerous tumor types. However, the role of miR-183 in gastric cancer pathology is unclear and requires investigation. In the present study, the miR-183 expression levels of gastric cancer cell lines and tissues obtained from gastric cancer patients were measured by reverse transcription quantitative polymerase chain reaction analysis. The effect of miR-183 on gastric cancer cell proliferation and invasion was evaluated using MTT, colony formation and Transwell assays. The target of miR-183 was identified and confirmed using a luciferase activity assay. The results revealed that miR-183 was significantly downregulated in gastric cancer cells compared with GES-1 normal gastric epithelial cells. In addition, miR-183 was reduced in gastric cancer tissues compared with adjacent normal tissues. The ectopic expression of miR-183 significantly inhibited gastric cancer cell proliferation, colony formation and invasion. Bmi-1 was also confirmed as a downstream target of miR-183 in the gastric cancer cells by western blot analysis and luciferase activity assays. In conclusion, miR-183 is downregulated in gastric cancer cells and tissues, and inhibits gastric cancer cell proliferation and invasion by targeting Bmi-1. Therefore, targeting miR-183 may be a potential therapeutic strategy in gastric cancer patients.

Based on the project of editing and monitoring system of CNC punch press,the work path optimizing problem of CNC punch press is discussed here.Firstly,an analysis of the basic principles of the route optimization was given,and Abstract it for the TSP problem.Then using high efficient genetic algorithm for solving the TSP problem and discuss the realization of the genetic algorithm.Next,in order to solve the problem that the genetic algorithm is easy to fall into local optimal solution,adding the catastrophic operator to kill the current of high-quality individual.catastrophic operator makes the individual that far from the optimal solution have sufficient space to evolve and makes the solution more close to the global optimal solution.To carry on the comparative analysis of the the genetic algorithm and the catastrophic genetic algorithm.The paper have come to the conclusion that the catastrophic genetic algorithm has more advantages in NC machining field.

Store-operated Ca2+ channels are a major Ca2+ entry pathway in nonexcitable cells, which drive various essential cellular functions. Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca2+ release-activated Ca2+ (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca2+ sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca2+ stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca2+ stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.

Hap1 was originally identified as a neuronal protein that interacts with huntingtin, the Huntington's disease (HD) protein. Later studies revealed that Hap1 participates in intracellular trafficking in neuronal cells and that this trafficking function can be adversely affected by mutant huntingtin. Hap1 is also present in pancreatic β-cells and other endocrine cells; however, the role of Hap1 in these endocrine cells remains unknown. Using the Cre-loxP system, we generated conditional Hap1 knockout mice to selectively deplete the expression of Hap1 in mouse pancreatic β-cells. Mutant mice with Hap1 deficiency in pancreatic β-cells had impaired glucose tolerance and decreased insulin release in response to intraperitoneally injected glucose. Using cultured pancreatic β-cell lines and isolated mouse pancreatic islets, we confirmed that decreasing Hap1 could reduce glucose-mediated insulin release. Electron microscopy suggested that there was a reduced number of insulin-containing vesicles docked at the plasma membrane of pancreatic islets in Hap1 mutant mice following intraperitoneal glucose injection. Glucose treatment decreased the phosphorylation of Hap1A in cultured β-cells and in mouse pancreatic tissues. Moreover, this glucose treatment increased Hap1's association with kinesin light chain and dynactin p150, both of which are involved in microtubule-dependent trafficking. These studies suggest that Hap1 is important for insulin release from β-cells via dephosphorylation that can regulate its intracellular trafficking function.

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca(2+) FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ∼11-fold change in dynamic range in response to Ca(2+) binding. The enhanced dynamic range for Ca(2+) concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.