Recryopreservation (recryo) is occasionally applied in clinical, while the underlying mechanism of impaired clinical outcomes after recryo remains unclear. In this study, frozen embryo transfer (FET) cycles of single blastocyst transfer in an academic reproductive medicine center were enrolled. According to the number of times blastocysts experienced cryopreservation, they were divided into the cryopreservation (Cryo) group and the Recryo group. Donated human blastocysts were collected and detected for mechanism exploration. It was found that recryo procedure resulted in impaired blastocyst developmental potential, including decreased implantation rate, reduced biochemical pregnancy rate, declined clinical pregnancy rate, higher early miscarriage rate, and lower live birth rate. Moreover, recryo led to impaired trophectoderm (TE) function, exhibiting lower human chorionic gonadotropin levels 12 days after FET. In addition, single-cell RNA sequencing showed that the expression of genes involved in cell adhesion and embryo development were altered. More specifically, activated endoplasmic reticulum (ER) pathway and induced apoptosis were further verified by immunofluorescence and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay involving in the recryo procedure. In conclusion, recryo could interfere with the process of blastocyst implantation by impairing TE function, affecting blastocyst adhesion, activating ER stress pathway and inducing apoptosis. It provides caution to embryologists about the potential risk of recryopreservation.

The beneficial effects of xylo-oligosaccharides (XOS) on the intestine have been widely reported, including anti-inflammation, antioxidant, maintenance of intestinal epithelial barrier, and treatment of intestinal injury. However, the specific mechanism of XOS in mitigating intestinal injury in weaned piglets remains unclear. Therefore, this study aimed to explore the specific mechanism of XOS in mitigating intestinal injury. The study is a complete randomized design with 24 weaned piglets in a 2 × 2 factorial arrangement that includes diet treatments (basal diet vs 0.02% XOS) and immunological challenge [saline vs lipopolysaccharide (LPS)]. All piglets were fed a basal diet or a XOS diet for 21 days. On day 22, all piglets received an injection of LPS or saline. In this study, dietary XOS increased jejunal villus height, reduced crypt depth and oxidative stress, and enhanced the gene and protein expression of Claudin-1, Occludin, and zonula occludens 1 (P < 0.05). The piglets fed the XOS diet had lower serum Diamine oxidase activity and D-lactic acid content (P < 0.05). In addition, dietary XOS regulates endoplasmic reticulum (ER)-mitochondria system function and the expression of key molecules, including mitochondrial dynamics dysfunction [mitofusin (Mfn)-1, optic atrophy 1, fission 1, and dynamin-related protein 1], ER stress [activating transcription factor 4 (ATF4), ATF6, C/EBP homologous protein, eukaryotic initiation factor 2α, glucose-regulated protein (GRP) 78, GRP94 and protein kinase R-like ER kinase] and the mitochondria-associated ER membranes (MAM) disorders (Mfn2, GRP75 and voltage-dlependent anion channel 1) (P < 0.05). Therefore, the findings to indicate that dietary XOS is effective against LPS-induced jejunal injury may be attributed to its ability to alleviate mitochondrial dynamics dysfunction, ER stress, and MAM disorders.

BACKGROUND: Glioblastoma (GB) is recognized as one of the most aggressive brain tumors, with a median survival of 14.6 months. However, there are still some patients whose survival time was greater than 3 years, and the biological reasons behind this clinical phenomenon arouse our research interests. By conducting proteomic analysis on tumor tissues obtained from GB patients who survived over 3 years compared to those who survived less than 1 year, we identified a significant upregulation of SelK in patients with shorter survival times. Therefore, we hypothesized that SelK may be an important indicator related to the occurrence and progression of GBM.
METHODS: Proteomics and immunohistochemistry from GB patients were analyzed to investigate the correlation between SelK and clinical prognosis. Cellular phenotypes were evaluated by cell cycle analysis, cell viability assays, and xenograft models. Immunoblots and co-immunoprecipitation were conducted to verify SelK-mediated ubiquitin-dependent degradation of CDK4.
RESULTS: SelK was found to be significantly upregulated in GB samples from short-term survivors (≤ 1 year) compared to those from long-term survivors (≥ 3 years), and its expression levels were negatively correlated with clinical prognosis. Knocking down of SelK expression reduced GB cell viability, induced G0/G1 phase arrest, and impaired the growth of transplanted glioma cells in nude mice. Down-regulation of SelK-induced ER stress leads to a reduction in the expression of SKP2 and an up-regulation of β-TrCP1 expression. Up-regulation of β-TrCP1, thereby accelerating the ubiquitin-dependent degradation of CDK4 and ultimately inhibiting the malignant proliferation of the GB cells.
CONCLUSIONS: This study discovered a significant increase in SelK expression in GB patients with poor prognosis, revealing a negative correlation between SelK expression and patient outcomes. Further mechanistic investigations revealed that SelK enhances the proliferation of GB cells by targeting the endoplasmic reticulum stress/SKP2/β-TrCP1/CDK4 axis.

Epilepsy is one of the most commonly-seen neurological disorders, and both endoplasmic reticulum stress (ERS) and oxidative stress (OS) have been demonstrated to be associated with epileptic seizures. As one of the three endogenous thiol-containing amino acids, cysteine (Cys) is recognized not only as an important biomarker of various biological processes but also widely used as a significant additive in the food industry. However, the exact role that Cys plays in ERS has not been well answered up to now. In this paper, we reported the first flavone-based fluorescent probe (namely BFC) with nice endoplasmic reticulum (ER)-targeting ability, which was capable of monitoring Cys in a fast response (3.0 min), large stokes shift (130 nm) and low detection limit (10.4 nM). The recognition mechanism of Cys could be attributed to the addition-cyclization reaction involving a Cys residue and an acrylate group, resulting in the release of the strong excited-state intramolecular proton transfer (ESIPT) emission molecule of benzoflavonol (BF). The low cytotoxicity and good biocompatibility of the probe BFC allowed for monitoring the fluctuation of endogenous Cys levels under both ERS and OS processes, as well as in zebrafish models of epilepsy. Quantitative determination of Cys with the probe BFC was also achieved in three different food samples. Additionally, a probe-immersed test strips integrated with a smartphone device was successfully constructed for on-site colorimetric detection of Cys. Undoubtedly, our work provided a valuable tool for tracking Cys levels in both an epilepsy model and real food samples.

Nonalcoholic fatty liver disease (NAFLD) is a liver disease causing different progressive pathological changes. Trimethylamine N-oxide (TMAO), a product of gut microbiota metabolism, is a specific agonist of the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway, one of the endoplasmic reticulum stress (ERS) pathways. TMAO has been associated with the occurrence and development of NAFLD based on the results of previous studies, but whether the simple consumption of TMAO can directly induce NAFLD and its underlying mechanism remain unclear. To investigate this question, we constructed an animal model in which adult male zebrafish were fed a controlled diet containing 1 % or 3 % TMAO for 20 weeks. Eventually, we observed that TMAO caused lipid accumulation, inflammatory infiltration, liver injury and liver fibrosis in zebrafish livers; meanwhile, the PERK signaling pathway was activated in the zebrafish livers. This finding was further confirmed in HepG2 cells and hepatic stellate cells models. In conclusion, this study found that TMAO directly induced different pathological states of NAFLD in zebrafish liver, and the activation of PERK pathway is an important mechanism, which may provide crucial strategies for the diagnosis and treatment of NAFLD.

The combination of therapy-induced immunogenic cell death (ICD) and immune checkpoint blockade can provide a mutually reinforced strategy to reverse the poor immunogenicity and immune escape behavior of tumors. In this work, a chimeric peptide-engineered immunostimulant (ER-PPB) is fabricated for endoplasmic reticulum (ER)-targeted photodynamic immunotherapy against metastatic tumors. Among which, the amphiphilic chimeric peptide (ER-PP) is composed of ER-targeting peptide FFKDEL, hydrophilic PEG8 linker and photosensitizer protoporphyrin IX (PpIX), which could be assembled with a PD-1/PD-L1 blocker (BMS-1) to prepare ER-PPB. After passively targeting at tumor tissues, ER-PPB will selectively accumulate in the ER. Next, the localized PDT of ER-PPB will produce a lot of ROS to destroy the primary tumor cells, while increasing the ER stress to initiate a robust ICD cascade. Moreover, the concomitant delivery of BMS-1 can impede the immune escape of tumor cells through PD-1/PD-L1 blockade, thus synergistically activating the immune system to combat metastatic tumors. In vitro and in vivo results demonstrate the robust immune activation and metastatic tumor inhibition characteristics of ER-PPB, which may offer a promising strategy for spatiotemporally controlled metastatic tumor therapy.

Periodontitis is featured as the periodontium\'s pathologic destruction caused by the host\'s overwhelmed inflammation. Omentin-1 has been reported to be aberrantly downregulated in patients with periodontitis, but the specific regulation of Omentin-1 during the pathogenesis of periodontitis remains unclear. In this study, human periodontal ligament stem cells (hPDLSCs) were stimulated by lipopolysaccharide (LPS) from Porphyromonas gingivalis to establish an in vitro inflammatory periodontitis model. hPDLSCs were treated with recombinant human Omentin-1 (250, 500 and 750ng/mL) for 3h before LPS stimulation. Results revealed that Omentin-1 significantly inhibited LPS-induced inflammation in hPDLSCs through reducing the production of proinflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6) and downregulating the expression of Cox2 and iNOS. Meanwhile, Omentin-1 significantly enhanced alkaline phosphatase (ALP) activity and Alizarin red-stained area, accompanied by increasing expression osteogenic markers BMP2, OCN and Runx2, confirming that Omentin-1 restores osteogenic differentiation in LPS-induced hPDLSCs. In addition, the conditioned medium (CM) from LPS-induced hPDLSCs was harvested to culture macrophages, which resulted in macrophage polarization towards M1, while CM from Omentin-1-treated hPDLSCs reduced M1 macrophages polarization and elevated M2 polarization. Furthermore, Omentin-1 also inhibited LPS-triggered endoplasmic reticulum (ER) stress in hPDLSCs, and additional treatment of the ER stress activator tunicamycin (TM) partially reversed the functions of Omentin-1 on inflammation, osteogenic differentiation and macrophages polarization. In summary, Omentin-1 exerted a protective role against periodontitis through inhibiting inflammation and enhancing osteogenic differentiation of hPDLSCs, providing a novelty treatment option for periodontitis.

With the development of Type 1 diabetes mellitus (T1DM), various complications can be caused. Hyperglycemia affects the microenvironment of cardiomyocytes, changes endoplasmic reticulum homeostasis, triggers unfolding protein response and eventually promotes myocardial apoptosis. However, insulin therapy alone cannot effectively combat the complications caused by T1DM. Forty adult beagles were randomly divided into five groups: control group, diabetes mellitus group, insulin group, insulin combined with NAC group, and NAC group. 24-hour blood glucose, 120-day blood glucose, 120-day body weight, and serum FMN content were observed, furthermore, hematoxylin-eosin staining, Periodic acid Schiff reagent staining, and Sirius red staining of the myocardium were evaluated. The protein expressions of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase 3, Bcl2, and Bax were detected. Results of the pathological section of myocardial tissue indicated that insulin combined with NAC therapy could improve myocardial pathological injury and glycogen deposition. Additionally, insulin combined with NAC therapy down-regulates the expression of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase3, and Bax. These findings suggest that NAC has a phylactic effect on myocardial injury in beagles with T1DM, and the mechanism may be related to the improvement of endoplasmic reticulum stress-induced apoptosis.

BACKGROUND: Endoplasmic reticulum stress (ERS) could be a strategy for treating malignant tumors. Moreover, long noncoding RNAs (lncRNAs) can promote tumorigenesis and progression, and forecast the prognosis of cancers. Nevertheless, the prognostic value of ERS-related lncRNAs has not been reported in lung adenocarcinoma (LUAD).
METHODS: The messenger RNA (mRNA), microRNA (miRNA) and lncRNA expression data related to LUAD were obtained in public databases (TCGA and GEO databases). Prognostic ERS-related differentially expressed lncRNAs (ERS-DELs) were obtained and used to build an ERS-related model by Cox regression analysis. Moreover, we further screened independent prognostic elements and built a nomogram. Furthermore, enrichment analysis of genes was conducted to investigate the functions. A lncRNA-miRNA-mRNA network was built to explore mechanism of lncRNAs. Finally, qRT-PCR was utilized to examine the expression levels of lncRNAs.
RESULTS: 30 ERS-DELs were identified, and an ERS-related signature was built based on AF131215.2, LINC00472, LINC01352, RP1-78O14.1, RP11-253E3.3, RP11-98D18.9, and SNHG12. Gene set enrichment analysis indicated that genes in the high-risk group were chiefly focused on the regulation of mRNA binding, and genes in the low-risk group were significantly focused on protein localization to cilia. A lncRNA-miRNA-mRNA network, containing 7 signature lncRNAs, 23 miRNAs, and 128 mRNAs, was also established. Eventually, quantitative real-time polymerase chain reaction was used to confirm that seven prognostic lncRNAs had a consistent expression with the analysis.
CONCLUSIONS: An ERS-related signature containing seven prognostic lncRNAs was built, which offered new thinking concerning the role of ERS-related lncRNAs in LUAD.

OBJECTIVE: Endoplasmic reticulum protein 29 (ERP29) is crucial for endoplasmic reticulum stress (ERS). M6A plays an important role in the progression of endometrial cancer (EC). The study investigated the role of ERS-related gene (ERP29) and m6A in EC.
METHODS: We screened ERS-related genes based on the GEO dataset, GSEA dataset and TCGA-UCEC database using WGCNA and two machine learning algorithms. The m6A-related GEO dataset was employed to identify the ERS-related hub genes with m6A. Expression of hub genes in different cell types were visualize through scRNA-seq data analyzing. Using qPCR, Western blot, and Immunohistochemical assays to detect the expression of ERP29, the effect of ERP29 on cancer cell proliferation was investigated through CCK8, EdU and clone formation experiments. M6A modifications were studied using m6A Dot blot and MeRIP-qPCR. Finally, we conducted rescue experiments.
RESULTS: Ten ERS-related hub genes with m6A were identified. ERP29 is highly expressed in EC. ERP29 knockdown inhibits EC cell proliferation. METTL3 overexpression increases the ERP29 mRNA m6A and decreases the expression of ERP29. Cycloleucine (Cyc), a nucleic acid methylation inhibitor, treatment reduces ERP29 mRNA m6A and increases the expression of ERP29. Cyc rescue the low expression of ERP29 caused by overexpression of METTL3 through m6A. ERP29 knockdown rescued the increased proliferation of EC cells caused by low m6A.
CONCLUSIONS: ERP29 is highly expressed in EC. m6A regulates ERP29 expression and affects the proliferation of endometrial cancer cells. This represents the premise for applying ERP29 and m6A modifications in diagnosing and treating EC.