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Background Extracellular heat shock protein 90α (eHSP90α) regulates diverse cellular processes such as wound healing, tumor metastasis, angiogenesis and cell differentiation etc. Our previous data show that lens epithelial cells can secret eHSP90α, and administration of eHSP90α exerts a critical regulation for lens regeneration by promoting the differentiation of lens epithelial cells (ECs) to lens fiber cells. Autophagy is proposed to regulate the eHSP90α secretion from lens epithelial cells. However, the regulatory mechanism remains unclear. Methods Lens ECs cell line SRA01/04, ex vivo cultured rat lens capsular bags and primary rat lens ECs were used in this study. The immunoblotting and qPCR were used for measuring the expression of proteins and mRNAs. The immunofluorescence staining assay was for testing the autophagosomes or protein co-localization. siRNA or CRISPR-Cas9 were used to knock down genes’ expression in cells in vitro. The ChIP assay was used to study the interaction of transcriptional factor to the promoter of target genes. The nuclear and cytoplasmic extraction assay was for testing the nuclear translocation of TFEB and TFE3. Results eHSP90α is secreted through the secretory autophagy pathway in lens epithelial cells in vitro or in the residual anterior ECs of capsular bags ex vivo. HSP90α interacts with adaptor protein TRIM16, and was recruited to the R-SNARE protein SEC22B on the surface of secretory autophagosome membrane for secretion. Silencing autophagic ATG7 or components of secretory autophagy pathways, such as TRIM16 , SEC22B and Q-SNARE STX3 , STX4 and SNAP23 downregulates HSP90α secretion. TGF-β2 upregulates the eHSP90α secretion via upregulating secretory autophagy pathway. TGF-β2 treatment results in the upregulation of the expression of ATG7, Beclin1, LC3B at both protein and mRNA levels through activating p38 and ERK1/2. In addition, TGF-β2 increases SEC22B expression through ERK1/2-induced expression and nuclear translocation of transcriptional factors TFEB and TFE3, which form the heterodimer binding to the promoter of SEC22B. Functionally, the upregulated eHSP90α can trigger TGF-β2-induced differentiation of lens ECs to fiber cells. Conclusions In lens cells, the eHSP90α’s secretion undertakes secretory autophagy pathway through interacting with TRIM16 and SEC22B. TGF-β2 elevates eHSP90α secretion through upregulating secretory autophagy pathway, which in turn promote TGF-β2-induced lens epithelial cells’ differentiation to lens fiber cells.

For autonomous vehicles driving in off-road environments, it is crucial to have a sensitive environmental perception ability. However, semantic segmentation in complex scenes remains a challenging task. Most current methods for off-road environments often have the problems of single scene and low accuracy. Therefore, this paper proposes a semantic segmentation network based on LiDAR called Multi-scale Augmentation Point-Cylinder Network (MAPC-Net). The network uses a multi-layer receptive field fusion module to extract features from objects of different scales in off-road environments. Gated feature fusion is used to fuse PointTensor and Cylinder for encoding and decoding. In addition, we use CARLA to build off-road environments for obtaining datasets, and employ linear interpolation to enhance the training data to solve the problem of sample imbalance. Finally, we design experiments to verify the excellent semantic segmentation ability of MAPC-Net in an off-road environment. We also demonstrate the effectiveness of the multi-layer receptive field fusion module and data augmentation.

The Glucocorticoid-induced posterior subcapsular cataracts (GIC) is a common complication of patients received glucocorticoid treatment in clinic. We find that dexamethasone (DEX) induces lens epithelial cells’ ferroptosis. DEX treatment increases intracellular ferroptosis signatures in lens epithelial cell line in vitro as well as in rat lens in vivo. The inhibition of ferroptosis by liproxstatin-1 reduces the incidence of DEX-induced rat GIC. Experimental evidence and expression profiling showed that DEX induces ferroptosis through upregulating tetraspanin CD82- controlled P53 expression. DEX-activated glucocorticoid receptors directly bind to the CD82 promoter, driving its transcriptional upregulation. CD82 expression is upregulated in the anterior capsular epithelium of GIC patients as well as in the DEX-treated rat lens and caused the cell death of anterior capsule. DEX treatment and Overexpression of CD82 in cells recapitulated ferroptotic signatures through P53 activation and GPX4/SLC7A11 suppression. Taken together, GIC is closely associated with the upregulation of CD82-P53-GPX4/SLC7A11 axis-mediated ferroptosis. Dexamethasone (DEX) induces ferroptosis in lens epithelial cells, resulting in the formation of posterior subcapsular cataracts (GIC). It upregulates CD82, which activates P53 and suppresses GPX4/SLC7A11, contributing to ferroptosis in GIC.

Background HSPB5 is an ATP-independent molecular chaperone that is induced by heat shock or other proteotoxic stresses. HSPB5 is cytoprotective against stress both intracellularly and extracellularly. It acts as a potential therapeutic candidate in ischemia-reperfusion and neurodegenerative diseases. Results In this paper, we constructed a recombinant plasmid that expresses and extracellularly secrets a HSPB5-Fc fusion protein (sHSPB5-Fc) at 0.42 μg/ml in CHO-K1 cells. This sHSPB5-Fc protein contains a Fc-tag at the C-terminal extension of HSPB5, facilitating protein-affinity purification. Our study shows that sHSPB5-Fc inhibits heat-induced aggregation of citrate synthase in a time and dose dependent manner in vitro. Administration of sHSPB5-Fc protects lens epithelial cells against cisplatin- or UVB-induced cell apoptosis. It also decreases GFP-Htt ex1 -Q74 insolubility, and reduces the size and cytotoxicity of GFP-Htt ex1 -Q74 aggregates in PC-12 cells. Conclusion This recombinant sHSPB5-Fc exhibits chaperone activity to protect cells against proteotoxicity.

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo . Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans , were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.

Background Liver cancer stem cells (LCSCs) significantly impact chemo-resistance and recurrence in liver cancer. Dopamine receptor D4 (DRD4) is known to enhance the cancer stem cell (CSC) phenotype in glioblastoma and correlates with poor prognosis in some non-central nervous system tumors; however, its influence on LCSCs remains uncertain. Methods To investigate the gene and protein expression profiles of DRD4 in LCSCs and non-LCSCs, we utilized transcriptome sequencing and Western blotting analysis. Bioinformatics analysis and immunohistochemistry were employed to assess the correlation between DRD4 expression levels and the pathological characteristics of liver cancer patients. The impact of DRD4 on LCSC phenotypes and signaling pathways were explored using pharmacological or gene-editing techniques. Additionally, the effect of DRD4 on the protein expression and intracellular localization of β-catenin were examined using Western blotting and immunofluorescence. Results DRD4 expression is significantly elevated in LCSCs and correlates with short survival in liver cancer. The expression and activity of DRD4 are positive to resistance, self renewal and tumorigenicity in HCC. Mechanistically, DRD4 stabilizes β-catenin and promotes its entry into the nucleus via activating the PI3K/Akt/GSK-3β pathway, thereby enhancing LCSC phenotypes. Conclusions Inhibiting DRD4 expression and activation offers a promising targeted therapy for eradicating LCSCs and relieve chemo-resistance.

SARS-Cov-2 infection, which has caused the COVID-19 global pandemic, triggers cellular senescence. In this study, we investigate the role of the SARS-COV-2 spike protein (S-protein) in regulating the senescence of RPE cells. The results showed that administration or overexpression of S-protein in ARPE-19 decreased cell proliferation with cell cycle arrest at the G1 phase. S-protein increased SA-β-Gal positive ARPE-19 cells with high expression of P53 and P21, senescence-associated inflammatory factors (e.g., IL-1β, IL-6, IL-8, ICAM, and VEGF), and ROS. Elimination of ROS by N-acetyl cysteine (NAC) or knocking down p21 by siRNA diminished S-protein-induced ARPE cell senescence. Both administrated and overexpressed S-protein colocalize with the ER and upregulate ER-stress-associated BIP, CHOP, ATF3, and ATF6 expression. S-protein induced P65 protein nuclear translocation. Inhibition of NF-κB by bay-11-7082 reduced S-protein-mediated expression of senescence-associated factors. Moreover, the intravitreal injection of S-protein upregulates senescence-associated inflammatory factors in the zebrafish retina. In conclusions, the S-protein of SARS-Cov-2 induces cellular senescence of ARPE-19 cells in vitro and the expression of senescence-associated cytokines in zebrafish retina in vivo likely by activating ER stress, ROS, and NF-κb. These results may uncover a potential association between SARS-cov-2 infection and development of AMD.

Extracellular heat shock protein 90[alpha] (eHSP90[alpha]) regulates diverse cellular processes such as wound healing, tumor metastasis, angiogenesis and cell differentiation etc. Our previous data show that lens epithelial cells can secret eHSP90[alpha], and administration of eHSP90[alpha] exerts a critical regulation for lens regeneration by promoting the differentiation of lens epithelial cells (ECs) to lens fiber cells. Autophagy is proposed to regulate the eHSP90[alpha] secretion from lens epithelial cells. However, the regulatory mechanism remains unclear. Lens ECs cell line SRA01/04, ex vivo cultured rat lens capsular bags and primary rat lens ECs were used in this study. The immunoblotting and qPCR were used for measuring the expression of proteins and mRNAs. The immunofluorescence staining assay was for testing the autophagosomes or protein co-localization. siRNA or CRISPR-Cas9 were used to knock down genes' expression in cells in vitro. The ChIP assay was used to study the interaction of transcriptional factor to the promoter of target genes. The nuclear and cytoplasmic extraction assay was for testing the nuclear translocation of TFEB and TFE3. eHSP90[alpha] is secreted through the secretory autophagy pathway in lens epithelial cells in vitro or in the residual anterior ECs of capsular bags ex vivo. HSP90[alpha] interacts with adaptor protein TRIM16, and was recruited to the R-SNARE protein SEC22B on the surface of secretory autophagosome membrane for secretion. Silencing autophagic ATG7 or components of secretory autophagy pathways, such as TRIM16, SEC22B and Q-SNARE STX3, STX4 and SNAP23 downregulates HSP90[alpha] secretion. TGF-β2 upregulates the eHSP90[alpha] secretion via upregulating secretory autophagy pathway. TGF-β2 treatment results in the upregulation of the expression of ATG7, Beclin1, LC3B at both protein and mRNA levels through activating p38 and ERK1/2. In addition, TGF-β2 increases SEC22B expression through ERK1/2-induced expression and nuclear translocation of transcriptional factors TFEB and TFE3, which form the heterodimer binding to the promoter of SEC22B. Functionally, the upregulated eHSP90[alpha] can trigger TGF-β2-induced differentiation of lens ECs to fiber cells. In lens cells, the eHSP90[alpha]'s secretion undertakes secretory autophagy pathway through interacting with TRIM16 and SEC22B. TGF-β2 elevates eHSP90[alpha] secretion through upregulating secretory autophagy pathway, which in turn promote TGF-β2-induced lens epithelial cells' differentiation to lens fiber cells.

Heat shock factor 4 controls the transcription of small heat shock proteins (e.g., HSP25, alpha B-cyrstallin, and r-crystallin), that play important roles in modulating lens proteostasis. However, the molecular mechanism underlying HSF4-mediated transcription is still unclear. Using yeast two hybrid, we found that HSF4 interacts with the ATP-dependent DEXD/H-box RNA helicase UAP56, and their interaction in lens epithelial cell line was further confirmed by GST-pull down assay. UAP56 is a vital regulator of pre-mRNA splicing and mature mRNA nuclear export. The immunofluorescence assay showed that HSF4 and UBA56 colocalize with each other in the nucleus of lens epithelial cells. Ectopic UAP56 upregulated HSF4-controlled HSP25 and alpha B-crystallin proteins expression, while knocking down UAP56 by shRNA reversed it. Moreover, UAP56 interacts with and facilitates the nuclear exportation of HSP25 and alpha B-crystallin mRNA without impacting their total mRNA expression level. In lens tissues, both UAP56 and HSF4 are expressed in the same nucleus of lens fiber cells, and their expression levels are simultaneously reduced with fiber cell maturation. Taken together, these data suggested that UAP56 is a novel regulator of HSF4 and might upregulate HSF4' s downstream mRNA maturation and nuclear exportation.