Inefficacy of anti-VEGF therapy reflected in VEGF-mediated photoreceptor degeneration.

Retinal neovascularization (RNV) is primarily driven by vascular endothelial growth factor (VEGF). However, current anti-VEGF therapies are limited by short half-lives and repeated injections, which reduce patient quality of life and increase medical risks. Additionally, not all patients benefit from anti-VEGF monotherapy, and some problems, such as unsatisfactory vision recovery, persist after long-term treatment. In this study, we constructed a recombinant adeno-associated virus (AAV), AAV2-SPLTH, which encodes an anti-VEGF antibody similar to bevacizumab, and assessed its effects in a doxycycline-induced Tet-opsin-VEGFA mouse model of RNV. AAV2-SPLTH effectively inhibited retinal leakage, RNV progression, and photoreceptor apoptosis in a Tet-opsin-VEGF mouse model. However, proteomic sequencing showed that AAV2-SPLTH failed to rescue the expression of phototransduction-related genes, which corresponded to reduced photoreceptor cell numbers. This study suggests that anti-VEGF monotherapy can significantly inhibit RNV to some extent but may not be enough to save visual function in the long term.

A novel disulfide death-related genes prognostic signature identifies the role of IPO4 in glioma progression.

BACKGROUND: "Disulfide death," a form of cellular demise, is triggered by the abnormal accumulation of intracellular disulfides under conditions of glucose deprivation. However, its role in the prognosis of glioma remains undetermined. Therefore, the main objective of this study is to establish prognostic signature based on disulfide death-related genes (DDRGs) and to provide new solutions in choosing the effective treatment of glioma. METHODS: The RNA transcriptome, clinical information, and mutation data of glioma samples were sourced from The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA), while normal samples were obtained from the Genotype-Tissue Expression (GTEx). DDRGs were compiled from previous studies and selected through differential analysis and univariate Cox regression analysis. The molecular subtypes were determined through consensus clustering analysis. Further, LASSO analysis was employed to select characteristic genes, and subsequently, a risk model comprising seven DDRGs was constructed based on multivariable Cox analysis. Kaplan-Meier survival curves were employed to assess survival differences between high and low-risk groups. Additionally, functional analyses (GO, KEGG, GSEA) were conducted to explore the potential biological functions and signaling pathways of genes associated with the model. The study also explored immune checkpoint (ICP) genes, immune cell infiltration levels, and immune stromal scores. Finally, the effect of Importin-4(IPO4) on glioma has been further confirmed through RT-qPCR, Western blot, and cell functional experiments. RESULTS: 7 genes associated with disulfide death were obtained and two subgroups of patients with different prognosis and clinical characteristics were identified. Risk signature was subsequently developed and proved to serve as an prognostic predictor. Notably, the high-risk group exhibited an immunosuppressive microenvironment characterized by a high concentration of M2 macrophages and regulatory T cells (Tregs). In contrast, the low-risk group showed lower half-maximal inhibitory concentration (IC50) values. Therefore, patients in the high-risk group may benefit more from immunotherapy, while patients in the low-risk group may benefit more from chemotherapy. In addition, in vitro experiments have shown that inhibition of the expression of IPO4 leads to a significant reduction in the proliferation, migration, and invasion of glioma cells. CONCLUSION: This study identified two glioma subtypes and constructed a prognostic signature based on DDRGs. The signature has the potential to optimize the selection of patients for immune- and chemotherapy and provided a potential therapeutic target for glioma.

Intravitreal Delivery of rAAV2-hSyn-hRS1 Results in Retinal Ganglion Cell-Specific Gene Expression and Retinal Improvement in the Rs1-KO Mouse.

X-linked retinoschisis (XLRS) is a monogenic recessive inherited retinal disease caused by defects in retinoschisin (RS1). It manifests clinically as retinal schisis cavities and a disproportionate reduction of b-wave amplitude compared with the a-wave amplitude. Currently there is no approved treatment. In the last decade, there has been major progress in the development of gene therapy for XLRS. Previous preclinical studies have demonstrated the treatment benefits of hRS1 gene augmentation therapy in mouse models. However, outcomes in clinical trials have been disappointing, and this might be attributed to dysfunctional assembly of RS1 complexes and/or the impaired targeted cells. In this study, the human synapsin 1 gene promoter (hSyn) was used to control the expression of hRS1 to specifically target retinal ganglion cells and our results confirmed the specific expression and functional assembly of the protein. Moreover, our results demonstrated that a single intravitreal injection of rAAV2-hSyn-hRS1 results in architectural restoration of retinal schisis cavities and improvement in vision in a mouse model of XLRS. In brief, this study not only supports the clinical development of the rAAV2-hSyn-hRS1 vector in XLRS patients but also confirms the therapeutic potential of rAAV-based gene therapy in inherited retinal diseases.

A Machine Learning-Based Approach to Predict Prognosis and Length of Hospital Stay in Adults and Children With Traumatic Brain Injury: Retrospective Cohort Study.

BACKGROUND: The treatment and care of adults and children with traumatic brain injury (TBI) constitute an intractable global health problem. Predicting the prognosis and length of hospital stay of patients with TBI may improve therapeutic effects and significantly reduce societal health care burden. Applying novel machine learning methods to the field of TBI may be valuable for determining the prognosis and cost-effectiveness of clinical treatment. OBJECTIVE: We aimed to combine multiple machine learning approaches to build hybrid models for predicting the prognosis and length of hospital stay for adults and children with TBI. METHODS: We collected relevant clinical information from patients treated at the Neurosurgery Center of the Second Affiliated Hospital of Anhui Medical University between May 2017 and May 2022, of which 80% was used for training the model and 20% for testing via screening and data splitting. We trained and tested the machine learning models using 5 cross-validations to avoid overfitting. In the machine learning models, 11 types of independent variables were used as input variables and Glasgow Outcome Scale score, used to evaluate patients' prognosis, and patient length of stay were used as output variables. Once the models were trained, we obtained and compared the errors of each machine learning model from 5 rounds of cross-validation to select the best predictive model. The model was then externally tested using clinical data of patients treated at the First Affiliated Hospital of Anhui Medical University from June 2021 to February 2022. RESULTS: The final convolutional neural network-support vector machine (CNN-SVM) model predicted Glasgow Outcome Scale score with an accuracy of 93% and 93.69% in the test and external validation sets, respectively, and an area under the curve of 94.68% and 94.32% in the test and external validation sets, respectively. The mean absolute percentage error of the final built convolutional neural network-support vector regression (CNN-SVR) model predicting inpatient time in the test set and external validation set was 10.72% and 10.44%, respectively. The coefficient of determination (R2) was 0.93 and 0.92 in the test set and external validation set, respectively. Compared with back-propagation neural network, CNN, and SVM models built separately, our hybrid model was identified to be optimal and had high confidence. CONCLUSIONS: This study demonstrates the clinical utility of 2 hybrid models built by combining multiple machine learning approaches to accurately predict the prognosis and length of stay in hospital for adults and children with TBI. Application of these models may reduce the burden on physicians when assessing TBI and assist clinicians in the medical decision-making process.

The role of the exocytic pathway in cell wall assembly in yeast.

The cell wall is a dynamic organelle which is tightly controlled for cell morphology, viability, and pathogenesis. It was previously shown that exocytosis is involved in the secretion of some components and enzymes of the cell wall. However, how the secretory pathway affects the cell wall integrity and assembly remains unclear. Here we show that the secretory pathway mutant (sec) cells were sensitive to cell wall antagonists in Saccharomyces cerevisiae, and they were lysed at restrictive conditions but can be rescued by osmotic stabilizers, indicating their cell walls were disrupted. Although glucans were reduced at the cell surface in sec mutants as speculated, the other two main cell wall components, chitins, and mannoproteins, were accumulated at the cell surface. We also found that both the protein level and the phosphorylation level of Slt2 increased in sec mutants. These results suggest that the exocytic pathway has a critical role in cell wall assembly. Our study will help to understand the mechanism of cell wall formation.

Hormesis of low-dose inhibition of pAkt1 (Ser473) followed by a great increase of proline-rich inositol polyphosphate 5-phosphatase (PIPP) level in oocytes.

Hormesis describes a biphasic dose-response relationship generally characterized by a low-dose excitement and a high-dose inhibition. This phenomenon has been observed in the regulation of cell, organ, and organismic level. However, hormesis has not reported in oocytes. In this study, we observed, for the first time, hormetic responses of PIPP levels in oocytes by inhibitor of Akt1 or PKCδ. The expression of PIPP was detected by qPCR, immunofluorescent (IF), and Western Blot (WB). To observe the changes of PIPP levels, we used the inhibitors against pAkt1 (Ser473) or PKCδ, SH-6 or sotrastaurin with low and/or high-dose, treated GV oocytes and cultured for 4 h, respectively. The results showed that PIPP expression was significantly enhanced when oocytes were treated with SH-6 or sotrastaurin 10 µM, but decreased with SH-6 or sotrastaurin 100 µM. We also examined the changes of PIPP levels when GV oocytes were treated with exogenous PtdIns(3,4,5)P3 or LY294002 for 4 h. Our results showed that PIPP level was enhanced much higher under the treatment of 0.1 µM PtdIns(3,4,5)P3 than that of 1 µM PtdIns(3,4,5)P3, which is consistent with the changes of PIPP when oocytes were treated with inhibitors of pAkt1 (Ser473) or PKCδ. In addition, with PIPP siRNA, we detected that down-regulated PIPP may affect distributions of Akt, Cdc25, and pCdc2 (Tyr15). Taken together, these results show that the relationships between PIPP and Akt may follow the principle of hormesis and play a key role during release of diplotene arrest in mouse oocytes.

The N-terminus of Sec3 is required for cell wall integrity in yeast.

The cell wall is essential for cell viability and pathogenesis of fungi. It was previously shown that the exocytosis landmark Sec3 is an effector of the cell wall integrity (CWI) master regulator Rho1 GTPase. However, disruption of the interaction between Sec3 and Rho1 did not inhibit exocytic secretion and cell growth. The physiological role of Sec3 in fungi is unclear. We have examined the growth, cell wall sensitivity, exocyst localization, and exocytic secretion of Sec3-binding deficient rho1 mutants and Rho1-binding deficient sec3 mutants. We found that the Sec3 N-terminal deletion mutant was defective in cell wall integrity. The cells harboring binding mutation between Rho1 and Sec3 N-terminus were sensitive to cell wall antagonists. We also found that the polarized localization of exocyst subunits was disrupted in these mutants. Our study demonstrates that the N-terminus of Sec3 mediates cell wall integrity in yeast. Pathogenic fungi may use similar regulatory mechanisms because components of the exocytic signaling pathways are conserved.

Sphingolipids are required for exocyst polarity and exocytic secretion in Saccharomyces cerevisiae.

BACKGROUND: Exocytosis is a process by which vesicles are transported to and fused with specific areas of the plasma membrane. Although several studies have shown that sphingolipids are the main components of exocytic compartments, whether they control exocytosis process is unclear. RESULTS: Here, we have investigated the role of sphingolipids in exocytosis by reducing the activity of the serine palmitoyl-transferase (SPT), which catalyzes the first step in sphingolipid synthesis in endoplasmic reticulum. We found that the exocyst polarity and exocytic secretion were impaired in lcb1-100 mutant cells and in wild type cells treated with myriocin, a chemical which can specifically inhibit SPT enzyme activity, suggesting that sphingolipids controls exocytic secretion. This speculation was further confirmed by immuno-fluorescence and electron microscopy results that small secretory vesicles were accumulated in lcb1-100 mutant cells. CONCLUSIONS: Taken together, our results suggest that sphingolipids are required for exocytosis. Mammals may use similar regulatory mechanisms because components of the exocytic secretion apparatus and signaling pathways are conserved.

Cyclin-dependent kinase-mediated phosphorylation of the exocyst subunit Exo84 in late G1 phase suppresses exocytic secretion and cell growth in yeast.

In eukaryotic cells, the growth rate is strictly regulated for proper progression of the cell cycle. In the budding yeast Saccharomyces cerevisiae, it was previously shown that cell growth dramatically slows down when the cells start budding at the G1/S transition. However, the molecular mechanism for this G1/S-associated growth arrest is unclear. In this study, using exocytic secretion, cyclin-dependent kinase (CDK) assay, immunoprecipitation, and microscopy, we demonstrate that the exocyst subunit Exo84, which is known to be phosphorylated in mitosis, can also be phosphorylated directly by Cdk1 in the late G1 phase. Of note, we found that the Cdk1-mediated Exo84 phosphorylation impairs exocytic secretion in the late G1 phase. Using conditional cdc mutants and phosphodeficient and phosphomimetic exo84 mutants, we further observed that Cdk1-phosphoryated Exo84 inhibits the exocyst complex assembly, exocytic secretion, and cell growth, which may be important for proper execution of the G1/S-phase transition before commitment to a complete cell cycle. Our results suggest that the direct Cdk1-mediated regulation of the exocyst complex critically contributes to the coordination of cell growth and cell cycle progression.

Upregulation of the lncRNA Meg3 induces autophagy to inhibit tumorigenesis and progression of epithelial ovarian carcinoma by regulating activity of ATG3.

Maternally expressed gene 3 (Meg3), a long non-coding RNA, has been reported to be associated with the pathogenesis of multiple malignancies. However, little is known regarding the role of Meg3 in epithelial ovarian cancer (EOC). In this study, we found that the expression of Meg3 was lower in epithelial ovarian carcinoma, and has potential to be considered as a biomarker for ovarian cancer. After transfecting the ovarian cancer cell lines OVCAR3 and A2780 with Meg3, phenotypic changes and autophagy-related molecules were examined. Upregulation of Meg3 inhibited cell proliferation, plate colony formation, induced cell cycle arrest in G2 phases, and promoted apoptosis. Observation of autophagosomes was performed by transmission electron microscopy. The expression levels of LC3-II, ATG3, LAMP1 were elevated, while SQSTM1/p62 expression declined. Upregulated expression of Meg3 also suppressed tumorigenesis in vivo in a xenograft mouse model through upregulating ATG3 expression. RIP (ribonucleoprotein immunoprecipitation) and RNA pull-down assays showed that Meg3 was co-immunoprecipitated with ATG3. In addition, Meg3 protected ATG3 mRNA from degradation following treatment with actinomycin D. Overall, our results suggest that the lncRNA Meg3 acts as a tumor suppressor in EOC by regulating ATG3 activity and inducing autophagy.

Deficiency of caspase 3 in tumor xenograft impairs therapeutic effect of measles virus Edmoston strain.

The oncolytic measles virus Edmonston (MV-Edm) strain shows considerable oncolytic activity against a variety of human tumors. In this study, we report MV-Edm is able to trigger apoptosis pathways in infected tumor cells and elucidate the roles of cellular apoptosis in the whole oncolytic process. We also show that activated caspase 3, a key executioner of apoptosis, plays key roles in the oncolytic virotherapy. Activated caspase 3 can accelerate viral replication in cervical cancer cells and enhance the killing effects of the virus. Deficiency of caspase 3 either in tumor cells or in tumor xenograft significantly desensitized tumor to oncolysis with MV-Edm. In the infected cells, caspase 3 regulates interferon α release, which can inhibit viral replication in neighboring tumor cells. We propose that caspase-3 activation enhances the oncolytic effects of MV-Edm, thus inhibiting tumor growth in mice.

Effects of the TAT peptide orientation and relative location on the protein transduction efficiency.

To understand the protein transduction domain (PTD)-mediated protein transduction behavior and to explore its potential in delivering biopharmaceutic drugs, we prepared four TAT-EGFP conjugates: TAT(+)-EGFP, TAT(-)-EGFP, EGFP-TAT(+) and EGFP-TAT(-), where TAT(+) and TAT(-) represent the original and the reversed TAT sequence, respectively. These four TAT-EGFP conjugates were incubated with HeLa and PC12 cells for in vitro study as well as injected intraperitoneally to mice for in vivo study. Flow cytometric results showed that four TAT-EGFP conjugates were able to traverse HeLa and PC12 cells with almost equal transduction efficiency. The in vivo study showed that the TAT-EGFP conjugates could be delivered into different organs of mice with different transduction capabilities. Bioinformatic analyses and CD spectroscopic data revealed that the TAT peptide has no defined secondary structure, and conjugating the TAT peptide to the EGFP cargo protein would not alter the native structure and the function of the EGFP protein. These results conclude that the sequence orientation, the spatial structure, and the relative location of the TAT peptide have much less effect on the TAT-mediated protein transduction. Thus, the TAT-fused conjugates could be constructed in more convenient and flexible formats for a wide range of biopharmaceutical applications.