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.

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.

RCAd-LTH-shPD-L1, a double-gene recombinant oncolytic adenovirus with enhanced antitumor immunity, increases lymphocyte infiltration and reshapes the tumor microenvironment.

BACKGROUND: With the successful development of modern immunotherapy, immune checkpoint inhibitors (ICIs) are currently considered potential therapeutic options for patients with cancer. However, the therapeutic potential of ICIs in human cancer is mainly limited by their systemic toxicity and low response rate, which suggests the necessity of local drug delivery with an effective vector and reshaping the immunosuppressive tumor microenvironment (TME) to enhance ICI therapy. Here, we constructed a novel double-gene recombinant oncolytic adenovirus named RCAd-LTH-shPD-L1 based on the RCAd virus platform armed with a DNA fragment encoding an anti-VEGF antibody and shRNA to inhibit PD-L1 expression. METHODS: The correct assembly of RCAd-LTH-shPD-L1 was characterized by analyzing its secretion, antigen specificity, and replication using western blotting, ELISA and quantitative PCR, respectively. The in vitro effects of RCAd-LTH-shPD-L1 on cell proliferation, vasculogenic, and cell migration were assessed. Antitumor effects and therapeutic mechanisms were evaluated in vivo using immunodeficient and humanized immune system mouse models. The TME was studied by ELISA, immunohistochemistry and flow cytometry. RESULTS: RCAd-LTH-shPD-L1 cells secreted anti-VEGF antibodies and inhibited the expression of PD-L1 in cancer cells. Moreover, RCAd-LTH-shPD-L1 exerted a specific cytotoxic effect on human cancer cells, but not on murine cancer cells or normal human cells. RCAd-LTH-shPD-L1 elicited a more potent antitumor effect in an immunodeficient mouse model and a humanized immune system mouse model than RCAd-shPD-L1, as demonstrated by the significant decrease in tumor growth. Furthermore, RCAd-LTH-shPD-L1 modulated the TME, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by downregulation of anoxic factor HIF-1α and angiogenesis marker CD31, upregulation of cytokine such as IFN-γ, IL-6 and IL-12. CONCLUSIONS: In summary, our data demonstrated that the localized delivery of anti-VEGF antibodies and shPD-L1 by engineered RCAd-LTH-shPD-L1 is a highly effective and safe strategy for cancer immunotherapy. Moreover, the data underscore the potential of combining local virotherapy and anti-angiogenic therapy with ICIs as an effective TME therapy for poorly infiltrating tumors.

AAV2-antiVEGFscFv gene therapy for retinal neovascularization.

Retinal neovascularization (NV) may lead to irreversible vision impairment, the main treatment for which is the inhibition of vascular endothelial growth factor (VEGF). Existing drugs show limited clinical benefits because of their high prices and short half-lives, which increase the financial burden and medical risks to patients. Gene therapy on the basis of adeno-associated viruses is a promising approach to overcome these limitations because of the nonintegrative nature, low immunogenicity, and potential long-term gene expression of adeno-associated viruses. In this study, we constructed a novel recombinant adeno-associated virus with the single-chain fragment variable (scFv) fragment of the anti-VEGF antibody, AAV2-antiVEGFscFv, consisting of the VH and VL structural domains of IgG. AAV2-antiVEGFscFv effectively inhibited NV, retinal leakage, and retinal detachment in oxygen-induced retinopathy (OIR) mice, Tet/opsin/VEGF double-transgenic mice, and VEGF-induced rabbit NV models. AAV2-antiVEGFscFv also significantly suppressed VEGF-induced inflammation. Furthermore, we showed that AAV2-antiVEGFscFv could be sustainably expressed for a prolonged period and exhibited low immunotoxicity in vivo. This study indicates that AAV2-antiVEGFscFv could be a potential approach for NV treatment and provides strong support for preclinical research.

Erratum: Cell-penetrating peptides enhance the transduction of adeno-associated virus serotype 9 in the central nervous system.

[This corrects the article DOI: 10.1016/j.omtm.2021.02.019.].

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.

Cell-penetrating peptides enhance the transduction of adeno-associated virus serotype 9 in the central nervous system.

Recombinant adeno-associated viruses (rAAVs) have been widely used in the gene therapy field for decades. However, because of the challenge of effectively delivering rAAV vectors through the blood-brain barrier (BBB), their applications for treatment of central nervous system (CNS) diseases are quite limited. In this study, we found that several cell-penetrating peptides (CPPs) can significantly enhance the in vitro transduction efficiency of AAV serotype 9 (AAV9), a promising AAV vector for treatment of CNS diseases, the best of which was the LAH4 peptide. The enhancement of AAV9 transduction by LAH4 relied on binding of the AAV9 capsid to the peptide. Furthermore, we demonstrated that the LAH4 peptide increased the AAV9 transduction in the CNS in vitro and in vivo after systemic administration. Taken together, our results suggest that CPP peptides can interact directly with AAV9 and increase the ability of this AAV vector to cross the BBB, which further induces higher expression of target genes in the brain. Our study will help to improve the applications of AAV gene delivery vectors for the treatment of CNS diseases.

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.

Application of CareDose 4D combined with Karl 3D technology in the low dose computed tomography for the follow-up of COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a highly infectious disease caused by the new coronavirus. Previous studies have shown that the chest CT examination plays an important role in the diagnosis and monitoring of COVID-19. However, some patients with COVID-19 had low white blood cell counts and reduced lymphocyte ratios. Multiple CT examinations may cause radiation damages as well as increase the apoptosis of peripheral blood lymphocytes. A new low-dose CT method should be developed because the regular CT may aggravate the disease. METHOD: Sixty cases were randomly divided into the study group (n = 30) and control group (n = 30). The lung window was reconstructed by Karl 3D iterative technique in the study group. The image quality was subjectively evaluated by two senior chest group diagnostic physicians using a 5-point double-blind method. The value of CT measurement and its standard deviation (SD) was used as an objective evaluation criteria. The volume of CT dose index (CTDIvol), dose length product (DLP) and effective dose (ED) from the two groups were compared and analyzed statistically. RESULT: There was no significant difference in the occurrence rates of ground glass opacities, consolidation, crazy-paving pattern, fiber cable shadow and axial interstitial thickening between the study group and control group (p > 0.05). In addition, no significant difference was found for the subjective score of overall image quality and image noise level (SD) between the two groups (p > 0.05). However, significant differences was found in CTDIvol, DLP, and ED between the study group and the control group (p < 0.05). The effective dose of the study group was reduced by 76% compared to the control group. CONCLUSION: CareDose 4D low-dose scanning combined with Karl 3D iterative reconstruction technology can not only greatly reduce the radiation dose, but also provide images that meet the diagnostic criteria of COVID-19, which can be used as a routine method for the follow-up of COVID-19 patients.

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.

Spatial and Translational Regulation of Exocyst Subunits by Cell Cycle in Budding Yeast.

BACKGROUND Previous studies have shown that exocyst complex is located at polarized growth sites at different cell cycle stages in budding yeast. But how cell cycle and the cyclin-dependent kinase, Cdk1, regulate the distribution of exocyst complex on the plasma membrane and the protein level of each exocyst subunit is not clear. MATERIAL AND METHODS Using budding yeast as a research material, regulation of cell cycle and Cdk1 on exocyst localization on the plasma membrane and on level of each exocyst subunit were examined by methods of cell biology and molecular biology. RESULTS Exocyst complex is located at growth sites on the plasma membrane in both budding and non-budding stages. Cdk1 activity is required for polarized distribution of exocyst complex in late G1, S and M phases, but not in cytokinesis stage. Cdk1 is not required for the assembly and localization of exocyst complex on plasma membrane. The protein level of Sec3 but not other exocyst subunits is regulated by the cell cycle. CONCLUSIONS Cdk1 activity is required for exocyst polarization before cytokinesis during the cell cycle progression, but not for its assembly and localization on the plasma membrane. Dynamic localization and protein level of the complex subunits are regulated by the cell cycle.

The Cdc42 effectors Gic1 and Gic2 regulate polarized post-Golgi secretion.

BACKGROUND: Cell polarity refers to spatial difference in morphology, structure, and function within different parts of a single cell, which plays important roles in a wide range of cellular processes. In eukaryotic cells, the small GTPase Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) are critical components for cell polarity and required for polarized exocytosis and cell growth. Previous data showed that the GTPase-interacting components, Gic1 and Gic2, control cell polarity through its binding with Cdc42 and PtdIns(4,5)P2 in the plasma membrane in budding yeast. However, whether the Gic proteins regulate polarized exocytosis is unknown. RESULTS: In this study, we found that Gic2 co-immunoprecipitates with the exocyst complex, suggesting Gic proteins may be involved in exocytosis. Although we could not show the direct interaction between Gic2 and exocyst, we found gic1Δgic2Δ are synthetically sick with sec3ΔN. We demonstrated that Gic1 and Gic2 are required for polarized exocytosis in a yeast strain harboring the N-terminal domain deletion of Sec3, which is also known as an effector of Cdc42 GTPase. Gic proteins are required for polarized localization of exocyst, growth, and efficient secretion in sec3∆N mutant. In addition, we found that the N-terminal domain of both Gic2 and Sec3 share the similar binding sites of Cdc42. Surprisingly, not all the Sec3/Gic binding deficient cdc42 mutants displayed defects of growth and secretion, indicating that disruption of Cdc42 binding with Gic proteins and Sec3 does not necessarily show secretion defects in cdc42 mutants. CONCLUSIONS: We conclude that Gic1/2 and Sec3 act in parallel to regulate polarized post-Golgi secretion, but this regulation is not solely controlled by their upstream factor Cdc42. Considering that N-terminal domain of Gic2 and Sec3 can bind to both Cdc42 and PtdIns(4,5)P2, the regulation of Gic protein and Sec3 on polarized secretion may also be controlled by PtdIns(4,5)P2. Further experiments need to be performed to test this hypothesis. Our findings provide important clues for understanding the molecular mechanism of cell polarity establishment in eukaryotic cells.

Author Correction: Cryo-EM structure of the exocyst complex.

In the version of this article originally published, the value given for electron dose in Table 1 was incorrect. This value was originally stated as 4.8 but should have been 50. The error has been corrected in the HTML and PDF versions of the article.

Cryo-EM structure of the exocyst complex.

The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 Å. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.

The role of Sec3p in secretory vesicle targeting and exocyst complex assembly.

During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.

Mitotic phosphorylation of Exo84 disrupts exocyst assembly and arrests cell growth.

The rate of eukaryotic cell growth is tightly controlled for proper progression through each cell cycle stage and is important for cell size homeostasis. It was previously shown that cell growth is inhibited during mitosis when cells are preparing for division. However, the mechanism for growth arrest at this stage is unknown. Here we demonstrate that exocytosis of a select group of cargoes was inhibited before the metaphase-anaphase transition in the budding yeast Saccharomyces cerevisiae. The cyclin-dependent kinase, Cdk1, when bound to the mitotic cyclin Clb2, directly phosphorylated Exo84, a component of the exocyst complex essential for exocytosis. Mitotic phosphorylation of Exo84 disrupted the assembly of the exocyst complex, thereby affecting exocytosis and cell surface expansion. Our study demonstrates the coordination between membrane trafficking and cell cycle progression and provides a molecular mechanism by which cell growth is controlled during the cell division cycle.

Nutrients and the Pkh1/2 and Pkc1 protein kinases control mRNA decay and P-body assembly in yeast.

Regulated mRNA decay is essential for eukaryotic survival but the mechanisms for regulating global decay and coordinating it with growth, nutrient, and environmental cues are not known. Here we show that a signal transduction pathway containing the Pkh1/Pkh2 protein kinases and one of their effector kinases, Pkc1, is required for and regulates global mRNA decay at the deadenylation step in Saccharomyces cerevisiae. Additionally, many stresses disrupt protein synthesis and release mRNAs from polysomes for incorporation into P-bodies for degradation or storage. We find that the Pkh1/2-Pkc1 pathway is also required for stress-induced P-body assembly. Control of mRNA decay and P-body assembly by the Pkh-Pkc1 pathway only occurs in nutrient-poor medium, suggesting a novel role for these processes in evolution. Our identification of a signaling pathway for regulating global mRNA decay and P-body assembly provides a means to coordinate mRNA decay with other cellular processes essential for growth and long-term survival. Mammals may use similar regulatory mechanisms because components of the decay apparatus and signaling pathways are conserved.

Genome-wide transcriptome analyses of silicon metabolism in Phaeodactylum tricornutum reveal the multilevel regulation of silicic acid transporters.

BACKGROUND: Diatoms are largely responsible for production of biogenic silica in the global ocean. However, in surface seawater, Si(OH)(4) can be a major limiting factor for diatom productivity. Analyzing at the global scale the genes networks involved in Si transport and metabolism is critical in order to elucidate Si biomineralization, and to understand diatoms contribution to biogeochemical cycles. METHODOLOGY/PRINCIPAL FINDINGS: Using whole genome expression analyses we evaluated the transcriptional response to Si availability for the model species Phaeodactylum tricornutum. Among the differentially regulated genes we found genes involved in glutamine-nitrogen pathways, encoding putative extracellular matrix components, or involved in iron regulation. Some of these compounds may be good candidates for intracellular intermediates involved in silicic acid storage and/or intracellular transport, which are very important processes that remain mysterious in diatoms. Expression analyses and localization studies gave the first picture of the spatial distribution of a silicic acid transporter in a diatom model species, and support the existence of transcriptional and post-transcriptional regulations. CONCLUSIONS/SIGNIFICANCE: Our global analyses revealed that about one fourth of the differentially expressed genes are organized in clusters, underlying a possible evolution of P. tricornutum genome, and perhaps other pennate diatoms, toward a better optimization of its response to variable environmental stimuli. High fitness and adaptation of diatoms to various Si levels in marine environments might arise in part by global regulations from gene (expression level) to genomic (organization in clusters, dosage compensation by gene duplication), and by post-transcriptional regulation and spatial distribution of SIT proteins.

The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover.

Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1 and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines. A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We conclude that eisosome formation and turnover are regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway. These data and previous data showing that endocytosis is regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway suggest that Pkh1 and -2 respond to changes in membrane sphingolipids and transmit this information to eisosomes via Pil1 phosphorylation. Eisosomes then control endocytosis to align the composition and function of the plasma membrane to match demand.