Identification of miRNA858 long-loop precursors in seed plants.

MicroRNAs (miRNAs) are a class of nonprotein-coding short transcripts that provide a layer of post-transcriptional regulation essential to many plant biological processes. MiR858, which targets the transcripts of MYB transcription factors, can affect a range of secondary metabolic processes. Although miR858 and its 187-nt precursor have been well studied in Arabidopsis (Arabidopsis thaliana), a systematic investigation of miR858 precursors and their functions across plant species is lacking due to a problem in identifying the transcripts that generate this subclass. By re-evaluating the transcript of miR858 and relaxing the length cut-off for identifying hairpins, we found in kiwifruit (Actinidia chinensis) that miR858 has long-loop hairpins (1,100 to 2,100 nt), whose intervening sequences between miRNA generating complementary sites were longer than all previously reported miRNA hairpins. Importantly, these precursors of miR858 containing long-loop hairpins (termed MIR858L) are widespread in seed plants including Arabidopsis, varying between 350 and 5,500 nt. Moreover, we showed that MIR858L has a greater impact on proanthocyanidin and flavonol levels in both Arabidopsis and kiwifruit. We suggest that an active MIR858L-MYB regulatory module appeared in the transition of early land plants to large upright flowering plants, making a key contribution to plant secondary metabolism.

Restricted responses of AcMYB68 and AcERF74/75 enhanced waterlogging tolerance in kiwifruit.

Most of kiwifruit cultivars (e.g. Actinidia chinensis cv. Donghong, "DH") were sensitive to waterlogging, thus, waterlogging resistant rootstocks (e.g. Actinidia valvata Dunn, "Dunn") were widely used for kiwifruit industry. Those different species provided ideal materials to understand the waterlogging responses in kiwifruit. Compared to the weaken growth and root activities in "DH", "Dunn" maintained the relative high root activities under the prolonged waterlogging. Based on comparative analysis, transcript levels of pyruvate decarboxylase (PDCs) and alcohol dehydrogenase (ADHs) showed significantly difference between these two species. Both PDCs and ADHs had been significantly increased by waterlogging in "DH", while they were only limitedly triggered by 2 days stress and subsided during the prolonged waterlogging in "Dunn". Thus, 19 differentially expressed transcript factors (DETFs) had been isolated using weighted gene co-expression network analysis combined with transcriptomics and transcript levels of PDCs and ADHs in waterlogged "DH". Among these DETFs, dual luciferase and electrophoretic mobility shift assays indicated AcMYB68 could bind to and trigger the activity of AcPDC2 promoter. The stable over-expression of AcMYB68 significantly up-regulated the transcript levels of PDCs but inhibited the plant growth, especially the roots. Moreover, the enzyme activities of PDC in 35S::AcMYB68 were significantly enhanced during the waterlogging response than that in wild type plants. Most interestingly, comparative analysis indicated that the expression patterns of AcMYB68 and the previously characterized AcERF74/75 (the direct regulator on ADHs) either showed no responses (AcMYB68 and AcERF74) or very limited response (AcERF75) in "Dunn". Taken together, the restricted responses of AcMYB68 and AcERF74/75 in "Dunn" endow its waterlogging tolerance.

AcHZP45 is a repressor of chlorophyll biosynthesis and activator of chlorophyll degradation in kiwifruit.

The degradation of chlorophyll during fruit development is essential to reveal a more 'ripe' color that signals readiness to wild dispersers of seeds and the human consumer. Here, comparative biochemical analysis of developing fruit of Actinidia deliciosa cv. Xuxiang ('XX', green-fleshed) and Actinidia chinensis cv. Jinshi No.1 ('JS', yellow-fleshed) indicated that variation in chlorophyll content is the major contributor to differences in flesh color. Four differentially expressed candidate genes were identified: the down-regulated genes AcCRD1 and AcPOR1 involved in chlorophyll biosynthesis, and the up-regulated genes AcSGR1 and AcSGR2 driving chlorophyll degradation. Prochlorophyllide and chlorophyllide, the metabolites produced by AcCRD1 and AcPOR1, progressively reduced in 'JS', but not in 'XX', indicating that chlorophyll biosynthesis was less active in yellow-fleshed fruit. AcSGR1 and AcSGR2 were verified to be involved in chlorophyll degradation, using both transient expression in tobacco and stable overexpression in kiwifruit. Furthermore, a homeobox-leucine zipper (HD-Zip II), AcHZP45, showed significantly increased expression during 'JS' fruit ripening, which led to both repressed expression of AcCRD1 and AcPOR1 and activated expression of AcSGR1 and AcSGR2. Collectively, the present study indicated that different dynamics of chlorophyll biosynthesis and degradation coordinate the changes in chlorophyll content in kiwifruit flesh, which are orchestrated by the key transcription factor AcHZP45.

A dramatic decline in fruit citrate induced by mutagenesis of a NAC transcription factor, AcNAC1.

Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co-expression network analysis using different kiwifruit cultivars, an Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual-luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR-Cas9-induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

P16INK4a Upregulation Mediated by SIX6 Defines Retinal Ganglion Cell Pathogenesis in Glaucoma.

Glaucoma, a blinding neurodegenerative disease, whose risk factors include elevated intraocular pressure (IOP), age, and genetics, is characterized by accelerated and progressive retinal ganglion cell (RGC) death. Despite decades of research, the mechanism of RGC death in glaucoma is still unknown. Here, we demonstrate that the genetic effect of the SIX6 risk variant (rs33912345, His141Asn) is enhanced by another major POAG risk gene, p16INK4a (cyclin-dependent kinase inhibitor 2A, isoform INK4a). We further show that the upregulation of homozygous SIX6 risk alleles (CC) leads to an increase in p16INK4a expression, with subsequent cellular senescence, as evidenced in a mouse model of elevated IOP and in human POAG eyes. Our data indicate that SIX6 and/or IOP promotes POAG by directly increasing p16INK4a expression, leading to RGC senescence in adult human retinas. Our study provides important insights linking genetic susceptibility to the underlying mechanism of RGC death and provides a unified theory of glaucoma pathogenesis.

The red flesh of kiwifruit is differentially controlled by specific activation-repression systems.

Anthocyanins are visual cues for pollination and seed dispersal. Fruit containing anthocyanins also appeals to consumers due to its appearance and health benefits. In kiwifruit (Actinidia spp.) studies have identified at least two MYB activators of anthocyanin, but their functions in fruit and the mechanisms by which they act are not fully understood. Here, transcriptome and small RNA high-throughput sequencing were used to comprehensively identify contributors to anthocyanin accumulation in kiwifruit. Stable overexpression in vines showed that both 35S::MYB10 and MYB110 can upregulate anthocyanin biosynthesis in Actinidia chinensis fruit, and that MYB10 overexpression resulted in anthocyanin accumulation which was limited to the inner pericarp, suggesting that repressive mechanisms underlie anthocyanin biosynthesis in this species. Furthermore, motifs in the C-terminal region of MYB10/110 were shown to be responsible for the strength of activation of the anthocyanic response. Transient assays showed that both MYB10 and MYB110 were not directly cleaved by miRNAs, but that miR828 and its phased small RNA AcTAS4-D4(-) efficiently targeted MYB110. Other miRNAs were identified, which were differentially expressed between the inner and outer pericarp, and cleavage of SPL13, ARF16, SCL6 and F-box1, all of which are repressors of MYB10, was observed. We conclude that it is the differential expression and subsequent repression of MYB activators that is responsible for variation in anthocyanin accumulation in kiwifruit species.

Status of visual impairment among children with special needs in rural China.

Introduction Previous studies have reported a high prevalence of visual defects in children with special needs. However, routine ocular examinations for these children in rural areas of China are lacking. This study aimed to evaluate the status of visual impairment (VI) in children at special education schools in rural China. Methods A total of 316 students from two special schools in Zunyi city, Guizhou province, were enrolled. Full ophthalmic examinations were performed, and gene-sequencing services were offered to potential patients. Results The mean age of the 316 participants was 12.27±3.49 years, and 75 showed abnormal ophthalmic manifestations on slit-lamp examination. Visual acuity (VA) was assessed in 232 eyes, and the mean VA (logarithm of the minimum angle of resolution, logMAR) was 0.27±0.34. Whole-exome sequencing (WES) identified 19 mutations in these children, which might explain their visual complaints. Children with Down syndrome had a significantly higher prevalence of ocular disorders than those without. Conclusion VI is common among children at special education schools in rural areas; however, routine screening and effective interventions have not been consistently implemented. Efforts should be made to address this issue in these already disadvantaged children.

An ethylene-hypersensitive methionine sulfoxide reductase regulated by NAC transcription factors increases methionine pool size and ethylene production during kiwifruit ripening.

Ethylene plays an important role in regulating fruit ripening by triggering dynamic changes in expression of ripening-associated genes, but the functions of many of these genes are still unknown. Here, a methionine sulfoxide reductase gene (AdMsrB1) was identified by transcriptomics-based analysis as the gene most responsive to ethylene treatment in ripening kiwifruit. The AdMsrB1 protein exhibits a stereospecific activity toward the oxidative stress-induced R enantiomer of methionine sulfoxide (MetSO), reducing it to methionine (Met). Stable overexpression of AdMsrB1 in kiwifruit significantly increased the content of free Met and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, and increased ethylene production. Dual-luciferase assays indicated that the AdMsrB1 promoter was not directly upregulated by ethylene treatment but was modulated by two ethylene-inducible NAM/ATAF/CUC transcription factors (AdNAC2 and AdNAC72) that bind directly to the AdMsrB1 promoter. Overexpression of AdNAC72 in kiwifruit not only enhanced AdMsrB1 expression, but also increased free Met and ACC content and ethylene production rates. This finding establishes an unexpected regulatory loop that enhances ethylene production and the concentration of its biosynthetic intermediates.

Lanosterol reverses protein aggregation in cataracts.

The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

Identification of a novel RPGR mutation associated with X-linked cone-rod dystrophy in a Chinese family.

BACKGROUND: Cone-rod dystrophy (CORD) is a group of inherited retinal dystrophies, characterized by decreased visual acuity, color vision defects, photophobia, and decreased sensitivity in the central visual field. Our study has identified a novel pathogenic variant associated with X-linked cone-rod dystrophy (XLCORD) in a Chinese family. METHODS: All six family members, including the proband, affected siblings, cousins and female carriers, have underwent thorough ophthalmic examinations. The whole exome sequencing was performed for the proband, followed by Sanger sequencing for spilt-sample validation. A mammalian expression vector (AAV-MCS) with mutated retinitis pigmentosa GTPase regulator (RPGR) sequence was expressed in HEK293 T cells. The mutated protein was verified by Western blotting and immunohistochemistry. RESULTS: A novel mutation in the RPGR gene (c.2383G > T, p.E795X) is identified to be responsible for CORD pathogenesis. CONCLUSIONS: Our findings have expanded the spectrum of CORD-associated mutations in RPGR gene and serve as a basis for genetic diagnosis for X-linked CORD.

Genome-wide analysis of coding and non-coding RNA reveals a conserved miR164-NAC regulatory pathway for fruit ripening.

Kiwifruit (Actinidia spp.) is a climacteric fruit with high sensitivity to ethylene, influenced by multiple ethylene-responsive structural genes and transcription factors. However, the roles of other post-transcriptional regulators (e.g. miRNAs) necessary for ripening remain elusive. High-throughput sequencing sRNAome, degradome and transcriptome methods were used to identify further contributors to ripening control in the kiwifruit (A. deliciosa cv 'Hayward'). Two NAM/ATAF/CUC domain transcription factors (AdNAC6 and AdNAC7), both predicted targets for miR164, showed significant upregulation by exogenous ethylene. Gene expression analysis and luciferase reporter assays indicated that Ade-miR164 and one of its precursor miRNAs (Ade-MIR164b) were repressed by ethylene treatment and negatively correlated with AdNAC6/7 expression. Subsequent analysis indicated that both AdNAC6 and AdNAC7 proteins are transcriptional activators and physically bind the promoters of AdACS1 (1-aminocyclopropane-1-carboxylate synthase), AdACO1 (1-aminocyclopropane-1-carboxylic acid oxidase), AdMAN1 (endo-ß-mannanase) and AaTPS1 (terpene synthase). Moreover, subcellular analysis indicated that the location of the AdNAC6/7 proteins was influenced by Ade-miR164. Multiple omics-based approaches revealed a novel regulatory link for fruit ripening that involved ethylene-miR164-NAC. The regulatory pathway for miR164-NAC is present in various fruit (e.g. Rosaceae fruit, citrus, grape), with implications for fruit ripening regulation.

Solitary fibrofolliculoma of the upper eyelid in a 68-year old female: a case report.

BACKGROUND: Fibrofolliculoma is a benign, perifollicular, connective tissue tumor, and it usually arises in the form of multiple lesions, but rarely as a solitary lesion. We report a case of solitary fibrofolliculoma on the eyelid. CASE PRESENTATION: A 68-year-old female presented with an asymptomatic mass on the right upper eyelid. The lesion appeared as a flesh-colored, dome-shaped, smooth nodule being the size of 5 × 5 × 4 mm, with eyelashes protruding from the surface, and located on the upper lid margin. Shave excision was performed, and the diagnosis of fibrofolliculoma was confirmed finally through histological exam. CONCLUSIONS: Solitary fibrofolliculomas rarely arises on the eyelid. However, it should be suspected when a flesh-colored and doom-shaped lesion of the eyelid is encountered. The benign tumor on the lid margin can be removed by shave biopsy.

DNA methylation markers for diagnosis and prognosis of common cancers.

The ability to identify a specific cancer using minimally invasive biopsy holds great promise for improving the diagnosis, treatment selection, and prediction of prognosis in cancer. Using whole-genome methylation data from The Cancer Genome Atlas (TCGA) and machine learning methods, we evaluated the utility of DNA methylation for differentiating tumor tissue and normal tissue for four common cancers (breast, colon, liver, and lung). We identified cancer markers in a training cohort of 1,619 tumor samples and 173 matched adjacent normal tissue samples. We replicated our findings in a separate TCGA cohort of 791 tumor samples and 93 matched adjacent normal tissue samples, as well as an independent Chinese cohort of 394 tumor samples and 324 matched adjacent normal tissue samples. The DNA methylation analysis could predict cancer versus normal tissue with more than 95% accuracy in these three cohorts, demonstrating accuracy comparable to typical diagnostic methods. This analysis also correctly identified 29 of 30 colorectal cancer metastases to the liver and 32 of 34 colorectal cancer metastases to the lung. We also found that methylation patterns can predict prognosis and survival. We correlated differential methylation of CpG sites predictive of cancer with expression of associated genes known to be important in cancer biology, showing decreased expression with increased methylation, as expected. We verified gene expression profiles in a mouse model of hepatocellular carcinoma. Taken together, these findings demonstrate the utility of methylation biomarkers for the molecular characterization of cancer, with implications for diagnosis and prognosis.

Transcriptome Analysis Identifies a Zinc Finger Protein Regulating Starch Degradation in Kiwifruit.

Ripening, including softening, is a critical factor in determining the postharvest shelf-life of fruit and is controlled by enzymes involved in cell wall metabolism, starch degradation, and hormone metabolism. Here, we used a transcriptomics-based approach to identify transcriptional regulatory components associated with texture, ethylene, and starch degradation in ripening kiwifruit (Actinidia deliciosa). Twelve differentially expressed structural genes, including seven involved in cell wall metabolism, four in ethylene biosynthesis, and one in starch degradation, and 14 transcription factors (TFs) induced by exogenous ethylene treatment and inhibited by the ethylene signaling inhibitor 1-methylcyclopropene were identified as changing in transcript levels during ripening. Moreover, analysis of the regulatory effects of differentially expressed genes identified a zinc finger TF, DNA BINDING WITH ONE FINGER (AdDof3), which showed significant transactivation on the AdBAM3L (ß-amylase) promoter. AdDof3 interacted physically with the AdBAM3L promoter, and stable overexpression of AdBAM3L resulted in lower starch content in transgenic kiwifruit leaves, suggesting that AdBAM3L is a key gene for starch degradation. Moreover, transient overexpression analysis showed that AdDof3 up-regulated AdBAM3L expression in kiwifruit. Thus, transcriptomics analysis not only allowed the prediction of some ripening-regulating genes but also facilitated the characterization of a TF, AdDof3, and a key structural gene, AdBAM3L, in starch degradation.

Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma.

An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive 'liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

Involvement of XBP1s in Blue Light-Induced A2E-Containing Retinal Pigment Epithelium Cell Death.

PURPOSE: Retinal pigment epithelium (RPE) cell dysfunction is essential to the development of retinal degenerative disease. This study was designed to investigate how spliced X-box-binding protein 1 (XBP1s) regulates different modes of RPE cell death in vitro. METHODS: Human ARPE19 cells were incubated with 25 µM N-retinylidene-N-retinylethanolamine (A2E) and irradiated with blue light. Expressions of glucose-regulated protein 78 (GRP78) and XBP1s were detected by real-time quantitative PCR and Western blot. STF-083010 was used to suppress XBP1s expression. ARPE19 cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling and flow cytometry. Receptor-interacting protein kinase-3 (RIP3) was detected by Western blot. Changes in the morphology of ARPE19 cells were identified by transmission electron microscopy. RESULTS: Blue light-induced A2E-containing ARPE19 cell damage caused a transient elevation of GRP78 and XBP1s, while RIP3 rose in the late stage. STF-083010 effectively inhibited XBP1s expression and brought about the aggravation of apoptosis together with an alleviation of RIP3 expression. Most of the dying cells exhibited apoptotic morphology. CONCLUSION: A2E, along with blue light, brought about apoptosis and necroptosis of ARPE19 cells, and XBP1s was transiently elevated. The suppression of XBP1s induced ARPE19 cell death by promoting apoptosis rather than necroptosis. XBP1s might play a role in the pathogenesis of retinal degenerative diseases.

Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial.

MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to <20/6400 were entered in a phase I open-label, dose-escalation trial. One eye of each patient (the worse-seeing eye in five subjects) received a submacular injection of the viral vector, first at a dose of 150 µl (5.96 × 10(10)vg; 2 patients) and then 450 µl (17.88 × 10(10)vg; 4 patients). Patients were followed daily for 10 days at 30, 60, 90, 180, 270, 365, 540, and 730 days post-injection. Collected data included (1) full ophthalmologic examination including best-corrected VA, intraocular pressure, color fundus photographs, macular spectral domain optical coherence tomography and full-field stimulus threshold test (FST) in both the study and fellow eyes; (2) systemic safety data including CBC, liver and kidney function tests, coagulation profiles, urine analysis, AAV antibody titers, peripheral blood PCR and ASR measurement; and (3) listing of ophthalmological or systemic adverse effects. All patients completed the 2-year follow-up. Subretinal injection of rAAV2-VMD2-hMERTK was associated with acceptable ocular and systemic safety profiles based on 2-year follow-up. None of the patients developed complications that could be attributed to the gene vector with certainty. Postoperatively, one patient developed filamentary keratitis, and two patients developed progressive cataract. Of these two patients, one also developed transient subfoveal fluid after the injection as well as monocular oscillopsia. Two patients developed a rise in AAV antibodies, but neither patient was positive for rAAV vector genomes via PCR. Three patients also displayed measurable improved visual acuity in the treated eye following surgery, although the improvement was lost by 2 years in two of these patients. Gene therapy for MERTK-related RP using careful subretinal injection of rAAV2-VMD2-hMERTK is not associated with major side effects and may result in clinical improvement in a subset of patients.

Joint Antiangiogenic Effect of ATN-161 and Anti-VEGF Antibody in a Rat Model of Early Wet Age-Related Macular Degeneration.

The wet form of age-related macular degeneration (AMD) is a leading cause of blindness among elderly Americans and is characterized by abnormal vessel growth, termed choroidal neovascularization (CNV). Integrin α5ß1 is a transmembrane receptor that binds matrix macromolecules and proteinases to stimulate angiogenesis. We recently demonstrated that integrin α5ß1 plays a critical role in the development of choroidal neovascularization. In this study, we determined the role and underlying mechanisms of integrin α5ß1 in angiogenesis in human choroidal endothelial cells and evaluated the antiangiogenic effects of delivering a combination therapy of ATN-161, an integrin α5ß1 inhibitor, and an anti-VEGF monoclonal antibody to rats with laser-induced CNV. Vascular endothelial growth factor (VEGF) is a signaling protein that stimulates vasculogenesis and angiogenesis through a pathway that is distinct from the integrin α5ß1 signaling pathway. Our results indicate that fibronectin binds to integrin α5ß1 and synergizes VEGF-induced angiogenesis via two independent signaling pathways, FN/integrin α5ß1/FAK/ERK1/2 and FN/integrin α5ß1/FAK/AKT. Integrin α5 knockdown by shRNA inhibits endothelial cell migration, tube formation, and proliferation, while ATN-161 only partially decreases integrin α5 function. Treatment with ATN-161 combined with anti-VEGF antibody showed joint effects in attenuating angiogenesis. In summary, our results provide the first evidence for the mechanisms by which integrin α5ß1 is involved in ocular pathological neovascularization in vivo, suggesting that dual inhibition of integrin α5ß1 and VEGF may be a promising novel therapeutic strategy for CNV in wet AMD.

Overview of Research Status in Castleman Disease.

Castleman disease (CD) is characterized by the proliferation of lymphoid tissue and encompasses a range of disorders that vary in clinical presentation, histopathological features, and therapeutic approaches. This article presents a comprehensive review of the current state of CD research, emphasizing the etiology, pathogenesis, clinical manifestations, diagnostic criteria, treatment options, and prognostic factors. CD is a relatively rare condition infrequently encountered in clinical practice. Certain subtypes of CD progress rapidly and pose a significant threat to patient health. Consequently, a timely and accurate diagnosis is crucial. This article aimed to equip clinicians and researchers with an updated and detailed understanding of CD, thereby enhancing the management of this complex condition.