A deep-learning pipeline for the diagnosis and discrimination of viral, non-viral and COVID-19 pneumonia from chest X-ray images.

Common lung diseases are first diagnosed using chest X-rays. Here, we show that a fully automated deep-learning pipeline for the standardization of chest X-ray images, for the visualization of lesions and for disease diagnosis can identify viral pneumonia caused by coronavirus disease 2019 (COVID-19) and assess its severity, and can also discriminate between viral pneumonia caused by COVID-19 and other types of pneumonia. The deep-learning system was developed using a heterogeneous multicentre dataset of 145,202 images, and tested retrospectively and prospectively with thousands of additional images across four patient cohorts and multiple countries. The system generalized across settings, discriminating between viral pneumonia, other types of pneumonia and the absence of disease with areas under the receiver operating characteristic curve (AUCs) of 0.94-0.98; between severe and non-severe COVID-19 with an AUC of 0.87; and between COVID-19 pneumonia and other viral or non-viral pneumonia with AUCs of 0.87-0.97. In an independent set of 440 chest X-rays, the system performed comparably to senior radiologists and improved the performance of junior radiologists. Automated deep-learning systems for the assessment of pneumonia could facilitate early intervention and provide support for clinical decision-making.

Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell Proliferation.

OBJECTIVE: Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. APPROACH AND RESULTS: To study the role of intermedin, we generated the IMD-KO (Adm2-/-) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/ß-arr1 (ß-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell-cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. CONCLUSIONS: Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.

Efficacy of Lenvatinib, a multitargeted tyrosine kinase inhibitor, on laser-induced CNV mouse model of neovascular AMD.

Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Although intravitreal injection of anti-VEGF antibodies and VEGF Trap have significant clinical benefits, the complications of intravitreal injection, drug resistance and patient compliance still need to be concerned. In this study, the effects of an orally administered multi-targeted tyrosine kinase inhibitor (Lenvatinib, E7080) were evaluated in vitro and in vivo on neovascular AMD mouse model. The results showed that E7080 effectively inhibited the proliferation, migration and tubule formation of human choroidal microvascular endothelial cells (HCMECs), and suppressed the angiogenesis of zebrafish subintestinal vessels without causing malformation. The anti-angiogenic effect of E7080 on the laser-induced choroidal neovascularization (CNV) mouse model by oral administration of 10 mg/kg/day was observed. The fluorescein angiography showed CNV leakage area in treatment group vs control group was 3.407 ±â€¯0.2939 vs 5.202 ±â€¯0.9001 (P = .0133) at day 7th post laser-induced CNV, 1.138 ±â€¯0.4334 vs 3.122 ±â€¯0.3466 (P = .0064) at day 14th, 1.401 ±â€¯0.6577 vs 2.781 ±â€¯0.9815 (P = .00262) at day 21th respectively. Moreover, pharmacokinetics analysis in rat retina showed that E7080 rapidly penetrated the blood-retina barrier to retina through oral administration. The T1/2 in retina was 3.81 ±â€¯0.77 h, the Tmax was 4.60 ±â€¯0.73 h, the AUC0-∞ was 110448.51 ± 18532.51 h*ng/g after a single dose administration analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In conclusion, our study suggested that orally administered E7080 can be a novel therapeutic strategy for neovascular AMD.

MicroRNA Expression Profile on Solid Subtype of Invasive Lung Adenocarcinoma Reveals a Panel of Four miRNAs to Be Associated with Poor Prognosis in Chinese Patients.

According to the reclassification of lung adenocarcinoma (LAC) proposed in 2011, solid predominant lung adenocarcinoma (SPA) has been associated with poor outcomes for LAC patients. However, the prognostic value of the presence of solid subtype remains unclear. Besides, there is little data about the roles of microRNA (miRNA) in solid subtype of LAC. In this study, 243 LAC patients were classified into solid subtype positive and negative groups (S+ LAC, n=134 and S- LAC, n=109) according to whether the solid subtype was more than 5% of the tumor component or not. We analyzed the relationship between solid subtype and patients' outcome by univariate and multivariate analyses. Solid subtype was proved to be significantly associated with the 5-year overall survival and played as an independent prognostic factor for stage I-III invasive LAC patients. Then miRNA microarray was used to identify differentially expressed miRNAs in solid subtype, resulting in 31 differential miRNAs. Quantitative reverse transcription-PCR (QRT-PCR) was used to validate 4 key miRNAs (miR-133b, miR-155-5p, miR-124-3p, miR-145-5p). Further, CCK-8 and transwell assays were performed to validate the impact of one dysregulated miRNA (miR-133b) on LAC cell function. Interestingly, while miR-133b could significantly inhibit the proliferation of A549 and SPC-A1, it showed no effect on the migration or invasion of LAC cell lines. These results suggest that solid subtype can exert independent prognostic impact on LAC patients, and 4 important dysregulated miRNAs in solid subtype of LAC may be involved in the malignancy of S+LAC, thus may further have clinical perspective for S+ LAC in the future.

Lens regeneration using endogenous stem cells with gain of visual function.

The repair and regeneration of tissues using endogenous stem cells represents an ultimate goal in regenerative medicine. To our knowledge, human lens regeneration has not yet been demonstrated. Currently, the only treatment for cataracts, the leading cause of blindness worldwide, is to extract the cataractous lens and implant an artificial intraocular lens. However, this procedure poses notable risks of complications. Here we isolate lens epithelial stem/progenitor cells (LECs) in mammals and show that Pax6 and Bmi1 are required for LEC renewal. We design a surgical method of cataract removal that preserves endogenous LECs and achieves functional lens regeneration in rabbits and macaques, as well as in human infants with cataracts. Our method differs conceptually from current practice, as it preserves endogenous LECs and their natural environment maximally, and regenerates lenses with visual function. Our approach demonstrates a novel treatment strategy for cataracts and provides a new paradigm for tissue regeneration using endogenous stem cells.

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.

Effects of adiponectin polymorphisms on the risk of advanced age-related macular degeneration.

OBJECTIVE: To determine the relationships between variants in adiponectin gene (ADIPOQ) with advanced forms of age-related macular degeneration (AMD) susceptibility. METHODS: A total of 189 advanced AMD patients and 168 controls were recruited. Seven tagging single-nucleotide polymorphisms in ADIPOQ were genotyped by the SNaPshot method. RESULTS: Alleles or genotypes of rs822396 distributed significantly differently in advanced AMD patients and controls. The minor allele G at rs822396 was associated with an increased risk of advanced AMD in a dominant model. Furthermore, haplotype analysis revealed that haplotypes AGGACCT and TGACCCC were significantly increased the advanced AMD susceptibility, whereas haplotypes AGAACGC, TGAACGT and TGACAGC had protective effects. CONCLUSION: ADIPOQ genetic variant rs822396 might affect an individual's susceptibility to AMD, making it efficient genetic biomarkers for early detection of AMD.

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.

Transcription Factor PAX6 (Paired Box 6) Controls Limbal Stem Cell Lineage in Development and Disease.

PAX6 is a master regulatory gene involved in neuronal cell fate specification. It also plays a critical role in early eye field and subsequent limbal stem cell (LSC) determination during eye development. Defects in Pax6 cause aniridia and LSC deficiency in humans and the Sey (Small eye) phenotype in mice (Massé, K., Bhamra, S., Eason, R., Dale, N., and Jones, E. A. (2007) Nature 449, 1058-1062). However, how PAX6 specifies LSC and corneal fates during eye development is not well understood. Here, we show that PAX6 is expressed in the primitive eye cup and later in corneal tissue progenitors in early embryonic development. In contrast, p63 expression commences after that of PAX6 in ocular adnexal and skin tissue progenitors and later in LSCs. Using an in vitro feeder-free culture system, we show that PAX6 knockdown in LSCs led to up-regulation of skin epidermis-specific keratins concomitant with differentiation to a skin fate. Using gene expression analysis, we identified the involvement of Notch, Wnt, and TGF-ß signaling pathways in LSC fate determination. Thus, loss of PAX6 converts LSCs to epidermal stem cells, as demonstrated by a switch in the keratin gene expression profile and by the appearance of congenital dermoid tissue.

Identify the signature genes for diagnose of uveal melanoma by weight gene co-expression network analysis.

AIM: To identify and understand the relationship between co-expression pattern and clinic traits in uveal melanoma, weighted gene co-expression network analysis (WGCNA) is applied to investigate the gene expression levels and patient clinic features. Uveal melanoma is the most common primary eye tumor in adults. Although many studies have identified some important genes and pathways that were relevant to progress of uveal melanoma, the relationship between co-expression and clinic traits in systems level of uveal melanoma is unclear yet. We employ WGCNA to investigate the relationship underlying molecular and phenotype in this study. METHODS: Gene expression profile of uveal melanoma and patient clinic traits were collected from the Gene Expression Omnibus (GEO) database. The gene co-expression is calculated by WGCNA that is the R package software. The package is used to analyze the correlation between pairs of expression levels of genes. The function of the genes were annotated by gene ontology (GO). RESULTS: In this study, we identified four co-expression modules significantly correlated with clinic traits. Module blue positively correlated with radiotherapy treatment. Module purple positively correlates with tumor location (sclera) and negatively correlates with patient age. Module red positively correlates with sclera and negatively correlates with thickness of tumor. Module black positively correlates with the largest tumor diameter (LTD). Additionally, we identified the hug gene (top connectivity with other genes) in each module. The hub gene RPS15A, PTGDS, CD53 and MSI2 might play a vital role in progress of uveal melanoma. CONCLUSION: From WGCNA analysis and hub gene calculation, we identified RPS15A, PTGDS, CD53 and MSI2 might be target or diagnosis for uveal melanoma.

Salidroside protects retinal endothelial cells against hydrogen peroxide-induced injury via modulating oxidative status and apoptosis.

Oxidative stress can cause injury in retinal endothelial cells. Salidroside is a strong antioxidative and cytoprotective supplement in Chinese traditional medicine. In this study, we investigated the effects of salidroside on H2O2-induced primary retinal endothelial cells injury. Salidroside decreased H2O2-induced cell death, and efficiently suppressed cellular ROS production, malondialdehyde generation, and cell apoptosis induced by H2O2 treatment. Salidroside induced the intracellular mRNA expression, protein expression, and enzymatic activities of catalase and Mn-SOD and increased the ratio of Bcl2/Bax. Our results demonstrated that salidroside protected retinal endothelial cells against oxidative injury through increasing the Bcl2/Bax signaling pathway and activation of endogenous antioxidant enzymes. This finding presents salidroside as an attractive agent with potential to attenuate retinopathic diseases.

CETP Gene may be Associated with Advanced Age-Related Macular Degeneration in the Chinese Population.

OBJECTIVES: This study aims to investigate whether variations in LIPC, CETP, ABCA1 and LPL, which are involved in high-density lipoprotein (HDL) metabolism, are associated with advanced age-related macular degeneration (AMD) in the Chinese population. DESIGN AND METHODS: A total of 119 Chinese patients with advanced AMD and 99 control individuals were recruited. Genomic DNA was extracted from peripheral blood leukocytes. Genotypes of seven single nucleotide polymorphisms (SNPs) including rs1061170 and rs1410996 in CFH, rs10490924 in HTRA1, rs10468017 in LIPC, rs3764261 in CETP, rs1883025 in ABCA1 and rs12678919 near LPL were determined by polymerase chain reaction (PCR) followed by allele-specific restriction enzyme digestion or SNaPshot. Unconditional logistic regression analyses were performed to generate a risk predictive model. RESULTS: We observed the frequency of allele A of rs3764261 in CETP to be significantly lower in advanced AMD after Bonferroni correction (15.5% in patients with AMD and 20.7% in controls; OR = 0.49, 95% CI: 0.29-0.85; p = 0.011). Furthermore, we found that it was also associated with reduced risk of both unilateral AMD (OR = 0.52, 95% CI: 0.28-0.98; p = 0.043) and bilateral AMD (OR = 0.45, 95% CI: 0.22-0.91; p = 0.026). Rs10468017 in LIPC, rs12678919 near LPL and rs1883025 in ABCA1 were not found to be associated with advanced AMD (all p > 0.05). CONCLUSION: Our data suggested that the allele A in rs3764261 in CETP gene may be associated with a decreased risk of advanced AMD in Chinese population.

Malattia leventinese/Doyne honeycomb retinal dystrophy in a chinese family with mutation of the EFEMP1 gene.

PURPOSE: To characterize the clinical features and molecular genetic findings in a Chinese pedigree with Malattia leventinese/Doyne honeycomb retinal dystrophy. METHODS: All patients underwent ophthalmologic examinations, including Snellen best-corrected visual acuity, fundus photography, fundus autofluorescence imaging, fundus fluorescein angiography, and optical coherence tomography. Genomic DNA was isolated from blood samples. All exons of EFEMP1 were amplified by polymerase chain reaction and sequenced. Possible structural and functional impacts of the protein because of amino acid substitution were predicted by bioinformatics analysis. RESULTS: A heterozygous missense mutation comprising C > T in exon 10 of EFEMP1 was identified in all patients of the pedigree; this resulted in an amino acid substitution at position 345 (Arg345Trp, R345W). Clinically, six patients from the Chinese family were ascertained with varying degrees of early onset drusen. Besides the drusen, choroidal neovascularization and retinal pigment epithelium changes were noted in some patients. Increased autofluorescence corresponding to the drusen was detected in the R345W mutation patients. Intrafamilial patients with Malattia leventinese/Doyne honeycomb retinal dystrophy seem to be phenotypically variable in visual loss, ophthalmoscopic findings, autofluorescence imaging, and optical coherence tomography changes. The amino acid change may have an effect on protein structure and function through bioinformatics analysis. CONCLUSION: The R345W mutation in EFEMP1 caused Malattia leventinese/Doyne honeycomb retinal dystrophy in a Chinese family. This is the first report, as per our knowledge, of the R345W mutation in EFEMP1 in a Chinese pedigree of this disease.

WNT7A and PAX6 define corneal epithelium homeostasis and pathogenesis.

The surface of the cornea consists of a unique type of non-keratinized epithelial cells arranged in an orderly fashion, and this is essential for vision by maintaining transparency for light transmission. Cornea epithelial cells (CECs) undergo continuous renewal from limbal stem or progenitor cells (LSCs), and deficiency in LSCs or corneal epithelium--which turns cornea into a non-transparent, keratinized skin-like epithelium--causes corneal surface disease that leads to blindness in millions of people worldwide. How LSCs are maintained and differentiated into corneal epithelium in healthy individuals and which key molecular events are defective in patients have been largely unknown. Here we report establishment of an in vitro feeder-cell-free LSC expansion and three-dimensional corneal differentiation protocol in which we found that the transcription factors p63 (tumour protein 63) and PAX6 (paired box protein PAX6) act together to specify LSCs, and WNT7A controls corneal epithelium differentiation through PAX6. Loss of WNT7A or PAX6 induces LSCs into skin-like epithelium, a critical defect tightly linked to common human corneal diseases. Notably, transduction of PAX6 in skin epithelial stem cells is sufficient to convert them to LSC-like cells, and upon transplantation onto eyes in a rabbit corneal injury model, these reprogrammed cells are able to replenish CECs and repair damaged corneal surface. These findings suggest a central role of the WNT7A-PAX6 axis in corneal epithelial cell fate determination, and point to a new strategy for treating corneal surface diseases.

Effects of nerve growth factor on nerve regeneration after corneal nerve damage.

The study aims to determine the relation between the effects of mouse nerve growth factor (mNGF) and nerve regeneration after corneal surgery nerve damage. Mechanical nerve injury animal model was established by LASIK (the excimer laser keratomileusis) surgery in 12 Belgian rabbits. mNGF and the balanced salt solution (BBS) were alternatively administered in the left and right eye two times every day for 8 weeks. The morphous and growth of the sub-basal nerve plexus and superficial stroma were observed by in vivo confocal microscopy at the end of weeks 1, 2, 4 and 8 after the surgery. The animal model is successfully established. The morphology and density of corneal nerve have been observed and demonstrated by confocal microscopy. A systematic administration of mNGF can significantly promote the nerve regeneration at the end of weeks 1, 2, 4 and 8, which comparing to the administration of balanced salt solution (P < 0.05). mNGF has effect on sub-basal nerve plexus and superficial stroma after corneal nerve damage which is caused by LASIK. The experimental results suggested that the mNGF may solve the problem of dry eye after LASIK.

RAD51 gene is associated with advanced age-related macular degeneration in Chinese population.

OBJECTIVES: This study aims to investigate whether variations in RAD51, B3GALTL, TNFRSF10A and REST-C4ORF14-POLR2B-IGFBP7 are associated with advanced forms of age-related macular degeneration (AMD) in Chinese population. DESIGN AND METHODS: A total of 119 Chinese patients with AMD and 99 control individuals were recruited. Genomic DNA was extracted from peripheral blood leukocytes. Seven single nucleotide polymorphisms (SNPs) from CFH, HTRA1, RAD51, B3GALTL, TNFRSF10A and REST-C4ORF14-POLR2B-IGFBP7 were genotyped by polymerase chain reaction (PCR) followed by allele-specific restriction enzyme digestion or SNaPshot. RESULTS: Rs10483810 in RAD51 was significantly associated with advanced AMD (P=0.045). Compared with the wild-type genotype GG, the odds ratio for the risk of advanced AMD was 4.92 (95% confidence interval: 1.04-23.36) for the heterozygous TG genotype. Moreover, the GT genotype at rs10483810 confers significantly increased risk of bilateral AMD compared to unilateral AMD (OR=12.04, 95% CI: 2.50-57.69, P=0.002). Rs13278062 in TNFRSF10A, rs1713985 in REST-C4ORF14-POLR2B-IGFBP7 and rs9542236 in B3GALTL were not found to be associated with AMD (all P>0.05). CONCLUSION: Our data suggested that the risk allele T of rs10483810 in RAD51 gene is associated with an increased risk of advanced AMD, especially bilateral AMD, in Chinese population.

TGFBI gene mutation analysis in a Chinese pedigree of Reis-Bücklers corneal dystrophy.

PURPOSE: To analyze transforming growth factor beta-induced (TGFBI) gene mutations in a Chinese pedigree with Reis-Bücklers dystrophy (RBCD). METHODS: In a four-generation Chinese family with Reis-Bücklers dystrophy, six members were patients and the rest were unaffected. All members of the family underwent complete ophthalmologic examinations. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database. The sequencing results were reconfirmed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: A single heterozygous C>T (R124C) point mutation was found in exon 4 of TGFBI in all six members of the pedigree affected with RBCD, but not in the unaffected members. CONCLUSIONS: Within this pedigree, RBCD segregates with the R124C variance, which is a known mutation for lattice corneal dystrophy type I. Therefore, along with G623D and R124L, the R124C mutation in TGFBI is also found to be responsible for RBCD.