Optimized protocol for high-titer lentivirus production and transduction of primary fibroblasts.

The lentivirus-short hairpin RNA (shRNA) system is a widely used tool for RNA interference. Multiple factors may affect the RNA interference efficiency during lentivirus production and transduction procedures. Thus, an optimized protocol is required to achieve high-titer lentivirus and efficient gene delivery. In the present study, lentivirus was produced by transfecting lentiviral transfer and packaging plasmids into HEK 293T cells. The factors affecting lentiviral titer were assessed, including lentiviral plasmid ratio, lentiviral transfer plasmid type, serum type for cell culture, transfection reagent-plasmid mixture incubation time, and the inoculation density of 293T cells for transfection. The high-titer lentivirus was achieved when plasmids were transfected at a molar ratio of 1:1:1:2, and the transfection reagent-plasmid mixture was replaced 6-8 h after transfection. The pLVX-shRNA2 lentiviral transfer plasmid was associated with the highest lentiviral titer, while both pLVX-shRNA2 and psi-LVRU6GP plasmids were associated with efficient RNA interference in target cells. The serum type for 293T cell culture affected the lentiviral titer significantly, while the inoculation density of 293T cells showed no influence on transfection efficiency or lentiviral titer. Moreover, the human primary fibroblasts infected with lentivirus, using the centrifugation method, achieved higher transduction efficiency than those infected with the non-centrifugation method. In conclusion, this study helped optimize lentiviral production and transduction procedures for more efficient gene delivery.

Autophagy facilitates age-related cell apoptosis-a new insight from senile cataract.

Age-related cell loss underpins many senescence-associated diseases. Apoptosis of lens epithelial cells (LECs) is the important cellular basis of senile cataract resulted from prolonged exposure to oxidative stress, although the specific mechanisms remain elusive. Our data indicated the concomitance of high autophagy activity, low SQSTM1/p62 protein level and apoptosis in the same LEC from senile cataract patients. Meanwhile, in primary cultured LECs model, more durable autophagy activation and more obvious p62 degradation under oxidative stress were observed in LECs from elder healthy donors, compared with that from young healthy donors. Using autophagy-deficiency HLE-B3 cell line, autophagy adaptor p62 was identified as the critical scaffold protein sustaining the pro-survival signaling PKCι-IKK-NF-κB cascades, which antagonized the pro-apoptotic signaling. Moreover, the pharmacological inhibitor of autophagy, 3-MA, significantly inhibited p62 degradation and rescued oxidative stress-induced apoptosis in elder LECs. Collectively, this study demonstrated that durable activation of autophagy promoted age-related cell death in LECs. Our work contributes to better understanding the pathogenesis of senescence-associated diseases.

Optimization of determinant factors associated with the efficiency of experimental autoimmune uveitis induction in C57BL/6 mice.

Background: Experimental autoimmune uveitis (EAU) is a widely used animal model for uveitis research. The C57BL/6 mouse strain is the most commonly used mouse strain in the research of genetic modification, but C57BL/6 mice are not sufficiently susceptible to EAU induction, partly due to experimental factors. This work aims to optimize relevant factors to improve the efficiency of EAU induction in C57BL/6 mice. Methods: To induce EAU, mice were immunized via intraperitoneal injection with pertussis (PTX) and subcutaneous injection with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20) emulsified with complete Freund's adjuvant (CFA). The severity of inflammation was assessed using several approaches. The relevant experimental factors were evaluated, including methods of emulsification and doses of peptide and PTX. Results: Uveitis occurred at 8-12 days after immunization and reached its peak at 18-20 days, while T helper type 17 (Th17) cells peaked earlier at 14-18 days after immunization. Based on clinical and histological scores, 500 µg of IRBP peptide was the optimal dose required to induce EAU. The PTX dose demonstrated no influence on EAU incidence, but potentially affected the severity of uveitis. A single injection of 1,000 ng of PTX induced the most severe EAU and the highest proportion of Th17 cells. Compared to extruded emulsion, sonicated emulsion produced a higher incidence, higher histological score, and a 2-day-earlier onset of EAU. Electron microscopy showed a significantly different microstructure between the 2 emulsions. Conclusions: This work optimized the protocols of EAU induction and obtained a high and stable induction rate with severe inflammation in the C57BL/6 mouse strain. Our results facilitate future experimental research involving uveitis.

Machine learning for predicting the treatment effect of orthokeratology in children.

Purpose: Myopia treatment using orthokeratology (ortho-k) slows myopia progression. However, it is not equally effective in all patients. We aimed to predict the treatment effect of ortho-k using a machine-learning-assisted (ML) prediction model. Methods: Of the 119 patients who started ortho-k treatment between January 1, 2019, and January 1, 2022, 91 met the inclusion criteria and were included in the model. Ocular parameters and clinical characteristics were collected. A logistic regression model with least absolute shrinkage and selection operator regression was used to select factors associated with the treatment effect. Results: Age, baseline axial length, pupil diameter, lens wearing time, time spent outdoors, time spent on near work, white-to-white distance, anterior corneal flat keratometry, and posterior corneal astigmatism were selected in the model (aera under curve: 0.949). The decision curve analysis showed beneficial effects. The C-statistic of the predictive model was 0.821 (95% CI: 0.815, 0.827). Conclusion: Ocular parameters and clinical characteristics were used to predict the treatment effect of ortho-k. This ML-assisted model may assist ophthalmologists in making clinical decisions for patients, improving myopia control, and predicting the clinical effect of ortho-k treatment via a retrospective non-intervention trial.

Effect of corneal incision features on anterior and posterior corneal astigmatism and higher-order aberrations after cataract surgery.

PURPOSE: To evaluate the influence of 2.2 mm clear corneal incision (CCI) features in surgically induced astigmatism (SIA) and higher-order aberrations (HOAs) after cataract surgery. METHODS: Right eyes of 92 subjects receiving 2.2 mm incision cataract surgery were involved. A total of 38 eyes were categorized as the intact incision group, and 54 eyes were the defective incision group. Pre- and postoperative (1 month and 6 months) corneal astigmatism and HOAs on anterior and posterior corneal surfaces, corneal volume, and corneal thickness (CT) were measured using Pentacam. The CCI features including incision length (IL), incision angles, distance from incision to central cornea (Dis-En/Ex), and CT at incision site were quantified using AS-OCT. RESULTS: The defective incision group showed shorter IL and larger incision angles [false discovery rate (FDR) - p < 0.05]. Changes in CT at incision site were more pronounced for the defective incision group (FDR - p < 0.05). Some SIA parameters were related to the certain specific CCI features, especially IL (FDR - p < 0.05). Both groups exhibited significant increased 6 mm posterior corneal tHOAs at 1 month (Bonferroni corrected - p < 0.01) and the defective incision group showed increased 6 mm posterior tHOAs at 6 months (Bonferroni corrected - p = 0.023). There were characteristic correlations between Zernike terms and CCI features including IL, CT, Dis-En/Ex, and incision angles at 1 month, especially over 6 mm zone. CONCLUSION: The CCI deformities can affect corneal recovery and induce more HOAs at 1 month postoperatively. Such effects became minor, but could persist until 6 months. The IL combined with Angle-En/Ex was important factor influencing CCI integrity and corneal optical quality.

Analyzing fundus images to detect diabetic retinopathy (DR) using deep learning system in the Yangtze River delta region of China.

BACKGROUND: This study aimed to establish and evaluate an artificial intelligence-based deep learning system (DLS) for automatic detection of diabetic retinopathy. This could be important in developing an advanced tele-screening system for diabetic retinopathy. METHODS: A DLS with a convolutional neural network was developed to recognize fundus images of referable diabetic retinopathy. A total data set of 41,866 color fundus images were obtained from 17 cities in the Yangtze River Delta Urban Agglomeration (YRDUA). Five experienced retinal specialists and 15 ophthalmologists were recruited to verify images. For training, 80% of the data set was used, and the other 20% served as the validation data set. To effectively understand the learning process, the DLS automatically superimposed a heatmap on the original image. The regions utilized by the DLS were highlighted for diagnosis. RESULTS: Using the local validation data set, the DLS achieved an area under the curve of 0.9824. Based on the manual screening criteria, an operating point was set at about 0.9 sensitivity to evaluate the DLS. Specificity was recorded at 0.9609 and sensitivity was 0.9003. The DLSs showed excellent reliability, repeatability, and high efficiency. After analyzing the misclassification, it was found that 88.6% of the false-positives were mild non-proliferative diabetic retinopathy (NPDR) whereas, 81.6% of the false-negatives were intraretinal microvascular abnormalities. CONCLUSIONS: The DLS efficiently detected fundus images from complex sources in the real world. Incorporating DLS technology in tele-screening will advance the current screening programs to offer a cost-effective and time-efficient solution for detecting diabetic retinopathy.

AI-Model for Identifying Pathologic Myopia Based on Deep Learning Algorithms of Myopic Maculopathy Classification and "Plus" Lesion Detection in Fundus Images.

Background: Pathologic myopia (PM) associated with myopic maculopathy (MM) and "Plus" lesions is a major cause of irreversible visual impairment worldwide. Therefore, we aimed to develop a series of deep learning algorithms and artificial intelligence (AI)-models for automatic PM identification, MM classification, and "Plus" lesion detection based on retinal fundus images. Materials and Methods: Consecutive 37,659 retinal fundus images from 32,419 patients were collected. After excluding 5,649 ungradable images, a total dataset of 32,010 color retinal fundus images was manually graded for training and cross-validation according to the META-PM classification. We also retrospectively recruited 1,000 images from 732 patients from the three other hospitals in Zhejiang Province, serving as the external validation dataset. The area under the receiver operating characteristic curve (AUC), sensitivity, specificity, accuracy, and quadratic-weighted kappa score were calculated to evaluate the classification algorithms. The precision, recall, and F1-score were calculated to evaluate the object detection algorithms. The performance of all the algorithms was compared with the experts' performance. To better understand the algorithms and clarify the direction of optimization, misclassification and visualization heatmap analyses were performed. Results: In five-fold cross-validation, algorithm I achieved robust performance, with accuracy = 97.36% (95% CI: 0.9697, 0.9775), AUC = 0.995 (95% CI: 0.9933, 0.9967), sensitivity = 93.92% (95% CI: 0.9333, 0.9451), and specificity = 98.19% (95% CI: 0.9787, 0.9852). The macro-AUC, accuracy, and quadratic-weighted kappa were 0.979, 96.74% (95% CI: 0.963, 0.9718), and 0.988 (95% CI: 0.986, 0.990) for algorithm II. Algorithm III achieved an accuracy of 0.9703 to 0.9941 for classifying the "Plus" lesions and an F1-score of 0.6855 to 0.8890 for detecting and localizing lesions. The performance metrics in external validation dataset were comparable to those of the experts and were slightly inferior to those of cross-validation. Conclusion: Our algorithms and AI-models were confirmed to achieve robust performance in real-world conditions. The application of our algorithms and AI-models has promise for facilitating clinical diagnosis and healthcare screening for PM on a large scale.

Development of deep learning-based detecting systems for pathologic myopia using retinal fundus images.

Globally, cases of myopia have reached epidemic levels. High myopia and pathological myopia (PM) are the leading cause of visual impairment and blindness in China, demanding a large volume of myopia screening tasks to control the rapid growing myopic prevalence. It is desirable to develop the automatically intelligent system to facilitate these time- and labor- consuming tasks. In this study, we designed a series of deep learning systems to detect PM and myopic macular lesions according to a recent international photographic classification system (META-PM) classification based on color fundus images. Notably, our systems recorded robust performance both in the test and external validation dataset. The performance was comparable to the general ophthalmologist and retinal specialist. With the extensive adoption of this technology, effective mass screening for myopic population will become feasible on a national scale.

Evaluation of ocular symptoms and tropism of SARS-CoV-2 in patients confirmed with COVID-19.

PURPOSE: The SARS-CoV-2 RNA has been detected in tears and conjunctival samples from infected individuals. Conjunctivitis is also reported in a small number of cases. We evaluated ocular symptoms and ocular tropism of SARS-CoV-2 in a group of patients with COVID-19. METHOD: Fifty-six patients infected with SARS-CoV-2 were recruited as subjects. Relevant medical histories were obtained from the electronic medical record system. Ocular history and ocular symptoms data were obtained by communicating directly with the subjects. The Ocular Surface Disease Index (OSDI) and Salisbury Eye Evaluation Questionnaire (SEEQ) were used to assess the anterior ocular surface condition before and after the onset of disease. RESULTS: Patients classified as severe COVID-19 cases were more likely to have hypertension compared to mild cases (p = 0.035). Of the 56 subjects, thirteen patients (23%) were infected in Wuhan, 32 patients (57%) were community-infected, 10 patients (18%) were unknown origin, 1 (2%) was a physician likely infected by a confirmed patient. Three patients wore face mask with precaution when contacting the confirmed patients. Fifteen (27%) had aggravated ocular symptoms, of which 6 (11%) had prodromal ocular symptoms before disease onset. The differences in mean scores of OSDI questionnaire and SEEQ between before and after onset of COVID-19 were all significant (p < 0.05 for both). CONCLUSIONS: Ocular symptoms are relatively common in COVID-19 disease and may appear just before the onset of respiratory symptoms. Our data provided the anecdotal evidences of transmission of SARS-CoV-2 via ocular surface.

Growth, antioxidant capacity, intestinal morphology, and metabolomic responses of juvenile Oriental river prawn (Macrobrachium nipponense) to chronic lead exposure.

Understanding the mechanisms of metal toxicity to organisms farmed for food may suggest mitigation strategies. We determined the 24-, 48-, 72-, and 96-h median lethal concentrations of lead in juvenile oriental river prawn (Macrobrachium nipponense). The prawns were then exposed to sub-lethal concentrations (13.13 and 26.26 µg/L) of lead for 60 days and growth, antioxidant enzyme activity, intestinal morphology, and metabolite profiles were assessed. Prawns exposed to 26.26 µg/L but not to 13.13 µg/L lead exhibited lower weight gain than controls. The lead burden in muscle was 0.067 and 0.25 µg/g of dry weight exposed to 13.13 and 26.26 µg/L, respectively. Levels of glutamic oxaloacetic transaminase and glutamic-pyruvic transaminase were not altered following exposure. Exposure increased malondialdehyde activity in the hepatopancreas and decreased superoxide dismutase and glutathione peroxidase activities. Catalase activity first increased and then decreased as lead concentrations increased. Some intestinal epithelial cells disassociated from the basement membrane in prawns exposed to 13.13 µg/L lead. Intestinal epithelial cells in prawns exposed to 26.26 µg/L lead separated completely from the basement membrane. Gas chromatography-mass spectrometry metabolomics assays showed the 13.13-µg/L exposure did not elicit significant metabolic alterations. Exposure to 26.26 µg/L lead differentially up-regulated 58 metabolites and down-regulated 21 metabolites. The metabolites identified were involved in galactose, purine, glutathione, and carbon metabolism, biosynthesis of amino acids and steroids, and neuroactive ligand-receptor interaction. These data indicate that chronic lead exposure can adversely affect growth, increase accumulation in muscle, impair intestinal morphology, and induce oxidant stress or neurotoxicity-related effects in M. nipponense.

Targeted Exome Sequencing of Congenital Cataracts Related Genes: Broadening the Mutation Spectrum and Genotype-Phenotype Correlations in 27 Chinese Han Families.

Congenital cataract is the most frequent inherited ocular disorder and the most leading cause of lifelong visual loss. The screening of pathogenic mutations can be very challenging in some cases, for congenital cataracts are clinically and genetically heterogeneous diseases. The aim of this study is to investigate the mutation spectrum and frequency of 54 cartaract-associated genes in 27 Chinese families with congenital cataracts. Variants in 54 cataract-associated genes were screened by targeted next-generation sequencing (NGS) and then validated by Sanger sequencing. We identified pathogenic variants in 62.96% (17/27) of families, and over 52.94% (9/17) of these variants were novel. Among them, three are splicing site mutations, four are nonsense mutations, seven are missense mutations, two are frame shift mutations and one is intronic mutation. This included identification of: complex ocular phenotypes due to two novel PAX6 mutations; progressive cortical cataract and lamellar cataract with lens subluxation due to two novel CRYGS mutations. Mutations were also found in rarely reported genes including CRYBA4, CRYBA2, BFSP1, VIM, HSF4, and EZR. Our study expands the mutation spectrum and frequency of genes responsible for congenital cataracts. Targeted next-generation sequencing in inherited congenital cataract patients provided significant diagnostic information.

Evaluation of dry eye after femtosecond laser-assisted cataract surgery.

PURPOSE: To compare dry-eye signs and symptoms after femtosecond laser-assisted cataract surgery and conventional phacoemulsification. SETTING: Eye Center of the 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou, China. DESIGN: Prospective consecutive nonrandomized comparative cohort study. METHODS: Consecutive patients who had femtosecond laser-assisted or phacoemulsification cataract surgery were assessed. Dry-eye markers including the ocular surface disease index (OSDI) and subjective symptom questionnaire, tear-film assessment using Keratograph 4 corneal topography, Schirmer testing I, and fluorescein staining were sequentially evaluated preoperatively and postoperatively at 1 day, 1 week, and 1 month. RESULTS: The study recruited 137 eyes (137 patients) with similar baseline characteristics. Most patients developed dry eye postoperatively. Subjective symptoms and fluorescein staining scores elevated from baseline, tear breakup time and Schirmer testing I values decreased postoperatively, which peaked at 1 week and did not return to baseline within 1 month. There were no significant differences between the 2 groups (all P > .05) except for a higher fluorescein staining score in the femtosecond group at 1 day (P = .001), 1 week (P = .047), and 1 month (P = .025). OSDI score and subjective symptoms were greater in the laser group at 1 week (P = .014 and P = .016, respectively). Subgroup analysis showed obvious worsening by fluorescein staining at 1 day (P = .016) and 1 month (P = .009) in preoperative dry-eye patients. CONCLUSIONS: Both methods worsened dry eye postoperatively. Femtosecond-assisted surgery had a higher risk for staining and dry-eye symptoms. Patients with preexisting dry eye who had femtosecond-assisted surgery had more severe ocular surface staining than those having conventional surgery. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.