Whole Genome Sequencing Highlights the Pathogenic Profile in Nocardia Keratitis.

Purpose: Nocardia keratitis is a serious and sight-threatening condition. This study aims to reveal the virulence and antimicrobial resistance gene profile of Nocardia strains using whole genome sequencing. Methods: Whole-genome sequencing was performed on 23 cornea-derived Nocardia strains. Together with genomic data from the respiratory tract and the environment, 141 genomes were then utilized for phylogenetic and pan-genome analyses, followed by virulence and antibiotic resistance analysis. The correlations between virulence genes and pathogenicity were experimentally validated, including the characteristics of Nocardia colonies and clinical and histopathological evaluations of Nocardia keratitis mice models. Results: Whole-genome sequencing of 141 Nocardia strains revealed a mean of 220 virulence genes contributed to bacterial pathogenesis. The mce gene family analysis led to the categorization of strains from the cornea into groups A, B, and C. The colonies of group C had the largest diameter, height, and fastest growth rate. The size of corneal ulcers and the clinical scores showed a significant increase in mouse models induced by group C. The relative expression levels of pro-inflammatory cytokines (CD4, IFN-γ, IL-6Rα, and TNF-α) in the lesion area exhibited an increasing trend from group A to group C. Antibiotic resistance genes (ARGs) spanned nine distinct drug classes, four resistance mechanisms, and seven primary antimicrobial resistance gene families. Conclusions: Whole genome sequencing highlights the pathogenic role of mce gene family in Nocardia keratitis. Its distribution pattern may contribute to the distinct characteristics of the growth of Nocardia colonies and the clinical severity of the mice models.

Acanthamoeba Keratitis in China: Genotypic and Clinical Correlations.

Purpose: To investigate the relationship between Acanthamoeba genotypes, clinical manifestations, and outcomes in Acanthamoeba keratitis (AK) patients. Methods: This retrospective study included 159 culture-confirmed AK patients. Patients' data were collected, including demographics, initial diagnosis, treatments, and clinical features. The genotype of Acanthamoeba was identified through sequencing the Diagnostic Fragment 3 (DF3) region in the small ribosomal subunit RNA genes. The phylogenetic tree was constructed using the ClustalW model and maximum likelihood method. Cases with "poor outcome" were defined based on specific clinical criteria, including corneal perforation, keratoplasty, other eye surgery, duration of anti-amoebic therapy ≥8.0 months, and final visual acuity ≤20/80. "Better outcome" cases were the remainder. The correlation between T4 subtypes, clinical phenotypes, and clinical prognosis were further analyzed. Results: In this study, AK was primarily attributed to the T4A genotype, with a positive correlation between geographical and genetic distances. The primary clinical associated with T4 subtypes was deep stromal infiltration. Results was also showed a significant association between T4 subtypes and clinical outcomes (P = 0.021). Further analysis revealed that T4C was closely associated with a better prognosis (P = 0.040) and T4D with worse outcomes (P = 0.013). Conclusions: In China, AK was predominantly caused by the T4A subtype. Geographical distance positively correlated with genetic distance. Clinical prognosis varied among different subtypes, notably in T4C and T4D. Translational Relevance: This study demonstrated the association between T4 subtypes and clinical phenotypes, as well as the effects of T4 subtypes on clinical prognosis.

Ferroptosis as a Potential Therapeutic Target for Reducing Inflammation and Corneal Scarring in Bacterial Keratitis.

Purpose: Bacterial keratitis (BK) is a serious ocular infection that can cause severe inflammation and corneal scarring, leading to vision loss. In this study, we aimed to investigate the involvement of ferroptosis in the pathogenesis of BK. Methods: Transcriptome analysis was performed to evaluate ferroptosis-related gene expression in human BK corneas. Subsequently, the ferroptosis in mouse models of Pseudomonas aeruginosa keratitis and corneal stromal stem cells (CSSCs) were validated. The mice were treated with levofloxacin (LEV) or levofloxacin combined with ferrostatin-1 (LEV+Fer-1). CSSCs were treated with lipopolysaccharide (LPS) or LPS combined Fer-1. Inflammatory cytokines, α-SMA, and ferroptosis-related regulators were evaluated by RT-qPCR, immunostaining, and Western blot. Iron and reactive oxygen species (ROS) were measured. Results: Transcriptome analysis revealed significant alterations in ferroptosis-related genes in human BK corneas. In the BK mouse models, the group treated with LEV+Fer-1 exhibited reduced inflammatory cytokines (MPO, TNF-α, and IFN-γ), decreased corneal scarring and α-SMA expression, and lower Fe3+ compared to the BK and LEV groups. Notably, the LEV+Fer-1 group showed elevated GPX4 and SLC7A11 in contrast to the BK and LEV group. In vitro, Fer-1 treatment effectively restored the alterations of ROS, Fe2+, GPX4, and SLC7A11 induced by LPS in CSSCs. Conclusions: Ferroptosis plays a crucial role in the pathogenesis of BK. The inhibition of ferroptosis holds promise for mitigating inflammation, reducing corneal scarring, and ultimately enhancing the prognosis of BK. Consequently, this study provides a potential target for innovative therapeutic strategies for BK, which holds immense potential to transform the treatment of BK.

Protective effects of Huang-Qi-Ge-Gen decoction against diabetic liver injury through regulating PI3K/AKT/Nrf2 pathway and metabolic profiling.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Qi-Ge-Gen decoction (HGD) is a traditional Chinese medicine prescription that has been used for centuries to treat "Xiaoke" (the name of diabetes mellitus in ancient China). However, the ameliorating effects of HGD on diabetic liver injury (DLI) and its mechanisms are not yet fully understood. AIM OF THE STUDY: To elucidate the ameliorative effect of HGD on DLI and explore its material basis and potential hepatoprotective mechanism. MATERIALS AND METHODS: A diabetic mice model was induced by feeding a high-fat diet and injecting intraperitoneally with streptozotocin (40 mg kg-1) for five days. After the animals were in confirmed diabetic condition, they were given HGD (3 or 12 g kg-1, i. g.) for 14 weeks. The effectiveness of HGD in treating DLI mice was evaluated by monitoring blood glucose and blood lipid levels, liver function, and pathological conditions. Furthermore, UPLC-MS/MS was used to identify the chemical component profile in HGD and absorption components in HGD-treated plasma. Network pharmacology and molecular docking were performed to predict the potential pathway of HGD intervention in DLI. Then, the results of network pharmacology were validated by examining biochemical parameters and using western blotting. Lastly, urine metabolites were analyzed by metabolomics strategy to explore the effect of HGD on the metabolic profile of DLI mice. RESULTS: HGD exerted therapeutic potential against the disorders of glucose metabolism and lipid metabolism, liver dysfunction, liver steatosis, and fibrosis in a DLI model mice induced by HFD/STZ. A total of 108 chemical components in HGD and 18 absorption components in HGD-treated plasma were preliminarily identified. Network pharmacology and molecular docking results of the absorbed components in plasma indicated PI3K/AKT as a potential pathway for HGD to intervene in DLI mice. Further experiments verified that HGD markedly reduced liver oxidative stress in DLI mice by modulating the PI3K/AKT/Nrf2 signaling pathway. Moreover, 19 differential metabolites between normal and DLI mice were detected in urine, and seven metabolites could be significantly modulated back by HGD. CONCLUSIONS: HGD could ameliorate diabetic liver injury by modulating the PI3K/AKT/Nrf2 signaling pathway and urinary metabolic profile.

In Vivo Corneal Confocal Microscopy for Detecting Corneal Nerve Fiber Changes in Patients with Different Types of Optic Neuritis: A Cross-Sectional Study.

PURPOSE: Optic neuritis (ON), a demyelinating disease of the central nervous system, is often a precursor manifestation of neuromyelitis optica spectrum disorders (NMOSD) or multiple sclerosis (MS). Reduced corneal nerve fiber counts have been found in patients with NMOSD or MS. This study aimed to observe and compare the corneal subbasal nerve plexus in patients with three types of ON and controls without ON using in vivo corneal confocal microscopy (IVCM). METHODS: Data were analyzed for 77 eyes of 48 patients with ON, grouped according to seropositivity for anti-aquaporin-4 IgG, myelin oligodendrocyte glycoprotein antibody, or no seropositivity, and 35 healthy eyes in the control group. Corneal parameters were quantified based on IVCM images. Visual function indicators were recorded, following which their correlations with IVCM parameters were analyzed. RESULTS: Significant differences in IVCM parameters were detected among the different groups. Reductions in corneal nerve fiber counts were negatively correlated with visual acuity. Corneal nerve fibers were significantly more damaged in the affected eye than in the unaffected eye in patients with ON. CONCLUSION: IVCM revealed corneal nerve fiber loss of varying degrees, depending on the type of ON. This indicates that, although ON primarily affects the central nervous system, peripheral nerves, such as the trigeminal nerve, which innervates the corneal subbasal nerve plexus may also be damaged in affected patients.

Corneal Epithelial Dendritic Cells: An Objective Indicator for Ocular Surface Inflammation in Patients with Obstructive Meibomian Gland Dysfunction?

PURPOSE: To examine whether corneal epithelial dendritic cells (CEDC) could serve as an indicator to distinguish obstructive meibomian gland dysfunction (MGD) with or without ocular surface inflammation (OSI). METHODS: We performed a case-control study on patients with diagnosed obstructive MGD between August 2017 and November 2019. RESULTS: 30 MGD cases and 25 healthy controls were recruited. The classification of MGD patients with and without OSI was based on the tear pro-inflammatory cytokine levels. Compared with the MGD without OSI and the control group, a higher CEDC density was detected in the MGD with OSI subgroup. The presence of >15.6 cells/mm2 CEDC had a sensitivity of 73% and specificity of 75% for the diagnosis of MGD with OSI. CONCLUSIONS: OSI is not present in all patients with obstructive MGD. Evaluation of CEDC density in the central cornea may help identify whether MGD is concomitant with OSI.

Changes in Care Associated With Integrating Medicare and Medicaid for Dual-Eligible Individuals.

Importance: There is growing interest in expanding integrated models, in which 1 insurer manages Medicare and Medicaid spending for dually eligible individuals. Fully integrated dual-eligible special needs plans (FIDE-SNPs) are one of the largest integrated models, but evidence about their performance is limited. Objective: To evaluate changes in care associated with integrating Medicare and Medicaid coverage in a FIDE-SNP in Pennsylvania. Design, Setting, and Participants: This cohort study using a difference-in-differences analysis compared changes in care between 2 cohorts of dual-eligible individuals: (1) an integration cohort composed of Medicare Dual Eligible Special Needs Plan enrollees who joined a companion Medicaid plan following a 2018 state reform mandating Medicaid managed care (leading to integration), and (2) a comparison cohort with nonintegrated coverage before and after the start of Medicaid managed care. Analyses were conducted between February 2022 and June 2023. Main Outcomes and Measures: Analyses examined outcomes in 4 domains: use of home- and community-based services (HCBS), care management and coordination, hospital stays and postacute care, and long-term nursing home stays. Results: The study included 7967 individuals in the integration cohort and 3832 individuals in the comparison cohort. In the integration cohort, the mean (SD) age at baseline was 63.3 (14.7) years, and 5268 individuals (66.1%) were female and 2699 (33.9%) were male. In the comparison cohort, the mean (SD) age at baseline was 64.8 (18.6) years, and 2341 individuals (61.1%) were female and 1491 (38.9%) were male. At baseline, integration cohort members received a mean (SD) of 2.83 (8.70) days of HCBS per month and 3.34 (3.56) medications for chronic conditions per month, and the proportion with a follow-up outpatient visit after a hospital stay was 0.47. From baseline through 3 years after integration, HCBS use increased differentially in the integration vs comparison cohorts by 0.61 days/person-month (95% CI, 0.28-0.94; P < .001). However, integration was not associated with changes in care management and coordination, including medication use for chronic conditions (-0.02 fills/person-month; 95% CI, -0.10 to 0.06; P = .65) or follow-up outpatient care after a hospital stay (-0.01 visits/hospital stay; 95% CI, -0.04 to 0.03; P = .61). Hospital stays did not change differentially between the cohorts. Unmeasured factors contributing to differential mortality limited the ability to identify changes in long-term nursing home stays associated with integration. Conclusions and Relevance: In this cohort study with a difference-in-differences analysis of 2 cohorts of individuals dually eligible for Medicare and Medicaid, integration was associated with greater HCBS use but not with other changes in care patterns. The findings highlight opportunities to strengthen how integrated programs manage care and a need to further evaluate their performance.

Extracellular Vesicle MicroRNAs From Corneal Stromal Stem Cell Enhance Stemness of Limbal Epithelial Stem Cells by Targeting the Notch Pathway.

Purpose: The limbal niche supports the self-renewal of limbal epithelial stem cells (LESCs). The corneal stromal stem cell (CSSC) is an important component in the niche that regulates the LESC phenotype. However, the intercellular communication between LESCs and CSSCs has yet to be elucidated. Methods: A traditional two-dimensional (2D) system, a direct three-dimensional (3D) system, and an indirect 3D coculture system of LESCs and CSSCs were used to elucidate the paracrine pathway effect of CSSCs on LESCs. To reveal the impact of CSSC derived extracellular vesicles (CSSC-EVs) on LESCs, GW4869 and CSSC-EVs were added separately to the LESC culture medium. The outgrowth rate, cell density, differentiation, and stemness maintenance were compared among these methods. The miRNAs in the CSSC-EVs were sequenced, and the targeted Notch pathway was further confirmed by RT‒qPCR and Western blotting. Results: Compared with 2D culture, both the direct and indirect 3D coculture systems yielded a higher outgrowth rate and expression of stem cell markers of LESCs. The phenotypes of LESCs cultivated using the two coculture approaches were also comparable. Nevertheless, GW4869 inhibited the effect of CSSCs on LESCs, and the addition of CSSC-EVs to the 2D culture system could increase cell density, and the proportion of p63αbright cells, which indicated that CSSC-EVs were crucial in regulating LESCs. Furthermore, the EV-AlixKD with reduced miRNA partly lost its regulating function. The abundant miRNAs in CSSC-EVs, such as hsa-miR-663b, hsa-miR-16-5p, and hsa-miR-1290, target the Notch pathway. The LESCs transfected with miR-663b had higher p63 expression via downregulating of the Notch pathway. Conclusions: CSSC-EV played an important role in promoting LESC proliferation and stemness maintenance by targeting Notch signaling via miRNAs, which will increase our understanding of the limbal niche and provide a potential new approach for LESC culture and the treatment of corneal epithelial disorders.

Pilot Study of a Motivational Interviewing Training on Practitioners' Skill Set for Patient Centered Communication.

PURPOSE: Increase practitioners' Motivational Interviewing (MI) skill set to develop intrinsic motivation and behavior change competencies. DESIGN: Pilot comparing intervention group with waitlist-control group. SETTING: Health Plan in northeast of U.S. INTERVENTION: Training program including a 3 day 19 hour MI training intensive, 1.5 hour follow-up skill-building trainings, and quality assurance review using an MI assessment tool paired with strengths-based mentoring. Cohort 1 (experimental group) received the intervention for 6-months and cohort 2 for 3 months (control group). MEASURES: Practitioners' MI skill set was assessed pre- and post-intervention using Motivational Interviewing Competency Assessment (MICA). Practitioners' MI knowledge and attitudes were assessed pre- and post-training using adapted Motivational Interviewing Knowledge and Attitudes Test (MIKAT). Active learning and confidence questionnaires were administered post-training. ANALYSIS: A generalized linear mixed model with repeated measures to analyze difference in MICA growth rates; paired T-test for MIKAT pre/post training analysis. Descriptive statistics for active learning and confidence. RESULTS: MICA scores significantly improved for both cohorts (P < .0001). Practitioners had significant increase in knowledge and attitudes for MI (P < .001) and confidence in using MI (P < .01). High levels of active learning were observed (93-100%). CONCLUSION: With planning, adequate resources/support, and iterative processes for adjustment, practitioners can improve their MI skill set in a short period of time with modest investment of practitioner resources.

Combination of UHPLC-Q Exactive-Orbitrap MS, Bioinformatics and Molecular Docking to Reveal the Mechanism of Huan-Lian-Jie-Du Decoction in the Treatment of Diabetic Encephalopathy.

Diabetic encephalopathy (DE) is a serious complication of diabetes, which affects patients' quality of life. We aimed to explore HLJDD in the treatment of DE by LC/MS and bioinformatics. UPLC-Q Exactive-Orbitrap MS was employed to clarify the compounds. The modules and hub targets of DE were gained from WGCNA. Subsequently, an Herb-Compound-Target network was constructed and enrichment analysis was used. In addition, a protein-protein interaction (PPI) network was constructed and molecular docking was used to verify the above analysis. As result, 138 compounds and 10 prototypes in brain were identified. In network pharmacology, 8 modules and 5692 hub targets were obtained from WGCNA. An Herb-Compound-Target network was constructed by 4 herbs, 10 compounds and 56 targets. The enrichment analysis showed that the treatment of DE with HLJDD involve oxidative stress and neuroprotection. Beside, SRC, JUN, STAT3, MAPK1 and PIK3R1 were identified and as hub targets of HLJDD in treating DE. Moreover, Molecular docking showed that five hub targets had strong affinity with the corresponding alkaloids. Therefore, we explored the underlying mechanisms of HLJDD in the treatment of DE and to provide the theoretical and scientific basis for subsequent experimental studies and clinical applications.

Infectious Keratitis After Keratorefractive Surgery: Update and Review of the Literature.

OBJECTIVES: To summarize the clinical manifestations, microbiological profile, treatment, and prognosis of corneal infections after different keratorefractive surgery. METHODS: To obtain relevant studies, English-language databases, including PubMed, Ovid Embase, Web of Science, and CLNAHL, were searched from January 1979 to March 2022. The fundamentals of the literature, clinical characteristics, pathogens, and treatments were retrieved for each included article. RESULTS: Eighty-four studies involving 306 infectious eyes were included in this review. Risk factors of potential infection included a history of blepharitis, contact lens usage, and contaminated surgical instruments. The mean onset time was 22.9±38.7 days (range: 1 day to 3 years). The most common organism isolated from infectious keratitis after keratorefractive surgery were Staphylococcus aureus , followed by Mycobacterium and coagulase-negative Staphylococcus . Most of the infections after refractive procedures were sensitive to medical treatment alone, and the ultimate best-corrected visual acuity after medical treatment was as follows: 20/20 or better in 82 cases (37.0%), 20/40 or better in 170 cases (76.5%), and worse than 20/40 in 52 cases (23.5%). Surgical interventions including flap lift, flap amputation, ring removal, and keratoplasty were performed in 120 eyes (44.5%). CONCLUSIONS: Most infections after keratorefractive surgery occur within a week, whereas more than half of the cases after laser-assisted in situ keratomileusis happen after about a month. Gram-positive cocci and mycobacterium are the most common isolates. Infections after LASIK, intracorneal ring (ICR) implantation, and small incision lenticule extraction, which primarily occur between the cornea layers, require irrigation of the tunnels or pocket with antibiotics.

Pterostilbene could alleviate diabetic cognitive impairment by suppressing TLR4/NF-кB pathway through microbiota-gut-brain axis.

Diabetic cognitive impairment (DCI) is a serious neurodegenerative disorder caused by diabetes, with chronic inflammation being a crucial factor in its pathogenesis. Pterostilbene is a well-known natural stilbene derivative that has excellent anti-inflammatory activity, suggesting its potential medicinal advantages for treating DCI. Therefore, this study is to explore the beneficial effects of pterostilbene for improving cognitive dysfunction in DCI mice. A diabetic model was induced by a high-fat diet plus streptozotocin (40 mg·kg-1 ) for consecutive 5 days. After the animals were confirmed to be in a diabetic state, they were treated with pterostilbene (20 or 60 mg·kg-1 , i.g.) for 10 weeks. Pharmacological evaluation showed pterostilbene could ameliorate cognitive dysfunction, regulate glycolipid metabolism disorders, improve neuronal damage, and reduce the accumulation of ß-amyloid in DCI mice. Pterostilbene alleviated neuroinflammation by suppressing oxidative stress and carbonyl stress damage, astrocyte and microglia activation, and dopaminergic neuronal loss. Further investigations showed that pterostilbene reduced the level of lipopolysaccharide, modulated colon and brain TLR4/NF-κB signaling pathways, and decreased the release of inflammatory factors, which in turn inhibited intestinal inflammation and neuroinflammation. Furthermore, pterostilbene could also improve the homeostasis of intestinal microbiota, increase the levels of short-chain fatty acids and their receptors, and suppress the loss of intestinal tight junction proteins. In addition, the results of plasma non-targeted metabolomics revealed that pterostilbene could modulate differential metabolites and metabolic pathways associated with inflammation, thereby suppressing systemic inflammation in DCI mice. Collectively, our study found for the first time that pterostilbene could alleviate diabetic cognitive dysfunction by inhibiting the TLR4/NF-κB pathway through the microbiota-gut-brain axis, which may be one of the potential mechanisms for its neuroprotective effects.

The global epidemiology and clinical diagnosis of Acanthamoeba keratitis.

Acanthamoeba keratitis is a rare parasitic infection of the cornea that can lead to permanent blindness if not diagnosed and treated promptly. We collected data on the incidences of Acanthamoeba keratitis from 20 countries and calculated an annual incidence of 23,561 cases, with the lowest rates in Tunisia and Belgium, and the highest in India. We analyzed 3755 Acanthamoeba sequences from the GenBank database across Asia, Europe, North America, South America, and Oceania and genotyped them into T1, T2, T3, T4, T5, T10, T11, T12, and T15. Many genotypes possess different characteristics, yet T4 is the most prevalent genotype. As efficient treatment against Acanthamoeba remains lacking, prevention from early diagnosis via staining, PCR, or in vivo confocal microscopy (IVCM) becomes significant for the condition's prognosis. IVCM is the most recommended approach for the early detection of Acanthamoeba. If IVCM is unavailable, PCR should be used as an alternative.

Time-Course Transcriptomic Analysis Reveals the Crucial Roles of PANoptosis in Fungal Keratitis.

Purpose: Fungal keratitis (FK) is a serious corneal infection with high morbidity. Host immune responses function as a double-edged sword by eradicating fungal pathogens while also causing corneal damage, dictating the severity, progression, and outcome of FK. However, the underlying immunopathogenesis remains elusive. Methods: Time-course transcriptome was performed to illustrate the dynamic immune landscape in a mouse model of FK. Integrated bioinformatic analyses included identification of differentially expressed genes, time series clustering, Gene Ontology enrichment, and inference of infiltrating immune cells. Gene expression was verified by quantitative polymerase chain reaction (qPCR), Western blot, or immunohistochemistry. Results: FK mice exhibited dynamic immune responses with concerted trends with clinical score, transcriptional alteration, and immune cell infiltration score peaking at 3 days post infection (dpi). Disrupted substrate metabolism, broad immune activation, and corneal wound healing occurred sequentially in early, middle, and late stages of FK. Meanwhile, dynamics of infiltrating innate and adaptive immune cells displayed distinct characteristics. Proportions of dendritic cells showed overall decreasing trend with fungal infection, whereas that of macrophages, monocytes, and neutrophils rose sharply in early stage and then gradually decreased as inflammation resolved. Activation of adaptive immune cells was also observed in late stage of infection. Furthermore, shared immune responses and activation of AIM2-, pyrin-, and ZBP1-mediated PANoptosis were revealed across different time points. Conclusions: Our study profiles the dynamic immune landscape and highlights the crucial roles of PANoptosis in FK pathogenesis. These findings provide novel insights into host responses to fungi and contribute to the development of PANoptosis-targeted therapeutics for patients with FK.

MicroRNAs of extracellular vesicles derived from mesenchymal stromal cells alleviate inflammation in dry eye disease by targeting the IRAK1/TAB2/NF-κB pathway.

PURPOSE: To investigate the efficacy and mechanisms of human umbilical cord-derived MSC-derived extracellular vesicles (hucMSC-EVs) in a mouse model of desiccation-induced dry eye disease (DED). METHODS: hucMSC-EVs were enriched by ultracentrifugation. The DED model was induced by desiccating environment combined with scopolamine administration. The DED mice were divided into the hucMSC-EVs group, fluorometholone (FML) group, PBS group, and blank control group. Tear secretion, corneal fluorescein staining, the cytokine profiles in tears and goblet cells, TUNEL-positive cell, and CD4+ cells were examined to assess therapeutic efficiency. The miRNAs in the hucMSC-EVs were sequenced, and the top 10 were used for miRNA enrichment analysis and annotation. The targeted DED-related signaling pathway was further verified by using RT‒qPCR and western blotting. RESULTS: Treatment with hucMSC-EVs increased the tear volume and maintained corneal integrity in DED mice. The cytokine profile in the tears of the hucMSC-EVs group presented with a lower level of proinflammatory cytokines than PBS group. Moreover, hucMSC-EVs treatment increased goblet cell density and inhibited cell apoptosis and CD4+ cell infiltration. Functional analysis of the top 10 miRNAs in hucMSC-EVs showed a high correlation with immunity. Among them, miR-125 b, let-7b, and miR-6873 were conserved between humans and mice and were associated with the IRAK1/TAB2/NF-κB pathway that was activated in DED. Furthermore, IRAK1/TAB2/NF-κB pathway activation and the abnormal expression of IL-4, IL-8, IL-10, IL-13, IL-17, and TNF-α were reversed by hucMSC-EVs. CONCLUSIONS: hucMSCs-EVs alleviate DED signs, suppress inflammation and restore homeostasis of the corneal surface by multitargeting the IRAK1/TAB2/NF-κB pathway via certain miRNAs.

SLAMF7/STAT6 Pathway Inhibits Innate Immune Response in Late-Stage Human Acanthamoeba Keratitis: A Comparative Transcriptome Analysis.

Acanthamoeba keratitis (AK) is a blinding corneal infection caused by the protozoan Acanthamoeba. The long-term course of AK suggests the host immunity could not kill Acanthamoeba rapidly. The immune status is still unclear in the late stage of AK. The comparative transcriptome analysis was made based on the bulk RNA sequencing of cornea tissues from AK patients and donors. Differentially expressed genes and enriched signaling pathways were calculated. CIBERSORT algorithm was used for immune infiltration analysis of cornea tissue between AK and normal controls. A total of 2668 differentially expressed genes, including 1477 upregulated genes and 1191 downregulated genes, were detected. Gene Ontology analysis revealed that the pathways were significantly enriched in leukocyte migration, regulation of T-cell activation, the external side of plasma membrane, collagen-containing extracellular matrix, immune receptor activity, and cytokine binding. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the pathways were significantly enriched in the cytokine-cytokine receptor interaction, hematopoietic cell lineage, and Staphylococcus aureus infection pathway. The immune infiltration profiles varied little between AK and normal controls. Compared with normal tissue, cornea tissue of AK contained a higher proportion of M0 macrophages and CD8 T cells, while resting memory CD4 T cells contributed to a relatively lower portion (p < 0.05). Finally, the expression levels of cell markers and SLAMF7/STAT6 pathway were confirmed by histopathology examinations, RT-qPCR, and Western blot.

Stepwise Optimization of Inducible Expression System for the Functional Secretion of Horseradish Peroxidase in Saccharomyces cerevisiae.

Horseradish peroxidase (HRP) is a plant-derived glycoprotein that can be developed as a food additive to cross-link proteins or biopolymers. Although Saccharomyces cerevisiae has advantages in the production of food-grade HRP, the low expressional level and inefficient secretion hindered its application values. After comparing the effects of constitutive and inducible expression on cell growth, the strength of HRP expression was roughly tuned by replacing core regions of the promoter in the GAL80-knockout strain and further finely tuned by terminator screening. Additionally, the most suitable signal peptide was selected, and the pre-peptide with pro-peptides was modified to balance the transport of HRP in the endoplasmic reticulum. The extracellular HRP activity of the best strain reached 13 506 U/L at the fermenter level, 330-fold higher than the previous result of 41 U/L in S. cerevisiae. The strategy can be applied to alleviate the inhibition of cell growth caused by the expression of toxic proteins and improve their secretion.

Development and multi-center validation of machine learning model for early detection of fungal keratitis.

BACKGROUND: Fungal keratitis (FK) is a leading cause of corneal blindness in developing countries due to poor clinical recognition and laboratory identification. Here, we aimed to identify the distinct clinical signature of FK and develop a diagnostic model to differentiate FK from other types of infectious keratitis. METHODS: We reviewed the electronic health records (EHRs) of all patients with suspected infectious keratitis in Beijing Tongren Hospital from January 2011 to December 2021. Twelve clinical signs of slit-lamp images were assessed by Lasso regression analysis and collinear variables were excluded. Three models based on binary logistic regression, random forest classification, and decision tree classification were trained for FK diagnosis and employed for internal validation. Independent external validation of the models was performed in a cohort of 420 patients from seven different ophthalmic centers to evaluate the accuracy, specificity, and sensitivity in real world. FINDINGS: Three diagnostic models of FK based on binary logistic regression, random forest classification, and decision tree classification were established and internal validation were achieved with the mean AUC of 0.916, 0.920, and 0.859, respectively. The models were well-calibrated by external validation using a prospective cohort including 210 FK and 210 non-FK patients from seven eye centers across China. The diagnostic model with the binary logistic regression algorithm classified the external validation dataset with a sensitivity of 0.907 (0.774, 1.000), specificity 0.899 (0.750, 1.000), accuracy 0.905 (0.805, 1.000), and AUC 0.903 (0.808, 0.998). INTERPRETATION: Our model enables rapid identification of FK, which will help ophthalmologists to establish a preliminary diagnosis and to improve the diagnostic accuracy in clinic. FUNDING: The Open Research Fund from the National Key Research and Development Program of China (2021YFC2301000) and the Open Research Fund from Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beijing Tongren Hospital, Beihang University &Capital Medical University (BHTR-KFJJ-202001) supported this study.

Trends of ocular fungal infections in North China (2001-2020).

OBJECTIVES: To analyze the epidemiologic features, culture positivity, the fungal spectrum of different sites of ocular infection in North China over 20 years from 2001 to 2020. METHODS: 11, 635 patients suspected of ocular fungal infection were reviewed. The demographic profile, fungal positive culture rate among different sites, the distribution, and trends of main pathogens among cornea and intraocular fluid were analyzed. RESULTS: Among 11, 635 samples, the positive culture rate of ocular fungal infection was 23.6%. Most of samples (83.1%) were from cornea, and their culture positivity was 26.9%. Fungal keratitis occurred more often during the harvesting season (October to December; 34.0%) than in other seasons (average: 22.0%). Fusarium sp. (53.2%), Aspergillus sp. (15.9%) and Alternaria sp. (12.5%) were the most common fungal species of ocular mycotic infections in the past two decades in north China. 2562 organisms were identified from cornea, of which 1443 (56.3%) were Fusarium sp., 403 (15.7%) and 329 (12.8%) were Aspergillus sp., and Alternaria sp., respectively. Of the 120 fungi isolated from the intraocular fluid, the most common was Aspergillus sp. (33.3%), followed by Fusarium sp. (24.2%) and Candida sp. (15.0%). CONCLUSIONS: Fusarium sp., Aspergillus sp. and Alternaria sp. were the most common organisms in cases of fungal keratitis, while Aspergillus sp., Fusarium sp. and Candida sp. were the most frequent isolates for fungal endophthalmitis.

Deep learning-based classification of infectious keratitis on slit-lamp images.

Background: Infectious keratitis (IK) is an ocular emergency caused by a variety of microorganisms, including bacteria, fungi, viruses, and parasites. Culture-based methods were the gold standard for diagnosing IK, but difficult biopsy, delaying report, and low positive rate limited their clinical application. Objectives: This study aims to construct a deep-learning-based auxiliary diagnostic model for early IK diagnosis. Design: A retrospective study. Methods: IK patients with pathological diagnosis were enrolled and their slit-lamp photos were collected. Image augmentation, normalization, and histogram equalization were applied, and five image classification networks were implemented and compared. Model blending technique was used to combine the advantages of single model. The performance of combined model was validated by 10-fold cross-validation, receiver operating characteristic curves (ROC), confusion matrix, Gradient-wright class activation mapping (Grad-CAM) visualization, and t-distributed Stochastic Neighbor Embedding (t-SNE). Three experienced cornea specialists were invited and competed with the combined model on making clinical decisions. Results: Overall, 4830 slit-lamp images were collected from patients diagnosed with IK between June 2010 and May 2021, including 1490 (30.8%) bacterial keratitis (BK), 1670 (34.6%) fungal keratitis (FK), 600 (12.4%) herpes simplex keratitis (HSK), and 1070 (22.2%) Acanthamoeba keratitis (AK). KeratitisNet, the combination of ResNext101_32x16d and DenseNet169, reached the highest accuracy 77.08%. The accuracy of KeratitisNet for diagnosing BK, FK, AK, and HSK was 70.27%, 77.71%, 83.81%, and 79.31%, and AUC was 0.86, 0.91, 0.96, and 0.98, respectively. KeratitisNet was mainly confused in distinguishing BK and FK. There were 20% of BK cases mispredicted into FK and 16% of FK cases mispredicted into BK. In diagnosing each type of IK, the accuracy of model was significantly higher than that of human ophthalmologists (p < 0.001). Conclusion: KeratitisNet demonstrates a good performance on clinical IK diagnosis and classification. Deep learning could provide an auxiliary diagnostic method to help clinicians suspect IK using different corneal manifestations.