Establishment of mouse model of neurotrophic keratopathy through TRPV1 neuronal ablation.

Neurotrophic keratopathy (NK) is a challenging disease with the reduced innervation to the cornea. To establish a genetic and stable mouse model of NK, we utilized the TRPV1-DTR mice with intraperitoneal injection of diphtheria toxin (DT) to selectively eliminate TRPV1 neurons. After DT administration, the mice exhibited robust ablation of TRPV1 neurons in the trigeminal ganglion, accompanied with reduced corneal sensation and nerve density, as well as the decreased calcitonin-gene-related peptide (CGRP) and substance P levels. According to disease progression of TRPV1 neuronal ablation, tear secretion was reduced from day 3, which followed by corneal epithelial punctate lesions from day 7. From day 11 to day 16, the mice exhibited persistent corneal epithelial defects and stromal edema. By day 21, corneal ulceration and stromal melting were observed with the abundant inflammatory cell infiltration, corneal neovascularization, and enhanced cell apoptosis. Moreover, subconjunctival injection of CGRP delayed the NK progression with the characteristics of reduced severe corneal epithelial lesions and corneal inflammation. In addition, the impairments of conjunctival goblet cells, lacrimal gland, and meibomian gland were identified by the diminished expression of MUC5AC, AQP5, and PPARγ, respectively. Therefore, these results suggest that the TRPV1-DTR mice may serve as a reliable animal model for the research of NK pathogenesis.

The inhibition of p38 MAPK blocked inflammation to restore the functions of rat meibomian gland epithelial cells.

Meibomian glands (MGs) are vital for ocular surface health. However, the roles of inflammation in the progression of meibomian gland dysfunction (MGD) are largely unknown. In this study, the roles of the inflammation factor interleukin-1ß (IL-1ß) via the p38 mitogen-activated protein kinases (MAPK) signaling pathway on rat meibomian gland epithelial cells (RMGECs) were explored. Eyelids from adult rat mice at 2 months and 2 years of age were stained with specific antibodies against IL-1ß to identify inflammation levels. RMGECs were exposed to IL-1ß and/or SB203580, a specific inhibitor of p38 MAPK signaling pathway, for 3 days. Cell proliferation, keratinization, lipid accumulation, and matrix metalloproteinases 9 (MMP9) expression were evaluated by MTT assay, polymerase chain reaction (PCR), immunofluorescence staining, apoptosis assay, lipid staining, and Western blot analyses. We found that IL-1ß was significantly higher in the terminal ducts of MGs in rats with age-related MGD than in young rats. IL-1ß inhibited cell proliferation, suppressed lipid accumulation and peroxisome proliferator activator receptor γ (PPARγ) expression, and promoted apoptosis while activating the p38 MAPK signaling pathway. Cytokeratin 1 (CK1), a marker for complete keratinization, and MMP9 in RMGECs were also up-regulated by IL-1ß. SB203580 effectively diminished the effects of IL-1ß on differentiation, keratinization, and MMP9 expression by blocking IL-1ß-induced p38 MAPK activation, although it also inhibited cell proliferation. The inhibition of the p38 MAPK signaling pathway blocked IL-1ß-induced differentiation reduction, hyperkeratinization, and MMP9 overexpression of RMGECs, which provides a potential therapy for MGD.

Ductal Hyperkeratinization and Acinar Renewal Abnormality: New Concepts on Pathogenesis of Meibomian Gland Dysfunction.

Meibomian gland dysfunction (MGD) is a functional and morphological disorder of the meibomian glands which results in qualitative or quantitative alteration in meibum secretion and is the major cause of evaporative dry eye (EDE). EDE is often characterized by tear film instability, increased evaporation, hyperosmolarity, inflammation, and ocular surface disorder. The precise pathogenesis of MGD remains elusive. It has been widely considered that MGD develops as a result of ductal epithelial hyperkeratinization, which obstructs the meibomian orifice, halts meibum secretion, and causes secondary acinar atrophy and gland dropout. Abnormal self-renewal and differentiation of the acinar cells also play a significant role in MGD. This review summarizes the latest research findings regarding the possible pathogenesis of MGD and provides further treatment strategies for MGD-EDE patients.

Atomically Dispersed Uranium Enables an Unprecedentedly High NH3 Yield Rate.

The low NH3 yield rate is a grand challenge for electrocatalytic N2 reduction to NH3. Herein, we report the first uranium single-atom catalyst (SAC) capable of catalyzing the electrochemical N2 reduction reaction (NRR). The uranium SAC features a low limiting potential (<0.5 V) and near-zero free energy changes for N2 adsorption and NH3 desorption. The integration of these merits enables the uranium SAC to afford an unprecedentedly high NH3 yield rate, 3-4 orders of magnitude higher than that of the Ru(0001) surface, which is widely recognized as an excellent NRR electrocatalyst. Further theoretical analysis reveals that the N2 reduction catalyzed by the uranium SAC is synergistically regulated by the d and f electrons of atomic uranium. This work proposes a promising solution (that is, atomically dispersed uranium) to the daunting challenge associated with the low NH3 yield rate, thus enabling the scalable production of NH3 via electrochemical N2 reduction.

Tissue-Engineered Corneal Endothelial Sheets Using Ultrathin Acellular Porcine Corneal Stroma Substrates for Endothelial Keratoplasty.

Tissue-engineered cornea endothelial sheets (TECES), created using a biocompatible thin and transparent carrier with corneal endothelial cells, could alleviate the shortage of donor corneas and provide abundant functional endothelial cells. In our previous clinical trials, the effectiveness and safety of the acellular porcine corneal stroma (APCS) applied in lamellar keratoplasty have been confirmed. In this study, we optimized the method to cut APCS into multiple 20 µm ultrathin lamellae by a cryostat microtome and investigated the feasibility of TECES by seeding rabbit corneal endothelial cells (RCECs) on ultrathin APCS. Cell adhesion, proliferation, and functional gene expression of RCECs on tissue-culture plastic and APCS of different thicknesses were compared. The results indicated that ultrathin lamellae were superior in increasing cell viability and maintaining cell functions. Analyzing with histology, electron microscopy, and immunofluorescence, we found that RCECs cultured on 20 µm ultrathin APCS for 5 days grew into a confluent monolayer with a density of 3726 ± 223 cells/mm2 and expressed functional biomarkers Na+/K+-ATPase and zonula occludens. After 14 days, RCECs formed an early stage of Descemet's membrane-like structure by synthesizing collagen IV and laminin. Human corneal endothelial cells were also used to further validate the supportive effect of ultrathin APCS on cells. The resulting constructs were flexible and tough enough to implant into rabbits' anterior chambers through small incisions. TECES adhered to the posterior corneal stroma, and the thickness of cornea gradually reduced to normal after grafting. These results indicate that the ultrathin APCS can serve as a tissue engineering carrier and might be a suitable alternative for endothelial cells expansion in endothelial keratoplasty.

Geographic distribution and time trend of human exposure of Di(2-ethylhexyl) phthalate among different age groups based on global biomonitoring data.

Despite being restricted by many authorities, di (2-ethylhexyl) phthalate (DEHP) is still widely detected in the environment and biospecimens. To indentify populations of high risk and evaluate the effects of DEHP restrictions, we elucidated the geographic distribution of DEHP exposure levels among pregnant women and different age groups, and compared the time trend of exposure levels with the time course of productions/restrictions. The estimated daily intake (EDI) was calculated based on biomonitoring data in published epidemiological studies, and then the group EDI (EDIG) was calculated for one particular population, region, or period by weighting EDIs by sample sizes. Overall, 144,965 samples from 45 nations were included, with the sampling time ranging from 1982 to 2017. Children had the highest exposure level (5.50 µg/kg bw/day) worldwide, while infants and pregnant women had low levels (2.13 and 1.89 µg/kg bw/day, respectively). The EDIGs varied considerably between countries, and the majority of corresponding hazard quotients were less than 1; however, the risk behind can not be ignored. In the general population, the DEHP exposure level showed a downtrend from 4.40 µg/kg bw/day before 2000 to 2.23 µg/kg bw/day in 2015-2017. In the European Union, the annual trend of DEHP EDIGs of children and adults fitted the production and consumption volume, and the EDIGs decreased more sharply in children. The EDIGs of children decreased with a delay along with the regulations on the use of DEHP. Cutting productions/consumptions and restrictions are effective to reduce DEHP exposure, but current efforts are far from enough on a worldwide scale.

Associations of urine metals and metal mixtures during pregnancy with cord serum vitamin D Levels: A prospective cohort study with repeated measurements of maternal urinary metal concentrations.

BACKGROUND: Vitamin D deficiency has been associated with the increased risk of many diseases, especially during early life. Exposure to some toxic metals may decrease vitamin D levels in adults and children in previous studies. However, less is known about the associations of maternal metals exposure during pregnancy with newborns' vitamin D status. OBJECTIVE: We conducted a prospective cohort study to investigate the relationships between urine metals and metal mixtures during pregnancy and newborns' vitamin D status. METHODS: Urine samples of 598 pregnant women were collected in each trimester and cord blood samples of newborns were collected at delivery. The concentrations of 20 metals in urine and 25-hydroxyvitamin D [25(OH)D] in cord serum were quantified. Generalized linear models were used to estimate the associations between individual metals and cord serum total 25(OH)D. We applied Bayesian Kernel Machine Regression (BKMR) to evaluate the mixture and interaction effects of urine metals. RESULTS: In individual metals analyses, we reported that a double increase in urine vanadium (V), cobalt (Co), and thallium (Tl) throughout pregnancy was associated with a 9.91% [95% confidence interval (CI): -18.58%, -0.30%], 11.42% (95% CI: -17.73%, -4.63%), and 12.64% (95% CI: -21.44%, -2.86%) decrease in cord serum total 25(OH)D, respectively. Exposures to the three metals during the whole pregnancy were also correlated to increased odds for newborns' vitamin D deficiency (<20 ng/mL) [odds ratio (95% CI): 1.80 (1.05, 3.10) for V, 1.88 (1.25, 2.82) for Co, and 1.90 (1.07, 3.38) for Tl]. BKMR analyses revealed a negative influence of metal mixtures (V+Co+Tl) on neonatal vitamin D status, as well as potential synergism between V and Co and between V and Tl. CONCLUSIONS: Our study provides evidence of negative impacts of maternal exposure to V, Co, and Tl during pregnancy on cord serum vitamin D levels at delivery. Potential synergism between V and Co and between V and Tl existed in their associations with cord serum total 25(OH)D.

Evidence of SARS-CoV-2 Transmission Through the Ocular Route.

PURPOSE: Currently, the coronavirus disease 2019 (COVID-19) pandemic is raging around the world. However, the transmission of its pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is not fully clear. It is still controversial whether the ocular transmission of SARS-CoV-2 exists. This review aimed to summarize the evidence of SARS-CoV-2 ocular transmission. METHODS: Online articles were searched till October 23, 2020 in Pubmed, Embase, and websites of World Health Organization, Centers for Disease Control and Prevention COVID-19, American Academy of Ophthalmology, and American Society of Cataract and Refractive Surgery under the search strategy of (((("COVID-19"[Mesh]) OR ("SARS-CoV-2"[Mesh])) OR (2019 novel coronavirus)) OR (2019-nCoV)) AND (((("Conjunctivitis"[Mesh]) OR (Ocular Surface)) OR ("Eye"[Mesh])) OR ("Ophthalmology"[Mesh])). The language was not restricted. After screening, 1445 records were excluded and 168 references original articles were finally included. RESULTS: Cells of ocular surface express both the receptor of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), offering molecular bases for the ocular susceptibility to SARS-CoV-2. Accumulated COVID-19 patients presented conjunctivitis as the initial or the only symptom. Whether COVID-19 patients had ocular symptoms or not, SARS-CoV-2 was detectable on the ocular surface, and the isolated virus was infectious, proving that the ocular surface can not only be a reservoir but also a source of contagion. SARS-CoV-2 may reach the ocular surface by hand-eye contact and aerosols. Once SARS-CoV-2 reaches the ocular surface, it may transfer to other systems through the nasolacrimal system or hematogenous metastasis. CONCLUSION: The ocular surface can serve as a reservoir and source of contagion for SARS-CoV-2. SARS-CoV-2 can be transmitted to the ocular surface through hand-eye contact and aerosols, and then transfer to other systems through nasolacrimal route and hematogenous metastasis. The possibility of ocular transmission of SARS-CoV-2 cannot be ignored.

Hedgehog Signaling Pathway Regulates the Proliferation and Differentiation of Rat Meibomian Gland Epithelial Cells.

Purpose: Meibomian glands play a vital role in maintaining ocular surface stability. This study aimed to investigate whether Hedgehog signaling is involved in the regulation of meibomian gland epithelial cells. Methods: Rat meibomian glands epithelial cells (RMGECs) were isolated from ducts and ductules, and then were cultivated to passage two on Matrigel coated wells in meibomian gland epithelial cells medium (MGECM). Cells were switched from MGECM to differentiation medium (DM) or DM added 10 µg/mL azithromycin (DM + AZM) when reached 50% to 60% confluence. The effects of the Smoothened (Smo) agonist (Smo agonist [SAG]) and antagonist (by cyclopamine) on RMGECs were analyzed using quantitative RT-PCR, cell proliferation analysis, immunofluorescence staining, and Nile red staining. Results: The Hedgehog receptor, Smo, and its downstream molecules, Glis, were expressed both in vivo and in vitro. Smo and Gli1 both decreased with the increase of differentiation in vitro. Smo antagonist, cyclopamine, reduced cell numbers, and the expression of Ki67 in MGECM, and promoted the expression of SREBP1 and lipid production in DM + AZM. Smo agonist, SAG, inhibited the expression of SREBP1 and lipid accumulation in DM + AZM but showed no significant effects on raising cell numbers and the expression of Ki67 in MGECM. Conclusions: The Hedgehog signaling pathway appears to play important roles in RMGECs proliferation and differentiation. This may provide a potential therapeutic way to treat meibomian gland dysfunction (MGD).

Subconjunctival Injection of Transdifferentiated Oral Mucosal Epithelial Cells for Limbal Stem Cell Deficiency in Rats.

Rat limbal niche cells (LNCs) have been proven to induce transdifferentiation of oral mucosal epithelial cells (OMECs) into corneal epithelial-like cells termed transdifferentiated oral mucosal epithelial cells (T-OMECs). This investigation aimed to evaluate the effect of subconjunctival T-OMEC injections on alkali-induced limbal stem cell deficiency (LSCD) in rats. LNCs were cocultured with OMECs in the Transwell system to obtain T-OMECs, with NIH-3T3 cells serving as a control. Subconjunctival injection of single T-OMEC or OMEC suspension was performed immediately after corneal alkali injury. T-OMECs were prelabeled with the fluorescent dye CM-DiI in vitro and tracked in vivo. Corneal epithelial defect, opacity, and neovascularization were quantitatively analyzed. The degree of corneal epithelial defect (from day 1 onward), opacity (from day 5 onward), and neovascularization (from day 2 onward) was significantly less in the T-OMEC group than in the OMEC group. Cytokeratin 12 (CK12), pigment epithelium-derived factor, and soluble fms-like tyrosine kinase-1 were expressed at a higher rate following T-OMEC injection. Some CM-DiI-labeled cells were found to be coexpressed with CK12, Pax6, and ΔNp63α in the corneal epithelium after subconjunctival injection. Subconjunctival injection of T-OMECs prevents conjunctival invasion and maintains a normal corneal phenotype, which might be a novel strategy in the treatment of LSCD.

A Comparison of Methods for Isolation of Limbal Niche Cells: Maintenance of Limbal Epithelial Stem/Progenitor Cells.

Purpose: Limbal niche cells (LNCs) play a vital role in the maintenance of limbal epithelial stem/progenitor cells (LESCs). Four methods have been reported to isolate and expand LNCs: digestion by collagenase alone (C-LNC), collagenase following dispase removal of the limbal epithelium (DC-LNC), dissection of dispase-isolated limbal epithelial sheets (D-LNC), and explant cultures of limbal stromal tissues (Ex-LNC). This study aimed to isolate LNCs using those four methods and to compare their capacity to maintain LESCs. Methods: LNCs were isolated from the rat corneal limbus by the following methods: C-LNC, DC-LNC, D-LNC, and Ex-LNC. Quantitative real-time PCR and immunofluorescence staining were used to analyze the expression of embryonic stem cell (ESC) markers. The ability to maintain LESCs was assessed on the basis of colony-forming capacity and the expression of progenitor, proliferation, and differentiation markers in three-dimensional (3D) Matrigel and Transwell systems. Notch signaling of LESCs supported by different LNCs in Transwell inserts was analyzed by quantitative real-time PCR. Results: DC-LNCs exhibited lower expression of CK12 during isolation and expansion. Among P4-expanded LNCs, DC-LNCs expressed significantly higher levels of Sox2, Oct4, Nanog, and N-cadherin than C-LNCs, D-LNCs, and Ex-LNCs. Compared with other LNCs, DC-LNCs were more effective in maintaining LESCs with higher holoclone-forming efficiency, greater expression of ΔNp63α and Ki67, and lower expression of CK12. DC-LNCs were also more capable of downregulating Notch signaling of LESCs. Conclusions: DC-LNCs were more effective in expressing ESC markers and maintaining LESCs compared to other LNCs. This study identifies an optimal method for the isolation of LNCs in tissue engineering and ocular surface reconstruction.

High dielectric ternary oxides from crystal structure prediction and high-throughput screening.

The development of new high dielectric materials is essential for advancement in modern electronics. Oxides are generally regarded as the most promising class of high dielectric materials for industrial applications as they possess both high dielectric constants and large band gaps. Most previous researches on high dielectrics were limited to already known materials. In this study, we conducted an extensive search for high dielectrics over a set of ternary oxides by combining crystal structure prediction and density functional perturbation theory calculations. From this search, we adopted multiple stage screening to identify 441 new low-energy high dielectric materials. Among these materials, 33 were identified as potential high dielectrics favorable for modern device applications. Our research has opened an avenue to explore novel high dielectric materials by combining crystal structure prediction and high throughput screening.

Early pregnancy exposure to benzotriazoles and benzothiazoles in relation to gestational diabetes mellitus: A prospective cohort study.

BACKGROUND: Benzotriazoles (BTRs) and benzothiazoles (BTHs) are emerging contaminants with potential insulin modulation activities. Pregnancy exposure to BTs (BTRs and BTHs) may be a risk factor for the development of gestational diabetes mellitus (GDM). However, epidemiological studies are limited. OBJECTIVES: We prospectively investigated the associations of exposure to BTs at early pregnancy with the blood glucose levels and the risks of GDM. METHODS: A prospective cohort of 1770 pregnant women who were free of diabetes at baseline was established between 2013 and 2015 in Wuhan, China. Urine samples collected at 13.1 ± 1.1 weeks of gestation were analyzed to estimate the exposure level of BTs. The diagnosis of GDM was based on a 75 g oral glucose tolerance test (OGTT) conducted at 26.4 ± 2.4 weeks of gestation. We examined the associations between urinary concentration of BTs and blood glucose levels by linear regression models. The associations of urinary BTs concentrations with the relative risk (RR) of GDM were evaluated by generalized estimating equations with Poisson regression. Effect modifications by fetus sex and pre-pregnancy body mass index (BMI) were further evaluated in the sensitivity analysis. RESULTS: A total of 147 (8.31%) pregnant women were diagnosed with GDM. Median concentrations of urinary BTs did not differ significantly between pregnant women with and without GDM. It was found that urinary levels of benzothiazole and 2-hydroxy-benzothiazole (2-OH-BTH) were positively associated with 2-hour blood glucose (p for trend < 0.050). Comparing the high exposure group with the low exposure group of 2-OH-BTH, the adjusted RR of GDM was 1.79 (95% CI = 1.18 to 2.69, p for trend = 0.012). Sensitivity analysis indicated that the positive association of the urinary 2-OH-BTH level with the RR of GDM remained significant among pregnant women who had a male fetus (RR = 1.76, 95% CI = 1.02 to 3.03, p for trend = 0.041) and those with a normal pre-pregnancy BMI (RR = 1.85, 95% CI = 1.09 to 3.11, p for trend = 0.034). CONCLUSIONS: These findings suggested that higher urinary level of 2-OH-BTH in early pregnancy was associated with impaired glucose homeostasis and the increased risk of GDM. The results underscore the need of follow-up studies to validate the findings and elucidate the underlying biological mechanism.

Electrides with Dinitrogen Ligands.

Electrides are a class of materials which contain excess electrons occupying the cavities in the crystal and playing the role of anions. To achieve electron-rich conditions, it usually requires a positive total formal charge in electride materials. However, the assignment of charges relies on a detailed analysis on chemical bonding. Herein, we present a survey on potential electrides which may be overlooked if no bonding analysis is performed. By applying various structure sampling techniques in conjunction with first-principles calculation, we predicted two compounds Ba2N2:e- and Li2Ca3N6:2e-, both of which are featured by the presence of dinitrogen ligands [N2], to be potential electrides. While Li2Ca3N6:2e- with [N2]2- ions has been synthesized in the past, its electride nature was discovered for the first time based on our high-throughput screening. On the other hand, Ba2N2:e- with [N2]3- ions is a new compound entirely from first-principles structure prediction. The different valence states of dinitrogen ligands identified in these two compounds suggest a novel route to tune the concentration and anisotropic properties of anionic interstitial electrons. Our discovery does not only establish a new class of inorganic electrides but also demonstrates the predictive power of modern crystal structure sampling techniques toward rational material design.