Exploring Immune Cell Diversity in the Lacrimal Glands of Healthy Mice: A Single-Cell RNA-Sequencing Atlas.

The lacrimal gland is responsible for maintaining the health of the ocular surface through the production of tears. However, our understanding of the immune system within the lacrimal gland is currently limited. Therefore, in this study, we utilized single-cell RNA sequencing and bioinformatic analysis to identify and analyze immune cells and molecules present in the lacrimal glands of normal mice. A total of 34,891 cells were obtained from the lacrimal glands of mice and classified into 18 distinct cell clusters using Seurat clustering. Within these cell populations, 26 different immune cell subpopulations were identified, including T cells, innate lymphocytes, macrophages, mast cells, dendritic cells, and B cells. Network analysis revealed complex cell-cell interactions between these immune cells, with particularly significant interactions observed among T cells, macrophages, plasma cells, and dendritic cells. Interestingly, T cells were found to be the main source of ligands for the Thy1 signaling pathway, while M2 macrophages were identified as the primary target of this pathway. Moreover, some of these immune cells were validated using immunohistological techniques. Collectively, these findings highlight the abundance and interactions of immune cells and provide valuable insights into the complexity of the lacrimal gland immune system and its relevance to associated diseases.

A preparation method for mRNA-LNPs with improved properties.

The properties of mRNA lipid nanoparticles (mRNA-LNPs), including size, empty particles, morphology, storage stability, and transfection potency, are critically dependent on the preparation methods. Here, a Two-step tangential-flow filtration (TFF) method was successfully employed to improve the properties of mRNA-LNPs during the preparation process. This method involves an additional ethanol removal step prior to the particle fusion process. Notably, this innovative approach has yielded mRNA-LNPs with larger particles, a reduced proportion of empty LNPs, optimized storage stability (at least 6 months at 2-8 °C), improved in vitro transfection efficiency, and minimized distribution in the heart and blood in vivo. In summary, this study represents the implementation of the innovative Two-step TFF method in the preparation of mRNA-LNPs. Our findings indicate substantial enhancements in the properties of our mRNA-LNPs, specifically with regard to the percentage of empty LNPs, stability, transfection efficiency, and in vivo distribution. These improvements have the potential to optimize their industrial applicability and expand their clinical use.

Mechanisms of Extraorbital Lacrimal Gland Aging in Mice: An Integrative Analysis of the Temporal Transcriptome.

Purpose: This study used high-throughput RNA sequencing (RNA-Seq) and bioinformatics analysis to investigate the altered transcriptome profile of aging lacrimal glands in mice that occurs over the course of a 24-hour cycle. Methods: Male C57BL/6J mice aged 12 weeks (young) and 20 months (aging) were housed in a pathogen-free setting with a 12-hour light/12-hour dark cycle. Throughout a 24-hour cycle, mouse extraorbital lacrimal glands (ELGs) were collected at eight time points at three-hour intervals. To prepare for the high-throughput RNA-Seq, whole mRNA was extracted. Differentially expressed genes (DEGs) in the young and aging groups were subjected to bioinformatic analysis based on diurnal patterns. Furthermore, the cell populations in which significant DEGs express and signaling pathways occur were validated at the single-cell RNA sequencing (scRNA-seq) level. Results: The total transcriptome composition was significantly altered in aging ELGs compared with that in young mouse ELGs at eight time points during the 24-hour cycle, with 864 upregulated and 228 downregulated DEGs, which were primarily enriched in inflammatory pathways. Further comparative analysis of the point-to-point transcriptome revealed that aging ELGs underwent alterations in the temporal transcriptome profile in several pathways, including the inflammation-related, metabolism-related, mitochondrial bioenergetic function-associated, synaptome neural activity-associated, cell processes-associated, DNA processing-associated and fibrosis-associated pathways. Most of these pathways occurred separately in distinct cell populations. Conclusions: Transcriptome profiles of aging lacrimal glands undergo considerable diurnal time-dependent changes; this finding offers a comprehensive source of information to better understand the pathophysiology of lacrimal gland aging and its underlying mechanisms.

Long-term high fructose intake promotes lacrimal gland dysfunction by inducing gut dysbiosis in mice.

The lacrimal gland is essential for maintaining ocular surface health through the secretion of the aqueous layer of the tear film. It is therefore important to explore the intrinsic and extrinsic factors that affect the structure and function of the lacrimal gland and the mechanisms underlying them. With the prevalence of Westernized diets characterized by high sugar and fat content, the susceptibility to many diseases, including ocular diseases, is increased by inducing dysbiosis of the gut microbiome. Here, we found that the composition, abundance, and diversity of the gut microbiome was significantly altered in mice by drinking 15% high fructose water for one month, as determined by 16S rRNA sequencing. This was accompanied by a significant increase in lipid deposition and inflammatory cell infiltration in the extraorbital lacrimal glands (ELGs) of mice. Transcriptome analysis based on bulk RNA-sequencing revealed abnormal activation of some of several metabolic and immune-related pathways. In addition, the secretory response to stimulation with the cholinergic receptor agonist pilocarpine was significantly reduced. However, when the composition and diversity of the gut microbiome of high fructose intake (HFI)-treated mice were improved by transplanting feces from normal young healthy mice, the pathological alterations in ELG structure, inflammatory cell infiltration, secretory function and transcriptome analysis described above were significantly reversed compared to age-matched control mice. In conclusion, our data suggest that prolonged HFI may cause pathological damage to the structure and function of the ELG through the induction of gut dysbiosis. Restoration of intestinal dysbiosis in HFI-treated mice by fecal transplantation has a potential role in ameliorating these pathological impairments.

High-fat intake reshapes the circadian transcriptome profile and metabolism in murine meibomian glands.

Background: Nutritional and food components reshape the peripheral clock and metabolism. However, whether food challenges affect the circadian clock and metabolism of meibomian glands (MGs) has not been fully explored. This study was designed to analyze alterations in the rhythmic transcriptome and metabolism of MGs of murine fed a balanced diet or a high-fat diet (HFD). Methods: Male C57BL/6J mice were maintained on a 12/12 h light/dark cycle and fed ad libitum on normal chow (NC) or HFD for 4 weeks. MGs were collected from sacrificed animals at 3-h intervals throughout a 24-h circadian cycle. The circadian transcriptome of MGs was analyzed via bioinformatics approaches using high-throughput RNA sequencing (RNA-seq). In addition, circadian oscillations of lipid components in MGs were analyzed. Results: Meibomian glands displayed robust transcriptome rhythmicity. HFD feeding significantly altered the circadian transcriptome profile of MGs-including composition and phase-and spatiotemporally affected the enriched signaling pathways. In addition, HFD feeding significantly altered the normal rhythmic oscillations of lipid components in MGs. Conclusion: Our data show that HFD significantly affects MGs' rhythmicity, which reveals a high sensitivity of MGs' clocks to lipid composition in food.

Screening marker genes of type 2 diabetes mellitus in mouse lacrimal gland by LASSO regression.

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and a relative deficiency of insulin. This study aims to screen T2DM-related maker genes in the mouse extraorbital lacrimal gland (ELG) by LASSO regression.C57BLKS/J strain with leptin db/db homozygous mice (T2DM, n = 20) and wild-type mice (WT, n = 20) were used to collect data. The ELGs were collected for RNA sequencing. LASSO regression was conducted to screen marker genes with the training set. Five genes were selected from 689 differentially expressed genes by LASSO regression, including Synm, Elovl6, Glcci1, Tnks and Ptprt. Expression of Synm was downregulated in ELGs of T2DM mice. Elovl6, Glcci1, Tnks, and Ptprt were upregulated in T2DM mice. Area under receiver operating curve of the LASSO model was 1.000(1.000-1.000) and 0.980(0.929-1.000) in the training set and the test set, respectively. The C-index and the robust C-index of the LASSO model were 1.000 and 0.999, respectively, in the training set, and 1.000 and 0.978, respectively, in the test set. In the lacrimal gland of db/db mice, Synm, Elovl6, Glcci1, Tnks and Ptprt can be used as marker genes of T2DM. Abnormal expression of marker genes is related to lacrimal gland atrophy and dry eye in mice.

Sleep Loss Causes Dysfunction in Murine Extraorbital Lacrimal Glands.

Purpose: Sleep loss markedly affects the structure and function of the lacrimal gland and may cause ocular surface disease as a common public health problem. This study aims to investigate the circadian disturbance caused by sleep loss leading to dysfunction of extraorbital lacrimal glands (ELGs). Methods: A mouse sleep deprivation (SD) model for sleep loss studies was built in C57BL/6J male mice. After four weeks, the ELGs were collected at three-hour intervals during a 24-hour period. The Jonckheere-Terpstra-Kendall algorithm was used to determine the composition, phase, and rhythmicity of transcriptomic profiles in ELGs. Furthermore, we compared the non-sleep-deprived and SD-treated mouse ELG (i) reactive oxygen species (ROS) by fluorescein staining, (ii) DNA damage by immunostaining for γ-H2Ax, and (iii) circadian migration of immune cells by immunostaining for CD4, CD8, γδ-TCR, CD64, and CX3CR1. Finally, we also evaluated (i) the locomotor activity and core body temperature rhythm of mice and (ii) the mass, cell size, and tear secretion of the ELGs. Results: SD dramatically altered the composition and phase-associated functional enrichment of the circadian transcriptome, immune cell trafficking, metabolism, cell differentiation, and neural secretory activities of mouse ELGs. Additionally, SD caused the ROS accumulation and consequent DNA damage in the ELGs, and the ELG dysfunction caused by SD was irreversible. Conclusions: SD damages the structure, function, and diurnal oscillations of ELGs. These results highlight comprehensive characterization of insufficient sleep-affected ELG circadian transcriptome that may provide a new therapeutic approach to counteract the effects of SD on ELG function.

High-Fat Nutritional Challenge Reshapes Circadian Signatures in Murine Extraorbital Lacrimal Glands.

Purpose: A high-fat diet (HFD) increases the risk of developing many systemic diseases; however, the effects of high fat intake on lacrimal gland functions and the molecular mechanisms underlying these effects are unknown. We explored the effects of an HFD on the circadian rhythms of the extraorbital lacrimal glands (ELGs). Methods: Male C57BL/6J mice maintained on a 12/12-hour light/dark cycle were fed an ad libitum HFD or normal chow (NC) for 2 weeks. The ELGs were collected from euthanized animals every 3 hours throughout the circadian cycle (24 hours). Using high-throughput RNA-sequencing (RNA-Seq), we studied the circadian transcriptomic profile of the ELGs. Circadian oscillations in cell size, secretion response, lipid deposition, and immune cell trafficking of the ELGs were also analyzed. Results: An HFD modulated the circadian transcriptomic profile of the ELGs, including the composition, phase, and amplitude of cyclical transcript oscillations, and affected the associated signaling pathways at spatiotemporal levels. HFD feeding significantly altered the normal rhythmic oscillations of ELG cell size, immune cell trafficking, secretion response, and lipid deposition. After dietary reversal in HFD-fed animals, the activity, core temperature, and lipid accumulation in lacrimal glands recovered partially to the level of NC-fed animals. However, the average cell size of the ELGs, the recruitment of immune cells, and the rhythm of lacrimal secretion did not return to the levels of the NC-fed group. Conclusions: HFD perturbation interferes with the cyclical transcriptomic profile, cell size, immune cell trafficking, and secretion function of the ELGs with a strikingly high sensitivity.

Epigenetic Regulation of PDX-1 in Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by hyperglycemia which is caused by insufficient insulin secretion or insulin resistance. Interaction of genetic, epigenetic and environmental factors plays a significant role in the development of T2DM. Several environmental factors including diet and lifestyle, as well as age have been associated with an increased risk for T2DM. It has been demonstrated that these environmental factors may affect global epigenetic status, and alter the expression of susceptible genes, thereby contributing to the pathogenesis of T2DM. In recent years, a growing body of molecular and genetic studies in diabetes have been focused on the ways to restore the numbers or function of ß-cells in order to reverse a range of metabolic consequences of insulin deficiency. The pancreatic duodenal homeobox 1 (PDX-1) is a transcriptional factor that is essential for the development and function of islet cells. A number of studies have shown that there is a significant increase in the level of DNA methylation of PDX-1 resulting in reduced activity in T2DM islets. The decrease in PDX-1 activity may be a critical mediator causing dysregulation of pancreatic ß cells in T2DM. This article reviews the epigenetic mechanisms of PDX-1 involved in T2DM, focusing on diabetes and DNA methylation, and discusses some potential strategies for the application of PDX-1 in the treatment of diabetes.

Molecular Dynamic Simulation of D-Mannitol Polymorphs in Solid State and in Solution Relating With Spontaneous Nucleation.

In this article, the polymorphic nucleation of D-mannitol was studied in depth by molecular simulating the solid state of different forms and the aqueous solution both in unsaturated and supersaturated conditions. In our previous work, it was found that different polymorphs of D-mannitol could nucleate in various supersaturations. Here in this work, we try to use molecular dynamic simulation as a tool to explore the system to microscopically understand the phenomenon. First, the molecular structure of the 3 D-mannitol polymorphs in the solid unit cell is studied, and it is found that the molecular conformation directly affects the stability of the polymorphs. Then, the properties of D-mannitol molecules in unsaturated and supersaturated aqueous solutions are investigated. The results reveal that D-mannitol molecules are mostly monomers in unsaturated aqueous solution, while dimers start to show as the increase of concentration and also in the supersaturated aqueous solutions. Moreover, the form of the dimer varies with the degree of supersaturation, which might be the reason why the final crystal forms are different under different supersaturations. It seems that the I-dimers in supersaturated aqueous solutions is beneficial to the formation of the delta form of D-mannitol, and the T-dimers might be likely to form alpha crystal form, while the H-type dimers should be more favorable for to nucleate the beta form.

Binding study of lysozyme with Al(III) using chemiluminescence analysis.

The binding behavior of lysozyme with Al(III) is described using luminol as a luminescence probe by flow injection-chemiluminescence (FI-CL) analysis. It was found that the CL intensity of the luminol-lysozyme reaction could be markedly enhanced by Al(III), and the increase in CL intensity was linear with the Al(III) concentration over the range 0.3-30.0 pg mL(-1) , with a detection limit of 0.1 pg mL(-1) (3σ). Based on the interaction model of lysozyme with Al(III), lg[(I - I0 )/(2I0 - I)] = lgK + nlg[M], the binding constant K = 6.84 × 10(6) L mol(-1) and the number of binding sites (n) = 0.76. The relative standard deviations were 3.2, 2.4 and 2.0% for 10.0, 20.0 and 30.0 pg mL(-1) Al(III) (n = 7), respectively. This new method was successfully applied to continuous, quantitative monitoring of picogram level Al(III) in human saliva following oral intake of compound aluminum hydroxide tablets. It was found that Al(III) in saliva reached a maximum of 101.2 ng mL(-1) at 3.0 h. The absorption rate constant ka , elimination rate constant k and half-life time t1/2 of Al(III) were 1.378 h(-1) , 0.264 h(-1) and 2.624 h, respectively.