Enhancing antioxidant levels and mitochondrial function in porcine oocyte maturation and embryonic development through notoginsenoside R1 supplementation.

This study examines the impact of Notoginsenoside R1 (NGR1), a compound from Panax notoginseng, on the maturation of porcine oocytes and their embryonic development, focusing on its effects on antioxidant levels and mitochondrial function. This study demonstrates that supplementing in vitro maturation (IVM) medium with NGR1 significantly enhances several biochemical parameters. These include elevated levels of glutathione (GSH), nuclear factor erythrocyte 2-related factor 2 (NRF2) and mRNA expression of catalase (CAT) and GPX. Concurrently, we observed a decrease in reactive oxygen species (ROS) levels and an increase in JC-1 immunofluorescence, mitochondrial distribution, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and nuclear NRF2 mRNA levels. Additionally, there was an increase in ATP production and lipid droplets (LDs) immunofluorescence. These biochemical improvements correlate with enhanced embryonic outcomes, including a higher blastocyst rate, increased total cell count, enhanced proliferative capacity and elevated octamer-binding transcription factor 4 (Oct4) and superoxide dismutase 2 (Sod2) gene expression. Furthermore, NGR1 supplementation resulted in decreased apoptosis, reduced caspase 3 (Cas3) and BCL2-Associated X (Bax) mRNA levels and decreased glucose-regulated protein 78 kD (GRP78) immunofluorescence in porcine oocytes undergoing in vitro maturation. These findings suggest that NGR1 plays a crucial role in promoting porcine oocyte maturation and subsequent embryonic development by providing antioxidant levels and mitochondrial protection.

Mangiferin improves early porcine embryonic development by reducing oxidative stress.

Mangiferin (MGN) is primarily found in the fruits, leaves, and bark of plants of the Anacardiaceae family, including mangoes. MGN exhibits various pharmacological effects, such as protection of the liver and gallbladder, anti-lipid peroxidation, and cancer prevention. This study aimed to investigate the effects of MGN supplementation during in vitro culture (IVC) on the antioxidant capacity of early porcine embryos and the underlying mechanisms involved. Porcine parthenotes in the IVC medium were exposed to different concentrations of MGN (0, 0.01, 0.1, and 1 µM). The addition of 0.1 µM MGN significantly increased the blastocyst formation rate of porcine embryos while reducing the apoptotic index and autophagy. Furthermore, the expression of antioxidation-related (SOD2, GPX1, NRF2, UCHL1), cell pluripotency (SOX2, NANOG), and mitochondria-related (TFAM, PGC1α) genes was upregulated. In contrast, the expression of apoptosis-related (CAS3, BAX) and autophagy-related (LC3B, ATG5) genes decreased after MGN supplementation. These findings suggest that MGN improves early porcine embryonic development by reducing oxidative stress-related genes.

Apigenin delays postovulatory oocyte aging by reducing oxidative stress through SIRT1 upregulation.

After ovulation, senescent oocytes inevitably experience reduced quality and defects in embryonic development. Apigenin (API) is a flavonoid with a wide range of pharmacological effects. Therefore, this study examined the protective effects of API on the quality of porcine oocytes during in-vitro ageing and the underlying mechanisms. The results showed that API treatment could reduce the activation rate after aging for 48 h. In addition, API significantly reduced reactive oxygen species, abnormal distribution of mitochondria, early apoptosis in ageing oocytes, increased glutathione, and mitochondrial adenosine triphosphate levels in ageing oocytes. Importantly, API increased the embryonic development rate in aged oocytes. We also examined molecular changes, finding decreased sirtuin 1 expression in in-vitro postovulatory oocytes, but API reversed this effect. Our results suggest that API attenuates the deterioration of oocyte quality during in-vitro ageing, possibly by reducing oxidative stress through the upregulation of sirtuin 1.

Nanovaccines: An effective therapeutic approach for cancer therapy.

Cancer vaccines hold considerable promise for the immunotherapy of solid tumors. Nanomedicine offers several strategies for enhancing vaccine effectiveness. In particular, molecular or (sub) cellular vaccines can be delivered to the target lymphoid tissues and cells by nanocarriers and nanoplatforms to increase the potency and durability of antitumor immunity and minimize negative side effects. Nanovaccines use nanoparticles (NPs) as carriers and/or adjuvants, offering the advantages of optimal nanoscale size, high stability, ample antigen loading, high immunogenicity, tunable antigen presentation, increased retention in lymph nodes, and immunity promotion. To induce antitumor immunity, cancer vaccines rely on tumor antigens, which are administered in the form of entire cells, peptides, nucleic acids, extracellular vesicles (EVs), or cell membrane-encapsulated NPs. Ideal cancer vaccines stimulate both humoral and cellular immunity while overcoming tumor-induced immune suppression. Herein, we review the key properties of nanovaccines for cancer immunotherapy and highlight the recent advances in their development based on the structure and composition of various (including synthetic and semi (biogenic) nanocarriers. Moreover, we discuss tumor cell-derived vaccines (including those based on whole-tumor-cell components, EVs, cell membrane-encapsulated NPs, and hybrid membrane-coated NPs), nanovaccine action mechanisms, and the challenges of immunocancer therapy and their translation to clinical applications.

3D hepatic organoid production from human pluripotent stem cells.

Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.

Production of Highly Uniform Midbrain Organoids from Human Pluripotent Stem Cells.

Brain organoids have been considered as an advanced platform for in vitro disease modeling and drug screening, but numerous roadblocks exist, such as lack of large-scale production technology and lengthy protocols with multiple manipulation steps, impeding the industrial translation of brain organoid technology. Here, we describe the high-speed and large-scale production of midbrain organoids using a high-throughput screening-compatible platform within 30 days. Micro midbrain organoids (µMOs) exhibit a highly uniform morphology and gene expression pattern with minimal variability. Notably, µMOs show dramatically accelerated maturation, resulting in the generation of functional µMOs within only 30 days of differentiation. Furthermore, individual µMOs display highly consistent responsiveness to neurotoxin, suggesting their usefulness as an in vitro high-throughput drug toxicity screening platform. Collectively, our data indicate that µMO technology could represent an advanced and robust platform for in vitro disease modeling and drug screening for human neuronal diseases.

Customized liver organoids as an advanced in vitro modeling and drug discovery platform for non-alcoholic fatty liver diseases.

Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) have presented a major and common health concern worldwide due to their increasing prevalence and progressive development of severe pathological conditions such as cirrhosis and liver cancer. Although a large number of drug candidates for the treatment of NASH have entered clinical trial testing, all have not been released to market due to their limited efficacy, and there remains no approved treatment for NASH available to this day. Recently, organoid technology that produces 3D multicellular aggregates with a liver tissue-like cytoarchitecture and improved functionality has been suggested as a novel platform for modeling the human-specific complex pathophysiology of NAFLD and NASH. In this review, we describe the cellular crosstalk between each cellular compartment in the liver during the pathogenesis of NAFLD and NASH. We also summarize the current state of liver organoid technology, describing the cellular diversity that could be recapitulated in liver organoids and proposing a future direction for liver organoid technology as an in vitro platform for disease modeling and drug discovery for NAFLD and NASH.

Application of a Magnetic Platform in α6 Integrin-Positive iPSC-TM Purification.

The emergence of induced pluripotent stem cell (iPSC) technology has provided a new approach to regenerating decellularized trabecular meshwork (TM) in glaucoma. We have previously generated iPSC-derived TM (iPSC-TM) using a medium conditioned by TM cells and verified its function in tissue regeneration. Because of the heterogeneity of iPSCs and the isolated TM cells, iPSC-TM cells appear to be heterogeneous, which impedes our understanding of how the decellularized TM may be regenerated. Herein, we developed a protocol based on a magnetic-activated cell sorting (MACS) system or an immunopanning (IP) method for sorting integrin subunit alpha 6 (ITGA6)-positive iPSC-TM, an example of the iPSC-TM subpopulation. We first analyzed the purification efficiency of these two approaches by flow cytometry. In addition, we also determined cell viability by analyzing the morphologies of the purified cells. To conclude, the MACS-based purification could yield a higher ratio of ITGA6-positive iPSC-TM and maintain a relatively higher cell viability than the IP-based method, allowing for the preparation of any iPSC-TM subpopulation of interest and facilitating a better understanding of the regenerative mechanism of iPSC-based therapy.

TNF-α/IL-1ß-licensed hADSCs alleviate cholestatic liver injury and fibrosis in mice via COX-2/PGE2 pathway.

BACKGROUND: Adipose tissue-derived stem cell (ADSC) transplantation has been shown to be effective for the management of severe liver disorders. Preactivation of ADSCs enhanced their therapeutic efficacy. However, these effects have not yet been examined in relation to cholestatic liver injury. METHODS: In the present study, a cholestatic liver injury model was established by bile duct ligation (BDL) in male C57BL/6 mice. Human ADSCs (hADSCs) with or without tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1ß) pretreatment were administrated into the mice via tail vein injections. The efficacy of hADSCs on BDL-induced liver injury was assessed by histological staining, real-time quantitative PCR (RT-qPCR), Western blot, and enzyme-linked immune sorbent assay (ELISA). In vitro, the effects of hADSC conditioned medium on the activation of hepatic stellate cells (HSCs) were investigated. Small interfering RNA (siRNA) was used to knock down cyclooxygenase-2 (COX-2) in hADSCs. RESULTS: TNF-α/IL-1ß preconditioning could downregulate immunogenic gene expression and enhance the engraftment efficiency of hADSCs. Compared to control hADSCs (C-hADSCs), TNF-α/IL-1ß-pretreated hADSCs (P-hADSCs) significantly alleviated BDL-induced liver injury, as demonstrated by reduced hepatic cell death, attenuated infiltration of Ly6G + neutrophils, and decreased expression of pro-inflammatory cytokines TNF-α, IL-1ß, C-X-C motif chemokine ligand 1 (CXCL1), and C-X-C motif chemokine ligand 2 (CXCL2). Moreover, P-hADSCs significantly delayed the development of BDL-induced liver fibrosis. In vitro, conditioned medium from P-hADSCs significantly inhibited HSC activation compared to that from C-hADSCs. Mechanistically, TNF-α/IL-1ß upregulated COX-2 expression and increased prostaglandin E2 (PGE2) secretion. The blockage of COX-2 by siRNA transfection reversed the benefits of P-hADSCs for PGE2 production, HSC activation, and liver fibrosis progression. CONCLUSION: In conclusion, our results suggest that TNF-α/IL-1ß pretreatment enhances the efficacy of hADSCs in mice with cholestatic liver injury, partially through the COX-2/PGE2 pathway.

Efficacy and Safety of Mesenchymal Stromal Cells Therapy for COVID-19 Infection: A Systematic Review and Meta-analysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an infectious respiratory disease prevalent worldwide with a high mortality rate, and there is currently no specific medicine to treat patients. OBJECTIVE: We aimed to assess the safety and efficacy of stem cell therapy for COVID-19 by providing references for subsequent clinical treatments and trials. METHOD: We systematically searched PubMed, Embase, Cochrane, and Web of Science, using the following keywords: "stem cell" or "stromal cell" and "COVID-19." Controlled clinical trials published in English until 24th August 2021 were included. We followed the PRISMA guidelines and used Cochrane Collaboration's tool for assessing the risk of bias. We analysed the data using a fixed-effect model. RESULTS: We identified 1779 studies, out of which eight were eligible and included in this study. Eight relevant studies consisted of 156 patients treated with stem cells and 144 controls (300 individuals in total). There were no SAEs associated with stem cell therapy in all six studies, and no significant differences in AEs (p = 0.09, I2 = 40%, OR = 0.53, 95% CI: 0.26 to 1.09) between the experimental group and control group were observed. Moreover, the meta-analysis found that stem cell therapy effectively reduced the high mortality rate of COVID-19 (14/156 vs. 43/144; p<0.0001, I2 = 0%, OR=0.18, 95% CI: 0.08 to 0.41). CONCLUSION: This study suggests that MSCs therapy for COVID-19 has shown some promising results in safety and efficacy. It effectively reduces the high mortality rate of COVID-19 and does not increase the incidence of adverse events.

Lineage-selective super enhancers mediate core regulatory circuitry during adipogenic and osteogenic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) can be differentiated into osteoblasts and adipocytes. During these processes, super enhancers (SEs) play important roles. Here, we performed comprehensive characterization of the SEs changes associated with adipogenic and osteogenic differentiation of hMSCs, and revealed that SEs changed more dramatically compared with typical enhancers. We identified a set of lineage-selective SEs, whose target genes were enriched with cell type-specific functions. Functional experiments in lineage-selective SEs demonstrated their specific roles in directed differentiation of hMSCs. We also found that some key transcription factors regulated by lineage-selective SEs could form core regulatory circuitry (CRC) to regulate each other's expression and control the hMSCs fate determination. In addition, we found that GWAS SNPs of osteoporosis and obesity were significantly enriched in osteoblasts-selective SEs or adipocytes-selective SEs, respectively. Taken together, our studies unveiled important roles of lineage-selective SEs in hMSCs differentiation into osteoblasts and adipocytes.

Hypoxic bone marrow mesenchymal stromal cells-derived exosomal miR-182-5p promotes liver regeneration via FOXO1-mediated macrophage polarization.

Mesenchymal stromal cells (MSCs) are attractive candidates for treating hepatic disorders given their potential to enhance liver regeneration and function. The paracrine paradigm may be involved in the mechanism of MSC-based therapy, and exosomes (Exo) play an important role in this paracrine activity. Hypoxia significantly improves the effectiveness of MSC transplantation. However, whether hypoxia preconditioned MSCs (Hp-MSCs) can enhance liver regeneration, and whether this enhancement is mediated by Exo, are unknown. In this study, mouse bone marrow-derived MSCs (BM-MSCs) and secreted Exo were injected through the tail vein. We report that Hp-MSCs promote liver regeneration after partial hepatectomy in mice through their secreted exosomes. Interestingly, MSC-Exo were concentrated in liver 6 h after administration and mainly taken up by macrophages, but not hepatocytes. Compared with normoxic MSC-Exo (N-Exo), hypoxic MSC-Exo (Hp-Exo) enhanced M2 macrophage polarization both in vivo and in vitro. Microarray analysis revealed significant enrichment of microRNA (miR)-182-5p in Hp-Exo compared with that in N-Exo. In addition, miR-182-5p knockdown partially abolished the beneficial effect of Hp-Exo. Finally, Hp-MSC-derived exosomal miR-182-5p inhibited theprotein expression of forkhead box transcription factor 1 (FOXO1) in macrophages, which inhibited toll-like receptor 4 (TLR4) expression and subsequently induced an anti-inflammatory response. These results highlight the therapeutic potential of Hp-Exo in liver regeneration and suggest that miR-182-5p from Hp-Exo facilitates macrophage polarization during liver regeneration by modulating the FOXO1/TLR4 signaling pathway.

Novel pterostilbene-loaded pro-phytomicelles: preclinical pharmacokinetics, distribution, and treatment efficacy against acetaminophen-induced liver injury.

A novel pro-phytomicelle formulation with small molecule phytochemicals as nanomaterials was developed for the oral delivery of pterostilbene (PTE) in our previous work. The present report was designed to preclinically evaluate the in vivo oral bioavailability, organ and tissue distribution, as well as the strengthened efficacy against acetaminophen-induced liver damage of this novel formulation, named PTE pro-phytomicelles. After oral administration in rats, an improvement in the area under the curve (AUC)0→t of the PTE pro-phytomicelles (34832.25 vs. 13115.72 ng ml-1 h) and an increase in their maximum plasma concentration (Cmax) (4.940 vs. 2.175 µg ml-1), as compared with those of bare PTE, were observed. The organ and tissue distribution further showed that the PTE pro-phytomicelles could effectively increase the concentration of PTE in all the tested organs and gastrointestinal segments. In the efficacy evaluation, the overdose of Acetaminophen induced severe liver injury in mice, and the oral administration of PTE pro-phytomicelles could efficiently suppress ALT and AST levels in serum, restore histopathological changes, and result in more effective improvement against liver damage via oxidative stress and inflammation cytokine inhibition. The mechanism through which high-mobility group box 1 (HMGB1) signaling was regulated was involved in this treatment. These results reveal that PTE pro-phytomicelles could achieve significantly improved in vivo profiles than bare PTE, and these pro-phytomicelles might provide a new concept and promising therapeutic potential in terms of PTE nanomedicines.

Electronic and Optical Properties of BP, InSe Monolayer and BP/InSe Heterojunction with Promising Photoelectronic Performance.

Two-dimensional (2D) materials provide a new strategy for developing photodetectors at the nanoscale. The electronic and optical properties of black phosphorus (BP), indium selenide (InSe) monolayer and BP/InSe heterojunction were investigated via first-principles calculations. The geometric characteristic shows that the BP, InSe monolayer and BP/InSe heterojunction have high structural symmetry, and the band gap values are 1.592, 2.139, and 1.136 eV, respectively. The results of band offset, band decomposed charge and electrostatic potential imply that the heterojunction structure can effectively inhibit the recombination of electron--hole pairs, which is beneficial for carrier mobility of photoelectric devices. Moreover, the optical properties, including refractive index, reflectivity, electron energy loss, extinction coefficient, absorption coefficient and photon optical conductivity, show excellent performance. These findings reveal the optimistic application potential for future photoelectric devices. The results of the present study provide new insight into challenges related to the peculiar behavior of the aforementioned materials with applications.

Novel luteolin@pro-phytomicelles: In vitro characterization and in vivo evaluation of protection against drug-induced hepatotoxicity.

A novel nanoformulation with the small molecule phytochemical dipotassium glycyrrhizinate as a nanomaterial was developed for the oral delivery of luteolin (Lut), a widely used phytochemical, but it suffered from poor water solubility and low oral bioavailability. This novel nanoformulation, named Lut@pro-phytomicelles, can be fabricated with a simple process. Lut@pro-phytomicelles can instantly dissolve into aqueous mediums and formulate through self-assembly a clear phytomicelle solution with a Lut encapsulation efficiency of 99.16 ± 0.90%, a small micelle size of 30.32 ± 0.12 nm, and a narrow polydispersity index of 0.138 ± 0.024. The optimized formulation demonstrated that Lut had solubility in up to 50 mg/ml of water as a result of its encapsulation within DG phytomicelles. Lut@pro-phytomicelles exhibited excellent characteristics, including good storage stability, a fast in vitro release profile, improvement in in vitro antioxidant activity, and high safety potential. In the oral bioavailability evaluation, a shorter Tmax, increased Cmax, and improved AUC0-t were obtained with Lut@pro-phytomicelles when compared to bare Lut. The distribution evaluation further showed that Lut@pro-phytomicelles could effectively increase the concentrations of Lut in all the tested organs and gastrointestinal segments. In the protection efficacy evaluation, 100 mg/kg Lut@pro-phytomicelles demonstrated strong effects against acetaminophen-induced hepatotoxicity. The mechanisms of inhibiting high-mobility group box 1 signaling and suppressing oxidative stress were involved in this strong treatment effect. These results showed that simple but novel Lut@pro-phytomicelles provided a new, promising nano-delivery system for Lut with a significantly improved in vivo profile.

Cationic Mechanosensitive Channels Mediate Trabecular Meshwork Responses to Cyclic Mechanical Stretch.

The trabecular meshwork (TM) is responsible for intraocular pressure (IOP) homeostasis in the eye. The tissue senses IOP fluctuations and dynamically adapts to the mechanical changes to either increase or decrease aqueous humor outflow. Cationic mechanosensitive channels (CMCs) have been reported to play critical roles in mediating the TM responses to mechanical forces. However, how CMCs influence TM cellular function affect aqueous humor drainage is still elusive. In this study, human TM (HTM) cells were collected from a Chinese donor and subjected to cyclically equiaxial stretching with an amplitude of 20% at 1 Hz GsMTx4, a non-selective inhibitor for CMCs, was added to investigate the proteomic changes induced by CMCs in response to mechanical stretch of HTM. Gene ontology enrichment analysis demonstrated that inhibition of CMCs significantly influenced several biochemical pathways, including store-operated calcium channel activity, microtubule cytoskeleton polarity, toll-like receptor signaling pathway, and neuron cell fate specification. Through heatmap analysis, we grouped 148 differentially expressed proteins (DEPs) into 21 clusters and focused on four specific patterns associated with Ca2+ homeostasis, autophagy, cell cycle, and cell fate. Our results indicated that they might be the critical downstream signals of CMCs adapting to mechanical forces and mediating AH outflow.

Novel carvedilol-loaded pro-phytomicelles: formulation, characterization and enhanced protective efficacy against acetaminophen-inducedliverinjury in mice.

The work describes a novel, small-molecule phytochemicals as nanomaterials based pro-micelles (pro-phytomicelles) drug delivery system, for oral delivery of carvedilol (CAR). This novel nanoformulation of CAR, named CAR pro-phytomicelles, was prepared with rebaudioside A (RA) and dipotassium glycyrrhizinate (DG) as mixed nanomaterials. The formulation was optimized, leading to a 502-fold increase in solubility of CAR in water as a result of encapsulation within mixed phytomicelles based on DG and RA. CAR pro-phytomicelles samples could be instantly dissolved into aqueous media to formulate clear phytomicelle solutions with CAR encapsulation efficiency of 99.67 ± 0.02 %, and small micelle size of 15.62 ± 0.27 nm. CAR pro-phytomicelles exhibited good storage stability, rapid in vitro release in simulated intestinal fluid, and improved in vitro antioxidant activity. CAR pro-phytomicelles had good biocompatibility. Protective efficacy evaluation revealed that acetaminophen overdose could induce high mortality and severe liver injury in mice, while CAR pro-phytomicelle treatment exhibited significant protective effect against acetaminophen overdose. This protective efficacy was due to a mechanism that involved the regulation of high-mobility group box 1 and its signaling-related proinflammatory cytokines. These results show that pro-phytomicelles could provide a new concept and promising therapeutics as nanomedicines for improving the activities of CAR against acetaminophen-induced liver injury.

Lineage-specific rearrangement of chromatin loops and epigenomic features during adipocytes and osteoblasts commitment.

Human mesenchymal stem cells (hMSCs) can be differentiated into adipocytes and osteoblasts. The processes are driven by the rewiring of chromatin architectures and transcriptomic/epigenomic changes. Here, we induced hMSCs to adipogenic and osteogenic differentiation, and performed 2 kb resolution Hi-C experiments for chromatin loops detection. We also generated matched RNA-seq, ChIP-seq and ATAC-seq data for integrative analysis. After comprehensively comparing adipogenesis and osteogenesis, we quantitatively identified lineage-specific loops and screened out lineage-specific enhancers and open chromatin. We reveal that lineage-specific loops can activate gene expression and facilitate cell commitment through combining enhancers and accessible chromatin in a lineage-specific manner. We finally proposed loop-mediated regulatory networks and identified the controlling factors for adipocytes and osteoblasts determination. Functional experiments validated the lineage-specific regulation networks towards IRS2 and RUNX2 that are associated with adipogenesis and osteogenesis, respectively. These results are expected to help better understand the chromatin conformation determinants of hMSCs fate commitment.

Corneal Epithelium-Derived Netrin-1 Alleviates Dry Eye Disease via Regulating Dendritic Cell Activation.

Purpose: To investigate the expression of corneal epithelium-derived netrin-1 (NTN-1) and its immunoregulatory function in dry eye disease (DED) using a DED mouse model. Methods: We generated DED mouse models with desiccating stress under scopolamine treatment. RNA sequencing was performed to identify differentially expressed genes (DEGs) in the corneal epithelium of DED mice. NTN-1 expression was analyzed via real-time PCR, immunofluorescence staining, and immunoblotting. The DED mice were then treated with recombinant NTN-1 or neutralizing antibodies to investigate the severity of the disease, dendritic cell (DC) activation, and inflammatory cytokine expression. Results: A total of 347 DEGs (292 upregulated and 55 downregulated) were identified in the corneal epithelium of DED mice: corneal epithelium-derived NTN-1 expression was significantly decreased in DED mice compared to that in control mice. Topical recombinant NTN-1 application alleviated the severity of the disease, accompanied by restoration of tear secretion and goblet cell density. In addition, NTN-1 decreased the number of DCs, inhibited the activation of the DCs and Th17 cells, and reduced the expression of inflammatory factors in DED mice. In contrast, blocking endogenous NTN-1 activity with an anti-NTN-1 antibody aggravated the disease, enhanced DC activation, and upregulated the inflammatory factors in the conjunctivae of DED mice. Conclusions: We identified decreased NTN-1 expression in the corneal epithelium of DED mice. Our findings elucidate the role of NTN-1 in alleviating DED and impeding DC activation, thereby indicating its therapeutic potential in suppressing ocular inflammation in DED.

Detection of Haemophilus influenzae by loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay.

BACKGROUND: Haemophilus influenzae was the most aggressive pathogen and formed a major cause of bacterial meningitis and pneumonia in young children and infants, which need medical emergency requiring immediate diagnosis and treatment. However, From isolation to identification of H. influenzae, the traditional diagnose strategy was time-consuming and expensive. Therefore, the establishment of a convenient, highly sensitive, and stable detection system is urgent and critical. RESULTS: In this study, we used a combined method to detect H. influenzae. Six specific primers were designed on the basis of outer membrane protein P6 gene sequence of H. influenzae. The reaction condition such as the optimum temperature was 65℃, and the optimum reaction time was 30 min, respectively. Through the loop-mediated isothermal amplification (LAMP) in combination with nanoparticle-based lateral flow biosensor (LFB), the sensitivity of LAMP-LFB showed 100 fg was the lowest genomic DNA templates concentration in the pure cultures. Meanwhile, the specificity of H. influenzae-LAMP-LFB assay showed the exclusive positive results, which were detected in H. influenzae templates. In 55 clinical sputum samples, 22 samples were positive with LAMP-LFB method, which was in accordance with the traditional culture and Polymerase Chain Reaction (PCR) method. The accuracy in diagnosing H. influenzae with LAMP-LFB could reach 100%, compared to culture and PCR method, indicating the LAMP-LFB had more advantages in target pathogen detection. CONCLUSIONS: Taken together, LAMP-LFB could be used as an effective diagnostic approach for H. influenzae in the conditions of basic and clinical labs, which would allow clinicians to make better informed decisions regarding patient treatment without delay.