Novel pretreatment nomograms based on pan-immune-inflammation value for predicting clinical outcome in patients with head and neck squamous cell carcinoma.

Background: The prognostic value of an effective biomarker, pan-immune-inflammation value (PIV), for head and neck squamous cell carcinoma (HNSCC) patients after radical surgery or chemoradiotherapy has not been well explored. This study aimed to construct and validate nomograms based on PIV to predict survival outcomes of HNSCC patients. Methods: A total of 161 HNSCC patients who underwent radical surgery were enrolled retrospectively for development cohort. The cutoff of PIV was determined using the maximally selected rank statistics method. Multivariable Cox regression and least absolute shrinkage and selection operator (LASSO) regression analyses were performed to develop two nomograms (Model A and Model B) that predict disease-free survival (DFS). The concordance index, receiver operating characteristic curves, calibration curves, and decision curve analysis were used to evaluate the nomograms. A cohort composed of 50 patients who received radiotherapy or chemoradiotherapy (RT/CRT) alone was applied for generality testing of PIV and nomograms. Results: Patients with higher PIV (≥123.3) experienced a worse DFS (HR, 5.01; 95% CI, 3.25-7.72; p<0.0001) and overall survival (OS) (HR, 5.23; 95% CI, 3.34-8.18; p<0.0001) compared to patients with lower PIV (<123.3) in the development cohort. Predictors of Model A included age, TNM stage, neutrophil-to-lymphocyte ratio (NLR), and PIV, and that of Model B included TNM stage, lymphocyte-to-monocyte ratio (LMR), and PIV. In comparison with TNM stage alone, the two nomograms demonstrated good calibration and discrimination and showed satisfactory clinical utility in internal validation. The generality testing results showed that higher PIV was also associated with worse survival outcomes in the RT/CRT cohort and the possibility that the two nomograms may have a universal applicability for patients with different treatments. Conclusions: The nomograms based on PIV, a simple but useful indicator, can provide prognosis prediction of individual HNSCC patients after radical surgery and may be broadly applicated for patients after RT/CRT alone.

Gasdermin D in macrophages drives orchitis by regulating inflammation and antigen presentation processes.

Inflammation in the testes induced by infection and autoimmunity contributes significantly to male infertility, a public health issue. Current therapies using antibiotics and broad-spectrum anti-inflammatory drugs are ineffective against non-bacterial orchitis and induce side effects. This highlights the need to explore the pathogenesis of orchitis and develop alternative therapeutic strategies. In this study, we demonstrated that Gasdermin D (GSDMD) was activated in the testes during uropathogenic Escherichia coli (UPEC)-induced acute orchitis, and that GSDMD in macrophages induced inflammation and affected spermatogenesis during acute and chronic orchitis. In testicular macrophages, GSDMD promoted inflammation and antigen presentation, thereby enhancing the T-cell response after orchitis. Furthermore, the pharmacological inhibition of GSDMD alleviated the symptoms of UPEC-induced acute orchitis. Collectively, these findings provide the first demonstration of GSDMD's role in driving orchitis and suggest that GSDMD may be a potential therapeutic target for treating orchitis.

Laser-Ignited Lipid Peroxidation Nanoamplifiers for Strengthening Tumor Photodynamic Therapy Through Aggravating Ferroptotic Propagation and Sustainable High Immunogenicity.

Photodynamic therapy (PDT) is extensively investigated for tumor therapy in the clinic. However, the efficacy of PDT is severely limited by the tissue penetrability of light, short effective half-life and radius of reactive oxygen species (ROS), and the weak immunostimulatory effect. In this study, a glutathione (GSH)-activatable nano-photosensitizer is developed to load with arachidonic acid (AA) and camouflage by erythrocyte membrane, which serves as a laser-ignited lipid peroxidation nanoamplifier (MAR). The photosensitive effect of MAR is recovered accompanied by the degradation in the tumor microenvironment and triggers the peroxidation of AA upon laser excitation. Interestingly, it aggravates the propagation of ferroptosis among cancer cells by driving the continuous lipid peroxidation chain reactions with the participation of the degradation products, ferrous ions (Fe2+), and AA. Consequently, even the deep-seated tumor cells without illumination also undergo ferroptosis owing to the propagation of ferroptotic signal. Moreover, the residual tumor cells undergoing ferroptosis still maintain high immunogenicity after PDT, thus continuously triggering sufficient tumor-associated antigens (TAAs) release to remarkably promote the anti-tumor immune response. Therefore, this study will provide a novel "all-in-one" nano-photosensitizer that not only amplifies the damaging effect and expands the effective range of PDT but also improves the immunostimulatory effect after PDT.

The preprogrammed anti-inflammatory phenotypes of CD11chigh macrophages by Streptococcus pneumoniae aminopeptidase N safeguard from allergic asthma.

BACKGROUND: Early microbial exposure is associate with protective allergic asthma. We have previously demonstrated that Streptococcus pneumoniae aminopeptidase N (PepN), one of the pneumococcal components, inhibits ovalbumin (OVA) -induced airway inflammation in murine models of allergic asthma, but the underlying mechanism was incompletely determined. METHODS: BALB/c mice were pretreated with the PepN protein and exposed intranasally to HDM allergen. The anti-inflammatory mechanisms were investigated using depletion and adoptive transfer experiments as well as transcriptome analysis and isolated lung CD11chigh macrophages. RESULTS: We found pretreatment of mice with PepN promoted the proliferation of lung-resident F4/80+CD11chigh macrophages in situ but also mobilized bone marrow monocytes to infiltrate lung tissue that were then transformed into CD11high macrophages. PepN pre-programmed the macrophages during maturation to an anti-inflammatory phenotype by shaping the metabolic preference for oxidative phosphorylation (OXPHOS) and also inhibited the inflammatory response of macrophages by activating AMP-activated protein kinase. Furthermore, PepN treated macrophages also exhibited high-level costimulatory signaling molecules which directed the differentiation into Treg. CONCLUSION: Our results demonstrated that the expansion of CD11chigh macrophages in lungs and the OXPHOS metabolic bias of macrophages are associated with reduced allergic airway inflammation after PepN exposure, which paves the way for its application in preventing allergic asthma.

The Feasibility Study of Intraoperative RLN Monitoring Using Cricothyroid Membrane-Inserted Needle Electrodes During Thyroid Surgery.

OBJECTIVE: This study evaluated the feasibility, stability, safety, and economy of cricothyroid membrane (CM)-inserted needle electrodes for recurrent laryngeal nerve monitoring. STUDY DESIGN: Parallel and controlled study. SETTING: Clinical research center for thyroid diseases of Shaanxi province. METHODS: A total of 64 patients in the needle electrodes group (104 recurrent laryngeal nerves [RLNs]) and 44 patients in the endotracheal tube (ETT)-based electrodes group (80 RLNs) underwent monitored thyroidectomy. The evoked electromyography (EMG) signals detected by the 2 electrodes were recorded and analyzed. The changes in EMG during Berry's ligament traction and tracheal displacement were compared. All patients underwent preoperative and postoperative laryngoscopy within 1 week. RESULTS: Both electrodes successfully recorded typical evoked laryngeal EMG waveforms from RLNs. The needle electrodes recorded relatively higher amplitudes and similar latencies compared to ETT-based electrodes. The evoked EMG signals attributed to needle electrodes could accurately predict the function of RLNs with 100% sensitivity and specificity. The reduction in the recorded amplitudes attributed to needle electrodes was higher than that observed with ETT-based electrodes during Berry's ligament traction or trachea displacement, whereas a similar increase in the latencies was recorded in the 2 groups. Particularly, Berry's ligament traction was more likely to lead to EMG amplitude reduction and latency prolongation. The needle electrodes group recorded 2 cases of minor bleeding on the CM. The needle electrodes were more cost-effective than ETT-based electrodes. CONCLUSION: The CM-inserted needle electrodes are feasible, stable, safe, and economical for RLN monitoring, and they provide an alternative novel intraoperative neural monitoring format for thyroid surgeons.

Survival benefits of perioperative chemoradiotherapy versus chemotherapy for advanced stage gastric cancer based on directed acyclic graphs.

The overall survival benefits of perioperative chemotherapy (PCT) and perioperative chemoradiotherapy (PCRT) for patients with locally advanced gastric cancer (GC) have not been fully explored. The aim of this study was to compare the benefits of PCT and PCRT in GC patients and determine the factors affecting survival rate using directed acyclic graphs (DAGs). The data of 1,442 patients with stage II-IV GC who received PCT or PCRT from 2000 to 2018 were retrieved from the Surveillance, Epidemiology, and End Results (SEER) database. First, the least absolute shrinkage and selection operator (LASSO) was used to identify possible influencing factors for overall survival. Second, the variables that were selected by LASSO were then used in univariate and Cox regression analyses. Third, corrective analyses for confounding factors were selected based on DAGs that show the possible association between advanced GC patients and outcomes and evaluate the prognosis. Patients who received PCRT had longer overall survival than those who received PCT treatment (P = 0.015). The median length of overall survival of the PCRT group was 36.5 (15.0 - 53.0) months longer than that of the PCT group (34.6 (16.0 - 48.0) months). PCRT is more likely to benefit patients who are aged ≤ 65, male, white, and have regional tumors (P<0.05). The multivariate Cox regression model showed that male sex, widowed status, signet ring cell carcinoma, and lung metastases were independent risk factors for a poor prognosis. According to DAG, age, race, and Lauren type may be confounding factors that affect the prognosis of advanced GC. Compared to PCT, PCRT has more survival benefits for patients with locally advanced GC, and ongoing investigations are needed to better determine the optimal treatment. Furthermore, DAGs are a useful tool for contending with confounding and selection biases to ensure the proper implementation of high-quality research.

CSF2 upregulates CXCL3 expression in adipocytes to promote metastasis of breast cancer via the FAK signaling pathway.

Recent studies have demonstrated that cancer-associated adipocytes (CAAs) in the tumor microenvironment are involved in the malignant progression of breast cancer. However, the underlying mechanism of CAA formation and its effects on the development of breast cancer are still unknown. Here, we show that CSF2 is highly expressed in both CAAs and breast cancer cells. CSF2 promotes inflammatory phenotypic changes of adipocytes through the Stat3 signaling pathway, leading to the secretion of multiple cytokines and proteases, particularly C-X-C motif chemokine ligand 3 (CXCL3). Adipocyte-derived CXCL3 binds to its specific receptor CXCR2 on breast cancer cells and activates the FAK pathway, enhancing the mesenchymal phenotype, migration, and invasion of breast cancer cells. In addition, a combination treatment targeting CSF2 and CXCR2 shows a synergistic inhibitory effect on adipocyte-induced lung metastasis of mouse 4T1 cells in vivo. These findings elucidate a novel mechanism of breast cancer metastasis and provide a potential therapeutic strategy for breast cancer metastasis.

In vivo characterization and analysis of glioblastoma at different stages using multiscale photoacoustic molecular imaging.

Simultaneous spatio-temporal description of tumor microvasculature, blood-brain barrier, and immune activity is pivotal to understanding the evolution mechanisms of highly aggressive glioblastoma, one of the most common primary brain tumors in adults. However, the existing intravital imaging modalities are still difficult to achieve it in one step. Here, we present a dual-scale multi-wavelength photoacoustic imaging approach cooperative with/without unique optical dyes to overcome this dilemma. Label-free photoacoustic imaging depicted the multiple heterogeneous features of neovascularization in tumor progression. In combination with classic Evans blue assay, the microelectromechanical system based photoacoustic microscopy enabled dynamic quantification of BBB dysfunction. Concurrently, using self-fabricated targeted protein probe (αCD11b-HSA@A1094) for tumor-associated myeloid cells, unparalleled imaging contrast of cells infiltration associated with tumor progression was visualized by differential photoacoustic imaging in the second near-infrared window at dual scale. Our photoacoustic imaging approach has great potential for tumor-immune microenvironment visualization to systematically reveal the tumor infiltration, heterogeneity, and metastasis in intracranial tumors.

Circular RNA hsa_circ_0003892 promotes the development of papillary thyroid carcinoma by regulating the miR-326/LASP1 axis.

BACKGROUND: Thyroid cancer is the most common malignancy of the endocrine system. Circular RNA (circRNA) is recognized as a key regulator of tumorigenesis in papillary thyroid carcinoma (PTC). Here this work focused on the mechanism of circRNA_0003892 (circ_0003892) in PTC progression. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to examine circ_0003892, microRNA-326 (miR-326) and LIM and SH3 protein 1 (LASP1) mRNA expression levels in PTC tissues and cell lines. Besides, cell counting kit-8 (CCK-8), EdU and transwell assays were conducted to detect the proliferative, migrative and invasive abilities of PTC cells, respectively. B The targeting relationships between miR-326 and circ_0003892 or LASP1 3'-UTR were verified by dual-luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. RESULTS: Circ_0003892 expression was raised in PTC tissues and cells, which was significantly interrelated with larger tumor size and extrathyroidal extension in PTC sufferers. Overexpression of circ_0003892 significantly promoted the malignant biological behaviors of PTC cells. Additionally, miR-326 was a downstream target of circ_0003892, and miR-326 overexpression weakened the promoting effect of circ_0003892 overexpression on the malignant progression of PTC. MiR-326 specifically inhibited LASP1. Circ_0003892 positively regulated LASP1 expression by targeting miR-326. CONCLUSION: Circ_0003892 up-regulates LASP1 expression and facilitates PTC progression via competitively binding to miR-326.

Hydrogen sulfide activatable metal-organic frameworks for Fluorescence Imaging-Guided Photodynamic Therapy of colorectal cancer.

Photodynamic therapy (PDT) is a promising alternative and palliative therapeutic strategy for colorectal cancer (CRC). A novel photosensitizer with higher selectivity for CRC and fewer side effects is vital for clinical application. Given that the overexpression of hydrogen sulfide (H2S) in CRC, it is expected to provide a selective stimulus for activatable photosensitizers that in respond to the specific microenvironment. Herein, we report a novel development of metal-organic frameworks (MOFs) composed of meso-Tetra (4-carboxyphenyl) porphine (TCPP) and ferric ion (Fe3+) through a facile one-pot process. Experiments both in vitro and in vivo reveal that the MOF is capable of depredating in response to the high content of H2S in tumor microenvironment of CRC. Accompanying with the degradation and release of TCPP, the fluorescence and photosensitivity effect is switched from "off" to "on", enabling the MOF to serve as a H2S activatable nano-photosensitizer for real-time fluorescence imaging-guided and targeted PDT of CRC.

Targeting EIF3C to suppress the development and progression of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma occurs in many parts of the pars nasalis pharyngis, and the pathological type is mainly squamous cell carcinoma. Because of the special position of nasopharynx, breathing, pronunciation and daily life will be seriously affected. At present, the research direction of nasopharyngeal carcinoma is mainly to explore the law of tumor cell proliferation and migration, study the molecular mechanism, master its biological behavior and clinical significance, try to find therapeutic targets, and further improve the level of tumor treatment. However, the pathologic structure and molecular mechanism of nasopharyngeal carcinoma have not been fully elucidated. In this study, the Lentivirus-mediated EIF3C shRNA vector (L.V-shEIF3C) was constructed to down-regulate the expression of EIF3C in human pharyngeal squamous carcinoma cell FaDu and the human nasopharyngeal carcinoma cell 5-8F, it was found that down-regulation of EIF3C could significantly inhibit the cell proliferation, promote cell apoptosis, induce cell cycle arrest, and inhibit the formation and growth of tumors in mouse models. This study provides strong evidence that EIF3C is a key gene driving the development and progression of head and neck cancer, which is of great significance for the diagnosis, prognosis or treatment of tumors, suggesting that EIF3C may become a valuable therapeutic development and intervention target.

Angelica Sinensis Polysaccharide Suppresses the Aging of Hematopoietic Stem Cells Through Sirt1/FoxO1 Signaling.

BACKGROUND: This study investigated the anti-aging effects of Angelica sinensis polysaccharide (ASP) on mouse hematopoietic stem cells (HSCs) and related mechanisms. METHODS: Seventy-two C57BL/6J mice were randomly divided into the following three groups (n = 24 per group): control group; aging group, in which mice were irradiated with X-ray uniformly to establish the aging model of HSCs; and ASP group, in which mice were given 200 mg/kg ASP during irradiation. HSCs were collected by immunomagnetic beads, transmission electron microscopy was used to examine the morphological changes in HSCs, SA-ß-Gal staining was used to detect the aging cells, immunofluorescence staining was used to detect the reactive oxygen species (ROS), and western blotting was used to evaluate the expression levels of sirtuin 1 (Sirt1) and forkhead box O1 (FoxO1). RESULTS: HSCs in the control group had an intact cytoplasmic structure and many mitochondria. In the aging group, HSCs had many vacuoles in the cytoplasm, and few and irregular mitochondria. In the ASP group, HSCs had a normal cytoplasmic structure and more mitochondria compared with the aging group. The aging group had a significantly higher positive rate of HSCs SA-ß-Gal staining and ROS production than the control group (p < 0.05), but had lower expression of Sirt1 and FoxO1 (p < 0.05). These patho(physio)logical changes were ameliorated by ASP treatment (all p < 0.05). CONCLUSIONS: ASP inhibits irradiation-induced oxidative stress and aging of HSCs at least in part by regulating the Sirt1/FoxO1 pathway, thereby delaying aging of HSCs in mice.

In Vitro Anti-Inflammatory Activity of Three Peptides Derived from the Byproduct of Rice Processing.

Inflammation is a contributing factor to the initiation and progression of many diseases, and some food-derived biofunctional peptides show high anti-inflammatory activity. In our previous study, we demonstrated that peptides derived from trypsin hydrolysis of rice protein show good immunological activity. In the present study, proteins of broken rice were extracted and identified by macroporous resin fractionation and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Subsequently, a bioinformatics prediction and in silico simulation approach was used to screen for peptides showing anti-inflammatory activity, including inhibition of the production of nitric oxide and proinflammatory cytokines (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) by lipopolysaccharide-stimulated RAW264.7 mice macrophages. Three peptides (DNIQGITKPAIR, IAFKTNPNSMVSHIAGK, and IGVAMDYSASSKR) that demonstrated the highest binding affinity were synthesized, and their in vitro anti-inflammatory activity was investigated. This is the first study that integrates LC-MS/MS identification and bioinformatics prediction for reporting the anti-inflammatory activity of anti-inflammatory peptides derived from broken rice protein. The study findings revealed that the peptides derived from the byproduct of rice milling could be potentially used as natural anti-inflammatory alternativities.

Cancer-associated adipocytes promote the invasion and metastasis in breast cancer through LIF/CXCLs positive feedback loop.

Cancer-associated adipocytes (CAAs), which are adipocytes transformed by cancer cells, are of great importance in promoting the progression of breast cancer. However, the underlying mechanisms involved in the crosstalk between cancer cells and adipocytes are still unknown. Here we report that CAAs and breast cancer cells communicate with each other by secreting the cytokines leukemia inhibitory factor (LIF) and C-X-C subfamily chemokines (CXCLs), respectively. LIF is a pro-inflammatory cytokine secreted by CAAs, which promotes migration and invasion of breast cancer cells via the Stat3 signaling pathway. The activation of Stat3 induced the secretion of glutamic acid-leucine-arginine (ELR) motif CXCLs (CXCL1, CXCL2, CXCL3 and CXCL8) in tumor cells. Interestingly, CXCLs in turn activated the ERK1/2/NF-κB/Stat3 signaling cascade to promote the expression of LIF in CAAs. In clinical breast cancer pathology samples, the up-regulation of LIF in paracancerous adipose tissue was positively correlated with the activation of Stat3 in breast cancer. Furthermore, we verified that adipocytes enhanced lung metastasis of breast cancer cells, and the combination of EC330 (targeting LIF) and SB225002 (targeting C-X-C motility chemokine receptor 2 (CXCR2)) significantly reduced lung metastasis of breast cancer cells in vivo. Our findings reveal that the interaction of adipocytes with breast cancer cells depends on a positive feedback loop between the cytokines LIF and CXCLs, which promotes breast cancer invasion and metastasis.

Doxorubicin-Loaded Metal-Organic Framework Nanoparticles as Acid-Activatable Hydroxyl Radical Nanogenerators for Enhanced Chemo/Chemodynamic Synergistic Therapy.

Doxorubicin (DOX) is a widely used first-line antitumor agent; however, acquired drug resistance and side effects have become the main challenges to effective cancer therapy. Herein, DOX is loaded into iron-rich metal-organic framework/tannic acid (TA) nanocomplex to form a tumor-targeting and acid-activatable drug delivery system (MOF/TA-DOX, MTD). Under the acidic tumor microenvironment, MTD simultaneously releases DOX and ferrous ion (Fe2+) accompanied by degradation. Apart from the chemotherapeutic effect, DOX elevates the intracellular H2O2 levels through cascade reactions, which will be beneficial to the Fenton reaction between the Fe2+ and H2O2, to persistently produce hydroxyl radicals (•OH). Thus, MTD efficiently mediates chemodynamic therapy (CDT) and remarkably enhances the sensitivity of chemotherapy. More encouragingly, the cancer cell killing efficiency of MTD is up to ~86% even at the ultralow equivalent concentration of DOX (2.26 µg/mL), while the viability of normal cells remained >88% at the same concentration of MTD. Taken together, MTD is expected to serve as drug-delivery nanoplatforms and •OH nanogenerators for improving chemo/chemodynamic synergistic therapy and reducing the toxic side effects.

Protective effects of NAC and salubrinal on apoptosis of retinal pigment epithelial cells induced by all-trans retinoic acid.

PURPOSE: Accumulation of endogenous all-trans retinoic acid (ATRA) plays a role in the degeneration of photoreceptor cells and retinal pigment epithelium (RPE) cells, contributing to age-related macular degeneration (AMD). This study attempted to investigate the influence of antioxidant N-acetylcysteine (NAC) and selective endoplasmic reticulum stress (ERS) inhibitor salubrinal on apoptosis of ARPE-19 cells induced by ATRA. METHODS: The RPE cell line (ARPE-19) was treated with ATRA, ATRA+NAC, ATRA+salubrinal or ATRA+NAC+salubrinal and the control was untreated. After 24 h of cell culture, the levels of apoptosis, multicaspase and reactive oxygen species (ROS) were detected by flow cytometry. Western blot analysis was employed to detect the expression of vascular endothelial growth factor-A (VEGF-A), C/EBP homologous protein (CHOP) and cleaved caspase-3 in the groups. RESULTS: The results of flow cytometry showed that NAC and salubrinal decreased the levels of apoptosis, ROS and multicaspase. ATRA increased VEGF-A levels associated with neovascularisation. NAC and salubrinal inhibited an increase in VEGF-A, CHOP and caspase-3 caused by ATRA in ARPE-19 cells. CONCLUSIONS: In ARPE-19 cells, the levels of ROS and ERS can be increased by ATRA, contributing to apoptosis, which can be effectively inhibited by NAC and salubrinal. Thus, ATRA may play an important role in the prevention, diagnosis and treatment of age-related macular degeneration.

Exosomes Derived from Mouse Adipose-Derived Mesenchymal Stem Cells Alleviate Benzalkonium Chloride-Induced Mouse Dry Eye Model via Inhibiting NLRP3 Inflammasome.

PURPOSE: The objective of the study was to investigate efficacy and mechanisms of mouse adipose-derived mesenchymal stem cell-derived exosomes (mADSC-Exos) in the benzalkonium chloride (BAC)-induced mouse dry eye model. METHODS: Exosomes in the mADSC culture supernatant were isolated by ultracentrifugation. Western blotting, nanoparticle tracking analysis, and transmission electron microscopy were used to characterize mADSC-Exos. An experimental mouse model of dry eye was established by instillation of 0.2% BAC. mADSC-Exos were administered following BAC treatment. The positive control group was treated with commercial eye drops (0.1% pranoprofen). Corneal fluorescein staining, tear secretion, and tear film break-up time (BUT) were evaluated, and histologic analysis of the cornea and conjunctiva was performed by hematoxylin and eosin and periodic acid-Schiff staining. Apoptosis in the corneal epithelium was detected with the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and by Western blotting. Levels of pro-inflammatory cytokines in the cornea and conjunctiva were evaluated by flow cytometry, and mRNA and protein levels of NLR family pyrin domain-containing 3 (NLRP3) pathway components were assessed by quantitative real-time PCR and Western blotting, respectively. RESULTS: mADSC-Exos were characterized as vesicles with a bilayer membrane. The particle size distribution peak was at 134 nm. mADSC-Exos specifically expressed cluster of differentiation (CD)9, CD63, and CD81. mADSC-Exos treatment repaired ocular surface damage. Additionally, mADSC-Exos inhibited cell apoptosis, decreased the levels of interleukin (IL)-1ß, IL-6, IL-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, and increased levels of the anti-inflammatory cytokine IL-10. Meanwhile, NLRP3 inflammasome activation and upregulation of caspase-1, IL-1ß, and IL-18 were reversed by mADSC-Exos. CONCLUSIONS: mADSC-Exos alleviate ocular surface inflammation, suggesting that it is a promising treatment for dry eye.

VEGF gene polymorphisms regulate human retinal vascular endothelial cell proliferation and apoptosis through ASF/SF2-associated alternative splicing.

This study investigated the effects of single nucleotide polymorphisms (SNPs) of the VEGF （vascular endothelial growth factor） gene, which are associated with susceptibility to age-related macular degeneration (AMD), on the expression of VEGF proteins (VEGF165 and VEGF165b) and their role in cell proliferation and apoptosis in human retinal vascular endothelial cells (hRVECs). Cell viability and VEGF165 and VEGF165b expressions were evaluated in hRVECs transfected with VEGF genes containing different SNPs (rs3025039, rs3025033, and rs10434). The Cell Counting Kit 8 assay, quantitative real-time PCR, western blotting, TUNEL assay, and enzyme-linked immunosorbent assay were used to examine the effects of VEGF gene SNPs on cell viability, VEGF165 and VEGF165b expressions, and cell apoptosis in hRVECs. The interaction and localization of the RNA-binding protein alternative splicing factor/splicing factor 2 (ASF/SF2) were assessed using RNA pull-down. Although VEGF165 expression decreased, VEGF165b levels increased significantly in hRVECs transfected with rs3025039, which decreased cell viability and induced apoptosis. The SNPs rs3025033 and rs10434 had no significant effects on VEGF165b protein production and apoptosis; however, they promoted cell proliferation. SNPs affected the interaction between RNA and ASF/SF2, a splicing factor for intron retention. Insulin-like growth factor-1 treatment induced the expression of VEGF165, but not VEGF165b, whereas SRPIN340 treatment, an inhibitor of ASF/SF2, increased VEGF165b protein levels. VEGF gene sequence variations affected hRVEC proliferation and apoptosis via alternative gene splicing. Thus, the regulation of splicing via ASF/SF2 could be a potential strategy in treating pathological neovascularization in patients with AMD.

Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing.

The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT).

Genetic polymorphisms in the PCNXL2 gene are risk factors for thyroid cancer in the Chinese population.

Background: Thyroid cancer is the most common endocrine malignancy and the fastest growing cancer worldwide. Thyroid cancer has the largest genetic component of all cancers. Previous genome-wide association studies indicated that genetic polymorphism in PCNXL2 is related to thyroid cancer susceptibility in European populations. This study aims to determine the influence of PCNXL2 polymorphisms on thyroid cancer risk in Chinese individuals. Methods: This case-control study identified four polymorphisms in PCNXL2 among 510 thyroid cancer cases and 509 healthy controls. The associations of PCNXL2 polymorphisms with thyroid cancer susceptibility were detected by calculating odds ratios. Multifactor dimensionality reduction was performed to detect the impact of SNP (single nucleotide polymorphism)-SNP interactions on the risk of thyroid cancer. Results: The study showed that rs10910660 in PCNXL2 was related to thyroid cancer susceptibility. Rs12129938 played a protective role in thyroid cancer susceptibility. Stratification analysis indicated that rs10910660 increased thyroid cancer risk at age >45 years. Rs12129938 enhanced susceptibility to thyroid cancer at age >45 years, while this SNP decreased thyroid cancer risk at age ≤45 years. Rs4649295 was associated with lower susceptibility to thyroid cancer at age ≤45 years. An association was observed between rs6424270 and rs12129938 with decreased susceptibility to thyroid cancer in women. Rs10910660 was related to thyroid cancer risk in men. The combination of rs6424270, rs10910660, rs12129938 and rs4649295 was the best model to predict thyroid cancer. Conclusion: This study suggests that PCNXL2 polymorphisms are risk factors for thyroid cancer in the Chinese population.