Xiaoqing: A Q&A model for glaucoma based on LLMs.

Glaucoma is one of the leading cause of blindness worldwide. Individuals affected by glaucoma, including patients and their family members, frequently encounter a deficit in dependable support beyond the confines of clinical environments. Seeking advice via the internet can be a difficult task due to the vast amount of disorganized and unstructured material available on these sites, nevertheless. This research explores how Large Language Models (LLMs) can be leveraged to better serve medical research and benefit glaucoma patients. We introduce Xiaoqing, a Natural Language Processing (NLP) model specifically tailored for the glaucoma field, detailing its development and deployment. To evaluate its effectiveness, we conducted two forms of experiments: comparative and experiential. In the comparative analysis, we presented 22 glaucoma-related questions in simplified Chinese to three medical NLP models (Xiaoqing LLMs, HuaTuo, Ivy GPT) and two general models (ChatGPT-3.5 and ChatGPT-4), covering a range of topics from basic glaucoma knowledge to treatment, surgery, research, management standards, and patient lifestyle. Responses were assessed for informativeness and readability. The experiential experiment involved glaucoma patients and non-patients interacting with Xiaoqing, collecting and analyzing their questions and feedback on the same criteria. The findings demonstrated that Xiaoqing notably outperformed the other models in terms of informativeness and readability, suggesting that Xiaoqing is a significant advancement in the management and treatment of glaucoma in China. We also provide a Web-based version of Xiaoqing, allowing readers to directly experience its functionality. The Web-based Xiaoqing is available at https://qa.glaucoma-assistant.com//qa.

Plasma exosomal miR-30a-5p inhibits osteogenic differentiation of bone marrow mesenchymal stem cells from a chronic unpredictable mild stress-induced depression rat model.

With rising society stress, depression-induced osteoporosis is increasing. However, the mechanism involved is unclear. In this study, we explored the effect of plasma exosomal miRNAs on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation in a chronic unpredictable mild stress (CUMS)-induced depression rat model. After 12 weeks of CUMS-induced depression, the pathological changes in the bone tissue and markers of osteogenic differentiation were tested by micro-computed tomography, hematoxylin-eosin staining, and quantitative real-time reverse transcription PCR (qRT-PCR). Plasma exosomes from rats were isolated and co-incubated with BMSCs for 14 d to detect the effect on osteogenic markers. Next-generation sequencing identified the miRNAs in the plasma exosomes, and the differential miRNAs were analyzed and verified by qRT-PCR. BMSCs were infected with lentivirus to upregulate miRNA-30a-5p and incubated in a medium that induced osteogenic differentiation for 14 d. The effect of miR-30a-5p on osteogenic differentiation was determined by qPCR and alizarin red staining. CUMS-induced depression rat model was established successfully, and exhibited reduced bone mass and damaged bone microstructure compared to that of the controls. The observed pathological changes suggested the occurrence of osteoporosis in the CUMS group, and the mRNA expression of osteogenic markers was also significantly reduced. Incubation of BMSCs with plasma exosomes from the CUMS group for 14 d resulted in a significant decrease in the expression of osteogenic markers. Twenty-five differentially expressed miRNAs in plasma exosomes were identified and upregulation of miR-30a-5p was observed to significantly inhibit the expression of osteogenic markers in BMSCs. Our findings contributed to a comprehensive understanding of the mechanism of osteoporosis caused by depression, and demonstrated the potential of miR-30a-5p as a novel biomarker or therapeutic target for the treatment of osteoporosis.

Identification of clinical characteristics biomarkers for rheumatoid arthritis through targeted DNA methylation sequencing.

OBJECTIVES: Rheumatoid arthritis (RA) is a complex disease with a challenging diagnosis, especially in seronegative patients. The aim of this study is to investigate whether the methylation sites associated with the overall immune response in RA can assist in clinical diagnosis, using targeted methylation sequencing technology on peripheral venous blood samples. METHODS: The study enrolled 241 RA patients, 30 osteoarthritis patients (OA), and 30 healthy volunteers control (HC). Fifty significant cytosine guanine (CG) sites between undifferentiated arthritis and RA were selected and analyzed using targeted DNA methylation sequencing. Logistic regression models were used to establish diagnostic models for different clinical features of RA, and six machine learning methods (logit model, random forest, support vector machine, adaboost, naive bayes, and learning vector quantization) were used to construct clinical diagnostic models for different subtypes of RA. Least absolute shrinkage and selection operator regression and detrended correspondence analysis were utilized to screen for important CGs. Spearman correlation was used to calculate the correlation coefficient. RESULTS: The study identified 16 important CG sites, including tumor necrosis factort receptor associated factor 5 (TRAF5) (chr1:211500151), mothers against decapentaplegic homolog 3 (SMAD3) (chr15:67357339), tumor endothelial marker 1 (CD248) (chr11:66083766), lysosomal trafficking regulator (LYST) (chr1:235998714), PR domain zinc finger protein 16 (PRDM16) (chr1:3307069), A-kinase anchoring protein 10 (AKAP10) (chr17:19850460), G protein subunit gamma 7 (GNG7) (chr19:2546620), yes1 associated transcriptional regulator (YAP1) (chr11:101980632), PRDM16 (chr1:3163969), histone deacetylase complex subunit sin3a (SIN3A) (chr15:75747445), prenylated rab acceptor protein 2 (ARL6IP5) (chr3:69134502), mitogen-activated protein kinase kinase kinase 4 (MAP3K4) (chr6:161412392), wnt family member 7A (WNT7A) (chr3:13895991), inhibin subunit beta B (INHBB) (chr2:121107018), deoxyribonucleic acid replication helicase/nuclease 2 (DNA2) (chr10:70231628) and chromosome 14 open reading frame 180 (C14orf180) (chr14:105055171). Seven CG sites showed abnormal changes between the three groups (P < 0.05), and 16 CG sites were significantly correlated with common clinical indicators (P < 0.05). Diagnostic models constructed using different CG sites had an area under the receiver operating characteristic curve (AUC) range of 0.64-0.78 for high-level clinical indicators of high clinical value, with specificity ranging from 0.42 to 0.77 and sensitivity ranging from 0.57 to 0.88. The AUC range for low-level clinical indicators of high clinical value was 0.63-0.72, with specificity ranging from 0.48 to 0.74 and sensitivity ranging from 0.72 to 0.88. Diagnostic models constructed using different CG sites showed good overall diagnostic accuracy for the four subtypes of RA, with an accuracy range of 0.61-0.96, a balanced accuracy range of 0.46-0.94, and an AUC range of 0.46-0.94. CONCLUSIONS: This study identified potential clinical diagnostic biomarkers for RA and provided novel insights into the diagnosis and subtyping of RA. The use of targeted deoxyribonucleic acid (DNA) methylation sequencing and machine learning methods for establishing diagnostic models for different clinical features and subtypes of RA is innovative and can improve the accuracy and efficiency of RA diagnosis.

Comprehensive profiling of cell subsets of gastric cancer and liver metastasis based on single cell RNA-sequencing analysis.

Background: Liver metastasis (Li) is one of the most common distant metastatic sites for gastric cancer. A deeper understanding of its mechanism of action from a bioinformatics perspective may provide new insights. Therefore, the aim of this study was to use single cell RNA sequencing (scRNA-seq) to evaluate cell subtypes and understand the molecular mechanism of gastric cancer Li. Methods: The scRNA-seq data GSE163558 of gastric cancer and Li were downloaded from Gene Expression Omnibus (GEO). Single cell data were analyzed by various R packages such as Seurat, CellChat, gene set variation analysis (GSVA), monocle, gene set enrichment analysis (GSEA), and survival analysis and the results were plotted by ggplot2, R4.1.1. Protein expression was confirmed by immunohistochemistry in an additional patient cohort. Results: The genes APOD, CXCL5, and JUN are involved in epithelial cell metastasis. The infiltration of cytotoxic CD8 T cells was more frequent in gastric primary tumors (PTs) than in Lis. IL7R high natural killer (NK) cells that had high TXNIP and RIPOR2 expression were located at the site of Li and corresponded to a favorable prognosis. NK cells with high TNFAIP3 expression were located at the PT site and corresponded to a poor prognosis. Furthermore, the gene expression of myeloid cells was different depending on their localization in the PT site or Li. MHC-I signaling pathway was found to be activated in the PT compared to MHC-II at the site of Li, as revealed by cell-cell interaction, and HLA-E-CD94/NKG2A of NK cells was only activated in the PT and not in the Li. Conclusions: The present study revealed the difference between Li and gastric PT by scRNA-seq, demonstrating the impact of partial gene expression on patient prognosis. Our study provides new insights into gastric cancer and Li.

Specific detection of Phytophthora parasitica by recombinase polymerase amplification (RPA) assays based on a unique multi-copy genomic sequence.

Phytophthora parasitica is a highly destructive oomycete plant pathogen that is capable of infecting a wide range of hosts including many agricultural cash crops, fruit trees, and ornamental garden plants. One of the most important diseases caused by P. parasitica worldwide is black shank of tobacco. Rapid, sensitive, and specific pathogen detection is crucial for early rapid diagnosis which can facilitate effective disease management. In this study, we used a genomics approach to identify repeated sequences in the genome of P. parasitica by genome sequence alignment, and identified a 203 bp P. parasitica-specific sequence, PpM34, that is present in 31-60 copies in the genome. The P. parasitica genome-specificity of PpM34 was supported by PCR amplification of 24 genetically diverse strains of P. parasitica, 32 strains representing twelve other Phytophthora species, one Pythium specie, six fungal species and three bacterial species, all of which are plant pathogens. Our PCR and real-time PCR assays showed that the PpM34 sequence was highly sensitive in specifically detecting P. parasitica. Finally, we developed a PpM34-based high-efficiency Recombinase Polymerase Amplification (RPA) assay, which allowed us to specifically detect as little as 1 pg of P. parasitica total DNA from both pure cultures and infected Nicotiana benthamiana at 39°C using a fluorometric thermal cycler. The sensitivity, specificity, convenience and rapidity of this assay represents a major improvement for early diagnosis of P. parasitica infection.

Circulating DNA methylation level of CXCR5 correlates with inflammation in patients with rheumatoid arthritis.

OBJECTIVES: To assess the differences in circulating DNA methylation levels of CXCR5 between rheumatoid arthritis (RA) and osteoarthritis (OA) and healthy controls (HC), and the correlation of methylation changes with clinical characteristics of RA patients. METHODS: Peripheral blood samples were collected from 239 RA patients, 30 patients with OA, and 29 HC. Target region methylation sequencing to the promoter region of CXCR5 was achieved using MethylTarget. The methylation level of cg04537602 and methylation haplotype were compared among the three groups, and the correlation between methylation levels and clinical characteristics of RA patients was performed by Spearman's rank correlation analysis. RESULTS: The methylation level of cg04537602 was significantly higher in the peripheral blood of RA patients compared with OA patients (p = 1.3 × 10-3 ) and in the HC group (p = 5.5 × 10- 4 ). The sensitivity was enhanced when CXCR5 methylation level combined with rheumatoid factor and anti-cyclic citrullinated peptide with area under curve (AUC) of 0.982 (95% confidence interval 0.970-0.995). The methylation level of cg04537602 in RA was positively correlated with C-reactive protein (CRP) (r = .16, p = .01), and in RA patients aged 60 years and above, cg04537602 methylation levels were positively correlated with CRP (r = .31, p = 4.7 × 10- 4 ), tender joint count (r = .21, p = .02), visual analog scales score (r = .21, p = .02), Disease Activity Score in 28 joints (DAS28) using the CRP level DAS28-CRP (r = .27, p = 2.1 × 10- 3 ), and DAS28-ESR (r = .22, p = .01). We also observed significant differences of DNA methylation haplotypes in RA patients compared with OA patients and HC, which was consistent with single-loci-based CpG methylation measurement. CONCLUSION: The methylation level of CXCR5 was significantly higher in RA patients than in OA and HC, and correlated with the level of inflammation in RA patients, our study establishes a link between CXCR5 DNA methylation and clinical features that may help in the diagnosis and disease management of RA patients.

Identification of key genes with prognostic value in gastric cancer by bioinformatics analysis.

Background: Gastric cancer (GC) is a digestive system tumor with high morbidity and mortality. It is urgently required to identify genes to elucidate the underlying molecular mechanisms. The aim of this study is to identify the key genes which may affect the prognosis of GC patients and be a therapeutic strategy for GC patients by bioinformatic analysis. Methods: The significant prognostic differentially expressed genes (DEGs) were screened out from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) datasets. The protein-protein interaction (PPI) network was established by STRING and screening key genes by MCODE and CytoNCA plug-ins in Cytoscape. Functional enrichment analysis, construction of a prognostic risk model, and nomograms verify key genes as potential therapeutic targets. Results: In total, 997 genes and 805 genes were related to the prognosis of GC in the GSE84437 and TCGA datasets, respectively. We define the 128 genes shared by the two datasets as prognostic DEGs (P-DEGs). Then, the first four genes (MYLK, MYL9, LUM, and CAV1) with great node importance in the PPI network of P-DEGs were identified as key genes. Independent prognostic risk analysis found that patients with high key gene expression had a poor prognosis, excluding their age, gender, and TNM stage. GO and KEGG enrichment analyses showed that key genes may exert influence through the PI3K-Akt pathway, in which extracellular matrix organization and focal adhesion may play important roles in key genes influencing the prognosis of GC patients. Conclusion: We found that MYLK, MYL9, LUM, and CAV1 are potential and reliable prognostic key genes that affect the invasion and migration of gastric cancer.

Functional characterization of a gamma-glutamyl phosphate reductase ProA in proline biosynthesis and promoting expression of type three secretion system in Ralstonia solanacearum.

Ralstonia solanacearum RSc2741 has been predicted as a gamma-glutamyl phosphate reductase ProA catalyzing the second reaction of proline formation from glutamate. Here, we experimentally demonstrated that proA mutants were proline auxotrophs that failed to grow in a minimal medium, and supplementary proline, but not glutamate, fully restored the diminished growth, confirming that ProA is responsible for the biosynthesis of proline from glutamate in R. solanacearum. ProA was previously identified as one of the candidates regulating the expression of genes for type three secretion system (T3SS), one of the essential pathogenicity determinants of R. solanacearum. Supplementary proline significantly enhanced the T3SS expression both in vitro and in planta, indicating that proline is a novel inducer of the T3SS expression. Deletion of proA substantially impaired the T3SS expression both in vitro and in planta even under proline-supplemented conditions, indicating that ProA plays additional roles apart from proline biosynthesis in promoting the expression of the T3SS genes. It was further revealed that the involvement of ProA in the T3SS expression was mediated through the pathway of PrhG-HrpB. Both the proA mutants and the wild-type strain grew in the intercellular spaces of tobacco leaves, while their ability to invade and colonize tobacco xylem vessels was substantially impaired, which was about a 1-day delay for proA mutants to successfully invade xylem vessels and was about one order of magnitude less than the wild-type strain to proliferate to the maximum densities in xylem vessels. It thus resulted in substantially impaired virulence of proA mutants toward host tobacco plants. The impaired abilities of proA mutants to invade and colonize xylem vessels were not due to possible proline insufficiency in the rhizosphere soil or inside the plants. All taken together, these results extend novel insights into the understanding of the biological function of ProA and sophisticated regulation of the T3SS and pathogenicity in R. solanacearum.

Identification of an Immune-Related Prognostic Risk Model in Glioblastoma.

Background: Glioblastoma (GBM) is the most common and malignant type of brain tumor. A large number of studies have shown that the immunotherapy of tumors is effective, but the immunotherapy effect of GBM is not poor. Thus, further research on the immune-related hub genes of GBM is extremely important. Methods: The GBM highly correlated gene clusters were screened out by differential expression, mutation analysis, and weighted gene co-expression network analysis (WGCNA). Least absolute shrinkage and selection operator (LASSO) and proportional hazards model (COX) regressions were implemented to construct prognostic risk models. Survival, receiver operating characteristic (ROC) curve, and compound difference analyses of tumor mutation burden were used to further verify the prognostic risk model. Then, we predicted GBM patient responses to immunotherapy using the ESTIMATE algorithm, GSEA, and Tumor Immune Dysfunction and Exclusion (TIDE) algorithm. Results: A total of 834 immune-related differentially expressed genes (DEGs) were identified. The five hub genes (STAT3, SEMA4F, GREM2, MDK, and SREBF1) were identified as the prognostic risk model (PRM) screened out by WGCNA and LASSO analysis of DEGs. In addition, the PRM has a significant positive correlation with immune cell infiltration of the tumor microenvironment (TME) and expression of critical immune checkpoints, indicating that the poor prognosis of patients is due to TIDE. Conclusion: We constructed the PRM composed of five hub genes, which provided a new strategy for developing tumor immunotherapy.

Semi-syntheses and interrogation of indole-substituted Aspidosperma terpenoid alkaloids.

We demonstrated here a series of Aspidosperma terpenoid alkaloids can be quickly prepared using semisynthesis from naturally sourced tabersonine, featuring multiple oxygen-based substituents on the indole ring such as hydroxy and methoxy groups. This panel of complex compounds enabled the exploration of indole modifications to optimize the indole alkaloids' anticancer activity, generating lead compounds (e.g., with C15-hydroxy, C16-methoxy, and/or C17-methoxy derivatizations) that potently inhibit cancer cell line growth in the single-digit micromolar range. These results can help guide the development of Aspidosperma terpenoid alkaloid therapeutics. Furthermore, this synthetic approach features late-stage facile derivatization on complex natural product molecules, providing a versatile path to indole derivatization of this family of alkaloids with diverse chemical functionalities for future medicinal chemistry and chemical biology discoveries.

Curcumin relieved the rheumatoid arthritis progression via modulating the linc00052/miR-126-5p/PIAS2 axis.

Curcumin, with its antioxidant, anti-inflammatory, and antitumor properties, is widely used in the treatment of bone disorders, including rheumatoid arthritis (RA). We investigated the effects of curcumin on fibroblast-like synoviocytes in RA and its underlying mechanism. mRNA and microRNA (miRNA) expression levels were determined using reverse transcription-quantitative polymerase chain reaction. Cellular functions were detected using cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Transwell, and flow cytometric assays. Enzyme-linked immunosorbent assay was performed to measure the cytokine release. Western blotting was used to determine the protein expression levels. An in vivo assay was performed to verify the role of linc00052 in RA. Curcumin promoted apoptosis and inhibited the growth, migration, and invasion of RA fibroblast-like synovial (RAFLS) cells. Curcumin treatment suppressed the inflammatory response of RAFLS cells. Moreover, curcumin increased linc00052 levels, and linc00052 knockdown reversed the effects of curcumin. Additionally, linc00052 functioned as a competing endogenous RNA to upregulate the expression of the protein inhibitor of activated STAT 2 (PIAS2) by sponging miR-126-5p. Curcumin inhibited the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. In vivo assays showed that curcumin decreased the arthritis score and improved inflammatory infiltration and synovial cell proliferation. These results reveal that curcumin protects against RA by regulating the inc00052/miR-126-5p/PIAS2 axis through JAK2/STAT3 signaling pathway.

Identification of Synovial Fibroblast-Associated Neuropeptide Genes and m6A Factors in Rheumatoid Arthritis Using Single-Cell Analysis and Machine Learning.

OBJECTIVES: Synovial fibroblasts (SFs) play an important role in the development and progression of rheumatoid arthritis (RA). However, the pathogenic mechanism of SFs remains unclear. The objective of this study was to investigate how neuropeptides and N6-methyladenosine (m6A) played an important role in the underlying pathogenic processes of SFs that contribute to the development of RA. METHODS: Single-cell RNA sequencing data were examined using single-cell analysis and machine learning. SF subgroups were identified based on the clustering and annotation results of the single-cell analysis. Moreover, cell-cell communication was used to analyse neuropeptide-related receptor and ligand pairs on the surface of SF cell membranes. Machine learning was used to explore the m6A factors acting on these neuropeptide genes. RESULTS: NPR3, GHR, BDKRB2, and CALCRL, four neuropeptide genes, were shown to be differently expressed among SF subgroups. Further investigation of receptor-ligand interactions found that NPR3 (in conjunction with NPPC, OSTN, NPPB, and NPPA) and GHR (in conjunction with GH1 and GH2) may have a role in SF interactions. As predicted by machine learning, IGFBP2 and METTL3 were identified as key factors regulating m6A of NPR3 and GHR. The expression levels and enrichment pathways of METTL3 and IGFBP2 were different among SF subgroups. CONCLUSIONS: Single-cell analysis and machine learning efficiently identified neuropeptide genes and m6A factors that perform important regulatory functions in RA. Our strategy may provide a basis for future studies to identify pathogenic cell subpopulations and molecular mechanisms in RA and other diseases.

Unprotected peptide macrocyclization and stapling via a fluorine-thiol displacement reaction.

We report the discovery of a facile peptide macrocyclization and stapling strategy based on a fluorine thiol displacement reaction (FTDR), which renders a class of peptide analogues with enhanced stability, affinity, cellular uptake, and inhibition of cancer cells. This approach enabled selective modification of the orthogonal fluoroacetamide side chains in unprotected peptides in the presence of intrinsic cysteines. The identified benzenedimethanethiol linker greatly promoted the alpha helicity of a variety of peptide substrates, as corroborated by molecular dynamics simulations. The cellular uptake of benzenedimethanethiol stapled peptides appeared to be universally enhanced compared to the classic ring-closing metathesis (RCM) stapled peptides. Pilot mechanism studies suggested that the uptake of FTDR-stapled peptides may involve multiple endocytosis pathways in a distinct pattern in comparison to peptides stapled by RCM. Consistent with the improved cell permeability, the FTDR-stapled lead Axin and p53 peptide analogues demonstrated enhanced inhibition of cancer cells over the RCM-stapled analogues and the unstapled peptides.

Arbutin attenuates ethanol-induced acute hepatic injury by the modulation of oxidative stress and Nrf-2/HO-1 signaling pathway.

Alcoholic liver disease (ALD) is a pervasive ailment due to the excessive consumption of alcohol and there is no operative drug for its treatment. The current exploration was intended to examine the hepatoprotective efficacy of arbutin against ethanol-provoked liver injury in rats via the modulation of the Nrf-2/HO-1 signaling cascade. Wistar rats were challenged with the 3 g/kg/day (40% v/v) of ethanol for 4 weeks to provoke the ALD and concomitantly supplemented with 40 mg/kg of arbutin. The liver function markers enzymes, inflammatory cytokines, and oxidative stress markers levels were scrutinized by using the respective assay kits. The mRNA expression of Nrf-2/HO-1 signaling proteins was studied by reverse-transcription polymerase chain reaction. The histological alterations of liver tissues were examined. HepG2 cells were used for the in vitro studies. The levels of oxidative stress markers and liver marker enzymes were examined by using kits. Reactive oxygen species (ROS) and apoptotic cell death was detected by using fluorescent staining. There were no major differences in the body weight and liver weight of experimental animals. Arbutin treatment appreciably reduced the liver marker enzymes, upregulated superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and the hydroxyl scavenging ability, and diminished the tumor necrosis factor-α and interleukin-6 levels in the serum of ethanol provoked animals. Arbutin triggered Nrf-2/HO-1 signaling cascade liver tissues of ethanol-provoked animals. Histological findings proved the preventing effects of arbutin. Arbutin did not demonstrate toxicity to the HepG2 cells. It reduced the aspartate aminotransferase and alanine aminotransferase, ROS, apoptotic cell death, lipid peroxidation and improved the antioxidants' levels in the ethanol-challenged HepG2 cells. In conclusion, our findings unveiled the hepatoprotective efficacy of arbutin against ethanol-provoked liver injury in rats. It could be a promising agent to treat alcoholic liver disease in the future.

Therapeutic Effects of (5R)-5-Hydroxytriptolide on Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via lncRNA WAKMAR2/miR-4478/E2F1/p53 Axis.

Rheumatoid arthritis (RA) is an autoimmune disease. Fibroblast-like synoviocytes (FLS) serve a major role in synovial hyperplasia and inflammation in RA. (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide derivative, shows promising therapeutic effects for RA and is now in phase II clinical trials in China. However, the underlying mechanism of LLDT-8 is still not fully understood. Here, we found that LLDT-8 inhibited proliferation and invasion of RA FLS, as well as the production of cytokines. Microarray data demonstrated that LLDT-8 upregulated the expression of long non-coding RNA (lncRNA) WAKMAR2, which was negatively associated with proliferation and invasion of RA FLS, as well as the production of pro-inflammatory cytokines. Knockdown of WAKMAR2 abolished the inhibitory effects of LLDT-8 on RA FLS. Mechanistically, WAKMAR2 sponged miR-4478, which targeted E2F1 and downstreamed p53 signaling. Rescue experiments indicated that the inhibitory effects of LLDT-8 on RA FLS were dependent on WAKMAR2/miR-4478/E2F1/p53 axis.

LncRNA NEAT1 regulates the proliferation and production of the inflammatory cytokines in rheumatoid arthritis fibroblast-like synoviocytes by targeting miR-204-5p.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, featured by erosive arthritis, which will eventually lead to deprivation normal functions of the joint and joint malformations. Continued illness also results in more serious complications, such as cardiovascular diseases and disability. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) function in various conditions, including RA. LncRNA NEAT1 was reported to promote migration and invasion in RA-FLSs, functioning as a promising diagnostic and therapeutic indicator in RA. The present work focused on the role of lncRNA NEAT1 in RA and the related mechanism. We collected the synovial tissue samples of 30 RA patients and 20 healthy controls. Moreover, RA fibroblast-like synoviocytes (RA-FLSs) cell line was bought and treated with tumor necrosis factor-α (TNF-α) to establish in vitro model of RA. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression of NEAT1 in synovial tissue and RA-FLSs. NEAT1 silencing plasmid were synthesized and co-trasnfected with miR-204-5p inhibitor into RA-FLSs. MTT and 5-Ethynyl-2'-deoxyuridine staining were used to assess cell proliferation. Flow cytometry and TUNEL assay were used to determine the cell apoptosis. miR-204-5p has been predicted as a target miRNA of NEAT1, and the interaction between NEAT1 and miR-204-5p was verified by dual-luciferase assay and RNA pull-down assay. qRT-PCR and enzyme-linked immunosorbent assay were used to determine the mRNA and protein concentration of interleukin-1ß and interleukin-6. Finally, western blot assay was applied to measure the effect of NEAT1 and on p53, NF-κB, and p-NF-κB expressions. We found that NEAT1 was up-regulated, and miR-204-5p was down-regulated in the RA patients' synovial tissue and TNF-α treated RA-FLSs. TNF-α increased NEAT1 level and decreased miR-204-5p level in RA-FLSs. There was no significant variance of p53 after transfected with NEAT1 in RA-FLSs. Meanwhile, Knockdown of NEAT1 attenuated TNF-α-induced RA-FLSs cell proliferation and inflammatory cytokine production while promoted cell apoptosis by targeting miR-204-5p through NF-κB pathway. These findings indicated that NEAT1 may be developed as a potential target for patients with RA.

Site-specific antibody fragment conjugates for targeted imaging.

This work details the use of amber suppression-mediated genetic incorporation of unnatural amino acids (UAAs), specifically p-azido-l-phenylalanine (pAzF) and p-acetyl-l-phenylalanine (pAcF), to develop site-specifically labeled antibody Fab fragments. These antibody fragment conjugates represent a novel class of imaging agents with optimal stability, efficacy, and pharmacological properties, which have demonstrated promising potential for probing and understanding the in vivo bio-distributions of protein targets of interest. This chapter provides general guidelines for preparing these Fab conjugates, and details of follow-up bioassays such as single-agent based positron emission tomography (PET) imaging of immune-checkpoint protein PD-L1, and the use of GCN4-mediated switchable antibody conjugates for near-infrared fluorescent imaging of cancer-related biomarkers.

Comparison of Hospital Anxiety and Depression Scale (HADS) and Zung Self-Rating Anxiety/Depression Scale (SAS/SDS) in Evaluating Anxiety and Depression in Patients with Psoriatic Arthritis.

BACKGROUND: This study aimed to compare the Hospital Anxiety and Depression Scale (HADS) and the Zung Self-Rating Anxiety/Depression Scale (SAS/SDS) in evaluating anxiety and depression in psoriatic arthritis (PsA) patients. METHODS: A total of 70 PsA patients were enrolled. Demographic and clinical characteristics were collected after enrollment. HADS-A and SAS were used to evaluate the anxiety of PsA patients, while HADS-D and SDS were used to evaluate the depression of PsA patients. RESULTS: Similar results were observed in detecting the rate of anxiety by HADS-A and SAS (27.1 vs. 21.4%, p = 0.424), and there was no difference in classifying the severity of anxiety by HADS-A and SAS (p = 0.347). The Spearman test also disclosed that HADS-A score was positively associated with SAS score (p <0.001). The rates of depression were similar by HADS-D and SDS (27.1 vs. 40.0%; p = 0.108). However, different results were observed in grading the severity of anxiety by HADS-D and SDS (p = 0.009), and no correlation was observed between HADS-D and SDS scores (p = 0.138). The consumption of time for HADS assessment was shorter than that for SAS/SDS assessment (p < 0.001). In addition, a positive correlation of HADS-A score with patients' global assessment (PGA) (p = 0.022) and fatigue scores (p = 0.028) was discovered, and HADS-D score was positively associated with PGA score (p = 0.019). SAS or SDS score presented less correlation with clinical features of PsA patients, which illuminated that only SAS score was positively associated with duration of psoriasis (p = 0.030). CONCLUSION: HADS seems to be a better option for anxiety and depression assessment than SAS/SDS in PsA patients.

A tumor-targeted immune checkpoint blocker.

To direct checkpoint inhibition to the tumor microenvironment, while avoiding systemic immune activation, we have synthesized a bispecific antibody [norleucine4, d-Phe7]-melanocyte stimulating hormone (NDP-MSH)-antiprogrammed cell death-ligand 1 antibody (αPD-L1) by conjugating a melanocyte stimulating hormone (α-MSH) analog to the antiprogrammed cell death-ligand 1 to (αPD-L1) antibody avelumab. This bispecific antibody can bind to both the melanocortin-1 receptor (MC1R) and to PD-L1 expressed on melanoma cells and shows enhanced specific antitumor efficacy in a syngeneic B16-SIY melanoma mouse model compared with the parental antibody at a 5 mg/kg dose. Moreover, the bispecific antibody showed increased infiltrated T cells in the tumor microenvironment. These results suggest that a tumor-targeted PD-L1-blocking bispecific antibody could have a therapeutic advantage in vivo, especially when used in combination with other checkpoint inhibitors.

Site-Specific Immuno-PET Tracer to Image PD-L1.

The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane- N, N', N″-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, 64Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.