BACKGROUND:Autophagy may be involved in the pathological process of steroid-induced necrosis of the femoral head(SINFH).Some studies have confirmed that circular RNAs(circRNAs)have a regulatory mechanism in SINFH;however,whether circCDR1as affects autophagy in SINFH has not been investigated. OBJECTIVE:To explore the level of autophagy and the regulatory mechanism of circCDR1as in SINFH. METHODS:Gene expression profiles of SINFH and control rats were extracted from the GSE26316 dataset and differential expression analysis was performed.Subsequently,the biological functions of differentially expressed genes were analyzed.Then,the target miRNAs of circCDR1as and the target genes of target miRNAs were predicted.Further,the target genes were compared with the differentially expressed genes to construct the regulatory network of circCDR1as.In addition,femoral head samples from patients with SINFH and healthy control individuals were collected.Bone marrow mesenchymal stem cells were also applied for cellular experiments and randomly divided into bone marrow mesenchymal stem cell group,model group(methylprednisolone-treated),model+si-NC group,and model+si-CDR1as group.RT-qPCR was used to detect the expression of circCDR1as and target genes in cells and tissue samples.Western blot was used to examine the expression of autophagy proteins.The luciferase reporter gene vectors,pmirGLO-CDR1as(WT),pmirGLO-RAF1(WT),pmirGLO-CDR1as(MUT),and pmirGLO-RAF1(MUT),were constructed and transfected into the cells.miR-7-5p mimic and mimic NC groups were established.The target-regulatory relationship of the circCDR1as network was detected. RESULTS AND CONCLUSION:A total of 1 283 differentially expressed genes were identified between the SINFH and control groups,which were mainly involved in apoptotic and autophagic signaling pathways.Prediction analysis revealed that circCDR1as targeted 6 miRNAs,which in turn regulated 305 target genes.Among these target genes,31 showed differential expression in SINFH.Among the differentially expressed target genes,RAF1,involved in autophagy,was selected as a key gene,leading to the construction of the circCDR1as/miR-7-5p/RAF1 regulatory network.Compared with the control group,circCDR1as,RAF1,and autophagy levels were upregulated in patients with SINFH and in hormone-induced bone marrow mesenchymal stem cells(P<0.05),while miR-7-5p expression was downregulated(P<0.05).Knockdown of circCDR1as significantly decreased cellular autophagy levels(P<0.05).Dual-luciferase reporter assays confirmed the targeting relationships between circCDR1as and miR-7-5p,as well as between miR-7-5p and RAF1.To conclude,the CircCDR1as/miR-7-5p/RAF1 may potentially promote SINFH through autophagy.Targeting circCDR1as is a potential strategy for partial autophagic repair in the treatment of SINFH.

This paper summarized Professor PENG Peichu's experience in the differentiation and treatment of prostate cancer in three phases and four stages. It is considered that prostatic cancer is categorized into root deficiency and branch excess， with depletion of healthy qi as the root， and the accumulation of cancer toxin as the minifestation. Clinical diagnosis and treatment of prostatic cancer can be divided into three phases and four stages according to the exuberance and decline of pathogenic and healthy qi and the changes of deficiency and excess of yin and yang. In the initial accumulation phase of cancer toxin （yang excess stage）， the key pathogenesis is the accumulation of dampness， heat and static blood， and internal generation of cancer toxin， and the treatment should be resolving toxins， fighting cancer and dispelling yang excess. In the phase of healthy qi deficiency and toxin accumulation （yin deficiency stage）， with the lung and kidney yin deficiency， dampness， heat and static toxin accumulation as the key pathogenesis， the treatment should be centered on mutual generation between metal and water to nourish kidney yin， supplemented with the method of clearing heat and draining dampness， activating blood and resolving toxins， for which self-made Nanbei Formula（南北方）is usually used. In the phase of yang deficiency and cold stagnation （yang deficiency stage and yin excess stage）， with the spleen and kidney yang deficiency， cold dampness stagnation， static heat and toxin accumulation as the key pathogenesis， the treatment should be warming and tonifying spleen and kidney to dissipate cold accumulation； for deficiency of both yin and yang， and excess pathogen obstruction， modified Yanghe Decoction（阳和汤） is recommended， while for yang deficiency， cold congealing and blood stasis， self-made Wenshen Sanjie Formula（温肾散结方） can be used， and for cold dampness binding with cancer toxin， and cold complex with heat， self-made Quanan Formula （泉安方） is advised.

mRNA vaccines and drugs enter host cells through delivery vectors and produce target proteins using the protein synthesis mechanism of cells. mRNA and target proteins can induce the body to produce innate immunity and adaptive immunity, and the target protein itself can also play a corresponding role. Tumor cells are inhibited and cleared under the above immune effects and target proteins. This article reviews the immunogenicity of mRNA, that is, the specific mechanism of stimulating the body to produce an immune response.At the same time, the main types of cells transfected by mRNA vaccine were briefly introduced. (1) Muscle cells, epidermal cells, dendritic cells and macrophages at the injection site; (2) immune cells in peripheral lymphoid organs;(3) liver cells and spleen cells, etc. Although transfected with a variety of cells, it is mainly enriched in immune cells and liver cells because immune cells express toll-like receptors and liver cells express low-density lipoprotein receptors. mRNA vaccines and drugs are mainly divided into non-replicating mRNA (nrmRNA),self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA) and circular RNA (circRNA).This article reviews how these 4 types of vaccines and drugs work, and compares their advantages and disadvantages. Due to its inherent immunogenicity, instability, and low delivery efficiency in vivo, mRNA vaccines and drugs have been unable to enter the clinic. This article describes in detail how to reformation and modify the 5'cap, 5'UTR, 3'UTR, ORF, 3'Poly(A) and some nucleotides of mRNA to eliminate its immunogenicity and instability. Due to the low efficiency of the delivery carrier, the researchers optimized it. This article briefly introduces the application of non-viral vectors and their targeting, specifically involving the mechanism of action of various types of delivery vectors and their advantages and disadvantages, and summarizes some of the current targeting vectors. Targeted carriers can improve the delivery efficiency of mRNA to specific tissues and prevent side effects of systemic exposure, such as liver injury. The specific methods of using mRNA vaccines and drugs to treat cancer are as follows: mRNA can be used to encode and transcribe tumor-associated antigens, tumor-specific antigens (TSAs), therapeutic antibodies, cytokines, tumor suppressors, oncolytic viruses, CRISPR-Cas9, CARs and TCRs, so as to play an anti-tumor role. In this paper, the specific mechanism of the above methods and the current research and development of corresponding mRNA vaccines and drugs are briefly reviewed. The successful development of the COVID-19 mRNA vaccine has brought mRNA technology to the attention of the world and brought new and effective means for the prevention and treatment of cancer. mRNA vaccines and drugs have the advantages of short development cycle, dual immune mechanism, safety, high efficiency and large-scale production. At the same time, there are also many areas that need further improvement, such as the development of ideal target TSAs, the in-depth development of saRNA, taRNA and circRNA, the development of targeted nano-delivery for different tissues and organs, the expansion of mRNA administration routes, and the development of mRNA that can be stably stored at room temperature or even high temperature. These problems need to be further studied and solved. In addition to cancer therapy, mRNA vaccines and drugs can also be used in the treatment of infectious diseases, genetic diseases, regenerative medicine and anti-aging. mRNA vaccines and drugs are a very promising platform, and we believe that they will benefit cancer patients in the near future.

The incidence of cancer is closely correlated with age,as 75％of non-small cell lung cancer(NSCLC)patients are aged at least 65 years.The availability of immune checkpoint inhibitors(ICIs)has altered the available NSCLC therapeutic pattern.Limited studies on elderly patients have demonstrated that ICIs as monotherapy provide substantial ben-efits for patients aged 65-75 years,showing no significant difference compared to younger patients.This benefit is also observed in combination with immune-combined chemotherapy or radiotherapy.For individuals older than 75 years,the survival effect was not evident,though.Immune-related adverse events(irAEs)with ICIs alone were similar in incidence across age catego-ries.Immune-combination chemotherapy resulted in a higher incidence of irAEs than chemotherapy alone,and patients ≥75 years of age were more likely to experience higher-grade irAEs.Besides the fact that immunosenescence in older patients influ-ences the immune milieu in a multifaceted manner,which in turn impacts the effectiveness of immunotherapy,the prognosis is also influenced by the Eastern Cooperative Oncology Group performance status(ECOG PS)score,among other factors.For certain individuals aged ≥75 years or in poor physical health,immunotherapy combined with low-intensity chemotherapy has emerged as a viable treatment option.However,there are fewer related studies,so there should be a conscious effort to increase the number of elderly patients enrolled in the trial and a comprehensive assessment to explore individualized treatment op-tions.To provide additional references and guidance for immunotherapy in elderly NSCLC patients and to propose new thera-peutic perspectives in combination with their characteristics,this review aims to summarize and analyze the pertinent studies on the application of programmed cell death protein 1(PD-1)/programmed cell death ligand 1(PD-L1)inhibitors in these patients.

Background and objective The malignant tumor that has the highest global morbidity and death rate is lung cancer,which primarily affects the elderly.The therapy landscape for non-small cell lung cancer(NSCLC)has trans-formed with the introduction of immune checkpoint inhibitors(ICIs).The purpose of this study was to compare the safety and efficacy of immune monotherapy and immunotheray combined with chemotherapy in patients with advanced NSCLC aged 75 years and above.Methods This study retrospectively analyzed 111 patients with advanced NSCLC who were at least 75 years old and received treatment at the First or Fifth Medical Centers of the People's Liberation Army General Hospital from January 2018 to October 2022.These patients underwent first-line or second-line treatment,with 70 receiving immunotherapy combined with chemotherapy and 41 receiving immunotherapy alone.Propensity score matching(PSM)was used to match the baseline characteristics of the patients,including age,Eastern Cooperative Oncology Group performance status(ECOG PS)score,and the number of treatment lines.The study endpoints included objective response rate(ORR),progression-free survival(PFS),overall survival(OS),and safety assessment.Results The median OS for the immunotherapy combined with chemother-apy group was 27.87 months,and the median PFS was 11.50 months.The median OS for the immune monotherapy group was 34.93 months,and the median PFS was 17.00 months.There were no significant differences in OS(P=0.722)and PFS(P=0.474)between the two groups,but a significant difference was observed in ORR(P=0.025).After PSM matching,each group comprised 27 patients.The median OS for the immunotherapy combined with chemotherapy group was 17.70 months,the median PFS was 8.97 months.The median OS for the immune monotherapy group was 17.87 months,and the median PFS was 11.53 months.No significant differences were observed in OS(P=0.635),PFS(P=0.878)and ORR(P=0.097).In terms of safety,the overall inci-dence of adverse events(AEs)before matching was 62.86％ in the immunotherapy combined with chemotherapy group,which was higher than 41.46％ in the immune monotherapy group(P=0.029),while there was no difference in the incidence of AEs of grade 3 or above between the two groups(P=0.221).After matching,AEs occurred in 17(62.96％ )patients in the immunotherapy combined with chemotherapy group and 13(48.15％ )in the immune monotherapy group.There were no significant differences in the overall incidence of AEs(P=0.273)or the incidence of grade 3 or above(P=0.299)between the two groups.Conclusion Im-munotherapy combined with chemotherapy does not significantly improve OS or PFS in patients with NSCLC aged 75 years and above when compared to immunotherapy alone,and this conclusion was further validated by the analysis after PSM.The safety assessment suggests that before matching,the incidence of AEs of any grade in the immunotherapy combined with chemotherapy group was higher.Still,the two groups had no difference in the incidence of AEs of grade 3 or above.Following matching,the tol-erability of the treatment was similar in both groups.According to the safety assessment,the unique circumstances and course of treatment for geriatric patients with advanced NSCLC should be considered.

The CRISPR/Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). The system forms an adaptive immune system in archaea and bacteria. The inherent defense mechanism enables these microorganisms to protect themselves against the invasion of foreign genetic material. The system functions of immune response including three main stages: adaptation, expression/maturation, and interference, each stage needs specific Cas proteins encoded by Cas gene located near the CRISPR sequences, along with other auxiliary proteins. In 2015, Zhang et al. reportedCas12a (Cpf1) as a member of the Class II type V CRISPR/Cas12a system, which possesses endonuclease activity. This finding holds great promise for its application in the field of biotechnology. In 2018, Doudna’s team first applied the CRISPR/Cas12a system for detecting HPV nucleic acid. The system comprises the following essential components in vitro detection: Cas12a, the crRNA sequence complementary to the target DNA, the PAM sequence, and the ssDNA reporter. Cas12a possesses a typical RuvC domain, displaying a canonical bilobed architecture that consists of a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe contains the REC1 and REC2 domains, and the NUC lobe includes RuvC, PAM-interacting (PI), Wedge (WED), and bridge helix (BH) domains. The mature crRNA for Cas12a has a length of 42-44 nt, consists of repeat sequence (19/20 nt) and spacer sequence (23-25 nt). The crRNA spacer sequence has been found to require a length of 18 nt to achieve complete cleavage activity in vitro. Additionally, mutation in the bases of crRNA can indeed affect the activity of Cas12a. The PAM sequence plays a critical role in the recognition and degradation of DNA by the CRISPR/Cas system, enabling the system to distinguish between self and non-self genomic materials. Cas12a can effectively target the spacer sequence downstream of a T-rich PAM sequence at the 5' end. LbCas12a and AsCas12a both recognize the PAM sequences of 5'-TTTN-3', while FnCas12a recognizes the PAM sequences of 5'-TTN-3'. All of these PAM sequences are located upstream on the non-template strand (NTS) at the 5' end. Cas12a (Cpf1), guided by the crRNA, binds to the target DNA by recognizing the PAM sequence. It exhibits the ability to induce arbitrary cleavage of ssDNA within the system while cleaving the target ssDNA or dsDNA. According to this feature, an array of nucleic acid detection methods has been developed for tumor detection and infection diagnostics, such as the DETECTR (RPA-CRISPR/Cas12a method) and HOLMES (PCR-CRISPR/Cas12a method) in 2018. Then, in 2019, Cas12aVDet (one-step detection method), where Cas12a protein was immobilized on the upper wall of the reaction tube. This not only prevented contamination from opening the tube but also reduced the detection reaction time. In 2021, the dWS-CRISPR (digital warm-start CRISPR) was developed as a one-pot detection method. It serves as an accurate approach for quantitatively detectingSARS-CoV-2 in clinical specimens. With the innovation of scientific technology, the high-sensitivity signal transduction technology has also been integrated with the CRISPR/Cas12a system, enabling direct detection of nucleic acids, and eliminating the need for nucleic acid amplification steps. Here, we elaborated the detection principles of CRISPR/Cas12a in in vitro detection. We discussed the different stages leading to the catalytic pathway of target DNA, and the practical applications of Cas12a in nucleic acid detection. These findings revealed a target interference mechanism that originates from the binding of Cas12a-guided RNA complex to complementary DNA sequences within PAM-dependent (dsDNA) regions. The crRNA-DNA binding activates Cas12a, enabling site-specific dsDNA cleavage and non-specific ssDNA trans-cleavage. The release of Cas12a ssDNase activity provides a novel approach to enhance the sensitivity and specificity of molecular diagnostic applications. Before these CRISPR/Cas12a-based nucleic acid detection methods can be introduced into clinical use, substantial work is still required to ensure the accuracy of diagnosis. Nevertheless, we believe that these innovative detection tools based on CRISPR/Cas will revolutionize future diagnostic technologies, particularly offering significant assistance in pathogen infection diagnosis for developing countries with relatively poor healthcare conditions and high prevalence of infectious diseases.

Objective To study the mechanism of the amelioration of renal injury in immunoglobulin A nephropathy(IgAN)rats by multi-glycosides of Tripterygium wilfordii(GTW)based on the sphingosine kinase 1(Sphk1)/sphingosine 1-phosphate receptor 2(S1PR2)signalling pathway.Methods An IgAN rat model was established by means of bovine serum albumin gavage+castor oil and carbon tetrachloride subcutaneous injection+lipopolysaccharide tail vein injection.The rats were randomly divided into the model,control and experimental groups,with 9 rats in each group,and 10 normal rats were taken as the blank group.In the control group,6.25 mg·kg-1·d-1 prednisone was given by gavage;in the experimental group,9.375 mg·kg-1·d-1GTW was given by gavage;and in the blank and model groups,0.5 mL·100 g-1·d-1 0.9％NaCl was given by gavage,and the drugs were administered to the rats once a day in each group.At the end of the 15th week,urine samples were collected and blood albumin(ALB),blood urea nitrogen(BUN),24 hour-urine protein quantification(24 h-UTP),and urine erythrocyte counts were determined in each group,and the expression levels of Sphk1/S1PR2 proteins in each group were detected by Western blotting.Results The renal pathological changes in the control and experimental groups were significantly reduced compared with those in the model group by hematoxylin-eosin staining and immunofluorescence.The levels of ALB in the blank,model,control and experimental groups were(32.49±2.23),(22.98±0.51),(26.01±1.33)and(26.53±1.92)g·L-1;the levels of BUN were(6.11±1.71),(13.75±2.96),(6.71±1.35)and(4.77±0.99)mmol·L-1;the levels of 24 h-UTP were(5.72±1.96),(9.12±2.15),(5.78±2.05)and(4.75±1.50)mg·24 h-1;the urine erythrocyte counts were(9.73±2.40),(14.62±2.60),(9.90±1.59)and(9.46±2.94)cell·μL-1;the relative expression levels of Sphk1 protein were 0.85±0.02,1.47±0.02,1.06±0.02 and 1.09±0.02;the relative expression levels of S1PR2 protein were 0.27±0.02,0.88±0.01,0.43±0.02,and 0.42±0.02,respectively.The above indexes in the model group were statistically significant when compared with those of the control group and the experimental group(all P＜0.01).Conclusion GTW may reduce the proliferation of mesangial cells by inhibiting the Sphk1/S1PR2 signalling pathway,thus attenuating kidney injury in IgAN rats.

AIM:To explore the correlation and consistency of three tear assessment methods in the diagnosis of dry eye, which include the Schirmer Ⅰ test(SⅠt), tear meniscus height(TMH)measurement and a new generation of tear detection technology-Strip meniscometry tube(SMTube).METHODS: A diagnostic test study. A total of 183 dry eye outpatients(183 right eyes)in the Dry Eye Center of Henan Eye Hospital were enrolled from May to June 2021. The SⅠt, TMH and SMTube were performed on all patients, and the correlation and consistency of the measurement results were analyzed.RESULTS:The ocular surface disease index(OSDI)of all included patients was 43.75(31.25, 58.33), noninvasive breakup time(NIBUT)was 7.26(4.97, 9.37)s, and the results of SⅠt, TMH and SMTube were 6(2, 12)mm/5min, 0.18(0.14, 0.22)mm and 5(3,8)mm/5s, respectively; The results of correlation analysis: TMH was positively correlated with SMTube(rs=0.751, P<0.001), however, SⅠt had no correlation with TMH(rs=0.139, P=0.060)and SMTube(rs=0.019, P=0.799). The results of consistency analysis: TMH showed good consistency with SMTube(Kappa=0.794, P=0.044), however, SⅠt showed poor consistency with TMH(Kappa=0.271, P=0.074)and SMTube(Kappa=0.193, P=0.070)respectively.CONCLUSION:SMTube is a new, simple and convenient tool for evaluating tear volume. It has a good correlation and consistency with TMH measurement results. Therefore, it can replace TMH measurement, but cannot replace SⅠt in clinical application.

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.
Rats ; Male ; Animals ; Diabetic Nephropathies/genetics* ; Interleukin-18/metabolism* ; Glycosides/pharmacology* ; Tripterygium ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Rats, Sprague-Dawley ; Caspase 1/metabolism* ; Pyroptosis ; Uridine Triphosphate/pharmacology* ; Kidney ; Valsartan/pharmacology* ; RNA, Messenger/metabolism* ; Diabetes Mellitus

OBJECTIVE: To investigate the mutational spectrum in young patients with diffuse large B-cell lymphoma (DLBCL) based on next generation sequencing (NGS), and to provide a basis for in-depth understanding of the molecular biological characteristics and accurate prognosis of young DLBCL. METHODS: From March 2009 to March 2021, 68 young DLBCL patients with complete initial diagnosis data from the Department of Hematology, The People's Hospital Xinjiang Uygur Autonomous Region were retrospectively analyzed, and their paraffin-embedded tissues were subjected to targeted sequencing analysis by NGS technology (including 475 Target genes), and the differences in gene mutation profiles and signaling pathways between high-risk patients with aaIPI ≥2 and low-intermediate risk patients with aaIPI <2 were compared. RESULTS: A total of 44 high-frequency mutation genes were detected in 68 young DLBCL patients. By comparing the high-frequency mutation genes in aaIPI high-risk group and low-intermediate risk group, it was found that CARD11 mutation in aaIPI high-risk group was significantly higher than that in low-intermediate risk group (P =0.002), while MGA mutation (P =0.037) only appeared in the aaIPI high-risk group, and SPEN mutation (P =0.004) only appeared in the aaIPI low-intermediate risk group. The high-frequency mutation genes and clinical indicators of the aaIPI high-risk group were included in the survival analysis, and the results showed that TP53 (P =0.009, P =0.027), POU2AF1 (P =0.003, P =0.006) and CCND3 (P =0.040, P =0.014) genes mutations were associated with worse PFS and OS, while B2M was associated with better PFS (P =0.014) and OS (P =0.013). Multivariate COX regression analysis showed that the TP53, POU2AF1 and CCND3 were independent risk factors for PFS（P ＝0.021，P =0.005，P =0.020） and OS（P ＝0.042，P =0.010，P =0.013）. CONCLUSION The aaIPI staging combination with molecular biology markers is more conducive to accurately judging the prognosis of young DLBCL patients. TP53, POU2AF1 and CCND3 mutations predict worse survival in the patients with the aaIPI high-risk group.
Humans ; Retrospective Studies ; Prognosis ; Lymphoma, Large B-Cell, Diffuse/genetics* ; Biomarkers ; Mutation ; High-Throughput Nucleotide Sequencing