CRISPR/Cas9-based therapeutics face significant challenges in penetrating the dense microenvironment of solid tumors, resulting in insufficient gene editing and compromised treatment efficacy. Current nanostrategies, which mainly focus on the paracellular pathway attempted to improve gene editing performance, whereas their efficiency remains uneven in the heterogenous extracellular matrix. Here, the nanoCRISPR system is prepared with self-cascading mechanisms for gene editing-mediated robust apoptosis and transcellular penetration. NanoCRISPR unlocks its self-cascade capability within the matrix metallopeptidase 2-enriched tumor microenvironment, initiating the transcellular penetration. By facilitating cellular uptake, nanoCRISPR triggers robust apoptosis in edited malignancies, promoting further transcellular penetration and amplifying gene editing in neighboring tumor cells. Benefiting from self-cascade between robust apoptosis and transcellular penetration, nanoCRISPR demonstrates continuous gene transfection/tumor killing performance (transfection/apoptosis efficiency: 1st round: 85%/84.2%; 2nd round: 48%/27%) and homogeneous penetration. In xenograft tumor-bearing mice, nanoCRISPR treatment achieves remarkable anti-tumor efficacy (∼83%) and significant survival benefits with minimal toxicity. This strategy presents a promising paradigm emphasizing transcellular penetration to enhance the effectiveness of CRISPR-based antitumor therapeutics.

Mesangial cell proliferation is an early pathological indicator of diabetic nephropathy (DN). Growing evidence highlights the pivotal role of paired-related homeobox 1 (Prrx1), a key regulator of cellular proliferation and tissue differentiation, in various disease pathogenesis. Notably, Prrx1 is highly expressed in mesangial cells under DN conditions. Both in vitro and in vivo studies have demonstrated that Prrx1 overexpression promotes mesangial cell proliferation and contributes to renal fibrosis in db/m mice. Conversely, Prrx1 knockdown markedly suppresses hyperglycemia-induced mesangial cell proliferation and mitigates renal fibrosis in db/db mice. Mechanistically, Prrx1 directly interacts with the Yes-associated protein 1 (YAP) promoter, leading to the upregulation of YAP expression. This upregulation promotes mesangial cell proliferation and exacerbates renal fibrosis. These findings emphasize the crucial role of Prrx1 upregulation in high glucose-induced mesangial cell proliferation, ultimately leading to renal fibrosis in DN. Therefore, targeting Prrx1 to downregulate its expression presents a promising therapeutic strategy for treating renal fibrosis associated with DN.

Inflammatory bowel disease (IBD) is a refractory disease characterized by chronic intestinal inflammation. Its typical pathological characteristics are the destruction of intestinal mucosal barrier and the imbalance of immune microenvironment. In severe cases, it can cause intestinal fibrosis and functional failure. Sulfation, a critical post-translational modification, plays an essential role in regulating intestinal homeostasis. Abnormal sulfation metabolism in the intestine of IBD patients can lead to the impairment of intestinal barrier function, immune system disorders and changes in the composition of microbiota. Consequently, targeting the sulfation pathway may be a novel therapeutic strategy for IBD. By supplementing exogenous sulfation substrates, regulating the activity of related key enzymes involved in sulfation or remodeling the microbiota, the level of intestinal sulfation can be effectively restored, thereby repairing the intestinal barrier function and inhibit the inflammatory response. This article systematically summarizes the pathogenic mechanisms of sulfation in IBD, providing a theoretical foundation for developing precision therapeutic strategies targeting the intestinal microenvironment.

Objective:To compare the application of cloning sequencing and third-generation nanopore sequencing technology in the identification of raw materials mixed in Tibetan patent medicine Shiliujianwei powder,and to pro-vide a reference for the establishment of the specific identification method for the formulation of medicinal prepara-tions using the raw powder of medicinal herbs or herbal pieces.Methods:By investigating the different concentra-tions of ExTaq enzyme,genomic DNA,upstream and downstream primers in the PCR amplification system,the suit-able PCR amplification system of genomic DNA universal primers for self-made Tibetan medicine Shiliujianwei powder was determined.The amplified products of Shiliujianwei powder were sequenced by two methods,the first was cloned and sequenced by Sanger sequencing technology,and the second was sequenced by third generation nanopore sequen-cing technology.Results:The addition of ExTaq enzyme,upstream and downstream primers and genomic DNA in 20 μL PCR amplification system of Shiliujianwei powder and single medicinal samples were determined to be 0.4,1.5 and 1 μL,respectively.The amplification products of the DNA from self-made Shiliujianwei powder were cloned and sequenced by Sanger sequencing technology,then only the ITS2 sequence of Carthami flos was obtained.The amplified products of self-made pomegranate Jianwei powder DNA were sequenced by three-generation nanopore se-quencing technology,and the ITS2 sequences of Granati semen,Carthami flos,Piperis longi fructus and Amomi fruc-tus rotundus were finally obtained,but the ITS2 sequence of cinnamomi cortex was not obtained.Conclusion:Both cloning sequencing and the third generation nanopore sequencing could solve the problem of overlapping peaks in the direct sanger sequencing for the universal primer amplification products of patent drugs.The third generation nano-pore sequencing was better than cloning sequencing in the sequencing of the universal primer amplification products of patent drugs,but there were still one raw material medicine that have not been detected.It is of certain reference val-ue for the molecular biological identification and DNA barcoding study of different raw materials in the patent medi-cine to establish the specific identification method of Tibetan patent medicine Shiliujianwei powder.

Objective To analyze the clinical distribution,drug resistance characteristics,molecular prevalence,and impact of immunocompromised status on the infection rate of carbapenem-resistant Enterobacteriaceae(CRE)isolated from pediatric respiratory tract samples(sputum,bronchoalveolar lavage fluid).Methods A retrospective analysis was conducted on clinical data of hospitalized children from 2019 to 2023.A total of 1 235 non-repetitive strains of Enterobacteriaceae bacteria were obtained from pediactric respiratory tract samples.Drug susceptibility testing,multilocus sequencing typing(MLST),and resistance-related gene sequencing were performed on 100 isolated CRE strains,to study the clinical distribution,drug resistance characteristics,and molecular prevalence of CRE,and to further analyze the impact of immunocompromised status on the respiratory CRE infection rate in children.Results From 2019 to 2023,the detection rate of CRE in 1 235 strains of Enterobacteriaceae bacteria was 8.10%(100/1 235).Among them,51.0%(51/100)of CRE were isolated from the intensive care unit(ICU),of which 33.0%(33/100)were isolated from the Surgical ICU,18.0%(18/100)of CRE was isolated from the Medical ICU;32.0%(32/100)of CRE were isolated from Department of Neonatology,with the majority(74.0%)isolated from infants under 6 months of age.Of all pediatric patients,85.0%recovered and were discharged after treatment.Immunocompromised status was identified as an independent risk factor for CRE infection.Among 100 strains of CRE,Carbapenem-resistant Klebsiella pneumoniae(CRKP)was the most commonly detected strain,accounting for 88.0%(88/100),followed by Escherichia coli at 6.0%(6/100)and Enterobacter cloacae at 4.0%(4/100).CRE strains were highly resistant to carbapenems and cephalosporins,with prevalent resistance to amikacin,ciprofloxacin,and levofloxacin.However,their resistance rates were relatively low to tigecycline,colistin,minocycline,ceftazidime/avibactam,meropenem/vaborbactam,and imipenem/relebactam.The screening results of carbapenem resistance genes showed that blaKPC-2 was the most prevalent gene(74.0%),followed by blaNDM-1(14.0%)and blaNDM-5(11.0%).Molecular typing showed that ST11 type CRE was the dominant type,comprising 72.0%(72/100)and was the primary epidemic clone.Conclusions CRKP is the most prevalent CRE strain isolated from pediatric respiratory tract samples,primarily identified in the ICU,Department of Neonatology,and among infants under 6 months of age.Immunocompromised status is an independent risk factor for respiratory CRE infection in children.CRE generally has high resistance to antibacterial drugs,with blaKPC-2 being the dominant resistance genotype,and ST11 as the predominant multilocus sequence type.Clinical management should account for these characteristics to implement timely interventions and rational therapeutic strategies.

Objective·To investigate the mechanisms by which different subtypes of G protein-coupled receptor kinases(GRKs)regulate the biased signaling transduction mediated by the muscarinic acetylcholine receptor 1(M1 receptor),focusing on their molecular effects in modulating the binding of the M1 receptor to the downstream heterotrimeric G protein(Gαq-Gβ1-Gγ2)andβ-arrestin 2(βarr2).Methods·By establishing a highly sensitive protein interaction detection system based on bioluminescence resonance energy transfer(BRET),six M1 receptor agonists/allosteric modulators were selected to measure the dynamic interactions between the M1 receptor and four GRK subtypes(GRK2/3/5/6),βarr2,and the G protein under stimulation.All BRET data were statistically quantified using the area under the curve(AUC)of the time-response curves.First,concentration-effect curves were established by treatment with gradient concentrations of agonists/allosteric modulators and AUC fitting,to comprehensively analyze the differences in efficacy between each agonist/allosteric modulator and the endogenous agonist acetylcholine chloride(ACh)in promoting the interactions of M1 receptor with GRK3/5,βarr2,and the G protein;next,GRKs were divided into two groups based on subtypes:GRK2/3 and GRK5/6.The maximum AUC values for the interaction between the M1 receptor and the two GRK groups under high concentrations were calculated respectively,to further evaluate the regulatory propensity of different types of GRKs on the binding strength of the M1 receptor to βarr2 or the G protein.Results·All six agonists/allosteric modulators effectively induced the association of the M1 receptor with GRK3,while simultaneousey inducing dissociation of the M1 receptor from GRK5.The allosteric modulator BQCA not only activated the M1 receptor alone and triggered its binding to downstream signaling proteins,but also,when co-treated with ACh,caused a significant leftward shift of the concentration-effect curves in the M1-G protein and M1-βarr2 systems,suggesting that its potentiation effect on ACh was mainly achieved by reducing the half-maximal effective concentration.A moderate positive correlation was observed between the maximum AUC values of M1-βarr2 and M1-G protein interactions induced by the seven groups of drug treatments(r=0.722,P=0.067).Further analysis showed that the ratio of the maximum AUC for M1-GRK2/3 interaction to that for M1-GRK5/6 interaction was also positively correlated with the ratio of the maximum AUC for M1-βarr2 interaction to that for M1-G protein interaction(r=0.760,P=0.047).Conclusion·The M1 receptor may be pre-coupled with GRK5/6 under basal conditions,and they dissociate upon receptor activation,suggesting that GRK5/6 may be involved in M1 receptor inactivation or signal reprogramming.The relative efficiency of the M1 receptor's interaction with different GRK subtypes determines its preference for downstream signaling pathways.

Objective:To conduct a scoping review of studies on the application of exercise interventions in the rehabilitation of patients with hemophilic arthropathy (HA) .Methods:A systematic search was conducted in PubMed, Web of Science, Cochrane Library, Embase, CINAHL, China National Knowledge Infrastructure, Wanfang Data, VIP, and China Biology Medicine disc. The search period was from the database inception to March 1, 2024. Relevant literature was screened, summarized, and analyzed.Results:A total of 31 studies were included. The study population mainly consisted of adult patients with moderate to severe hemophilia suffering from elbow, knee, and ankle arthropathy, who were in a stable bleeding phase without coagulation factor inhibitors. Exercise types included resistance training, aerobic exercise, balance/proprioceptive exercises, and flexibility training. The exercise intensity was predominantly low to moderate. The duration of each session was mostly 30-60 minutes, with a frequency of 2-5 times per week and a total intervention period of 4-12 weeks. Exercises were mostly conducted in medical institutions under face-to-face supervision. Exercise interventions led to varying degrees of improvement in mobility, balance/proprioceptive function, joint function, cardiopulmonary fitness, psychological well-being, and quality of life.Conclusions:Exercise interventions for patients with hemophilic arthropathy are diverse, demonstrating safety, feasibility, and efficacy. Future research should focus on a multidisciplinary, exercise-centered rehabilitation approach, standardizing evaluation criteria, and dynamically monitoring the rehabilitation process to develop standardized exercise rehabilitation protocols.

Inflammatory bowel disease (IBD) is a refractory disease characterized by chronic intestinal inflammation. Its typical pathological characteristics are the destruction of intestinal mucosal barrier and the imbalance of immune microenvironment. In severe cases, it can cause intestinal fibrosis and functional failure. Sulfation, a critical post-translational modification, plays an essential role in regulating intestinal homeostasis. Abnormal sulfation metabolism in the intestine of IBD patients can lead to the impairment of intestinal barrier function, immune system disorders and changes in the composition of microbiota. Consequently, targeting the sulfation pathway may be a novel therapeutic strategy for IBD. By supplementing exogenous sulfation substrates, regulating the activity of related key enzymes involved in sulfation or remodeling the microbiota, the level of intestinal sulfation can be effectively restored, thereby repairing the intestinal barrier function and inhibit the inflammatory response. This article systematically summarizes the pathogenic mechanisms of sulfation in IBD, providing a theoretical foundation for developing precision therapeutic strategies targeting the intestinal microenvironment.

Objective·To investigate the mechanisms by which different subtypes of G protein-coupled receptor kinases(GRKs)regulate the biased signaling transduction mediated by the muscarinic acetylcholine receptor 1(M1 receptor),focusing on their molecular effects in modulating the binding of the M1 receptor to the downstream heterotrimeric G protein(Gαq-Gβ1-Gγ2)andβ-arrestin 2(βarr2).Methods·By establishing a highly sensitive protein interaction detection system based on bioluminescence resonance energy transfer(BRET),six M1 receptor agonists/allosteric modulators were selected to measure the dynamic interactions between the M1 receptor and four GRK subtypes(GRK2/3/5/6),βarr2,and the G protein under stimulation.All BRET data were statistically quantified using the area under the curve(AUC)of the time-response curves.First,concentration-effect curves were established by treatment with gradient concentrations of agonists/allosteric modulators and AUC fitting,to comprehensively analyze the differences in efficacy between each agonist/allosteric modulator and the endogenous agonist acetylcholine chloride(ACh)in promoting the interactions of M1 receptor with GRK3/5,βarr2,and the G protein;next,GRKs were divided into two groups based on subtypes:GRK2/3 and GRK5/6.The maximum AUC values for the interaction between the M1 receptor and the two GRK groups under high concentrations were calculated respectively,to further evaluate the regulatory propensity of different types of GRKs on the binding strength of the M1 receptor to βarr2 or the G protein.Results·All six agonists/allosteric modulators effectively induced the association of the M1 receptor with GRK3,while simultaneousey inducing dissociation of the M1 receptor from GRK5.The allosteric modulator BQCA not only activated the M1 receptor alone and triggered its binding to downstream signaling proteins,but also,when co-treated with ACh,caused a significant leftward shift of the concentration-effect curves in the M1-G protein and M1-βarr2 systems,suggesting that its potentiation effect on ACh was mainly achieved by reducing the half-maximal effective concentration.A moderate positive correlation was observed between the maximum AUC values of M1-βarr2 and M1-G protein interactions induced by the seven groups of drug treatments(r=0.722,P=0.067).Further analysis showed that the ratio of the maximum AUC for M1-GRK2/3 interaction to that for M1-GRK5/6 interaction was also positively correlated with the ratio of the maximum AUC for M1-βarr2 interaction to that for M1-G protein interaction(r=0.760,P=0.047).Conclusion·The M1 receptor may be pre-coupled with GRK5/6 under basal conditions,and they dissociate upon receptor activation,suggesting that GRK5/6 may be involved in M1 receptor inactivation or signal reprogramming.The relative efficiency of the M1 receptor's interaction with different GRK subtypes determines its preference for downstream signaling pathways.

Objective:To compare the application of cloning sequencing and third-generation nanopore sequencing technology in the identification of raw materials mixed in Tibetan patent medicine Shiliujianwei powder,and to pro-vide a reference for the establishment of the specific identification method for the formulation of medicinal prepara-tions using the raw powder of medicinal herbs or herbal pieces.Methods:By investigating the different concentra-tions of ExTaq enzyme,genomic DNA,upstream and downstream primers in the PCR amplification system,the suit-able PCR amplification system of genomic DNA universal primers for self-made Tibetan medicine Shiliujianwei powder was determined.The amplified products of Shiliujianwei powder were sequenced by two methods,the first was cloned and sequenced by Sanger sequencing technology,and the second was sequenced by third generation nanopore sequen-cing technology.Results:The addition of ExTaq enzyme,upstream and downstream primers and genomic DNA in 20 μL PCR amplification system of Shiliujianwei powder and single medicinal samples were determined to be 0.4,1.5 and 1 μL,respectively.The amplification products of the DNA from self-made Shiliujianwei powder were cloned and sequenced by Sanger sequencing technology,then only the ITS2 sequence of Carthami flos was obtained.The amplified products of self-made pomegranate Jianwei powder DNA were sequenced by three-generation nanopore se-quencing technology,and the ITS2 sequences of Granati semen,Carthami flos,Piperis longi fructus and Amomi fruc-tus rotundus were finally obtained,but the ITS2 sequence of cinnamomi cortex was not obtained.Conclusion:Both cloning sequencing and the third generation nanopore sequencing could solve the problem of overlapping peaks in the direct sanger sequencing for the universal primer amplification products of patent drugs.The third generation nano-pore sequencing was better than cloning sequencing in the sequencing of the universal primer amplification products of patent drugs,but there were still one raw material medicine that have not been detected.It is of certain reference val-ue for the molecular biological identification and DNA barcoding study of different raw materials in the patent medi-cine to establish the specific identification method of Tibetan patent medicine Shiliujianwei powder.