Recent data suggest that vascular endothelial growth factor receptor inhibitor (VEGFRi) can enhance the anti-tumor activity of the anti-programmed cell death-1 (anti-PD-1) antibody in colorectal cancer (CRC) with microsatellite stability (MSS). However, the comparison between this combination and standard third-line VEGFRi treatment is not performed, and reliable biomarkers are still lacking. We retrospectively enrolled MSS CRC patients receiving anti-PD-1 antibody plus VEGFRi (combination group, n=54) or VEGFRi alone (VEGFRi group, n=32), and their efficacy and safety were evaluated. We additionally examined the immune characteristics of the MSS CRC tumor microenvironment (TME) through single-cell and spatial transcriptomic data, and an MSS CRC immune cell-related signature (MCICRS) that can be used to predict the clinical outcomes of MSS CRC patients receiving immunotherapy was developed and validated in our in-house cohort. Compared with VEGFRi alone, the combination of anti-PD-1 antibody and VEGFRi exhibited a prolonged survival benefit (median progression-free survival: 4.4 vs. 2.0 months, P=0.0024; median overall survival: 10.2 vs. 5.2 months, P=0.0038) and a similar adverse event incidence. Through single-cell and spatial transcriptomic analysis, we determined ten MSS CRC-enriched immune cell types and their spatial distribution, including naive CD4⁺ T, regulatory CD4⁺ T, CD4⁺ Th17, exhausted CD8⁺ T, cytotoxic CD8⁺ T, proliferated CD8⁺ T, natural killer (NK) cells, plasma, and classical and intermediate monocytes. Based on a systemic meta-analysis and ten machine learning algorithms, we obtained MCICRS, an independent risk factor for the prognosis of MSS CRC patients. Further analyses demonstrated that the low-MCICRS group presented a higher immune cell infiltration and immune-related pathway activation, and hence a significant relation with the superior efficacy of pan-cancer immunotherapy. More importantly, the predictive value of MCICRS in MSS CRC patients receiving immunotherapy was also validated with an in-house cohort. Anti-PD-1 antibody combined with VEGFRi presented an improved clinical benefit in MSS CRC with manageable toxicity. MCICRS could serve as a robust and promising tool to predict clinical outcomes for individual MSS CRC patients receiving immunotherapy.
Humans ; Colorectal Neoplasms/drug therapy* ; Male ; Female ; Immunotherapy ; Middle Aged ; Aged ; Tumor Microenvironment/immunology* ; Retrospective Studies ; Microsatellite Instability ; Transcriptome ; Single-Cell Analysis ; Programmed Cell Death 1 Receptor/immunology* ; Gene Expression Profiling ; Immune Checkpoint Inhibitors/therapeutic use* ; Adult ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors*

Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic

In this study, fluvoxamine maleate sustained-release pellet system tablets were prepared and were used to evaluate their release behaviors in vitro. Fluvoxamine maleate pellets were prepared using centrifugal-spherization method and coated by fluidized bed as bottom-spray. The multi-unit sustained-release pellets and appropriate excipients for prescription volumes were mixed uniformly and then compressed to tablets. Screening and determining the optimal formulation of drug loaded pellets through L8 (2⁴) Taguchi experiment. Using Minitab software to design a DOE experiment with 2⁴ partial factors, including material temperature, fan speed, atomization pressure, and spray rate to optimize the bottom spray coating process. Taking monostearate glycerol ester with a particle size of 24-40 mesh as the main diluent for tableting to relieve the delamination phenomenon between pellets and excipients during tablet pressing and reduce mechanical damage to the coating film. By examining the powder fluidity indexes such as angle of repose, bulk density, tapped density, and Hausner ratio of mixed particles, it was found that the flowability and compressibility are good and suitable for direct compression. Evaluate the basic properties of the sustained-release tablets, investigate the in vitro release behavior and study the release mechanism. The results of in vitro release test showed that the self-made sustained-release tablets could disintegrate into independent pellet units in phosphate buffer at pH 6.8 and release slowly within 24 h, which conformed to the first-order drug release model. The fluvoxamine maleate sustained-release pellet system tablets meet the requirements of preparation design and has a great commercial prospect.

Pyran- and furanocoumarins are key representatives of tetrahydropyrans and tetrahydrofurans, respectively, exhibiting diverse physiological and medical bioactivities. However, the biosynthetic mechanisms for their core structures remain poorly understood. Here we combined multiomics analyses of biosynthetic enzymes in Peucedanum praeruptorum and in vitro functional verification and identified two types of key enzymes critical for pyran and furan ring biosynthesis in plants. These included three distinct P. praeruptorum prenyltransferases (PpPT1-3) responsible for the prenylation of the simple coumarin skeleton 7 into linear or angular precursors, and two novel CYP450 cyclases (PpDC and PpOC) crucial for the cyclization of the linear/angular precursors into either tetrahydropyran or tetrahydrofuran scaffolds. Biochemical analyses of cyclases indicated that acid/base-assisted epoxide ring opening contributed to the enzyme-catalyzed tetrahydropyran and tetrahydrofuran ring refactoring. The possible acid/base-assisted catalytic mechanisms of the identified cyclases were theoretically investigated and assessed using site-specific mutagenesis. We identified two possible acidic amino acids Glu303 in PpDC and Asp301 in PpOC as vital in the catalytic process. This study provides new enzymatic tools in the epoxide formation/epoxide-opening mediated cascade reaction and exemplifies how plants become chemically diverse in terms of enzyme function and catalytic process.

Objective:To report the results of national surveillance on the distribution and antimicrobial resistance profile of clinical Gram-negative bacteria isolates from bloodstream infections in China in 2022.Methods:The clinical isolates of Gram-negative bacteria from blood cultures in member hospitals of national bloodstream infection Bacterial Resistant Investigation Collaborative System（BRICS）were collected during January 2022 to December 2022. Antibiotic susceptibility tests were conducted by agar dilution or broth dilution methods recommended by Clinical and Laboratory Standards Institute（CLSI）. WHONET 5.6 and SPSS 25.0 software were used to analyze the data.Results:During the study period，9 035 strains of Gram-negative bacteria were collected from 51 hospitals，of which 7 895（87.4%）were Enterobacteriaceae and 1 140（12.6%）were non-fermenting bacteria. The top 5 bacterial species were Escherichia coli（ n=4 510，49.9%）， Klebsiella pneumoniae（ n=2 340，25.9%）， Pseudomonas aeruginosa（ n=534，5.9%）， Acinetobacter baumannii complex（ n=405，4.5%）and Enterobacter cloacae（ n=327，3.6%）. The ESBLs-producing rates in Escherichia coli， Klebsiella pneumoniae and Proteus spp. were 47.1%（2 095/4 452），21.0%（427/2 033）and 41.1%（58/141），respectively. The prevalence of carbapenem-resistant Escherichia coli（CREC）and carbapenem-resistant Klebsiella pneumoniae（CRKP）were 1.3%（58/4 510）and 13.1%（307/2 340）；62.1%（36/58）and 9.8%（30/307）of CREC and CRKP were resistant to ceftazidime/avibactam combination，respectively. The prevalence of carbapenem-resistant Acinetobacter baumannii（CRAB）complex was 59.5%（241/405），while less than 5% of Acinetobacter baumannii complex was resistant to tigecycline and polymyxin B. The prevalence of carbapenem-resistant Pseudomonas aeruginosa（CRPA）was 18.4%（98/534）. There were differences in the composition ratio of Gram-negative bacteria in bloodstream infections and the prevalence of main Gram-negative bacteria resistance among different regions，with statistically significant differences in the prevalence of CRKP and CRPA（ χ2=20.489 and 20.252， P<0.001）. The prevalence of CREC，CRKP，CRPA，CRAB，ESBLs-producing Escherichia coli and Klebsiella pneumoniae were higher in provinicial hospitals than those in municipal hospitals（ χ2=11.953，81.183，10.404，5.915，12.415 and 6.459， P<0.01 or <0.05），while the prevalence of CRPA was higher in economically developed regions（per capita GDP ≥ 92 059 Yuan）than that in economically less-developed regions（per capita GDP <92 059 Yuan）（ χ2=6.240， P=0.012）. Conclusions:The proportion of Gram-negative bacteria in bloodstream infections shows an increasing trend，and Escherichia coli is ranked in the top，while the trend of CRKP decreases continuously with time. Decreasing trends are noted in ESBLs-producing Escherichia coli and Klebsiella pneumoniae. Low prevalence of carbapenem resistance in Escherichia coli and high prevalence in CRAB complex have been observed. The composition ratio and antibacterial spectrum of bloodstream infections in different regions of China are slightly different，and the proportion of main drug resistant bacteria in provincial hospitals is higher than those in municipal hospitals.

Aim To explore the mechanism of oxiracetam promoting neurogenesis and migration in rats with cer-ebral infarction through stromal cell-derived factor-1α(SDF-1α)/C-X-C chemokine receptor 4(CXCR4)pathway.Methods 100 SD rats were randomly divided into control group,cerebral ischemia(CI)group,oxiracetam(200 mg/kg)group,and oxiracetam(200 mg/kg)+AMD3100(5 mg/kg)group,with 25 rats in each group.Electrocoagulation was used to create rat model of local permanent cerebral infarction.After 1,7 and 14 days of modeling,neurological deficits were scored,TTC staining was used to detect the volume of cerebral infarction,Nissl staining was used to detect cell surviv-al in the infarcted area,Western blot was used to detect SDF-1α and CXCR4 protein levels in ischemic zone.After 1～7 days of modeling,BrdU(50 mg/kg)was continuously injected intraperitoneally.After 14 days,immunofluorescence double staining was used to detect the number of BrdU+Nestin+and BrdU+DCX+cells in the SVZ region.5 days before modeling,retroviruses carrying GFP were injected into the SVZ region.After 14 days,immunofluorescence double stai-ning was used to detect the number of GFP+DCX+,GFP+MAP-2+and GFP+GFAP+cells in infarction area.C17.2 cells were divided into control group,oxygen-glucose deprivation(OGD)group,oxiracetam(final concentration:200 mg/L)group,and oxiracetam(final concentration:200 mg/L)+AMD3100(final concentration:100 μmol/L)group.OGD was used to create cell CI model.After 12 hours,immunofluorescence double staining was used to detect the number of Br-dU+/Nestin+and BrdU+/MAP-2+cells,Transwell experiment was used to detect cell migration,Western blot was used to detect SDF-1α and CXCR4 protein levels in cell culture supernatant.Results Animal experiment results showed:compared with control group,mNSS score in CI group was increased,cerebral infarction volume was increased,the number of surviving cells in infarcted area was decreased,SDF-1α and CXCR4 protein levels were increased,the number of GFP+DCX+,GFP+MAP-2+and GFP+GFAP+cells in SVZ region were increased(P＜0.05);compared with CI group,mNSS score in oxiracetam group was decreased,cerebral infarction volume was decreased,the number of surviving cells in infarc-ted area was increased,SDF-1α and CXCR4 protein levels were increased,the number of GFP+DCX+,GFP+MAP-2+and GFP+GFAP+cells in SVZ region were increased,the number of GFP+DCX+,GFP+MAP-2+and GFP+GFAP+cells in in-farcted area were increased(P＜0.05);compared with oxiracetam group,mNSS score in oxiracetam+AMD3100 group was increased,cerebral infarction volume was increased,the number of surviving cells in infarcted area was decreased,CXCR4 protein level was decreased,the number of GFP+DCX+,GFP+MAP-2+and GFP+GFAP+cells in the SVZ region were de-creased(P＜0.05).Cell experiment results showed:compared with control group,the number of BrdU+/Nestin+and Br-dU+/MAP-2+cells in OGD group were increased,the number of cell migration,SDF-1α and CXCR4 protein levels in cell culture supernatant were increased(P＜0.05);compared with OGD group,the number of BrdU+/Nestin+and BrdU+/MAP-2+cells in oxiracetam group were increased,the number of cell migration,SDF-1α and CXCR4 protein levels in cell culture supernatant were increased(P＜0.05);compared with oxiracetam group,the number of BrdU+/Nestin+and BrdU+/MAP-2+cells in oxiracetam+AMD3100 group were decreased,the number of cell migration,CXCR4 protein level in cell culture supernatant were decreased(P＜0.05).Conclusion Oxiracetam may promote the migration of neural stem cells from the SVZ region to the ischemic zone,promoting neurogenesis and functional recovery in rats with cerebral infarction by activating SDF-1α/CXCR4 pathway.

Background::A phase II trial on recombinant human tenecteplase tissue-type plasminogen activator (rhTNK-tPA) has previously shown its preliminary efficacy in ST elevation myocardial infarction (STEMI) patients. This study was designed as a pivotal postmarketing trial to compare its efficacy and safety with rrecombinant human tissue-type plasminogen activator alteplase (rt-PA) in Chinese patients with STEMI.Methods::In this multicenter, randomized, open-label, non-inferiority trial, patients with acute STEMI were randomly assigned (1:1) to receive an intravenous bolus of 16 mg rhTNK-tPA or an intravenous bolus of 8 mg rt-PA followed by an infusion of 42 mg in 90 min. The primary endpoint was recanalization defined by thrombolysis in myocardial infarction (TIMI) flow grade 2 or 3. The secondary endpoint was clinically justified recanalization. Other endpoints included 30-day major adverse cardiovascular and cerebrovascular events (MACCEs) and safety endpoints.Results::From July 2016 to September 2019, 767 eligible patients were randomly assigned to receive rhTNK-tPA ( n = 384) or rt-PA ( n = 383). Among them, 369 patients had coronary angiography data on TIMI flow, and 711 patients had data on clinically justified recanalization. Both used a –15% difference as the non-inferiority efficacy margin. In comparison to rt-PA, both the proportion of patients with TIMI grade 2 or 3 flow (78.3% [148/189] vs. 81.7% [147/180]; differences: –3.4%; 95% confidence interval [CI]: –11.5%, 4.8%) and clinically justified recanalization (85.4% [305/357] vs. 85.9% [304/354]; difference: –0.5%; 95% CI: –5.6%, 4.7%) in the rhTNK-tPA group were non-inferior. The occurrence of 30-day MACCEs (10.2% [39/384] vs. 11.0% [42/383]; hazard ratio: 0.96; 95% CI: 0.61, 1.50) did not differ significantly between groups. No safety outcomes significantly differed between groups. Conclusion::rhTNK-tPA was non-inferior to rt-PA in the effect of improving recanalization of the infarct-related artery, a validated surrogate of clinical outcomes, among Chinese patients with acute STEMI.Trial registration::www.ClinicalTrials.gov (No. NCT02835534).

Objective To investigate the effect and molecular mechanism of tRF-1:30-Gln-CTG-4(tRF-1:30)on the expression of inflammatory factors in high glucose(HG)-induced renal tubular epithelial cells(RTECs).Methods RTECs were divided into the control group,the HG group,the HG+tRF-1:30 mimic group,the HG+tRF-1:30 negative control(NC)group,the HG+si-IKZF2 group and the HG+si-NC group.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression levels of tRF-1:30,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1)and IKAROS family zinc finger protein 2(IKZF2).Enzyme-linked immunosorbent assay(ELISA)was used to detect levels of TNF-α,IL-6 and MCP-1.Protein expression of IKZF2 was detected by Western blot assay.Dual-luciferase reporter assay was used to detect the targeting relationship between tRF-1:30 and IKZF2.Results The expression levels of inflammatory factors were elevated in HG-induced RTECs,and the expression level of tRF-1:30 was decreased(P＜0.05).Overexpression of tRF-1:30 significantly decreased expression levels of inflammatory factors in HG-induced RTECs(P＜0.05),and the expression level of IKZF2 was significantly increased(P＜0.05).Further knockdown of IKZF2 can inhibit the release of inflammatory factors,and the expression level of IKZF2 was down-regulated after overexpression of tRF-1:30.Double luciferase reporting experiment further verified the possible targeting relationship between tRF-1:30 and IKZF2.Conclusion Overexpression of tRF-1:30 inhibits the expression of inflammatory factors in HG-induced RTECs by target binding and negatively regulating the expression of IKZF2.

Objective To investigate the early diagnostic value of serum double cortin-like kinase 1(DCLK1),latent transforming growth factor binding protein 2(LTBP2)combined with transvaginal real-time shear wave elastography(SWE)for cervical cancer.Methods A total of 155 patients with cervical lesions treated in our hospital from August 2021 to December 2022 were selected as the research objects.Seventy-five patients with cervical cancer(the cervical cancer group)and 80 patients with cervical intraepithelial neoplasia(the CIN group)were diagnosed by surgical pathology,another 80 volunteers without cervical related diseases who participated in physical examinations at our hospital during the same period were selected as the control group.All subjects of each groups underwent serum DCLK1 and LTBP2 levels detection and transvaginal SWE examination.Receiver operating characteristic(ROC)curve was used to analyze the diagnostic value of serum DCLK1 and LTBP2 for cervical cancer;the diagnostic value of serum DCLK1 and LTBP2 combined with transvaginal SWE for cervical cancer was analyzed by fourfold table analysis.Results The serum levels of DCLK1 and LTBP2 of patients in the cervical cancer group were obviously higher than those in the CIN group and the control group(P<0.05).Compared with before surgery,the serum levels of DCLK1 and LTBP2 1,3,and 6 months after surgery in cervical cancer patients gradually decreased,and there were statistical differences in pairwise comparisons at each time point(P<0.05).The area under the curve of serum DCLK1 and LTBP2 for diagnosing cervical cancer were 0.868 and 0.754,respectively,with sensitivity of 86.67% and 78.67%,specificity of 81.25% and 60.00%,and optimal cutoff values of 1.49 ng/mL and 19.02 μg/mL.The maximum and average elastic modulus of lesion tissue in the cervical cancer group were obviously higher than those in the CIN group(P<0.05).The positive rates of serum DCLK1,serum LTBP2,and transvaginal SWE in the diagnosis of cervical cance were 86.67%,78.67%,and 82.67%,respectively,which were consistent with the gold standard(Kappa=0.624,0.501,0.673,P<0.001),and the positive rate of the combination of them in the diagnosis of cervical cancer was 97.33%,which was highly consistent with the gold standard(Kappa=0.846,P<0.001).The sensitivity and accuracy of the three combined diagnosis were obviously higher than those of the single indicator diagnosis of serum DCLK1,LTBP2,and transvaginal SWE,the specificity of the three combined diagnosis was obviously higher than that of the single indicator diagnosis of serum DCLK1 and LTBP2,the misdiagnosis rate of the three combined diagnosis was obviously lower than that of the single indicator diagnosis of serum DCLK1 and LTBP2,and the differences were all statistically significant(P<0.05).Conclusion Serum DCLK1 and LTBP2 combined with transvaginal SWE has high application value in the diagnosis of cervical cancer,which can further improve the sensitivity and accuracy of diagnosis,and reduce the misdiagnosis rate.