Objective:To analyze the effect of nucleotide sequence variants in the 5′ untranslated region (5′UTR) of Helicobacter pylori (Hp) cagA on mRNA secondary structure, as well as its regulatory role in cytotoxin-associated gene A (CagA) protein expression and bacterial virulence. Methods:The upstream nucleotide sequence of cagA was amplified by polymerase chain reaction (PCR) from 37 Hp strains, and the PCR products were sequenced. MEGA 5.0 software and RNAfold prediction software were used to analyze the nucleotide sequence variants of cagA 5′UTR and the changes in mRNA secondary structure of this region, respectively. Western blot was used to detect the expression level of CagA protein in Hp strains, and the regulatory effect of cagA 5′UTR variants on the difference in CagA protein expression was analyzed. An Hp-infected AGS cell model was established to evaluate bacterial adhesion rate; quantitative PCR (qPCR) was used to analyze the mRNA transcription levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α); enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of IL-8 and TNF-α proteins. Results:Nucleotide sequence alignment of cagA 5′UTR from 37 Hp strains showed that sequence differences were mainly concentrated in the -53motif, -10motif, + 34motif, and + 86motif regions. mRNA secondary structure prediction analysis revealed three types based on the StemB stem-loop structure: type Ⅰ (no StemB stem-loop), type ⅡA (StemB stem-loop with 3-4 base partial pairing), and type ⅡB (StemB stem-loop with 5 base full pairing). Western blot analysis showed that the CagA protein expression level was the highest in type Ⅰ Hp strains (1.72±0.29) and the lowest in type ⅡB strains (0.81±0.26), with a statistically significant difference between the two types ( P=0.030). The adhesion rate of type Ⅰ Hp strains to AGS cells was (52.90±11.17)%, which was higher than that of type Ⅱ strains [(21.27±6.16)%]. qPCR results showed that the mRNA transcription levels of IL-8 and TNF-α in AGS cells induced by type Ⅰ Hp strains were higher than those induced by type Ⅱ strains (140.23±24.47 vs 76.16±8.76, P=0.069; 55.20±9.04 vs 21.26±6.16, P=0.036). ELISA analysis further indicated that the secretion levels of IL-8 and TNF-α proteins in AGS cells induced by type Ⅰ Hp strains were also higher than those induced by type Ⅱ strains [(344.66±62.62)pg/ml vs (302.13±66.27)pg/ml, P=0.665; (131.04±4.94)pg/ml vs (79.17±11.32)pg/ml, P=0.014]. Conclusions:The cagA 5′UTR region of Hp strains exhibits significant nucleotide sequence variants. Hp strains with no StemB stem-loop (type Ⅰ) in the mRNA secondary structure show significantly increased CagA protein expression and higher bacterial pathogenic potential.

Objective: To investigate the therapeutic effect of patchouli alcohol on mice with lung-heat syndrome based on the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/nuclear factor-kappa B(NF-κB) signaling pathway. Methods: First, network pharmacology was used to predict the potential targets of patchouli alcohol in the treatment of lung-heat syndrome, and a "component-disease-key target" network was constructed for pathway analysis. Then, 40 BALB/c mice were assigned to the normal, lung-heat model, honeysuckle, and low-dose and high-dose patchouli alcohol groups. All groups, except the blank group, were intranasally infected with 50 μL (103 TCID50) of influenza virus solution. After two hours of infection, mice were treated once a day for seven consecutive days. The therapeutic mechanism of patchouli alcohol was explored by measuring pulmonary inflammatory factors, the PI3K/Akt/NF-κB pathway, hypothalamic fever markers (PGE2, cAMP, cGMP levels), rectal temperature, and tissue energy metabolism. Results: Network pharmacology identified 135 target genes related to patchouli alcohol and lung-heat syndrome, with the key targets being STAT3, H1F1A, and NF-κB1. In animal experiments, patchouli alcohol significantly alleviated influenza virus-induced lung inflammatory damage in mice with lung-heat syndrome, inhibited the expression of TNF-α and IL-6 in lung tissues(P<0.01), and suppressed the activation of the PI3K/Akt/NF-κB pathway. It also reduced hypothalamic levels of PGE2 and cAMP(P<0.01), suppressed the increase in rectal temperature, significantly decreased liver glycogen and pyruvate levels(P<0.01), and increased the activities of SDH, LDH, and Na+ -K+ -ATPase in the liver(P<0.01) Conclusion Patchouli alcohol improves the symptoms of lung-heat syndrome in mice by inhibiting the activation of the PI3K/Akt/NF-κB pathway, reducing proinflammatory cytokines and inflammatory damage, and regulating hypothalamic fever markers and energy metabolism.

Human NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavoenzyme expressed at high levels in multiple solid tumors, making it an attractive target for anticancer drugs. Bioactivatable drugs targeting NQO1, such as β-lapachone (β-lap), are currently in clinical trials for the treatment of cancer. β-Lap selectively kills NQO1-positive (NQO1⁺) cancer cells by inducing reactive oxygen species (ROS) via catalytic activation of NQO1. In this study, we demonstrated that cryptotanshinone (CTS), a naturally occurring compound, induces NQO1-dependent necrosis without affecting NQO1 activity. CTS selectively kills NQO1⁺ cancer cells by inducing NQO1-dependent necrosis. Interestingly, CTS directly binds to NQO1 but does not activate its catalytic activity. In addition, CTS enables activation of JNK1/2 and PARP, accumulation of iron and Ca²⁺, and depletion of ATP and NAD⁺. Furthermore, CTS selectively suppressed tumor growth in the NQO1⁺ xenograft models, which was reversed by NQO1 inhibitor and NQO1 shRNA. In conclusion, CTS induces NQO1-dependent necrosis via the JNK1/2/iron/PARP/NAD⁺/Ca²⁺ signaling pathway. This study demonstrates the non-enzymatic function of NQO1 in inducing cell death and provides new avenues for the design and development of NQO1-targeted anticancer drugs.

Objective:To analyze the effect of nucleotide sequence variants in the 5′ untranslated region (5′UTR) of Helicobacter pylori (Hp) cagA on mRNA secondary structure, as well as its regulatory role in cytotoxin-associated gene A (CagA) protein expression and bacterial virulence. Methods:The upstream nucleotide sequence of cagA was amplified by polymerase chain reaction (PCR) from 37 Hp strains, and the PCR products were sequenced. MEGA 5.0 software and RNAfold prediction software were used to analyze the nucleotide sequence variants of cagA 5′UTR and the changes in mRNA secondary structure of this region, respectively. Western blot was used to detect the expression level of CagA protein in Hp strains, and the regulatory effect of cagA 5′UTR variants on the difference in CagA protein expression was analyzed. An Hp-infected AGS cell model was established to evaluate bacterial adhesion rate; quantitative PCR (qPCR) was used to analyze the mRNA transcription levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α); enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of IL-8 and TNF-α proteins. Results:Nucleotide sequence alignment of cagA 5′UTR from 37 Hp strains showed that sequence differences were mainly concentrated in the -53motif, -10motif, + 34motif, and + 86motif regions. mRNA secondary structure prediction analysis revealed three types based on the StemB stem-loop structure: type Ⅰ (no StemB stem-loop), type ⅡA (StemB stem-loop with 3-4 base partial pairing), and type ⅡB (StemB stem-loop with 5 base full pairing). Western blot analysis showed that the CagA protein expression level was the highest in type Ⅰ Hp strains (1.72±0.29) and the lowest in type ⅡB strains (0.81±0.26), with a statistically significant difference between the two types ( P=0.030). The adhesion rate of type Ⅰ Hp strains to AGS cells was (52.90±11.17)%, which was higher than that of type Ⅱ strains [(21.27±6.16)%]. qPCR results showed that the mRNA transcription levels of IL-8 and TNF-α in AGS cells induced by type Ⅰ Hp strains were higher than those induced by type Ⅱ strains (140.23±24.47 vs 76.16±8.76, P=0.069; 55.20±9.04 vs 21.26±6.16, P=0.036). ELISA analysis further indicated that the secretion levels of IL-8 and TNF-α proteins in AGS cells induced by type Ⅰ Hp strains were also higher than those induced by type Ⅱ strains [(344.66±62.62)pg/ml vs (302.13±66.27)pg/ml, P=0.665; (131.04±4.94)pg/ml vs (79.17±11.32)pg/ml, P=0.014]. Conclusions:The cagA 5′UTR region of Hp strains exhibits significant nucleotide sequence variants. Hp strains with no StemB stem-loop (type Ⅰ) in the mRNA secondary structure show significantly increased CagA protein expression and higher bacterial pathogenic potential.

ObjectiveTo explore the impact of subjective vertical perception impairment after stroke on visuospatial cognition, balance, walking and activities of daily living, to investigate the mediating role of visuospatial cognition and lateropulsion. MethodsFrom February to December, 2023, 96 stroke patients were selected from the Rehabilitation Medicine Center of the First Affiliated Hospital of Nanjing Medical University. They were divided into vertical perception impairment group (n = 53) and non-vertical perception impairment group (n = 43). They were assessed with National Institutes of Health Stroke Scale (NIHSS), the Scale for Contraversive Pushing (SCP), Burke Lateropulsion Scale (BLS), Line Bisection Test (LBT), Line Cancellation Test (LCT), Star Cancellation Test (SCT), Subjective Visual Vertical (SVV), Berg Balance Scale (BBS), Holden Functional Ambulation Categories (FAC) and Barthel Index (BI). ResultsScores of SVV orientation, SVV uncertainty, NIHSS, SCT, SCP, BLS, BBS, FAC and BI were worse in the vertical perception impairment group than in the non-vertical perception impairment group (|t| > 2.414, Z = -3.481, P < 0.05). Scores of SVV were correlated with SCT, BLS and BBS (|r| ≥ 0.273, P < 0.05). After controlling for age and gender, SVV orientation score did not directly impact BBS score (β = -0.011, P = 0.920). However, it exerted a partial mediating effect through BLS (effect = -0.173, 95%CI -0.278 to -0.076) and a chain-mediated effect through SCT and BLS (effect = -0.073, 95%CI -0.137 to -0.027), impacting BBS score. ConclusionSubjective vertical perception impairment results in poorer visuospatial cognition, balance, walking and activities of daily living in stroke patients. This influence on balance function is mediated through the mediating effects of visuospatial cognition and lateropulsion.

Objective To explore the clinical application value of MRI fat quantification parameter[verte-bral bone marrow fat fraction(FF)]combined with 25-hydroxyvitamin D[25(OH)D]in predicting fracture risk in patients with osteoporosis.Methods A total of 90 patients with osteoporosis who were admitted to the hospital from January 2023 to April 2024 were selected as the subjects.Among them,50 patients with osteoporotic vertebral com-pression fractures were included in the fracture group,and 40 patients without fractures were included in the control group.All patients underwent the iterative decomposition of water and fat with echo asymmetry and least-squares estimation(IDEAL-IQ)method of MRI to measure the FF of each vertebra among L1-5 and the average FF of L1-5.Serum 25(OH)D level was detected by electrochemiluminescence method.FF and serum 25(OH)D levels of the two groups were compared.The correlation of FF,25(OH)D and bone mineral density(BMD)was analyzed.Multi-variate logistic regression analysis was conducted to screen the risk factors for fracture in patients with osteoporosis.Receiver operating characteristic(ROC)curves were used to evaluate the predictive value of FF,25(OH)D,and their combination for fracture in patients with osteoporosis.Results Patients in the fracture group were older than those in the control group.BMD and serum 25(OH)D level were lower than those of the control group(P<0.05).The FF of L2 and average FF of L1-5 in the fracture group were higher than those in the control group(P<0.05).Correlation analysis results showed that the FF of L2 and the average FF of L1-5 were negatively correlated with BMD(P<0.05),while serum 25(OH)D level was positively correlated with BMD(P<0.05).Multivariate logistic regression analysis showed that age and FF of L2 were independent risk factors for fracture in patients with osteoporo-sis,while BMD and 25(OH)D were protective factors(P<0.05).ROC curves indicated that the AUC values of FF of L2 and 25(OH)D for predicting fracture were 0.714(95%CI:0.606～0.822)and 0.774(95%CI:0.672～0.876).The AUC of joint prediction was 0.923(95%CI:0.867～0.978),which was significantly larger than that of separate prediction(P<0.05).Conclusions FF of L2 and serum 25(OH)D are related to fracture in patients with osteopo-rosis.Age and BMD are factors influencing the occurrence of fracture in patients with osteoporosis.FF of L2 and 25(OH)D have certain predictive value for fracture risk in patients with osteoporosis,and combined detection of the two can improve predictive efficiency.

Objective To investigate the mechanism of 2,6-dimethoxy-1,4-benzoquinone(DMQ),an active ingredients in fermented wheat germ extract,for inhibiting NLRP3 inflammasome activation and alleviating septic shock in mice.Methods Cultured murine bone marrow-derived macrophages(BMDM)stimulated with lipopolysaccharide(LPS)were treated with DMQ,followed by treatment with Nigericin,ATP,and MSU for activating the canonical NLRP3 inflammasome;the non-canonical NLRP3 inflammasome was activated by intracellular transfection of LPS,and AIM2 inflammasome was activated using Poly A:T.In human monocytic THP-1 cells,the effect of Nigericin on inflammasome activation products was examined using Western blotting and ELISA.Co-immunoprecipitation was performed to explore the mechanism of DMQ-induced blocking of NLRP3 inflammasome activation.In a male C57BL/6J mouse model of LPS-induced septic shock treated with 20 and 40 mg/kg DMQ,the levels of IL-1β and TNF-α in the serum and peritoneal lavage fluid were determined using ELISA,and the survival time of the mice within 36 h was observed.Results Treatment with DMQ effectively inhibited LPS-induced activation of canonical NLRP3 inflammasome in mouse BMDM and human THP-1 cells and also inhibited non-canonical NLRP3 inflammasome activation in mouse BMDM,but produced no significant effect on AIM2 inflammasome activation.DMQ significantly blocked the binding between ASC and NLRP3.In the mouse models of septic shock,DMQ treatment significantly reduced the levels of IL-1β in the serum and peritoneal fluid and obviously prolonged survival time of the mice.Conclusion DMQ can effectively block ASC-NLRP3 interaction to inhibit NLRP3 inflammasome activation and alleviate LPS-induced septic shock in mice.

Objective To investigate the mechanism of 2,6-dimethoxy-1,4-benzoquinone(DMQ),an active ingredients in fermented wheat germ extract,for inhibiting NLRP3 inflammasome activation and alleviating septic shock in mice.Methods Cultured murine bone marrow-derived macrophages(BMDM)stimulated with lipopolysaccharide(LPS)were treated with DMQ,followed by treatment with Nigericin,ATP,and MSU for activating the canonical NLRP3 inflammasome;the non-canonical NLRP3 inflammasome was activated by intracellular transfection of LPS,and AIM2 inflammasome was activated using Poly A:T.In human monocytic THP-1 cells,the effect of Nigericin on inflammasome activation products was examined using Western blotting and ELISA.Co-immunoprecipitation was performed to explore the mechanism of DMQ-induced blocking of NLRP3 inflammasome activation.In a male C57BL/6J mouse model of LPS-induced septic shock treated with 20 and 40 mg/kg DMQ,the levels of IL-1β and TNF-α in the serum and peritoneal lavage fluid were determined using ELISA,and the survival time of the mice within 36 h was observed.Results Treatment with DMQ effectively inhibited LPS-induced activation of canonical NLRP3 inflammasome in mouse BMDM and human THP-1 cells and also inhibited non-canonical NLRP3 inflammasome activation in mouse BMDM,but produced no significant effect on AIM2 inflammasome activation.DMQ significantly blocked the binding between ASC and NLRP3.In the mouse models of septic shock,DMQ treatment significantly reduced the levels of IL-1β in the serum and peritoneal fluid and obviously prolonged survival time of the mice.Conclusion DMQ can effectively block ASC-NLRP3 interaction to inhibit NLRP3 inflammasome activation and alleviate LPS-induced septic shock in mice.

Objective:To construct a chronic obstructive pulmonary disease (COPD) assessment test (CAT) score prediction model based on a deep network fused with air data, and to explore its significance.Methods:From February 2015 to December 2017, the outdoor environmental monitoring air data near the residential area of the patients with COPD from the Respiratory Outpatient Clinics of Peking University Third Hospital, Peking University People′s Hospital and Beijing Jishuitan Hospital were collected and the daily air pollution exposure of patients was calculated. The daily CAT scores were recorded continuously. The CAT score of the patients in the next week was predicted by fusing the time series algorithm and neural network to establish a model, and the prediction accuracy of the model was compared with that of the long short-term memory model (LSTM), the LSTM-attention model and the autoregressive integrated moving average model (ARIMA).Results:A total of 47 patients with COPD were enrolled and followed up for an average of 381.60 days. The LSTM-convolutional neural networks (CNN)-autoregression (AR) model was constructed by using the collected air data and CAT score, and the root mean square error of the model was 0.85, and the mean absolute error was 0.71. Compared with LSTM, LSTM-attention and ARIMA, the average prediction accuracy was improved by 21.69%.Conclusion:Based on the air data in the environment of COPD patients, the fusion deep network model can predict the CAT score of COPD patients more accurately.

A new variant of concern for SARS-CoV-2,Omicron(B.1.1.529),was designated by the World Health Organization on November 26,2021.This study analyzed the viral genome sequenc-ing data of 108 samples collected from patients infected with Omicron.First,we found that the enrichment efficiency of viral nucleic acids was reduced due to mutations in the region where the primers anneal to.Second,the Omicron variant possesses an excessive number of mutations compared to other variants circulating at the same time(median:62 vs.45),especially in the Spike gene.Mutations in the Spike gene confer alterations in 32 amino acid residues,more than those observed in other SARS-CoV-2 variants.Moreover,a large number of nonsynonymous mutations occur in the codons for the amino acid residues located on the surface of the Spike protein,which could potentially affect the replication,infectivity,and antigenicity of SARS-CoV-2.Third,there are 53 mutations between the Omicron variant and its closest sequences available in public databases.Many of these mutations were rarely observed in public databases and had a low muta-tion rate.In addition,the linkage disequilibrium between these mutations was low,with a limited number of mutations concurrently observed in the same genome,suggesting that the Omicron vari-ant would be in a different evolutionary branch from the currently prevalent variants.To improve our ability to detect and track the source of new variants rapidly,it is imperative to further strengthen genomic surveillance and data sharing globally in a timely manner.