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*

BACKGROUND:Osteoporosis is a high-risk factor for dental implant treatment.The preparation of tissue engineering scaffolds with sustained-release Alendronate and its application in oral implant surgery for osteoporosis patients is a current hot topic in oral bone tissue engineering research.OBJECTIVE:To prepare alendronate/chitosan/polyvinyl alcohol composite hydrogel film and characterize its sustained release properties.METHODS:(1)Chitosan/polyvinyl alcohol composite hydrogel films with varying mass ratios(mass ratios of chitosan and polyvinyl alcohol were 3:7,5:5,and 7:3,respectively),chitosan and polyvinyl alcohol hydrogel films were prepared using a physical crosslinking method.By characterizing the morphology,water contact angle,mechanical properties,swelling rate,and cell compatibility of the hydrogel film,a suitable hydrogel film was screened as a carrier of alendronate.(2)Chitosan/polyvinyl alcohol composite hydrogel films containing 0,0.4,1.2,and 2.0 mg/L alendronate were prepared,and rat bone marrow mesenchymal stem cells were co-cultured with the four groups of hydrogel films.The cytocompatibility of the hydrogel films was detected by CCK-8 assay and CD44 immunofluorescence staining.The drug-loaded chitosan/polyvinyl alcohol composite hydrogel films were immersed in PBS.The drug release performance of the composite hydrogel films was detected by ultraviolet-visible spectroscopy.RESULTS AND CONCLUSION:(1)The characterization results showed that with the increase of the mass of polyvinyl alcohol in the hydrogel film,the structural density of the hydrogel film increased,the porosity decreased,the water contact angle(all within 90°),the elongation at break and the compressive strength increased,and the equilibrium swelling rate decreased.It had no effect on the proliferation of rat bone marrow mesenchymal stem cells.Chitosan hydrogel film could promote the adhesion of rat bone marrow mesenchymal stem cells.The 3:7 and 5:5 ratio composite hydrogel films did not affect the adhesion of rat bone marrow mesenchymal stem cells.Polyvinyl alcohol hydrogel film and 7:3 ratio composite hydrogel film inhibited the adhesion of rat bone marrow mesenchymal stem cells.Based on the above results,the 5:5 composite hydrogel film was selected as the carrier of alendronate.(2)CCK-8 assay results showed that the composite hydrogel film containing 0.4 and 1.2 mg/L alendronate had no cytotoxicity.CD44 immunofluorescence staining results showed that the composite hydrogel film containing 0.4 mg/L alendronate did not affect the adhesion of rat bone marrow mesenchymal stem cells,while the composite hydrogel film containing 1.2 and 2.0 mg/L alendronate inhibited the adhesion of rat bone marrow mesenchymal stem cells.Chitosan/polyvinyl alcohol composite hydrogel film containing 0.4,1.2,and 2.0 mg/L alendronate released alendronate in the first 6 hours,and then released alendronate evenly and stably,with a release time of more than 96 hours.(3)The results showed that the chitosan/polyvinyl alcohol composite hydrogel film with a ratio of 5:5 had good physical and chemical properties and cytocompatibility,and could be used as a sustained-release carrier of alendronate.

OBJECTIVE To explore the improvement effect and mechanism of salidroside on radiation-induced parotid gland injury in rats. METHODS Rats were randomly assigned into normal group， radiation group， salidroside low-dose （salidroside-L， 50 mg/kg） group， salidroside high-dose （salidroside-H， 100 mg/kg） group， and salidroside-H+inhibitor （100 mg/kg salidroside+0.1 µmol/kg H-89） group， with 10 rats in each group. Except for the normal group， rats in the other groups were subjected to radiation exposure to establish a model of radiation-induced parotid gland injury. Rats in each group were intraperitoneally injected with the corresponding drug or normal saline， once a day， for 40 consecutive days. After the last administration， the levels of reactive oxygen species （ROS）， cyclic adenosine monophosphate （cAMP）， superoxide dismutase （SOD）， and amylase in serum were detected； the pathological changes of parotid gland tissue were observed； the apoptosis rate of parotid gland tissue cells， the expression levels of B-cell lymphoma-2 （Bcl-2） and its associated X protein （Bax）， mRNA expression levels of interleukin-6 （IL- 6） and tumor necrosis factor-α （TNF-α）， the protein expression levels of type Ⅲ collagen （Col Ⅲ）， vasoactive intestinal peptide （VIP）， and the phosphorylation level of protein kinase A （PKA） in parotid gland tissue were determined. RESULTS Compared with normal group， the levels of ROS， amylase， apoptosis rate， Bax expression level， mRNA expression levels of IL-6 and TNF- α， and protein expression level of Col Ⅲ in the radiation group were significantly increased， while the levels of cAMP， SOD， Bcl-2 expression level， VIP protein expression level and PKA phosphorylation level were significantly decreased （P＜0.05）. Compared with radiation group， the above indicators in the salidroside-L group and salidroside-H group were significantly improved （P＜0.05）， and the improvement in the salidroside-H group was more significant （P＜0.05）； inhibitor H-89 significantly reversed the changes in the above indicators of the salidroside-H group （P＜0.05）. CONCLUSIONS Salidroside can effectively alleviate radiation-induced parotid gland injury in rats， and its mechanism may be related to the activation of the VIP-cAMP pathway.

Objective To investigate the status quo of indwelling needles use in the hospitals at all lev-els in Chongqing city,and to evaluate the normalization and safety of indwelling needles use by referring to Nursing Practice Standards for Intravenous Therapy.Methods A self-designed questionnaire was used in July 2023 to conduct a cross-sectional survey on the use of indwelling needles in the hospitals at all levels in Chongqing city.Results A total of 30 807 patients in 77 hospitals were included,among them 62.67%used indwelling needles.The closed type indwelling needles accounted for 88.87%and the safety type indwelling needles accounted for 41.74%.Non-needle infusion connectors accounted for 37.78%,and steel needle con-nected with heparin caps accounted for 51.69%.95.66%of puncture site was in the upper limb,the non-cor-rosive drug infusion accounted for 87.57%,and the complications occurrence rate was 2.46%.Among the 6 419 surveyed nurses,75.51%removed indwelling needles based on clinical indications.There were statistically significant differences in the usage rates of closed type indwelling needles and non-needle infusion connectors among hospitals at different levels(P<0.05).There were also statistically significant differences in the inci-dence rates of complication and phlebitis(P<0.05).Conclusion The overall application of indwelling needles in the hospitals at all levels in Chongqing city is generally standardized,but the use of safety type indwelling needles and needle-free infusion connectors still needs to be improved.

Objective To investigate the expression and clinical significance of serum microRNA-144-3p(miR-144-3p)and microRNA-126-3p(miR-126-3p)in patients with intracerebral hemorrhage(ICH).Methods From February 2022 to April 2023,120 ICH patients who underwent treatment at our hospital were recruited as the ICH group.They were separated into a good prognosis group and a poor prognosis group based on the modified Rankin Scale(mRS)score After 6 months treatment Additionally,110 healthy individuals who underwent physical examination at our hospital were included as the control group.Clinical data of patients were collected and analyzed.Enzyme linked immunosorbent assay(ELISA)was applied to detect serum levels of tumor necrosis factor-α(TNF-α)and vascular endothelial growth factor(VEGF)indicators.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was applied to detect the expression levels of serum miR-144-3p and miR-126-3p.Pearson method was applied to analyze the correlation between serum miR-144-3p,miR-126-3p,TNF-α,and VEGF levels in ICH patients.Multivariate Logistic regression was applied to analyze the factors affecting the prognosis of ICH patients.ROC curve was applied to analyze the predictive value of serum miR-144-3p and miR-126-3p for the prognosis of ICH patients.Results In the control group,the serum miR-144-3p was 1.02±0.02,the TNF-α was(19.15±6.79)pg/ml,the VEGF level was(1.05±0.18)ng/ml,and the serum miR-126-3p level was 1.04±0.05.The values in the ICH group were 2.12±0.23,(48.32±8.43)pg/ml,(6.56±1.49)ng/ml,and(0.53±0.12),respectively.There was a statistically significant difference between the two groups(P＜0.05).Pearson analysis results showed that serum miR-144-3p was positively correlated with TNF-α and VEGF levels in ICH patients(r=0.496,0.542,P＜0.05),while serum miR-126-3p was negatively correlated with TNF-α and VEGF levels(r=-0.493,-0.526,P＜0.05).There were significant differences in the serum levels of miR-144-3p,miR-126-3p,TNF-α,VEGF,and NIHSS score between different prognostic groups(P＜0.05).Multivariate logistic analysis revealed that serum miR-144-3p,miR-126-3p,TNF-α,and VEGF were all factors affecting the poor prognosis of ICH patients(P＜0.05).The area under the curve(AUC)of serum miR-144-3p,miR-126-3p,and combined prediction of prognosis in ICH patients was 0.851,0.886,and 0.952,respectively,the combined prediction of the two was more valuable(Zcombination-miR-144-3p=3.371,Zcombination-miR-126-3p=2.791,P＜0.05).Conclusion The expression of miR-144-3p in the serum of ICH patients increases,while the expression of miR-126-3p decreases.The two may serve as biological markers for predicting the prognosis of ICH patients,and the combination of the two is more valuable in predicting the prognosis of ICH patients.

Objective To investigate the expression and clinical significance of serum microRNA-144-3p(miR-144-3p)and microRNA-126-3p(miR-126-3p)in patients with intracerebral hemorrhage(ICH).Methods From February 2022 to April 2023,120 ICH patients who underwent treatment at our hospital were recruited as the ICH group.They were separated into a good prognosis group and a poor prognosis group based on the modified Rankin Scale(mRS)score After 6 months treatment Additionally,110 healthy individuals who underwent physical examination at our hospital were included as the control group.Clinical data of patients were collected and analyzed.Enzyme linked immunosorbent assay(ELISA)was applied to detect serum levels of tumor necrosis factor-α(TNF-α)and vascular endothelial growth factor(VEGF)indicators.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was applied to detect the expression levels of serum miR-144-3p and miR-126-3p.Pearson method was applied to analyze the correlation between serum miR-144-3p,miR-126-3p,TNF-α,and VEGF levels in ICH patients.Multivariate Logistic regression was applied to analyze the factors affecting the prognosis of ICH patients.ROC curve was applied to analyze the predictive value of serum miR-144-3p and miR-126-3p for the prognosis of ICH patients.Results In the control group,the serum miR-144-3p was 1.02±0.02,the TNF-α was(19.15±6.79)pg/ml,the VEGF level was(1.05±0.18)ng/ml,and the serum miR-126-3p level was 1.04±0.05.The values in the ICH group were 2.12±0.23,(48.32±8.43)pg/ml,(6.56±1.49)ng/ml,and(0.53±0.12),respectively.There was a statistically significant difference between the two groups(P＜0.05).Pearson analysis results showed that serum miR-144-3p was positively correlated with TNF-α and VEGF levels in ICH patients(r=0.496,0.542,P＜0.05),while serum miR-126-3p was negatively correlated with TNF-α and VEGF levels(r=-0.493,-0.526,P＜0.05).There were significant differences in the serum levels of miR-144-3p,miR-126-3p,TNF-α,VEGF,and NIHSS score between different prognostic groups(P＜0.05).Multivariate logistic analysis revealed that serum miR-144-3p,miR-126-3p,TNF-α,and VEGF were all factors affecting the poor prognosis of ICH patients(P＜0.05).The area under the curve(AUC)of serum miR-144-3p,miR-126-3p,and combined prediction of prognosis in ICH patients was 0.851,0.886,and 0.952,respectively,the combined prediction of the two was more valuable(Zcombination-miR-144-3p=3.371,Zcombination-miR-126-3p=2.791,P＜0.05).Conclusion The expression of miR-144-3p in the serum of ICH patients increases,while the expression of miR-126-3p decreases.The two may serve as biological markers for predicting the prognosis of ICH patients,and the combination of the two is more valuable in predicting the prognosis of ICH patients.

BACKGROUND:Osteoporosis is a high-risk factor for dental implant treatment.The preparation of tissue engineering scaffolds with sustained-release Alendronate and its application in oral implant surgery for osteoporosis patients is a current hot topic in oral bone tissue engineering research.OBJECTIVE:To prepare alendronate/chitosan/polyvinyl alcohol composite hydrogel film and characterize its sustained release properties.METHODS:(1)Chitosan/polyvinyl alcohol composite hydrogel films with varying mass ratios(mass ratios of chitosan and polyvinyl alcohol were 3:7,5:5,and 7:3,respectively),chitosan and polyvinyl alcohol hydrogel films were prepared using a physical crosslinking method.By characterizing the morphology,water contact angle,mechanical properties,swelling rate,and cell compatibility of the hydrogel film,a suitable hydrogel film was screened as a carrier of alendronate.(2)Chitosan/polyvinyl alcohol composite hydrogel films containing 0,0.4,1.2,and 2.0 mg/L alendronate were prepared,and rat bone marrow mesenchymal stem cells were co-cultured with the four groups of hydrogel films.The cytocompatibility of the hydrogel films was detected by CCK-8 assay and CD44 immunofluorescence staining.The drug-loaded chitosan/polyvinyl alcohol composite hydrogel films were immersed in PBS.The drug release performance of the composite hydrogel films was detected by ultraviolet-visible spectroscopy.RESULTS AND CONCLUSION:(1)The characterization results showed that with the increase of the mass of polyvinyl alcohol in the hydrogel film,the structural density of the hydrogel film increased,the porosity decreased,the water contact angle(all within 90°),the elongation at break and the compressive strength increased,and the equilibrium swelling rate decreased.It had no effect on the proliferation of rat bone marrow mesenchymal stem cells.Chitosan hydrogel film could promote the adhesion of rat bone marrow mesenchymal stem cells.The 3:7 and 5:5 ratio composite hydrogel films did not affect the adhesion of rat bone marrow mesenchymal stem cells.Polyvinyl alcohol hydrogel film and 7:3 ratio composite hydrogel film inhibited the adhesion of rat bone marrow mesenchymal stem cells.Based on the above results,the 5:5 composite hydrogel film was selected as the carrier of alendronate.(2)CCK-8 assay results showed that the composite hydrogel film containing 0.4 and 1.2 mg/L alendronate had no cytotoxicity.CD44 immunofluorescence staining results showed that the composite hydrogel film containing 0.4 mg/L alendronate did not affect the adhesion of rat bone marrow mesenchymal stem cells,while the composite hydrogel film containing 1.2 and 2.0 mg/L alendronate inhibited the adhesion of rat bone marrow mesenchymal stem cells.Chitosan/polyvinyl alcohol composite hydrogel film containing 0.4,1.2,and 2.0 mg/L alendronate released alendronate in the first 6 hours,and then released alendronate evenly and stably,with a release time of more than 96 hours.(3)The results showed that the chitosan/polyvinyl alcohol composite hydrogel film with a ratio of 5:5 had good physical and chemical properties and cytocompatibility,and could be used as a sustained-release carrier of alendronate.

In order to establish a rapid,specific and sensitive detection method for Streptococcus equi subspecies zooepidemicus(SEZ)and to understand the infection status of SEZ in horses ente-ring China,specific primers were designed and synthesized based on the conserved gene comB of standard strain SEZ(ATCC 43079)in this work.Then,the pMD19-T-comB recombinant plasmid was constructed and used as a standard positive template.After that,the fluorescence-based quantitative PCR(qPCR)detection method based on SYBR Green Ⅰ dye was established.Totally,477 equine entry serum samples from 6 countries,including Netherlands,Belgium,Japan,Germa-ny,Argentina and New Zealand,during 2018 to 2023,were randomly selected and detected for SEZ by the qPCR method.Results showed that the established qPCR method had specific amplification for only SEZ,which illustrated a good specificity.Sensitivity test of the method showed that the limited detection amount was 4.58 X101 copies/μL.And the repeatability test showed that the coef-ficient of variation of intra-batch repeatability was less than 0.5％,while the inter-batch repeat-ability was less than 3.0％,which indicated good repeatability and high stability.Retrospective a-nalysis showed that totally 11 of 477 positive samples were detected,with a relatively low positive rate of 2.31％(11/477).Among them,all the 40 samples from Netherlands in 2018 were negative(0/40).In the samples of 2019,one positive was detected from Belgium(1/20),while all other 36 samples which form Japan and Germany were negative.In the samples of 2021,three samples(3/34)from Japan and one sample(1/20)from Argentina were positive,and all the other 40 samples from the Netherlands were negative.In the samples of 2022,76 samples from Netherlands were all negative.While in the 2023,5(5/126)of 126 samples from Netherlands and one(1/88)of 88 from New Zealand were found positive with SEZ.To summarize,The SYBR Green Ⅰ qPCR method for the diagnosis of SEZ was successfully established,and it could provide necessary technical support for the rapid quarantine of China's entry-exit and port departments,as well as the epidemiological investigation of the disease.

In order to understand the prevalence of Akabane disease(AKAD)in Guizhou Province and the molecular characteristics of the isolates,the whole-genome sequence of a strain of Akabane virus(AKAV)from a bovine AKAD-positive sample was determined and analyzed.The genotype and genetic variation of the strain were also explored.Based on the conserved S sequence,a fluores-cence quantitative PCR(qPCR)detection method was established and applied for the investigation of AKAV infection status in four large-scale beef cattle farms of Guizhou.Results showed that the S,M and L fragments of the bovine strain were highly homologous to the Tianjin strain(TJ2016/China/2016)and the Australian strain(JaLAB39/Australia/1959),where they were in the same evolutionary branch and belonged to genotype Ⅱ.Sensitivity assay found that the lowest detection limit was 2.5 X 101 copies/μL.Specificity assay showed the established method detected only AKAV with no amplification on bovine bluetongue virus(BLUV),Pasteurella multocida(PM),bovine infectious rhinotracheitis virus(IBRV)and bovine Mycoplasma bovis.The variation coefficients of inter-and intra batches in the repeatability test were both lower than 2.26％.These findings illus-trated that the established qPCR method had high sensitivity,good specificity and repeatability.A total of 298 serum samples from 4 large-scale beef cattle farms in Qianxi City and Huangping County of Guizhou Province were collected and tested for AKAV by the method.Out of 298 sam-ples,25 positive samples(25/298)were detected as positive with a positive rate of 8.39％.In sum-mary,this work provided the reference data for a deep understanding of the molecular prevalence of AKAV in Guizhou Province and laid foundation for the prevention and control of AKAD.

Objective To systematically evaluate the incidence rate of low-level viremia(LLV)in chronic hepatitis B(CHB)patients and related influencing factors,and to provide evidence-based medicine evidence for effective intervention and prevention of LLV in clinical practice.Methods This study was conducted according to the PRISMA guideline,with a PROSPERO registration number of CRD42023455304.CNKI,Wanfang Data,VIP,SinoMed,PubMed,Embase,Web of Science,and the Cochrane library were searched for observational studies on LLV and related influencing factors in CHB patients published up to July 21,2023.Stata 16.0 software was used to perform the meta-analysis.Results A total of 12 articles were included,with a total sample size of 3408 cases,among whom there were 1181 patients with LLV.The meta-analysis showed that the incidence rate of LLV was 32.8%(95%confidence interval[CI]:27.6%—38.3%)in treatment-experienced CHB patients.High HBsAg quantification(odds ratio[OR]=2.107,95%CI:1.782—2.491,P<0.001),positive HBeAg(OR=3.258,95%CI:2.629—4.038,P<0.001),high HBV DNA level at baseline(OR=1.286,95%CI:1.157—1.430,P<0.001),and history of entecavir treatment(OR=3.089,95%CI:1.880—5.074,P<0.001)were risk factors for LLV;duration of antiviral therapy≥3 years(OR=0.175,95%CI:0.093—0.331,P<0.001)and high alanine aminotransferase level at baseline(OR=0.985,95%CI:0.978—0.992,P<0.001)were protective factors against LLV.The sensitivity analysis showed no significant change in effective value,suggesting that the results of the meta-analysis were relatively stable.The funnel plot of the studies included was basically symmetrical,and the results of the Egger's test and the Begg's test suggested that there was no obvious publication bias in the articles included.Conclusion Clinicians should guide decision making based on the influencing factors for LLV and related clinical evidence,so as to reduce long-term clinical risks and avoid adverse outcomes.