Bovine neosporosis,a significant disease affecting the livestock industry,is caused by the protozoan parasite Neospora caninum(N.caninum).The current absence of efficacious vaccines and therapeutics necessitates the exploration of novel interventions.Cordycepin,a bioactive nucleo-side derived from Cordyceps militaris,has garnered attention for its diverse pharmacological properties.This study endeavors to elucidate the inhibitory effects of cordycepin on N.caninum in-fection.The cytotoxicity of cordycepin to bovine macrophages was assessed using the CCK-8 assay to ascertain a non-toxic concentration range.The impact of cordycepin on the N.caninum burden within bovine macrophages was evaluated using qPCR analysis and immunofluorescence assays.Additionally,the modulation of cytokine,interferon,and defensin expression induced by N.cani-num in the presence of cordycepin was examined through qRT-PCR analysis.The results showed that cordycepin exhibited negligible cytotoxicity to bovine macrophages at concentrations ranging from 2.812 5 to 180.000 0 μmol/L,compared to the control group.At concentrations of 5.625 0,11.250 0,and 22.500 0 μmol/L,cordycepin significantly reduced the intracellular N.caninum loads and diminished the intensity of intracellular parasite fluorescence.While N.caninum infection downregulated the expression of pro-inflammatory cytokines such as TNF-α,IL-1β,and IL-6,cordycepin treatment robustly induced their expression.Furthermore,although cordycepin treatment reduced the expression levels of IFN-α,IFN-β,and IFN-γ that were upregulated by N.caninum infection,it maintained substantial expression levels.In conclusion,cordycepin demon-strates a promising resistance against N.caninum infection,suggesting its potential as a therapeu-tic agent for the treatment of bovine neosporosis.

Bovine neosporosis,a significant disease affecting the livestock industry,is caused by the protozoan parasite Neospora caninum(N.caninum).The current absence of efficacious vaccines and therapeutics necessitates the exploration of novel interventions.Cordycepin,a bioactive nucleo-side derived from Cordyceps militaris,has garnered attention for its diverse pharmacological properties.This study endeavors to elucidate the inhibitory effects of cordycepin on N.caninum in-fection.The cytotoxicity of cordycepin to bovine macrophages was assessed using the CCK-8 assay to ascertain a non-toxic concentration range.The impact of cordycepin on the N.caninum burden within bovine macrophages was evaluated using qPCR analysis and immunofluorescence assays.Additionally,the modulation of cytokine,interferon,and defensin expression induced by N.cani-num in the presence of cordycepin was examined through qRT-PCR analysis.The results showed that cordycepin exhibited negligible cytotoxicity to bovine macrophages at concentrations ranging from 2.812 5 to 180.000 0 μmol/L,compared to the control group.At concentrations of 5.625 0,11.250 0,and 22.500 0 μmol/L,cordycepin significantly reduced the intracellular N.caninum loads and diminished the intensity of intracellular parasite fluorescence.While N.caninum infection downregulated the expression of pro-inflammatory cytokines such as TNF-α,IL-1β,and IL-6,cordycepin treatment robustly induced their expression.Furthermore,although cordycepin treatment reduced the expression levels of IFN-α,IFN-β,and IFN-γ that were upregulated by N.caninum infection,it maintained substantial expression levels.In conclusion,cordycepin demon-strates a promising resistance against N.caninum infection,suggesting its potential as a therapeu-tic agent for the treatment of bovine neosporosis.

[Purpose]To investigate the potential causal relationships between serum levels of trace elements and head and neck cancers.[Methods]Single nucleotide polymorphism(SNP)of oral cancer,oropharyngeal cancer,laryngeal cancer and thyroid cancer,associated with calcium,copper,iron,magnesium,zinc,were obtained from genome-wide association studies(GWAS).A two-sample bidirectional Mendelian randomization(MR)analysis was performed using the inverse variance weighting(IVW)method by calculating odds ratio(OR)and 95％confidence interval(CI).Pleiotropy was assessed using MR-PRESSO and MR-Egger regression,and sensitivity analysis was conducted via the"leave-one-out"method.[Results]IVW analysis revealed a causal association between serum magnesium levels and the incidence of oral cancer(OR=0.976,95％CI:0.956～0.997,P=0.025),also between thyroid cancer and serum calcium levels(OR=1.008,95％CI:1.001～1.015,P=0.023).No significant causal associations were observed between other trace ele-ments and head and neck cancers(all P＞0.05).[Conclusion]This MR study suggests that serum magnesium levels serve as a protective factor against oral cancer,while thyroid cancer leads to el-evated serum calcium levels.

Background and purpose:In recent years,deep inspiration breath hold(DIBH)based on surface-guided radiation therapy(SGRT)has emerged as one of the most promising techniques in breast cancer radiotherapy.Conventionally,cone-beam CT(CBCT)is used before treatment to verify and correct patient positioning deviations.However,patient position may change during treatment,and pre-fraction CBCT cannot fully reflect real-time positional deviations.Therefore,this study aimed to introduce intra-fraction CBCT into left-sided breast DIBH radiotherapy to monitor and correct real-time deviations,verify whether patients achieve the desired position during treatment,and evaluate the clinical value of intra-fraction CBCT in left-breast radiotherapy.Methods:Clinical data from postoperative left-sided breast cancer patients treated at Fudan University Shanghai Cancer Center between June 2023 and January 2024 were collected.Patients with satisfactory cardiopulmonary function and stable DIBH compliance were included.Pre-fraction CBCT scans were performed before treatment,and data were recorded.During treatment,the gantry was rotated to-180° to trigger kV-level intra-fraction CBCT scanning.After gantry repositioning to the starting angle of the tangential field,MV beam delivery commenced.Intra-fraction CBCT scans were acquired for positional registration,and deviations were compared with pre-fraction CBCT results.Results:A total of 30 eligible patients were included,with 150 pairs of pre-fraction and intra-fraction CBCT images analyzed per patient.The mean registration deviations for intra-fraction CBCT were 26%,52%,and 51%lower than pre-fraction CBCT in the X,Y,and Z axes,respectively(P＜0.05),demonstrating significantly reduced real-time deviations.The probability of intra-fraction deviations being＜6 mm was 100.0%,100.0%,and 99.3%for the X,Y,and Z axes,respectively.Correlation analysis revealed a weak correlation between intra-fraction and pre-fraction deviations in the X-axis(P＜0.05),while no significant correlation was observed for the Y and Z axes(P＞0.05).Conclusion:Intra-fraction CBCT effectively monitors and corrects real-time positional deviations in left-sided breast DIBH radiotherapy,serving as a valuable supplement to conventional pre-fraction CBCT.It minimally impacts treatment efficiency while significantly improving positional accuracy,offering clinical and practical benefits for left-breast cancer radiotherapy.

Background and purpose:In recent years,deep inspiration breath hold(DIBH)based on surface-guided radiation therapy(SGRT)has emerged as one of the most promising techniques in breast cancer radiotherapy.Conventionally,cone-beam CT(CBCT)is used before treatment to verify and correct patient positioning deviations.However,patient position may change during treatment,and pre-fraction CBCT cannot fully reflect real-time positional deviations.Therefore,this study aimed to introduce intra-fraction CBCT into left-sided breast DIBH radiotherapy to monitor and correct real-time deviations,verify whether patients achieve the desired position during treatment,and evaluate the clinical value of intra-fraction CBCT in left-breast radiotherapy.Methods:Clinical data from postoperative left-sided breast cancer patients treated at Fudan University Shanghai Cancer Center between June 2023 and January 2024 were collected.Patients with satisfactory cardiopulmonary function and stable DIBH compliance were included.Pre-fraction CBCT scans were performed before treatment,and data were recorded.During treatment,the gantry was rotated to-180° to trigger kV-level intra-fraction CBCT scanning.After gantry repositioning to the starting angle of the tangential field,MV beam delivery commenced.Intra-fraction CBCT scans were acquired for positional registration,and deviations were compared with pre-fraction CBCT results.Results:A total of 30 eligible patients were included,with 150 pairs of pre-fraction and intra-fraction CBCT images analyzed per patient.The mean registration deviations for intra-fraction CBCT were 26%,52%,and 51%lower than pre-fraction CBCT in the X,Y,and Z axes,respectively(P＜0.05),demonstrating significantly reduced real-time deviations.The probability of intra-fraction deviations being＜6 mm was 100.0%,100.0%,and 99.3%for the X,Y,and Z axes,respectively.Correlation analysis revealed a weak correlation between intra-fraction and pre-fraction deviations in the X-axis(P＜0.05),while no significant correlation was observed for the Y and Z axes(P＞0.05).Conclusion:Intra-fraction CBCT effectively monitors and corrects real-time positional deviations in left-sided breast DIBH radiotherapy,serving as a valuable supplement to conventional pre-fraction CBCT.It minimally impacts treatment efficiency while significantly improving positional accuracy,offering clinical and practical benefits for left-breast cancer radiotherapy.

[Purpose]To investigate the potential causal relationships between serum levels of trace elements and head and neck cancers.[Methods]Single nucleotide polymorphism(SNP)of oral cancer,oropharyngeal cancer,laryngeal cancer and thyroid cancer,associated with calcium,copper,iron,magnesium,zinc,were obtained from genome-wide association studies(GWAS).A two-sample bidirectional Mendelian randomization(MR)analysis was performed using the inverse variance weighting(IVW)method by calculating odds ratio(OR)and 95％confidence interval(CI).Pleiotropy was assessed using MR-PRESSO and MR-Egger regression,and sensitivity analysis was conducted via the"leave-one-out"method.[Results]IVW analysis revealed a causal association between serum magnesium levels and the incidence of oral cancer(OR=0.976,95％CI:0.956～0.997,P=0.025),also between thyroid cancer and serum calcium levels(OR=1.008,95％CI:1.001～1.015,P=0.023).No significant causal associations were observed between other trace ele-ments and head and neck cancers(all P＞0.05).[Conclusion]This MR study suggests that serum magnesium levels serve as a protective factor against oral cancer,while thyroid cancer leads to el-evated serum calcium levels.

Background and purpose:Radiotherapy is an important means to treat pelvic tumors.It is important to improve the accuracy of radiotherapy and enhance the effectiveness.This study aimed to compare and analyze the influence of positioning errors during the radiotherapy of patients using surface guided radiotherapy(SGRT)automatic positioning versus traditional surface marking positioning.Methods:We performed a retrospective analysis of 50 patients with pelvic tumors who received radiotherapy and had simple positioning fixation methods at the Fudan University Shanghai Cancer Center from April 2023 to April 2024.A total of 25 patients were treated using SGRT(observation group),and 25 patients were treated using traditional surface marking(control group).After positioning according to the two positioning techniques,cone beam computed tomography(CBCT)images were collected.These data were compared with the initial reference computed tomography(CT)images within 6 dimensions.The correction error values of the six dimensions were recorded.We calculated the averages,standard deviations,and the number of cases and probabilities in different interval distributions.SPSS 27.0 was used to perform statistical analysis of the data from the two groups of patients.We also analyzed the correlation of patients in the observation group within the 6 dimensions.Results:The non-parametric test showed significant differences in only the LNG and PITCH directions(P＜0.05).The number of cases(probability)in the observation group with errors less than 3 mm and less than 1 were 108(86.4%),93(74.4%),108(86.4%),and 96(76.8%),111(88.8%),107(85.6%),respectively.In the control group,the numbers were 113(90.4%),107(85.6%),118(94.4%),and 71(56.8%),95(76.0%),110(88.0%),respectively.The Spearman correlation analysis showed that in the observation group,the left-right direction had a negative correlation with pitch,roll and yaw,while all other directions were positively correlated.Statistically significant positive correlations(P＜0.05)were found between the superior-inferior and pitch directions,left-right and roll directions,as well as pitch and roll directions,with positive correlation coefficients(r=0.270,r=0.301,and r=0.247).Conclusion:The application of SGRT automatic positioning can achieve the effect of regular surface marking line positioning,has advantages in rotation direction adjustment,and can replace surface marking line positioning where conditions permit.

Objective:To investigate the clinical effect of free fibular flap of great toe without vein in repairing soft tissue defect of finger pulp.Methods:From June 2020 to January 2021, 8 cases of finger pulp soft tissue defect were repaired with free flap of fibular side of great toe without vein, including 6 males and 2 females, with an average age of 32(18-42) years old. The defect area of finger pulp was 1.0 cm×0.5 cm-1.8 cm×0.8 cm. The proper digital artery and digital nerve of the affected finger were anastomosed. All flaps did not carry reflux veins. The donor site of the toe was directly sutured. Postoperative routine anti infection, anti spasm and anticoagulant treatment was performed. Postoperative outpatient regular follow-up.Results:All flaps survived without vascular crisis, and the donor and recipient sites healed in the first stage. All patients were followed-up for an average of 6.4(4-12) months. At the last follow-up, the TPD was 6-8 mm, the hand function was evaluated according to the Trial Standard of Upper Limb Function Evaluation of Hand Surgery Society of Chinese Medical Association. Seven cases were excellent and 1 case was good.Conclusion:The free fibular flap of the great toe can be used to repair the small wound of finger pulp without carrying the draining vein, the flap can still survive. And it can provide a certain reference for the failure of anatomic separation of the draining vein.

Objective:To investigate the effect of BMAL1 gene on the proliferation, migration and invasion ability of radiation-resistant nasopharyngeal carcinoma cell line (5-8FR) and the molecular mechanism. Methods:A multi-target click model was constructed for radiation-resistant nasopharyngeal carcinoma cell line 5-8FR by low-dose fractionated irradiation, and the results of clone formation assay were used to fit the multi-target click model and calculate the sensitization ratio of radiotherapy. The expression levels of PI3K/Akt/MMP-2/9 signaling pathway-related proteins in 5-8FR and control 5-8F cell lines were detected by Western blot. The overexpression and knockdown vectors of BMAL1 gene were constructed and transfected with 5-8F and 5-8F cell lines, respectively. The BMAL1 gene overexpression (pcDNA-BMAL1) and its control (pcDNA) and interference (BMAL1-shRNA) and control (con-shRNA) cell lines were stably transfected with nasopharyngeal carcinoma cell line 5-8F and radiation-resistant cell line 5-8FR, respectively. Western blot was performed to verify the infection efficiency and detect the changes of PI3K/Akt/MMP-2/9 signaling pathway-related proteins after overexpression or interference of BMAL1 gene in both groups of cells. CCK-8 assay, cell scratch test and Transwell chamber test were conducted to investigate the proliferation, migration and invasion capabilities of 5-8FR cell line after overexpression or interference of BMAL1 gene. Results:BMAL1 gene expression was down-regulated, and those of PI3K/Akt pathway proteins and downstream related molecules of MMP-2 and MMP-9 were up-regulated, and TIMP-2 and TIMP-1 expression was down-regulated in nasopharyngeal carcinoma radiation-resistant cell lines. Overexpression of BMAL1 gene inhibited the expression of PI3K/Akt pathway proteins and downstream related molecules of MMP-2 and MMP-9, promoted the expression of TIMP-2 and TIMP-1, and inhibited the proliferation, migration and invasion capabilities of radiation-resistant nasopharyngeal carcinoma cells, while interference with BMAL1 gene yielded the opposite results. Conclusions:BMAL1 gene can reverse the expression of PI3K/Akt/MMP-2/9 signaling pathway-related proteins in radiation-resistant nasopharyngeal carcinoma cell lines and inhibit the proliferation, migration and invasion capabilities of radiation-resistant nasopharyngeal carcinoma cell lines.

Objective:To explore the relationship between expression levels of CLOCK mRNA and protein and the clinical characteristics of patients with nasopharyngeal carcinoma.Methods:The frozen tissue specimens from 33 patients with nasopharyngeal carcinoma in the Affiliated Tumor Hospital of Guizhou Medical University from 2018 to 2019 were collected. Seventeen cases of tissue specimens from patients with nasopharyngeal chronic inflammation in the Affiliated Hospital of Guizhou Medical University in 2019 were collected. From 2008 to 2014, 68 cases of formalin-fixed paraffin-embedding (FFPE) nasopharyngeal carcinoma tissue and 37 cases of FFPE nasopharyngeal chronic inflammation tissue were collected from the Affiliated Tumor Hospital of Guizhou Medical University. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot (WB) were used to detect the mRNA and protein expression levels of CLOCK. The nasopharyngeal carcinoma cells including CNE1, CNE2, 5-8F and the normal nasopharyngeal epithelial cell NP69 were cultured. qRT-PCR was used to detect the expression level of CLOCK mRNA in each cell line at the time points of ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22. The cosine method was used to fit the rhythm of CLOCK gene in nasopharyngeal carcinoma. The protein expression of CLOCK protein was detected by using immunohistochemical method in 68 cases of nasopharyngeal carcinoma and 37 cases of nasopharyngeal chronic inflammation tissue. Survival was analyzed by Kaplan-Meier method and Log rank test, and the influencing factors was analyzed by Cox regression model.Results:The expression levels of CLOCK mRNA in CNE1, CNE2 and 5-8F cells (0.63±0.07, 0.91±0.02 and 0.33±0.04, respectively) were lower than that in NP69 cell (1.00±0.00, P<0.05). The expression levels of CLOCK protein in CNE1, CNE2 and 5-8F cells (0.79±0.06, 0.57±0.05 and 0.74±0.10, respectively) were lower than that of NP69 cells (1.00±0.00, P<0.05). The expressions of CLOCK mRNA in nasopharyngeal carcinoma cells including CEN1, CNE2, 5-8F and normal nasopharyngeal epithelial cell NP69 were different at different time points, with temporal fluctuations. The fluctuation periods of CLOCK mRNA in CNE1, CNE2, 5-8F, and NP69 cells were 16, 14, 22 and 24 hours, respectively. The peak and trough times were ZT10: 40 and ZT18: 40, ZT10 and ZT3, ZT14: 30 and ZT3: 30, ZT12: 39 and ZT0: 39, respectively. CLOCK mRNA and protein expression levels in nasopharyngeal carcinoma tissues (0.37±0.20 and 0.20±0.26, respectively) were lower than those in nasopharyngeal chronic inflammation tissues (1.00±0.00 and 0.51±0.41, respectively, P<0.05). The 1, 3, and 5-year survival rates of patients in the CLOCK protein high expression group (CLOCK protein expression level ≥ 0.178) were 96.2%, 92.1%, and 80.1%, respectively, which were higher than those in the low expression group (CLOCK protein expression level <0.178, 92.9% , 78.6% and 57.1%, respectively, P=0.009). The 1, 3, and 5-year progression-free survival (PFS) rates of patients in the CLOCK protein high expression group were 96.2%, 87.8%, and 87.7%, respectively, which were higher than those in the low expression group (92.7%, 82.2%, and 70.8%, respectively, P=0.105). Compared with the low-expression group (100.0%, 96.9%, and 90.0%, respectively), the 1, 3, and 5-year recurrence-free survival rates of patients in the CLOCK protein high expression group (100.0%, 95.7%, and 95.7%, respectively) were not statistically significant ( P=0.514). Compared with the low-expression group (92.7%, 82.2%, and 79.3%), the 1, 3, and 5-year survival rates without metastasis in the CLOCK protein high expression group (96.2%, 92.0%, and 92.0%, respectively) were not statistically significant ( P=0.136). CLOCK protein expression and T stage were independent prognostic factors of overall survival ( P<0.05). Conclusions:The expression of CLCOK is downregulated in the nasopharyngeal carcinoma cell and nasopharyngeal carcinoma tissues. Clock gene CLOCK is rhythmically expressed in the nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells. Compared with normal nasopharyngeal epithelial cells, the fluctuation period of CLOCK in nasopharyngeal carcinoma cells is shortened. The overall survival of patients in the CLOCK protein high expression group is better than that of low expression group. The expression of CLOCK protein is an independent influencing factor for overall survival. CLOCK gene may be a potential tumor suppressor gene in the nasopharyngeal carcinoma.