Objective To establish a fluorescent recombinase-aided amplification (RAA) assay for detection of Strongyloides stercoralis nucleic acid and to preliminarily evaluate its performance. Methods Six sets of specific primers targeting S. stercoralis 18S ribosomal RNA (18S rRNA) gene and one fluorescent probe were designed and synthesized. The optimal primer-probe set was determined through systematic screening and optimization to establish the fluorescent RAA assay. The assay was evaluated using S. stercoralis genomic DNA at concentrations of 100, 10, and 1 pg/μL, and 100, 10, and 1 fg/μL, as well as recombinant pUC57 plasmids containing the target gene fragments at 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², 1 × 10¹, 1 × 10⁰ copies/reaction, to determine the analytical sensitivity. Genomic DNA from Ascaris lumbricoides, Ancylostoma duodenale, Enterobius vermicularis, Angiostrongylus cantonensis, Trichinella spiralis, Clonorchis sinensis, Schistosoma japonicum, and Taenia saginata was used to assess assay specificity. A total of 25 stool samples from patients suspected of S. stercoralis infection were tested by the modified Baermann funnel technique, PCR, and the established fluorescent RAA assay. The sensitivity, specificity, concordance rate and their 95% confidence intervals (CI) of these three techniques were estimated, and agreement between methods was evaluated using the Kappa coefficient. Results Exo-4 was identified as the optimal primer set screened from the six primer sets, and the best amplification performance was achieved when the final concentrations of the forward and reverse primers were 0.44 μmol/L and a probe concentration was 0.20 μmol/L. The limit of detection of the fluorescent RAA assay was 100 fg/μL for genomic DNA of S. stercoralis and 1 × 10⁰ copies/reaction for recombinant plasmids. Specific fluorescence signals were detected within 5 min, with no cross-reactivity observed with A. lumbricoides, A. duodenale, E. vermicularis, A. cantonensis, T. spiralis, C. sinensis, S. japonicum, or T. saginata. Among the 25 clinical stool samples from patients suspected of S. stercoralis infections, the modified Baermann funnel technique and fluorescent RAA assay detected 19 positives and 6 negatives, whereas PCR detected 18 positives and 7 negatives. The fluorescent RAA assay showed a sensitivity of 100.00% [95% CI: (82.35%, 100.00%)], specificity of 100.00% [95% CI: (54.07%, 100.00%)], concordance rate of 100.00% [95% CI: (86.28%, 100.00%)], and a Kappa coefficient of 1.00 [95% CI: (1.00, 1.00)] (P < 0.001) relative to the modified Baermann funnel technique, and a sensitivity of 100.00% [95% CI: (81.47%, 100.00%)], specificity of 85.71% [95% CI: (42.13%, 99.64%)], concordance rate of 96.00% [95% CI: (79.65%, 99.90%)], and a Kappa coefficient of 0.90 [95% CI: (0.70, 1.00)] (P < 0.001). Positive amplification products emitted green fluorescence under a portable blue-light device, enabling visual interpretation of results. Conclusions The fluorescent RAA assay established in this study is rapid, highly sensitive, and highly specific. It enables detection of S. stercoralis nucleic acid under isothermal conditions and allows visual interpretation of results, providing a novel tool for rapid clinical diagnosis and field screening of S. stercoralis infections.

Objective To investigate the regulatory role of the programmed cell death protein 1 (PD-1) and its ligand programmed cell death protein ligand 1 (PD-L1) signaling on the subtypes and functions of natural killer (NK) cells at the maternal-fetal interface during the second trimester in mice following Toxoplasma gondii infection during the first trimester. Methods Twelve 6- to 8-week-old female mice of the C57BL/6J strain were divided into a control group and an infection group, of 6 mice in each group. On the 6.5th day of pregnancy (Gd6.5), each pregnant mouse in the infection group was intraperitoneally injected with 150 tachyzoites of the Toxoplasma gondii PRU strain, while mice in the control group were injected with an equal volume of physiological saline. On the 12.5th day of pregnancy (Gd12.5), uterus and placenta tissues were sampled from pregnant mice for pathological observations, and the mRNA expression levels of PD-1, PD-L1, and tumor necrosis factor-α (TNF-α) were quantified in uterus and placenta tissues. The PD-1 and DX5 expression was measured on NK cells at the maternal-fetal interface using flow cytometry. In addition, the in vitro JEG-3 trophoblast cells and NK-92MI cells co-culture system was established as the control group, and the addition of T. gondii tachyzoites in the co-culture system served as the infection group. The PD-1, PD-L1, and DX5 mRNA expression was quantified in cells using real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) assay, and the TNF-α concentration was measured in the cell culture supernatant using enzyme-linked immunosorbent assay (ELISA). Results On Gd12.5, clear and intact cellular structures of placental decidual tissues were seen in pregnant mice in the control group, with no remarkable abnormal changes found in the uterine columnar epithelial cells, and inflammatory cell infiltration and blood stasis at varying degrees were found in uterine and placental tissues from pregnant mice in the infection group. The relative PD-1, PD-L1, and TNF-α mRNA expression was (1.004 ± 0.004), (1.001 ± 0.001), and (1.001 ± 0.001) in uterine tissues from pregnant mice in the control group and (2.480 ± 0.720), (3.355 ± 0.920), and (2.391 ± 0.073) in the infection group, respectively. The relative PD-1, PD-L1, and TNF-α mRNA expression was (1.007 ± 0.010), (1.006 ± 0.006), and (1.001 ± 0.001) in the uterine tissues in the control group and (6.948 ± 1.918), (3.225 ± 1.034), and (1.536 ± 0.150) in the infection group, respectively. The relative PD-1, PD-L1, and TNF-α mRNA expression was higher in both the uterine (t = 3.55, 4.43 and 33.02, all P values < 0.05) and placental tissues (t = 5.36, 3.72 and 6.18, all P values < 0.05) in the infection group than in the control group. Flow cytometry showed that the proportions of PD-1⁺ NK cells, PD-1⁺ DX5⁺ NK cells, and DX5⁺ NK cells were (12.200 ± 1.082)%, (9.373 ± 7.728)%, and (44.000 ± 4.095)% in uterine tissues from pregnant mice in the control group, and (21.733 ± 1.630)%, (18.767 ± 1.242)%, and (73.367 ± 0.611)% in the infection group, respectively. The proportions of PD-1⁺ NK cells, PD-1⁺ DX5⁺ NK cells, and DX5⁺ NK cells were (1.100 ± 0.510)%, (2.277 ± 1.337)%, and (96.167 ± 2.831)% in placental tissues from mice in the control group, and (26.867 ± 9.722)%, (23.433 ± 6.983)%, and (82.467 ± 2.248)% in the infection group, respectively. The proportions of PD-1⁺ NK cells (t = 8.45, P < 0.05) and DX5⁺ NK cells (t = 12.29, P < 0.05) were higher in uterine tissues from pregnant mice in the infection group than in the control group, and no significant difference was seen in the proportion of PD-1⁺ DX5⁺ NK cells (Z = -1.09, P > 0.05). The proportions of PD-1⁺ NK cells (t = 4.58, P < 0.05) and PD-1⁺ DX5⁺ NK cells (t = 5.15, P < 0.05) were higher in placental tissues from pregnant mice in the infection group than in the control group, while the proportion of DX5⁺ NK cells was lower in the infection group than in the control group (t = -6.56, P < 0.05). RT-qPCR assay revealed that the relative PD-1, PD-L1, and DX5 mRNA expression was (1.010 ± 0.005), (1.002 ± 0.003), and (1.001 ± 0.001) in the JEG-3 cells and NK92MI cells co-culture system and (3.638 ± 1.258), (0.397 ± 0.158), and (4.267 ± 1.750) in the control group, and ELISA measured that the TNF-α concentration was higher in the cell culture supernatant in the infection group [(22.056 ± 3.205) pg/mL] than in the control group [(12.441 ± 0.001) pg/mL] (t = 5.20, P < 0.05). The PD-1(t = 3.62, P < 0.05) and DX5 mRNA expression (t = 3.23, P < 0.05) was higher in the infection group than in the control group, and the PD-L1 mRNA expression was lower in the infection group than in the control group (t = -6.63, P < 0.05). Conclusions Following T. gondii infection, both PD-L1 expression and PD-1 expression on DX5⁺ NK cells at the maternal-fetal interface are upregulated in mice during the second trimester; however, the proportion of DX5⁺ NK cells decreases. These findings suggest that PD-1/PD-L1 signaling may suppress NK cell functions by modulating DX5⁺ NK cell subsets.

Objective:To investigate the effect of combined use of midazolam and remifentanil without muscle relaxant on tracheal intubation in female patients undergoing breast surgery.Methods:A retrospective analysis was performed on 40 female patients with breast disease who underwent tracheal intubation and general anesthesia at The First Affiliated Hospital of Zhengzhou University between January 2023 and June 2023. These patients were divided into a control group ( n = 20) and an observation group ( n = 20) based on whether muscle relaxants were applied at the time of intubation. The control group received intravenous rocuronium bromide, whereas the observation group did not use muscle relaxants. Both groups were intravenously administered midazolam (0.1 mg/kg) and remifentanil (4 μg/kg) prior to tracheal intubation. The intubation conditions were evaluated based on factors such as the ease of inserting the laryngoscope and the patient's response to intubation, including coughing. Results:There were no statistically significant differences in age, height, and body mass between the two groups (all P > 0.05). The excellent rate of intubation conditions was significantly lower in the observation group compared with the control group [45% (9/20) vs. 85% (17/20), χ2 = 7.03, P = 0.008). The good rate of intubation conditions was significantly higher in the observation group compared with the control group [40% (8/20) vs. 5% (1/20), χ2 = 7.03, P < 0.05]. There was no statistically significant difference in the excellent and good rates of intubation conditions between the observation and control groups [85% (17/20) vs. 90% (18/20), χ2 = 0.23, P > 0.05]. No significant difference in intraoperative awareness score was observed between the observation and control groups [(2.59 ± 0.44) points vs. (2.61 ± 0.31) points, P > 0.05]. None of the patients in either group exhibited any episodes of arrhythmias. Furthermore, no adverse reactions such as muscle stiffness, nausea, vomiting, or skin itching were observed in either group following the surgical procedure. Conclusion:Without the use of muscle relaxants, intravenous administration of midazolam at 0.1 mg/kg and remifentanil at 4 μg/kg for tracheal intubation in female patients undergoing breast surgery can offer excellent intubation conditions, ensuring that the patient remains unconscious throughout the surgical procedure.

Strongyloides stercoralis is an opportunistic pathogenic parasite that can cause severe strongyloidiasis and even death among immunocompromised individuals. Previous clinical studies have reported cases co-infected with S. stercoralis and other pathogens, such as parasites, viruses, bacteria and fungi. This review summarizes strongyloidiasis patients co-infected with pathogens, and analyzes the impact of co-infection on strongyloidiasis, so as to provide insights into the reduction of the morbidity and mortality of disorders associated with S. stercoralis infections.

Objective To investigate the dynamic expression of cluster of differentiation 47 (CD47) and its ligands signaling regulatory protein α (SIRPα) and thrombospondin-1 (TSP-1) in mice infected with Toxoplasma gondii in the second and third trimesters.. Methods C57BL/6J mice (6 to 8 weeks old) were used for modeling T. gondii infection in the first trimester, and the pregnant mice were randomly divided into the normal control and infection groups, of 10 mice in each group. Pregnant mice in the infection group were intraperitoneally injected with 150 T. gondii tachyzoites on gestational day (Gd) 6.5, while pregnant mice in the normal control group were intraperitoneally injected with the same volume of physiological saline at the same time. The uterine and placental specimens were collected from all pregnant mice on Gd12.5 and Gd18.5, and the pregnant outcomes were recorded. The pathological damages of mouse uterine and placental specimens were observed using hematoxylin-eosin (HE) staining on Gd12.5 and Gd18.5. The relative expression of CD47, SIRPα, TSP-1, surface antigen 1 (SAG1), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-13 mRNA was quantified in mouse uterine and placental specimens using real-time fluorescence quantitative PCR (qPCR) assay, and the CD47, SIRPα, TSP-1 expression was determined in mouse uterine and placental specimens using immunohistochemical staining. Results As compared with those in the normal control group, the pregnant mice in the infection group showed back arching, bristling, trembling and listlessness during pregnancy, and several mice presented virginal bleeding and abortion. Pathological examinations showed inflammatory cell infiltration, congestion and necrosis in uterine and placental specimens of pregnant mice in the infection group, a higher abortion rate of pregnant mice was seen in the infection group than in the normal control group on Gd12.5 (χ² = 20.405, P < 0.001) and Gd18.5 (χ² = 28.644, P < 0.001). qPCR assay showed significant differences in the expression of CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 genes in mouse placental specimens between the normal control and infection groups on Gd12.5 and Gd18.5 [F′ (F) = 37.511, 29.337, 97.343, 53.755, 67.188, 21.145, 8.658 and 13.930, all P values < 0.001]. Higher CD47, SIRPα and TSP-1 gene expression was quantified in mouse placental specimens in the infection group than in the normal control group on Gd12.5 (all P values < 0.01), and lower CD47, SIRPα and TSP-1 gene expression was quantified in the infection group than in the normal control group on Gd18.5 (all P values < 0.001), while higher SAG1 gene expression was detected in placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01). In addition, higher INF-γ and IL-2 expression and lower IL-4 and IL-13 expression was detected in mouse placental specimens in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001), and there were significant differences in the CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 gene expression in uterine specimens of pregnant mice between the normal control and infection groups on Gd12.5 and Gd18.5 [H(F′ and F) = 14.951, 25.977, 18.711, 48.595, 39.318, 14.248 and 15.468, all P values < 0.01], and higher CD47 and TSP-1 expression was detected in mouse uterine specimens in the infection group than in the control group on Gd12.5 and Gd18.5 (all P values < 0.01); however, no significant difference was found in the SIRPα expression (P > 0.05). Higher SAG1 expression was detected in uterine specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01), and higher INF-γ and IL-2 gene expression and lower IL-4 and IL-13 gene expression was found in the placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001). Spearman correlation analysis showed that the CD47 gene expression correlated positively with IFN-γ (r_s = 0.735, P < 0.05) and IL-2 (r_s = 0.655, P < 0.05) and negatively with IL-4 (r_s = −0.689, P < 0.05) and IL-13 expression (r_s = −0.795, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd12.5, and the CD47 gene expression correlated negatively with IFN-γ (r_s = −0.745, P < 0.05) and IL-2 expression (r_s = −0.816, P < 0.05) and positively with IL-4 (r_s = 0.704, P < 0.05) and IL-13 (r_s = 0.802, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd18.5. Immunohistochemical staining showed mild CD47, SIRPα and TSP-1 expression in uterine and placental specimens of pregnant mice in the normal control group on Gd12.5 and Gd18.5, strong CD47, SIRPα and TSP-1 expression in the placental specimens of pregnant mice in the infection group on Gd12.5 and strong CD47 and TSP-1 expression in the uterine specimens of pregnant mice in the infection group on Gd12.5. Conclusions T. gondii infection in the first trimester may cause abnormal expression of CD47 and its ligands SIRPα and TSP-1 in the maternal-fetal interface of pregnant mice in the second and third trimesters, which may be associated with the immune escape of T. gondii at the maternal-fetal interface.

Objective To predict the structure and antigenic epitope of the Strongyloides stercoralis serine protease inhibitor 1 (Ss-SRPN-1) protein using bioinformatics tools, and to construct prokaryotic expression plasmids for expression of recombinant Ss-SRPN-1 protein, so as to provide the basis for unraveling the function of the Ss-SRPN-1 protein. Methods The amino acid sequence of the Ss-SRPN-1 protein was downloaded from the NCBI database, and the physicochemical properties, structure and antigenic epitopes of the Ss-SRPN-1 protein were predicted using bioinformatics tools, including ExPASy, SWISS-MODEL and Protean. Primers were designed according to the nucleotide sequences of Ss-SRPN-1, and the Ss-SRPN-1 gene was amplified, cloned and sequenced with genomic DNA extracted from the infective third-stage larvae of S. stercoralis as a template. The Ss-SRPN-1 protein sequence was cloned into the pET28a (+) expression vector and transformed into Escherichia coli BL21 (DE) cells for induction of the recombinant Ss-SRPN-1 protein expression. The recombinant Ss-SRPN-1 protein was then purified and identified using Western blotting and mass spectrometry. Results Bioinformatics analysis showed that the Ss-SRPN-1 protein, which was composed of 372 amino acids and had a molecular formula of C₁₉₄₈H₃₀₄₆N₄₈₈O₅₇₅S₁₆, was a stable hydrophilic protein, and the subcellular localization of the protein was predicted to be extracellular. The Ss-SRPN-1 protein was predicted to contain 11 dominant B-cell antigenic epitopes and 20 T-cell antigenic epitopes. The Ss-SRPN-1 gene with a length of 1 119 bp was successfully amplified, and the recombinant plasmid pET28a (+)/Ss-SRPN-1 was constructed and transformed into E. coli BL21(DE) cells. The expressed recombinant Ss-SRPN-1 protein had a molecular weight of approximately 43 kDa, and was characterized as a Ss-SRPN-1 protein. Conclusions The recombinant Ss-SRPN-1 protein has been expressed successfully, and this recombinant protein may be a potential vaccine candidate against strongyloidiasis.

In order to fulfill the new requirement of educational and teaching reform in the new era, the Teaching and Research Section of Parasitology in Guangxi Medical University have constructed online courses of Parasites Invasion— Human Parasitology on the platform of Xueyin Online, providing resources in professional learning and general education. In the construction concept, we recombine teaching content to give consideration to both scientificity and interest. In terms of the curriculum content, the curriculum includes micro lecture, PPT, in-lass quiz, case analysis and other diversified teaching resources. In the curriculum management, teachers interact with students in real time with the help of various functions of the platform to fully play the role of supervision. In the assessment, teachers pay attention to students' learning process and carry out comprehensive evaluation. Since the online course was put into operation, good teaching effects has been achieved.

Objective To estimate the diagnostic value of cytology, DNA-ICM(DNA-image cytometry),cytology combined with DNA-ICM for pancreatic malignancy,and to explore the cut-off value for DNA-ICM. Methods Patients with suspicious pancreatic malignancy were retrospectively identified. In total,145 EUS-FNA specimens acquired from 140 separate patients were examined by cytology and DNA-ICM. Diagnostic values among cytology, DNA-ICM and the combination of the techniques in detecting pancreatic malignancy were compared. Results Compared with cytology, DNA-ICM had a lower sensitivity (63.0% VS 82.4%)and accuracy(69.7% VS 85.5%). After combining the techniques, the diagnostic value for pancreatic malignancy significantly improved compared with that by cytology(0.941 VS 0.912, P=0.007 0)or DNA-ICM only(0.941 VS 0.815, P<0.000 1). By using the Youden index, the cut-off value for DNA-ICM to detect pancreatic malignancy was one cell with DI(DNA index)≥2.5. Notably,with this standard, the sensitivity and accuracy of DNA-ICM significantly increased to 72.3% and 77.2%, and those of the combined techniques increased to 91.6% and 93.1%, respectively. Conclusion Automated DNA-ICM is an objective and effective method for pancreatic malignancy. Although DNA-ICM has a lower diagnostic value than that of conventional cytology, an improved value was obtained after combining the techniques.

Objective To explore the value of applying flipped classroom in human parasitology. Methods Totally 430 students of 5-year program were divided into experimental group and control group. The experimental class received human parasitology teaching through flipped classroom teaching mode, while the control class received traditional teaching. The effect of teaching was evaluated by questionnaire and examination. The data were analyzed through t-test. Result Meanwhile, statistical difference was found in aver age score of total between experiment group and control group [(68.2 ±8.6) vs. (66.6 ±11.0), P=0.032]. There was also statistical difference in average score of comprehensive analysis [(16.4±3.2) vs. (16.1 ±3.9), P=0.038]. Questionnaire survey of satisfaction showed that 191 students of experimental class (90.95%) felt new teaching mode could improve autonomous learning ability, 199 students (94.76%) in-creased interest in learning;185 students (88.10%) had more interactive with teachers, 178 students (84.76%) enhanced cooperation between st udents, 186 students (88.57%) approved of small group discussion learning and 165 students (78.57%) had no extra burden. Conclusion Flipped classroom teaching mode can improve students' autonomous learning ability and cultivate their abilities of independent thinking, cooperation, criti-cism, innovation, analyzing and solving problems. Thus this new teaching mode is worthy of reference and popularization.

Objective To evaluate the therapeutic effect of hydrogen-rich saline on allergic contact dermatitis (ACD) in mice and to explore its underlying mechanisms.Methods Forty mice were equally divided into 4 groups:control group,control treatment group,ACD group and ACD treatment group.ACD was induced by repetitive topical application of dinitrofluorobene (DNFB) to the left ear of mice on day 1,2 and 5.Hydrogen-rich saline was intraperitoneally given to the mice in the ACD treatment group at a dose of 5 ml/kg per day from day 1 to 5.On day 6,the mice were sacrificed,ear tissue was removed from them and subjected to measurement of thickness and weight,detection of tumor necrosis factor (TNF)-α,interleukin (IL)-6,IL-17 and interferon (IFN)-γ expression by enzyme linked immunosorbent assay,as well as numeration of inflammatory cells in lesions after hematoxylin-eosin (HE) staining.Data were processed by SPSS 18.0 software,and statistical analysis was carried out by one-way analysis of variance and least significant difference (LSD) procedure.Results Compared with the control group,the ACD group showed a significant increase in lesion score (7.33 ± 1.53 vs.0,P ＜ 0.05),differences in the thickness ((0.73 ± 0.15) mm vs.(0.13 ± 0.05) mm,P ＜ 0.05) and swelling degree (expressed as tissue weight:(18.67 ± 3.05) mg vs.(3.33 ± 1.52) mg,P ＜ 0.05) between the left and right ear,expressions of TNF-α ((1475.52 ± 233.81) pg/mg vs.(239.01 ± 52.39) pg/mg,P＜ 0.05),IL-6 ((184.65 ± 78.39) pg/mg vs.(42.28 ± 17.64) pg/mg,P＜ 0.05),IL-17 ((628.56 ± 201.44) pg/mg vs.(127.58 ± 50.28) pg/mg,P＜ 0.05) and IFN-γ ((197.72 ± 37.81) pg/mg vs.(24.57 ± 8.31) pg/mg,P ＜ 0.05),and the number of inflammatory cells per square millimetre in the left ear tissue (752.00 ± 166.06 vs.127.33 ± 77.18,P ＜ 0.05).However,hydrogen-rich saline treatment induced a statistical decrease in all of these parameters in the ACD treatment group compared with the ACD group,including lesion score (3.33 ± 0.58,P ＜ 0.05),difference in thickness ((0.46 ± 0.11) mm,P ＜ 0.05) and swelling degree ((11.00 ± 2.64) mg,P ＜ 0.05),expressions of TNF-α ((817.72 ± 101.13) pg/mg,P＜ 0.05),IL-6 ((95.86 ± 36.65) pg/mg,P＜ 0.05),IL-17 ((373.38 ± 126.74) pg/mg,P＜ 0.05),IFN-γ ((63.31± 17.38) pg/mg,P ＜ 0.05) and the number of inflammatory cells per square millimetre (384.00 ± 97.35,P ＜0.05).Conclusion Hydrogen may inhibit the release of inflammatory factors in ACD.