Objective:To construct a recombinant gene of single-chain fragment variable(scFv)against the SARS-CoV-2 spike protein(S protein).Methods:Using genetic engineering antibody technology,BALB/c mice were immunized with SARS-CoV-2 S protein antigen.The total RNA of the spleen was extracted to obtain the heavy chain(VH)and light chain(VL)gene sequences of the antibody variable region(V).Through multiple-primer PCR amplification and screening,the corresponding VH and VL gene sequences were obtained.After purification and recovery,the VH and VL primer genes of anti-SARS-CoV-2 S protein with restriction enzyme cutting sites and the VH and VL genes were successfully connected by the overlap extension PCR method to obtain the scFv recombinant gene.The sequencing results were compared and analyzed in the gene bank by using BLAST on the NCBI website.Results:The VH and VL genes of anti-SARS-CoV-2 S protein were successfully obtained,and scFv was obtained by connecting through the overlap extension PCR technique(SOE-PCR).The BLSAT analysis of scFv showed that its gene homology was greater than 80%.The indirect ELISA detection results showed that there was obvious binding in the prokaryotic expression supernatant.Conclusion:This study provides a technical basis for the diagnosis and treatment of SARS-CoV-2 and the research and development of new related antibodies.

Objective To investigate the cIinicopathological features and immunohistochemical expression of gastric hyperplastic polyps(GHPs)with dysplasia/adenocarcinoma.Methods A retrospective analysis of 24 cases(44 polyps)that were diagnosed as GHPs with dysplasia/adenocarcinoma in our hospital from January 2020 to December 2024 was reviewed,and clinical,histomorphological,immunophenotypic and follow-up data were analyzed.Results There were 20 female and 4 male cases,with a mean age of(65.5±7.9)(range 56～76)years.Among 44 polyps,3 occurred in the antrum of the stomach,1 in the gastric horn,and 40 in the fundus/body.Among the polyps,32 cases were diagnosed as high-grade dysplasia,4 cases as low-grade dysplasia,4 cases as coexistence of low-grade+high-grade dysplasia,2 cases as mucinous adenocarcinoma,1 cases as poorly differentiated adenocarcinoma,and 1 cases as signet-ring cell carcinoma.The histological manifestations of 23 cases of background mucosa were autoimmune metaplastic atrophic gastritis(AMAG).the P53 of 8 polyps showed a mutant expression pattern.Through MUC5/MUC6/MUC2/CD10 joint examination,33 cases showed gastric type(25 cases of which were foveal epithelium type),4 cases were intestinal type,5 cases were mixed gastrointestinal type,and 2 cases were non-gastrointestinal type.Conclusion The neoplastic transformation of GHPs is closely related to AMAG.It is necessary for clinicians and pathologists to strengthen their evaluation of background mucosa,to achieve early detection,early diagnosis and early treatment.

Objective:To explore the diagnostic efficacy of methylene blue staining combined with light transmission method (termed as light transmission and staining) in endoscopic submucosal dissection (ESD) specimens of early gastric cancer.Methods:A retrospective study was conducted on 75 specimens of early gastric cancer treated with ESD at Beijing Friendship Hospital, Capital Medical University from October 2021 to August 2023. Under a stereomicroscope, magnified observation and transmitted light transmission and staining observation were performed to compare the demarcation line (DL), irregular microvascular pattern (IMVP) and irregular microsurface pattern (IMSP) of the lesion, and the differences among histological types were compared. Furthermore, the false positive rate of surgical margin, the detection rate of undifferentiated cancer and multifocal lesions were compared against the 88 controls processed by traditional method.Results:Using the light transmission and staining method, DL, IMVP and IMSP were detected in 96.0% (72/75), 89.3% (67/75), and 98.7% (74/75), which was higher than 72.0% (54/75), 6.7% (5/75), and 26.7% (20/75) by using the magnified observation ( χ 2=8.036, P<0.001; χ 2=0.640, P<0.001; χ 2=0.369, P<0.001). There was no statistical difference in the coincidence rate of endoscopy and pathology between differentiated type, undifferentiated type and mixed type [92.2% (59/64), 50.0% (1/2), 77.8% (7/9), χ 2=5.145, P=0.055]. Compared to traditional methods, light transmission and staining could increase the detection rate of undifferentiated cancer [14.7% (11/75) VS 4.5% (4/88), χ 2=4.964, P=0.026] and reduce the false positive rate of surgical margins [1.3% (1/75) VS 11.4% (10/88), χ 2=4.585, P=0.032], but showed no statistical difference in the detection rate of multifocal lesions [5.3% (4/75) VS 0.0% (0/88), χ 2=2.841, P=0.094]. Conclusion:Light transmission and staining enhances pathological recognition of DL, IMVP and IMSP during specimen processing, improving detection of undifferentiated cancer and reducing false positive of margin.

Objective To investigate the cIinicopathological features and immunohistochemical expression of gastric hyperplastic polyps(GHPs)with dysplasia/adenocarcinoma.Methods A retrospective analysis of 24 cases(44 polyps)that were diagnosed as GHPs with dysplasia/adenocarcinoma in our hospital from January 2020 to December 2024 was reviewed,and clinical,histomorphological,immunophenotypic and follow-up data were analyzed.Results There were 20 female and 4 male cases,with a mean age of(65.5±7.9)(range 56～76)years.Among 44 polyps,3 occurred in the antrum of the stomach,1 in the gastric horn,and 40 in the fundus/body.Among the polyps,32 cases were diagnosed as high-grade dysplasia,4 cases as low-grade dysplasia,4 cases as coexistence of low-grade+high-grade dysplasia,2 cases as mucinous adenocarcinoma,1 cases as poorly differentiated adenocarcinoma,and 1 cases as signet-ring cell carcinoma.The histological manifestations of 23 cases of background mucosa were autoimmune metaplastic atrophic gastritis(AMAG).the P53 of 8 polyps showed a mutant expression pattern.Through MUC5/MUC6/MUC2/CD10 joint examination,33 cases showed gastric type(25 cases of which were foveal epithelium type),4 cases were intestinal type,5 cases were mixed gastrointestinal type,and 2 cases were non-gastrointestinal type.Conclusion The neoplastic transformation of GHPs is closely related to AMAG.It is necessary for clinicians and pathologists to strengthen their evaluation of background mucosa,to achieve early detection,early diagnosis and early treatment.

Objective:To construct a recombinant gene of single-chain fragment variable(scFv)against the SARS-CoV-2 spike protein(S protein).Methods:Using genetic engineering antibody technology,BALB/c mice were immunized with SARS-CoV-2 S protein antigen.The total RNA of the spleen was extracted to obtain the heavy chain(VH)and light chain(VL)gene sequences of the antibody variable region(V).Through multiple-primer PCR amplification and screening,the corresponding VH and VL gene sequences were obtained.After purification and recovery,the VH and VL primer genes of anti-SARS-CoV-2 S protein with restriction enzyme cutting sites and the VH and VL genes were successfully connected by the overlap extension PCR method to obtain the scFv recombinant gene.The sequencing results were compared and analyzed in the gene bank by using BLAST on the NCBI website.Results:The VH and VL genes of anti-SARS-CoV-2 S protein were successfully obtained,and scFv was obtained by connecting through the overlap extension PCR technique(SOE-PCR).The BLSAT analysis of scFv showed that its gene homology was greater than 80%.The indirect ELISA detection results showed that there was obvious binding in the prokaryotic expression supernatant.Conclusion:This study provides a technical basis for the diagnosis and treatment of SARS-CoV-2 and the research and development of new related antibodies.

Objective:To explore the diagnostic efficacy of methylene blue staining combined with light transmission method (termed as light transmission and staining) in endoscopic submucosal dissection (ESD) specimens of early gastric cancer.Methods:A retrospective study was conducted on 75 specimens of early gastric cancer treated with ESD at Beijing Friendship Hospital, Capital Medical University from October 2021 to August 2023. Under a stereomicroscope, magnified observation and transmitted light transmission and staining observation were performed to compare the demarcation line (DL), irregular microvascular pattern (IMVP) and irregular microsurface pattern (IMSP) of the lesion, and the differences among histological types were compared. Furthermore, the false positive rate of surgical margin, the detection rate of undifferentiated cancer and multifocal lesions were compared against the 88 controls processed by traditional method.Results:Using the light transmission and staining method, DL, IMVP and IMSP were detected in 96.0% (72/75), 89.3% (67/75), and 98.7% (74/75), which was higher than 72.0% (54/75), 6.7% (5/75), and 26.7% (20/75) by using the magnified observation ( χ 2=8.036, P<0.001; χ 2=0.640, P<0.001; χ 2=0.369, P<0.001). There was no statistical difference in the coincidence rate of endoscopy and pathology between differentiated type, undifferentiated type and mixed type [92.2% (59/64), 50.0% (1/2), 77.8% (7/9), χ 2=5.145, P=0.055]. Compared to traditional methods, light transmission and staining could increase the detection rate of undifferentiated cancer [14.7% (11/75) VS 4.5% (4/88), χ 2=4.964, P=0.026] and reduce the false positive rate of surgical margins [1.3% (1/75) VS 11.4% (10/88), χ 2=4.585, P=0.032], but showed no statistical difference in the detection rate of multifocal lesions [5.3% (4/75) VS 0.0% (0/88), χ 2=2.841, P=0.094]. Conclusion:Light transmission and staining enhances pathological recognition of DL, IMVP and IMSP during specimen processing, improving detection of undifferentiated cancer and reducing false positive of margin.

Objective:To investigate the application value of magnetic resonance imaging(MRI) in evaluating the efficacy of anti-PD-1 combined with neoadjuvant therapy for microsatellite stability (MSS)/proficient mismatch repair (pMMR) locally advanced rectal cancer (LARC).Methods:The prospective single-arm phase Ⅱ study was conducted. The clinicopathological data of 37 patients with MSS/pMMR LARC who were admitted to Beijing Friendship Hospital of Capital Medical University from April 2021 to September 2022 were collected. All patients underwent anti-PD-1 combined with neoadjuvant therapy and radical total mesorectal excision. Observation indicators: (1) enrolled pati-ents; (2) MRI and pathological examination; (3) concordance analysis of MRI examination reading; (4) evaluation of MRI examination. Measurement data with normal distribution were represented as Mean± SD. Count data were expressed as absolute numbers or percentages. Linear weighted κ value was used to evaluate the concordance of radiologist assessment. Sensitivity, negative predictive value, accuracy, overstaging rate and understaging rate were used to evaluate the predictive value. Results:(1) Enrolled patients. A total of 37 eligible patients were screened out, including 21 males and 16 females, aged (61±11)years. MRI examination was performed before and after combined therapy, and pathological examination was performed after radical resection. (2) MRI and pathological examination of patients. Among the 37 patients, MRI before combined therapy showed 0, 0, 5, 24 and 8 cases in stage T0, T1, T2, T3 and T4, 10, 17 and 10 cases in stage N0, N1 and N2, 28 and 9 cases of positive and negative extramural vascular invasion (EMVI), 4 and 33 cases of positive and negative mesorectal fascia (MRF), respectively. MRI examination after combined therapy showed 15, 4, 7, 10 and 1 cases in stage T0, T1, T2, T3 and T4, 34, 2 and 1 cases in stage N0, N1 and N2, 9 and 28 cases of positive and negative EMVI, 1 and 36 cases of positive and negative MRF. There were 16, 13, 8 and 0 cases of tumor regression grading (TRG) 0, 1, 2 and 3, respectively. Postoperative pathological examination showed 18, 4, 3, 11, 1 cases in stage T0, T1, T2, T3, T4, 33, 3, 1 cases in stage N0, N1, N2, positive and negative EMVI and unknown data in 1, 35, 1 cases, positive and negative circumferential margin in 0 and 37 cases, grade 0, grade 1, grade 2, grade 3 of American Joint Committee on Cancer TRG in 18, 9, 8, 2 cases, respectively. Pathological complete response rate was 48.6%(18/37) and approximate pathological complete response rate was 24.3%(9/37). (3)Concordance analysis of MRI examination reading. The κ value of T staging and N staging on MRI before combined therapy was 0.839 ( P<0.05) and 0.838 ( P<0.05), respectively. The κ value of T staging and N staging on MRI after combined therapy was 0.531 ( P<0.05) and 0.846 ( P<0.05), respectively. The κ value of EMVI and MRF was 0.708 ( P<0.05) and 0.680 ( P<0.05) before combined therapy, and they were 0.561 ( P<0.05) and 1.000 ( P<0.05) after combined therapy, respectively. The κ value of TRG 3-round reading for TRG was 0.448 ( P<0.05). (4) Evaluation of MRI examination. ① MRI evaluation of T and N staging. The accuracy of MRI examination after combined therapy for distinguishing stage T0 was 75.7%[28/37, 95% confidence interval ( CI) as 62.2%-89.2%], the understaging rate was 8.1%(3/37, 95% CI as 0-18.9%), the overstaging rate was 16.2%(6/37, 95% CI as 5.4%-29.7%). The accuracy of MRI examination for distinguishing stage T0-T2 was 86.5%(32/37, 95% CI as 73.0%-97.3%), its understaging rate and overstaging rate were 8.1%(3/37, 95% CI as 0-18.9%) and 5.4% (2/37, 95% CI as 0-13.5%), respectively. The accuracy of MRI examination for distinguishing N staging was 91.9%(34/37, 95% CI was 81.1%-100.0%), its understaging rate and overstaging rate were 5.4%(2/37, 95% CI as 0-13.5%) and 2.7%(1/37, 95% CI as 0-8.1%), respectively. Among 18 patients in pathological stage T0, the overstaging rate of MRI was 33.3%(6/18). All the 4 patients in pathological stage T1 and 3 pati-ents in pathological stage T2 had correct diagnosis. There were 3 cases with understaging among 12 patients in pathological stage T3-T4. Among the 37 patients in pathological stage N0-N2, 34 cases had correct diagnosis, 1 case was overstaged as stage N1 due to a round mesorectal lymph node with short diameter as 6 mm, and 2 cases were diagnosed as stage N0 due to the small lymph nodes with the maximum short diameter as 3 mm. ② MRI evaluation of EMVI and MRF. The accuracy, sensitivity and negative predictive value of MRI for evaluating EMVI were 86.5%(32/37, 95% CI as 75.0%-97.2%), 100.0% and 100.0%, respectively, and the overestimation rate of EMVI was 13.9%(5/36, 95% CI as 2.8%-25.0%), and no underestimation occurred. Of 35 pathologically negative EMVI patients, a rate of 14.3%(5/35) of patients were positive on MRI. The main reason for overestaging was that thickened fibrous tissue outside the rectal wall was mistaken for vascular invasion. The accuracy of MRI for evaluating MRF was 97.3%(36/37, 95% CI as 91.9%-100.0%), and 1 case (1/37, 2.7%, 95% CI as 0-8.1%) was overestimated as positive MRF due to misdiagnosis of pararectal MRF lymph nodes. The negative predictive value of MRI for assessing MRF was 100.0%. ③ MRI evaluation of TRG. The accuracy, understaging and overstaging rates of MRI for evaluating pathological TRG 0 were 78.4%(29/37, 95% CI as 64.9%-91.9%), 8.1%(3/37, 95% CI as 0-18.9%), 13.5%(5/37, 95% CI as 5.4%-27.0%), respectively. The accuracy, understaging and overstaging rates of MRI for evaluating pathological TRG 0-1 were 89.2%(33/37, 95% CI as 78.4%-97.3%), 8.1%(3/37, 95% CI as 0-18.9%), 2.7%(1/37, 95% CI as 0-8.1%), respectively. Of the 18 patients with pathologic complete response, 5 cases were diagnosed as pathological TRG 1 and 13 cases as pathological TRG 0. One near-pCR patient was assessed as pathological TRG 2. Two patients with pathological TRG 3 were incorrectly diagnosed on MRI. Conclusions:Anti-PD-1 combined with neoadjuvant therapy can downstage the LARC pati-ents with MSS/pMMR. MRI is effective in predicting T staging, N staging, EMVI, MRF and TRG. However, overstaging should be prevented.

Purpose To investigate the expression pattern of podoplanin in the periglandular fibroblasts and cancer-associ-ated fibroblasts of early-stage colorectal cancer,to analyze its re-lationship with clinicopathological features,and to explore its significance in clinicopathological diagnosis.Methods A total of 166 cases of early-stage colorectal cancer were enrolled.The deepest invasion tissue samples from 166 cases were explored with immunohistochemistry for expression profiling of podopla-nin.According to the expression pattern of analyze in the stroma,Pattern A showed that the periglandular fibroblasts were pole-ori-ented and uniformly expressed podoplanin,and stronger than CAFs around the glands,periglandular fibroblasts completely sur-round the glands;Pattern B showed that the periglandular fibro-blasts were loss of cell polarity,and the staining intensity was less than or equal to CAFs around the glands.And the correlation was also analyzed between the two expression patterns and clini-copathological features.Results Among 166 cases,55 cases were the Pattern A of podoplanin expression and 111 cases were the Pattern B.Monofactor analysis showed statistically significant differences in lymphatic vessel invasion,vascular invasion,bud-ding grade,differentiation degree,infiltration pattern and Kiku-chi classification between the two groups(P＜0.05).Multivari-ate analysis identified that infiltration pattern was an independent factor between the two groups(P＜0.05).Conclusion Podo-planin expression patterns are different in the stroma of early colorectal cancer and correlated with risk factors for lymph nodes metastasis.In clinicopathological diagnosis,podoplanin can be used as an auxiliary diagnostic tool to improve the consistency of pathologists'judgment of infiltration forms.

Objective To investigate the effects of Huanglian Jiedu Decoction Aqueous Extract(HJDAE)on the gut microbiota structure and colon metabolites of rats using 16S rRNA technology and non targeted metabolomics technology.Methods SD rats were continuously gavaged with HJDAE for 7 days and then Euthanized under anesthesia to extract the colon contents of the rats,,the contents of the rats'colon were taken,and 16S rRNA high-throughput sequencing was performed on the gut microbiota of the rats'colon segments using the Illumina Miseq platform.The differential microbiota,differential metabolites,and related pathways were analyzed in combination with the fecal non targeted metabonomics method.Results The results showed that HJDAE could affect the structure of gut microbiota in rats while maintaining a relatively stable proportion of various phyla of gut microbiota,and had significant promoting and inhibitory effects on multiple bacterial genera.Among them,it had the strongest inhibitory effect on Lactobacillus and Limosilactobacillus,and the most obvious promoting effect on Clostridium.Analyzing the data from two sets of experimental results,76 differential metabolites and 70 potential biomarkers were found.Among them,the top ten differential metabolites and their differences were xylose,acetic acid,propionic acid,and dimethylglycine with reduced production,while the production of palmitoleic acid,proline,and T-α-MCA,T-β-MCA,3-DHCA,HCA increased.The eight strongest metabolic pathways involved are Propanoate Metabolism,Butanoate Metabolism,TCA cycle,Alanine-Aspartate-Glutamate Metabolism,D-Glutamine and D-Glutamate Metabolism,Primary Bile Acid Biosynthesis,Nitrogen Metabolism,Valine-Leucine-Isoleucine Biosythesis,all of which are closely related to bile acid production.Conclusion The HJDAE promotes the production of primary bile acids by promoting the metabolism of sugars,amino acids,and fatty acids in rats.At the same time,the HJDAE causes a large number of Clostridium species to proliferate,and multiple Clostridium species metabolize primary bile acids into secondary bile acids,jointly leading to positive regulation of bile acid metabolism.

ObjectiveTo explore the impact of Gegen Qinliantang（GQT） on the fecal short-chain fatty acids（SCFAs） metabolism in antibiotic-associated diarrhea（AAD） through targeted metabolomics. MethodA total of 240 SD rats were randomly divided into six groups（n=40， half male and half female）， including blank group， model group， bifidobiogen group（0.15 g·kg^-1）， and GQT high-， medium-， and low-dose groups（10.08， 5.04， 2.52 g·kg^-1）， except for the blank group， clindamycin（250 mg·kg^-1） was given to all groups by gavage for modeling every day for 7 d. After successful modeling， each administered group was gavaged with the corresponding dose of the drug， and the blank and model groups were gavaged with an equal volume of normal saline solution， 1 time/d， for 14 d. At 0， 3， 7， 14 d after the drug intervention， eight rats were randomly selected from each group， respectively. Gas chromatography-time-of-flight mass spectrometry（GC-TOF-MS） was used to perform targeted metabolomic analysis of SCFAs in the feces of rats， and partial least squares-discriminant analysis（PLS-DA） was applied to compare the differences in metabolic profiles between groups at different treatment times， and to compare the changes in the contents of SCFAs in rat feces between groups. ResultPLS-DA results showed that the blank group could be clearly distinguishable from the model group， with GQT exhibiting a closer proximity to the blank group after 7 d of treatment. After further analyzing the composition of SCFAs， it was found that the proportion of acetic acid increased and the proportions of butyric acid， valeric acid， hexanoic acid and isovaleric acid decreased in the model group compared with the blank group. After the treatment with GQT， the proportions of butyric acid， isobutyric acid， valeric acid， and isovaleric acid increased， and the proportions of acetic acid， propionic acid and caproic acid decreased. Subsequent differential analysis revealed that GQT could significantly improve the content of butyric acid， and had a certain retrogressive effect on the contents of valeric acid and hexanoic acid. ConclusionThe medium dose group of GQT can improve the contents of SCFAs in AAD feces after 7 days of treatment， which may be related to the improvement of the composition ratio of SCFAs and the contents of butyric acid， valeric acid and caproic acid.