Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Improper diving decompression can desaturate inert gases to form bubbles in the body and may lead to decompression sickness.In the process of decompression sickness,bubbles can directly or indirectly induce changes in platelet activation and coagulation.These abnormal changes play an important role in the rapid onset and continuous injury of decompression sickness.By systematically reviewing the relevant literatures,this article summarizes the effects of diving decompression on platelets,discusses the advantages and disadvantages of current prevention and treatment strategies,and outlines future research directions on the relationship between diving decompression and platelets.

Objective:To investigate the clinical efficacy of pancreatic cancer surgery with arterial resection.Methods:The clinicopathological and follow-up data of 135 patients undergoing pancreatectomies with arterial resection in Pancreas Center, the First Affiliated Hospital of Nanjing Medical University from Sep 2013 to Dec 2023 were retrospectively analyzed.Results:There were 77 males and 58 females, with age [ M( IQR)] of 63 (14) years old. Among the 135 patients, 122 (90.4%) were distal pancreatectomies, 8 (5.9%) were pancreaticoduodenectomies, 4 (3.0%) were total pancreatectomies and 1 (0.7%) was resection for local recurrence after distal pancreatectomy. There were 120 (88.9%) celiac axis resections, 11 (8.1%) hepatic artery resections, 1 (0.7%) superior mesenteric artery resection and 3 (2.2%) other artery resections. Simultaneous portal vein-superior mesenteric vein or organ resection accounted for 26.7% (36/135) and 29.6% (40/135),respectively. The median blood loss was 300 (300) ml and the median operation time was 275 (105) minutes. The 90-day mortality rate was 7.4% (10/135). The overall morbidity rate was 70.4% (95/135) while the major morbidity rate was 18.5% (25/135). Postoperative hemorrhage occurred in 8.9% (12/135), clinically relevant postoperative pancreatic fistula in 57.0% (77/135), bile leak in 0.74% (1/135), delayed gastric emptying in 9.6% (13/135), liver failure in 3.7% (5/135) and transient liver enzyme elevation in 44.4% (60/135). All of the 135 cases were confirmed as pancreatic cancer histologically, including 54.6% (71/130) moderately differentiated, 45.4% (59/130) poorly differentiated and no for well differentiated. The median tumor size was 4.5 (2.3) cm. The median number of harvested lymph nodes was 14 (13) and the percentage of N0, N1 and N2 according to AJCC 8th staging system was 27.1% (36/133), 52.6% (70/133) and 20.3% (27/133), respectively. The R 0 resection was achieved in 40 of 123 cases (32.5%), whose margins of specimens were assessed circumferentially based on the 1mm rule. The median overall survival time (MST) after surgery was 22.5 months, and the median progress-free survival time was 16.1 months. The overall survival rate at 1-, 2- and 5-year was 71.5%, 45.1% and 11.3%, respectively. The MST of patients who received no adjuvant therapy, chemotherapy after surgery was 8.4 months, 25.3 months, respectively. Conclusions:Pancreatectomy with arterial resection is generally safe and feasible. Survival outcome improves significantly when combined with adjuvant chemotherapy.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective:To investigate the clinical efficacy of pancreatic cancer surgery with arterial resection.Methods:The clinicopathological and follow-up data of 135 patients undergoing pancreatectomies with arterial resection in Pancreas Center, the First Affiliated Hospital of Nanjing Medical University from Sep 2013 to Dec 2023 were retrospectively analyzed.Results:There were 77 males and 58 females, with age [ M( IQR)] of 63 (14) years old. Among the 135 patients, 122 (90.4%) were distal pancreatectomies, 8 (5.9%) were pancreaticoduodenectomies, 4 (3.0%) were total pancreatectomies and 1 (0.7%) was resection for local recurrence after distal pancreatectomy. There were 120 (88.9%) celiac axis resections, 11 (8.1%) hepatic artery resections, 1 (0.7%) superior mesenteric artery resection and 3 (2.2%) other artery resections. Simultaneous portal vein-superior mesenteric vein or organ resection accounted for 26.7% (36/135) and 29.6% (40/135),respectively. The median blood loss was 300 (300) ml and the median operation time was 275 (105) minutes. The 90-day mortality rate was 7.4% (10/135). The overall morbidity rate was 70.4% (95/135) while the major morbidity rate was 18.5% (25/135). Postoperative hemorrhage occurred in 8.9% (12/135), clinically relevant postoperative pancreatic fistula in 57.0% (77/135), bile leak in 0.74% (1/135), delayed gastric emptying in 9.6% (13/135), liver failure in 3.7% (5/135) and transient liver enzyme elevation in 44.4% (60/135). All of the 135 cases were confirmed as pancreatic cancer histologically, including 54.6% (71/130) moderately differentiated, 45.4% (59/130) poorly differentiated and no for well differentiated. The median tumor size was 4.5 (2.3) cm. The median number of harvested lymph nodes was 14 (13) and the percentage of N0, N1 and N2 according to AJCC 8th staging system was 27.1% (36/133), 52.6% (70/133) and 20.3% (27/133), respectively. The R 0 resection was achieved in 40 of 123 cases (32.5%), whose margins of specimens were assessed circumferentially based on the 1mm rule. The median overall survival time (MST) after surgery was 22.5 months, and the median progress-free survival time was 16.1 months. The overall survival rate at 1-, 2- and 5-year was 71.5%, 45.1% and 11.3%, respectively. The MST of patients who received no adjuvant therapy, chemotherapy after surgery was 8.4 months, 25.3 months, respectively. Conclusions:Pancreatectomy with arterial resection is generally safe and feasible. Survival outcome improves significantly when combined with adjuvant chemotherapy.

Objective To investigate the valuation of ceramide(Cer),trigly ceride-glucose index(TyG index)and residual cho-lesterol(RC)levels on intracranial and extracranial atherosclerotic stenosis in elderly patients with ischemic stroke(IS).Methods A total of 447 elderly patients with IS who were hospitalized in the Affiliated Hospital of Xuzhou Medical University from January 2022 to June 2023 were selected.According to the digital subtraction angiography(DSA)results,the patients were divided into non-stenosis group(n=144)and stenosis group(n=303).The stenosis group was further divided into moderate stenosis group(n=121),severe stenosis and occlusion group(n=182).The general information and baseline biochemical data of all patients were collected.High per-formance liquid chromatography tandem mass spectrometry was used to detect serum Cer,and TyG index and RC level were calculated.Spearman method was used to analyze the correlation between Cer,TyG index,RC and intracranial and extracranial atherosclerotic steno-sis.Multivariate Logistic regression was used to analyze the risk factors of intracranial and extracranial atherosclerotic stenosis.Results Compared with the non-stenosis group,Cer,TyG index,RC,Lp(a),and glycosylated hemoglobin(HbA1c)and other indexes were significantly increased in the stenosis group(P＜0.05).Multivariate Logistic regression analysis showed that Cer16:0,Cer18:0,Cer24:1,Cer24:1/Cer24:0,Cer18:0/Cer24:0,Cer16:0/Cer24:0 were independent risk factors for intracranial and extracranial atherosclerotic stenosis in elderly patients with IS.Spearman correlation analysis showed that Cer24 was negatively correlated with intracranial and ex-tracranial atherosclerotic stenosis(r=-0.122,P＜0.05).The results of receiver operating characteristic curve showed that,the areas under the curve of Cer24:1,Cer24:1/Cer24:0,Cer18:0/Cer24:0,Cer16:0/Cer24:0,RC,TyG index in the diagnosis of intracranial and extracranial atherosclerotic stenosis in elderly patients with IS were 0.832,0.846,0.826,0.829,0.578,0.586(P＜0.05).Conclusion The serum levels of serum Cer,TyG index and RC are higher in elderly patients with IS,which may be the risk factors for intracranial and extracranial atherosclerotic stenosis in elderly patients with IS.

Objective To investigate the correlation of ceramides(Cer),triglyceride-glucose(TyG)index,residual cholesterol(RC)and intracranial atherosclerotic stenosis(ICAS)in elderly with ischemic stroke(IS).Methods A total of 209 elderly IS patients admitted in the Affiliated Hospi-tal of Xuzhou Medical University from January to December 2022 were recruited,and according to intracranial artery stenosis,they were divided into stenosis group(n=122)and non-stenosis group(n=87).TyG index and RC levels were calculated after testing different types of Cer.Spearman correlation analysis and logistic regression analysis were used.Results Compared with the non-stenosis group,the stenosis group had significantly larger proportions of higher levels of LP(a),HbA1c,TyG index,RC and Cer16∶0,Cer18∶0,Cer24∶1,Cer16∶0/Cer24∶0,Cer24∶1/24∶0 and Cer18∶0/24∶0,and lower Cer24∶ 0 level(P＜0.05,P＜0.01).The levels of Cer16∶0,Cer18∶0,Cer24∶1,Cer16∶0/24∶0,Cer18∶0/24∶0,Cer24∶1/24∶0,LP(a),HbA1c,TyG index and RC were positively correlated with(r=0.290,0.343,0.383,0.436,0.434,0.481,0.166,0.248,0.140,0.204,P＜0.05,P＜0.01),and Cer24∶0 was negatively with ICAS(r=-0.247,P＜0.01).Cer18∶0,Cer24∶1,Cer16∶0/Cer24∶0,Cer18∶0/Cer24∶0 and RC were important influencing factors for ICAS(P＜0.05,P＜0.01).Conclusion The levels of Cer,TyG index and RC are higher in elderly IS patients,and these indicators might be the risk factors for ICAS.

Purpose To investigate the challenges associ-ated with HER2-low in breast cancer across hospitals of varying regions and grades in China.Drawing on the findings from this investigation,this study will integrate relevant clinical guide-lines,consensus statements,and literature advancements from both domestic and international sources to offer insights for de-veloping guidelines for HER2 detection in Chinese breast cancer cases.Methods The questionnaire implementation method was Tencent WeChat as the carrier and online survey in the form of electronic questionnaire.Results According to the annual HER2 testing results of various medical institutions,compared with HER2 immunohistochemistry(IHC)scores of 3+,HER2 IHC scores of 1+or 2+significantly increased in the proportion range of over 20％(P＜0.001).A total of 854 pathologists provided effective feedback for this survey,55.6％reported dif-ficulties in distinguishing between a score of 0 and 1+in certain cases,primarily due to issues related to staining percentage(83.4％)and the absence of an objective standard for weak staining(78.3％).Regarding testing procedures,417(50.5％)medical institutions used only HER2 IHC 3+as an external control,whereas 139(16.8％)set up a gradient of ex-ternal controls for HER2 IHC scores of 0,1+,2+,and 3+.68.5％of pathologists routinely adhered to the microscope mag-nification rule to assist in HER2 scoring.51.9％of pathologists support a three-category reporting model for HER2 status(nega-tive/low/positive).In cases exhibiting heterogeneity,56.3％of pathologists would accept reporting the percentages of different HER2 scores(0/1+/2+/3+).Conclusion The proportion of breast cancer patients with HER2-low is relatively high in China.Currently,there is no consensus on quality control stand-ards for the pathological diagnosis and testing procedures for HER2-low.Key diagnostic challenges include defining the lower limit of interpretation standards,standardizing testing proce-dures,establishing external controls,refining interpretation methods,and standardizing reporting practices.Given the publi-cation of the DESTINY-Breast06 study results during the revision period,there is an urgent need to develop a new edition of the Chinese guidelines for HER2 testing in breast cancer to provide clearer and more specific strategies.

Purpose To examine the contemporary state of pathological diagnosis for granulomatous lobular mastitis(GLM)in China.Methods Pathologists from across the nation were invited to engage in an online questionnaire survey,which ad-dressed pertinent topics such as the frequency of GLM diagnosis cases,the modalities of pathology reporting,and advancements in etiological research.The collected data were analyzed utili-zing the x2 test or Fisher's exact test.Results A survey of 702 pathologists included 26.9％(189/702)chief physicians,43.3％(304/702)deputy chief physician,26.1％(183/702)attending physicians,and 3.7％(26/702)residents.Partici-pants were from tertiary general hospitals(77.6％,545/702),traditional Chinese medicine hospitals(7.7％,54/702),ma-ternal and child specialty hospitals(6.1％,43/702),and on-cology specialty hospitals(4.0％,28/702).In 2023,there were over 45,000 mastitis diagnoses.A significantly higher per-centage of chief physicians(60.8％,115/189)and deputy chief physicians(52.6％,160/304)could diagnose specific mastitis types compared to attending and resident physicians(x2=23.537,P＜0.001),with"GLM"being the most common diagnosis(55.1％,81/147).In diagnostic work,78.06％(548/702)of pathologists consider patient clinical information,19.2％(135/702)screen specimens for pathogens using special staining,only 6.0％(42/702)of pathologists indicated that their hospitals routinely perform tissue culture for pathogens,with corynebacterium being the most common.Additionally,11.4％(80/702)reported that clinicians regularly test serum sex hormones,most frequently for elevated prolactin,while 7.1％(50/702)check autoimmune status,frequently detecting antibodies without clinical symptoms.Chief physicians had a significantly better understanding of the etiology of GLM com-pared to other physicians(x2=11.969,P=0.003;P=0.007).A majority of pathologists indicated a lack of guidelines for GLM pathology diagnosis(49.4％,347/702)and report standards(76.4％,536/702).Nearly all(97.4％)agreed on the need to establish a consensus for GLM diagnosis.Conclu-sion GLM pathological diagnosis is currently inconsistent and lacks standardization,highlighting the urgent need for a guiding consensus for pathologists.

Purpose To investigate the challenges associ-ated with HER2-low in breast cancer across hospitals of varying regions and grades in China.Drawing on the findings from this investigation,this study will integrate relevant clinical guide-lines,consensus statements,and literature advancements from both domestic and international sources to offer insights for de-veloping guidelines for HER2 detection in Chinese breast cancer cases.Methods The questionnaire implementation method was Tencent WeChat as the carrier and online survey in the form of electronic questionnaire.Results According to the annual HER2 testing results of various medical institutions,compared with HER2 immunohistochemistry(IHC)scores of 3+,HER2 IHC scores of 1+or 2+significantly increased in the proportion range of over 20％(P＜0.001).A total of 854 pathologists provided effective feedback for this survey,55.6％reported dif-ficulties in distinguishing between a score of 0 and 1+in certain cases,primarily due to issues related to staining percentage(83.4％)and the absence of an objective standard for weak staining(78.3％).Regarding testing procedures,417(50.5％)medical institutions used only HER2 IHC 3+as an external control,whereas 139(16.8％)set up a gradient of ex-ternal controls for HER2 IHC scores of 0,1+,2+,and 3+.68.5％of pathologists routinely adhered to the microscope mag-nification rule to assist in HER2 scoring.51.9％of pathologists support a three-category reporting model for HER2 status(nega-tive/low/positive).In cases exhibiting heterogeneity,56.3％of pathologists would accept reporting the percentages of different HER2 scores(0/1+/2+/3+).Conclusion The proportion of breast cancer patients with HER2-low is relatively high in China.Currently,there is no consensus on quality control stand-ards for the pathological diagnosis and testing procedures for HER2-low.Key diagnostic challenges include defining the lower limit of interpretation standards,standardizing testing proce-dures,establishing external controls,refining interpretation methods,and standardizing reporting practices.Given the publi-cation of the DESTINY-Breast06 study results during the revision period,there is an urgent need to develop a new edition of the Chinese guidelines for HER2 testing in breast cancer to provide clearer and more specific strategies.