In mass spectrometry-based proteomics experiments,achieving high-throughput and efficientproteolytic digestion is crucial to ensure optimal protein cleavage and enhance the depth of protein identi-fication (including the number of identified proteins and the coverage of protein amino acid sequences) .Trypsin is the most widely used protease in mass spectrometry-based proteomics due to its ability to spe-cifically cleave the carboxyl terminus of arginine and lysine.However,it was found that Trypsin has some missed enzymatic efficiency for the cleavage of lysine residues.Therefore,in actual proteomics sample preparation,a combination of Trypsin and LysC will be used to ensure adequate cleavage of lysine resi-dues.Our study revealed that the commonly employed LysC-Trypsin tandem cleavage method exerts an impact on the enzymatic cleavage of protein samples by Trypsin due to the subsequent cleavage of Trypsin by initially added LysC.Consequently,we adjusted the order of LysC and Trypsin tandem digestion,with Trypsin cleavage being performed first followed by the addition of LysC to target any missed lysine resi-dues.We comprehensively compared and analyzed three distinct sequential digestion methods,namely Trypsin-Trypsin (T-T),LysC-Trypsin (L-T),and Trypsin-LysC (T-L),in terms of their effects on pro-tein sample preparation quality.The results demonstrated that the Trypsin-LysC sequential digestion ap-proach not only minimizes missed protein lysine/arginine cleavage sites without increasing experimental costs,at the same time yielding peptides with a moderate amino acid sequence length.The use of Tryp-sin-LysC digestion enhances the adsorption and separation of peptide samples in RP-HPLC,as well as improves the depth of protein detection and amino acid sequence coverage during tandem mass spectrome-try analysis.This research work offers a novel technical solution and serves as a valuable reference for proteome sample preparation.

Objectives: To construct a nomogram for prediction of intrahepatic cholangiocarcinoma (ICC) lymph node metastasis based on inflammation-related markers,and to conduct its clinical verification. Methods: Clinical and pathological data of 858 ICC patients who underwent radical resection were retrospectively collected at 10 domestic tertiary hospitals in China from January 2010 to December 2018. Among the 508 patients who underwent lymph node dissection,207 cases had complete variable clinical data for constructing the nomogram,including 84 males,123 females,109 patients≥60 years old,98 patients<60 years old and 69 patients were pathologically diagnosed with positive lymph nodes after surgery. Receiver operating characteristic curve was drawn to calculate the accuracy of preoperative imaging examinations to determine lymph node status,and the difference in overall survival time was compared by Log-rank test. Partial regression squares and statistically significant preoperative variables were screened by backward stepwise regression analysis. R software was applied to construct a nomogram,clinical decision curve and clinical influence curve,and Bootstrap method was used for internal verification. Moreover,retrospectively collecting clinical information of 107 ICC patients with intraoperative lymph node dissection admitted to 9 tertiary hospitals in China from January 2019 to June 2021 was for external verification to verify the accuracy of the nomogram. 80 patients with complete clinical data but without lymph node dissection were divided into lymph node metastasis high-risk group and low-risk group according to the score of the nomogram among the 858 patients. Log-rank test was used to compare the overall survival of patients with or without lymph node metastasis diagnosed by pathology. Results: The area under the curve of preoperative imaging examinations for lymph node status assessment of 440 patients was 0.615,with a false negative rate of 62.8% (113/180) and a false positive rate of 14.2% (37/260). The median survival time of 207 patients used to construct a nomogram with positive or negative postoperative pathological lymph node metastases was 18.5 months and 27.1 months,respectively (P<0.05). Five variables related to lymph node metastasis were screened out by backward stepwise regression analysis,which were combined calculi,neutrophil/lymphocyte ratio,albumin,liver capsule invasion and systemic immune inflammation index,according to which a nomogram was constructed with concordance index(C-index) of 0.737 (95%CI: 0.667 to 0.806). The C-index of external verification was 0.674 (95%CI:0.569 to 0.779). The calibration prediction curve was in good agreement with the reference curve. The results of the clinical decision curve showed that when the risk threshold of high lymph node metastasis in the nomogram was set to about 0.32,the maximum net benefit could be obtained by 0.11,and the cost/benefit ratio was 1∶2. The results of clinical influence curve showed that when the risk threshold of high lymph node metastasis in the nomogram was set to about 0.6,the probability of correctly predicting lymph node metastasis could reach more than 90%. There was no significant difference in overall survival time between patients with high/low risk of lymph node metastasis assessed by the nomogram and those with pathologically confirmed lymph node metastasis or without lymph node metastasis (Log-rank test:P=0.082 and 0.510,respectively). Conclusion: The prediction accuracy of preoperative nomogram for ICC lymph node metastasis based on inflammation-related markers is satisfactory,which can be used as a supplementary method for preoperative diagnosis of lymph node metastasis and is helpful for clinicians to make personalized decision of lymph node dissection for patients with ICC.

OBJECTIVE: To investigate whether Omicron BA.1 breakthrough infection after receiving the SARS-CoV-2 vaccine could create a strong immunity barrier. METHODS: Blood samples were collected at two different time points from 124 Omicron BA.1 breakthrough infected patients and 124 controls matched for age, gender, and vaccination profile. Live virus-neutralizing antibodies against five SARS-CoV-2 variants, including WT, Gamma, Beta, Delta, and Omicron BA.1, and T-lymphocyte lymphocyte counts in both groups were measured and statistically analyzed. RESULTS: The neutralizing antibody titers against five different variants of SARS-CoV-2 were significantly increased in the vaccinated population infected with the Omicron BA.1 variant at 3 months after infection, but mainly increased the antibody level against the WT strain, and the antibody against the Omicron strain was the lowest. The neutralizing antibody level decreased rapidly 6 months after infection. The T-lymphocyte cell counts of patients with mild and moderate disease recovered at 3 months and completely returned to the normal state at 6 months. CONCLUSION Omicron BA.1 breakthrough infection mainly evoked humoral immune memory in the original strain after vaccination and hardly produced neutralizing antibodies specific to Omicron BA.1. Neutralizing antibodies against the different strains declined rapidly and showed features similar to those of influenza. Thus, T-lymphocytes may play an important role in recovery.
Humans ; Antibodies, Neutralizing ; Prospective Studies ; SARS-CoV-2 ; Breakthrough Infections ; COVID-19 Vaccines ; COVID-19 ; T-Lymphocytes ; China/epidemiology* ; Antibodies, Viral

Single-cell sequencing (SCS) sequences the genetic information of a single cell to better understand the differences amongst cells and reveal the unique changes of each cell type. The specific analysis of cell subsets at the single-cell level can accurately evaluate tumor cells and microenvironment cells to reveal the complexity of molecular components and the difference from the corresponding components in non-malignant tissues. Lymphoma is highly heterogeneous, some have unknown pathological types, etiology and poor prognosis. SCS is helpful to clarify the molecular mechanisms of lymphomagenesis and pathological staging, and guide clinical practice. This article reviews SCS and its application in lymphoma.

Objective: To establish a survival prediction model based on the independent prognostic factors of long-term prognosis after laparoscopic liver resection(LLR) for intrahepatic cholangiocarcinoma(ICC). Methods: The clinical and pathological data of 351 consecutive patients with ICC who received radical LLR in 13 Chinese medical centers from August 2010 to May 2021 were collected retrospectively. There were 190 males and 161 females,aged(M(IQR)) 61(14)years(range:23 to 93 years). The total cohort was randomly divided into a training dataset(264 cases) and a validation dataset(87 cases). The patients were followed up by outpatient service or telephone,and the deadline for follow-up was October 2021. Based on the training dataset,the multivariate Cox proportional hazards regression model was used to screen the independent influencing factors of long-term prognosis to construct a Nomogram model. The Nomogram model's discrimination,calibration,and clinical benefit were evaluated through internal and external validation,and an assessment of the overall value of two groups was made through the use of a receiver operating characteristic(ROC) curve. Results: There was no significant difference in clinical and pathological characteristics and long-term survival results between the training and validation datasets(all P>0.05). The multivariate Cox analysis showed that CA19-9,CA125,conversion to laparotomy during laparoscopic surgery,and lymph node metastasis were independent prognostic factors for ICC patients after LLR(all P<0.05). The survival Nomogram was established based on the independent prognostic factors obtained from the above screening. The ROC curve showed that the area under the curve of 1, 3 and 5-year overall survival rates of patients in the training dataset were 0.794(95%CI:0.721 to 0.867),0.728(95%CI:0.618 to 0.839) and 0.799(95%CI:0.670 to 0.928),and those in the validation dataset were 0.787(95%CI:0.660 to 0.915),0.831(95%CI:0.678 to 0.983) and 0.810(95%CI:0.639 to 0.982). Internal and external validation proved that the model exhibited a certain discrimination,calibration,and clinical applicability. Conclusion: The survival Nomogram model based on the independent influencing factors of long-term prognosis after LLR for ICC(including CA19-9,CA125,conversion to laparotomy during laparoscopic surgery,and lymph node metastasis) exhibites a certain differentiation,calibration,and clinical practicability.
Bile Duct Neoplasms/surgery* ; Bile Ducts, Intrahepatic/pathology* ; CA-19-9 Antigen ; Cholangiocarcinoma/diagnosis* ; Female ; Humans ; Laparoscopy ; Lymphatic Metastasis ; Male ; Nomograms ; Prognosis ; Retrospective Studies

Hematopoiesis starts from the embryo and runs through the entire life of a living body, which is a multi-stage and complex dynamic process that is regulated by multiple pathways, involving a variety of cells and hematopoietic anatomical locations. During the development of the mammalian hematopoietic system, the currently known hematopoietic anatomical locations mainly include yolk sac, aorta-gonad-mesonephros, fetal liver, bone marrow, and thymus. The first three are mainly responsible for hematopoiesis during the embryonic and fetal period, while bone marrow is the main place for postnatal hematopoiesis, and thymus is mainly responsible for the differentiation of T lymphocytes. Integrating flow cytometry, in vitro cell culture and in vivo animal transplantation models, early researchers conducted an in-depth analysis of the differentiation pathways of hematopoietic cells. However, due to technical limitations, it is difficult to track the single-cell hematopoietic activity of hematopoietic organs. Transcriptome sequencing at the single-cell level provides researchers with a unique perspective, making it possible to draw the most detailed cell fate transition maps of the hematopoietic development of living organisms, and providing new ideas for the diagnosis and treatment of hematological tumors. In this article, we reviewed the research progress in the use of large-scale single-cell transcriptome sequencing in the field of physiological hematopoiesis in recent years.
Technology

OBJECTIVE： To establish content determination method of Triptolide-glycyrrhetic acid compound microemulsion， optimize the formula and investigate its physicochemical properties and release rate in vitro. METHODS： The content of Triptolide- glycyrrhetic acid compound microemulsion was determined by UPLC. The determination was performed on ACQUITY UPLC BEH C18 column with mobile phase consisted of 0.1％ formic acid aqueous solution-acetonitrile （gradient elution） at the flow rate of 0.4 mL/min. The column temperature was 40 ℃. The detection wavelength was set at 218 nm， and sample size was 5 μL. Pseudo-ternary phase diagrams were drawn by water titration method. Using oil phase， surfactants and co-surfactants as index， the formula was optimized， and in intro release characteristics was investigated by in vitro release test. RESULTS： The linear range of triptolide and glycyrrhetinic acid were 1-40 μg/mL（r＝0.999 7） and 10-400 μg/mL（r＝0.999 8）， respectively. The limits of quantitation were 0.5 and 0.8 μg/mL； the limits of detection were 0.1 and 0.2 μg/mL. RSDs of precision， stability and reproducibility tests were all less than 2％. Average recoveries were 100.32％-101.15％ （RSD＝0.36％， n＝6）， 99.78％-101.42％ （RSD＝0.59％，n＝6）. The optimal formula included that medium chain triglyceride as oil phase， polyethylene glycol hydroxy stearate as surfactants， ethanol as co-surfactants， water as water phase， the proportion of them was 8 ∶  28 ∶ 14 ∶ 50. The obtained microemulsion was O/W type， being transparent and clear， with average diameter， average polydispersity index and average viscosity of （62.38±3.44） nm， 0.096±0.001 and （26.84±1.10） mPa·S. Within 24 h， cumulative release rates of triptolide and glycyrrhetinic acid in obtained microemulsion were 99.8％ and 99.7％ （in PBS pH 2.0）， 99.3％ and 99.4％ （in PBS pH 7.4）， 98.9％ and 98.4％ （in PBS pH 9.0）， respectively. Triptolide and glycyrrhetinic acid released faster in the single microemulsion than in the compound microemulsion. CONCLUSIONS： Established content determination method is simple and stable. The optimized formula is stable and feasible. Obtained iriptolide-glycyrrhetinic acid compound microemulsion show better sustained-release effect than sigle microemulsion.

This paper systematically reviews the latest and relevant literatures and policy documents on the in-tegrated health services in Canada in recent years. Therefore, it summarizes the practice and mode of integrated serv-ice delivery in Ontario, Alberta and Quebec wherein the integration among health organization, health service team, and a series of health services are included. The contributing factors and impeding factors ( the barriers) of organiza-tional integration and specific integration strategy were summarized. Finally, according to the actual practical situa-tion, it is proposed that China should adhere to the government-led approach in promoting the integration of health services, and give a full play to the positive role of the market mechanism. Through strengthening the network man-agement and group service of primary health services, emphasis will be put on health services of population groups and specific diseases. Therefore, integration will be regarded as a strategic priority, increasing incentives and boos-ting promotion of nursing personnel on the process of Integrated Service Delivery, building the health information sys-tem that is conducive to integration in order to continuously advance Hierarchical Diagnosis and bridge the fragmented service system. This will help in providing residents with personalized, convenient, comprehensive, and continuous health services.

Objective To explore the method of WeChat medication education for patients used by pharmacists,and to investigate the effect of WeChat medication education at Peking university third hospital.Methods Totally 30 patients who used warfarin at Peking university third hospital were enrolled.A series of subscriptions concerning warfarin medication education were sent to patients and/or their caregivers by WeChat regularly.The medication knowledge score after/before the intervention were assessed.Results The score of patients' warfarin medication knowledge before intervention was (14.20 ± 1.97) points,and after that the score was (16.63 ± 1.74) points,showing an improvenent with significance (P ＜ 0.001).Questions about adverse events of warfarin were most likely to be answered by mistake and the score of relevant questions was only (0.97 ± 0.80) points and (1.07 ± 0.81) points,respectively (P ＞ 0.05) before and after intervention.Conclusion It is of great significance for pharmacists to provide warfarin medication education.It is proved effective to improve patients' recognition of warfarin medication by means of WeChat subscriptions.

Salvianolic acids and tanshinones are main hydrophilic and lipophilic extracts from Salvia Miltiorrhiza with significant anti-pulmonary fibrosis effects. The aim of this study was to prepare a co-micronized salvianolic acids-tanshinones composite powder for inhalation using a planetary ball mill. The micronization process parameters were optimized by central composite design (CCD) and response surface methodology (RSM). Treatment time, rotation speed and the ball/sample weight ratio were selected as the independent variables, and the volume fraction of particle size in 1-5 μm was taken as the dependent variable. The powder properties were evaluated by scanning electron microscopy (SEM), laser diffraction and X-ray powder diffraction (XRPD). The powder flow and hygroscopicity were determined with repose angle, compressibility index and critical relative humidity(CRH). According to the results, the salvianolic acids-tanshinones composite powder produced in optimal conditions had a narrow and unimodal particle size distribution and a smaller D₅₀ of 2.33 μm. The volume fraction of particle size in 1-5 μm was 80.82%. The repose angle was (50.60±1.13) °, and the critical relative humidity is about 77%. After being micronized, the particle size significantly reduced, and the number of amorphous substances slightly increased, with no significant changes in powder flow and hygroscopicity. These findings indicate that the grinding method with a planetary ball mill can be used to co-micronize various components with different properties and prepare composite drug powders for dry powder inhalation.