ObjectiveTo establish the specific chromatogram and quantitative analysis of multi-components by single-marker（QAMS） based on linear calibration using two reference substances（LCTRS）， explore the consistency between Hedyotis Herba dispensing granules and standard decoction， and evaluate the quality of the dispensing granules. MethodsHigh performance liquid chromatography（HPLC） specific chromatogram was established based on 15 batches of Hedyotis Herba standard decoction and 10 batches of the dispensing granules， and LCTRS was used to locate chromatographic peaks. The actual retention times of 7 characteristic peaks in the specific chromatogram was measured on 24 different types of C₁₈ columns， taking deacetyl asperulosidic acid and asperulosidic acid as the dual standard compounds， the retention times of the other 5 characteristic peaks were predicted and validated. Based on this， QAMS was developed to determine the contents of four components（deacetyl asperulosidic acid， deacetyl asperulosidic acid methyl ester， asperulosidic acid， and p-coumaric acid）. Then， the relative correction factors of deacetyl asperulosidic acid， deacetyl asperulosidic acid methyl ester and p-coumaric acid were calculated using the reference peak of asperulosidic acid in the dual standard compounds， and each component was quantified accordingly. Finally， the consistency between the dispensing granules and standard decoction was assessed by taking extract rate of the standard decoction， consistency of the specific chromatograms， contents and transfer rates of the indicator components as indexes， and the quality of the dispensing granules was evaluated. ResultsThere were 7 common peaks in the characteristic chromatogram of samples of Hedyotis Herba standard decoction and the dispensing granules， and four of them were identified by reference standards， namely deacetyl asperulosidic acid（peak 1）， deacetyl asperulosidic acid methyl ester（peak 3）， asperulosidic acid（peak 6） and p-coumaric acid（peak 7）. The similarity between the dispensing granules and the standard decoction was >0.9. The absolute deviation in the predicted retention time for each component by LCTRS was lower than that of the relative retention time method. The extract rate of the 15 batches of Hedyotis Herba standard decoction ranged from 7.89% to 14.60%， the contents of deacetyl asperulosidic acid， deacetyl asperulosidic acid methyl ester， asperulosidic acid and p-coumaric acid were 6.62-19.70， 3.83-17.99， 1.57-6.69， 1.62-4.52 mg·g^-1， and the transfer rates of these components from decoction pieces to the standard decoction were 22.89%-39.60%， 34.03%-62.24%， 24.25%-43.70%， and 40.58%-73.71%， respectively. The extract rate， index component contents and transfer rates from decoction pieces to the three batches of Hedyotis Herba dispensing granules（P1-P3）， produced by manufacturer A， were similar to those of the standard decoction prepared from the same batch of decoction pieces， and all fell within the specified range. The contents of the 4 indicator components in 7 batches of the dispensing granules（P4-P10） from manufacturers B-E were all within the range of the content converted from the standard decoction based on the quantity of the dispensing granules. ConclusionThe established specific chromatogram and QAMS based on LCTRS are reasonable and reliable. Based on the evaluation indicators of standard decoction yield， consistency of specific chromatograms， contents and transfer rates of the four index components， the 10 batches of Hedyotis Herba dispensing granules from various manufacturers have exhibited good consistency with the standard decoction， indicating that the current production process is relatively reasonable.

Based on advancements in modern medical research regarding the intricate connection between the endocrine and exocrine functions of the pancreas， as well as the relationship between pancreatic functions and traditional Chinese medicine （TCM） spleen system， this paper discussed the categorization of the pancreas. It is proposed that the pancreas is neither a true zang organ nor a fu organ， but possessed the attributes of an extraordinary fu-organ and can be classified under the spleen. The spleen governs transportation and transformation， ascent of the clear and dispersion of essence， which encompasses the endocrine and exocrine functions， and pancreatic enzymes and glucose-regulating hormones form the material basis for the spleen's function of dispersing essence. Diseases of the pancreas exhibit characteristics of both zang-organ deficiency and fu-organ excess， so treatment should simultaneously supplement zang-organ disease and regulate fu-organ disease when pancreas showing endocrine and exocrine co-morbidities， with focus on restoring the pancreas （spleen）'s dispersing essence function. Therapeutic strategies include supplementing spleen qi， nourishing spleen yin to strengthen spleen earth， unblocking spleen collaterals， raising spleen yang， and removing spleen turbidity to support the spleen's dispersing essence function， so as to replenish the essential qi of zang-fu organs， ensure their distribution throughout the body， and improve the endocrine and exocrine functions of the pancreas.

Background::The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis.Method::The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors— Gata4, Foxa3, and Hnf1a—or alternatively, the expression of two transcription factors, Gata4 and Foxa3. In vivo, we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-Cre ER; Cas9 fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination. Results::Activation of only two factors, Gata4 and Foxa3, via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. Conclusion::CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

Ghrelin is a hormone produced by the stomach that regulates energy metabolism after acting on the central nervous system.Cocaine amphetamine-regulated transcriptional peptide(CART)neurons participate in the regulation of feeding behavior and energy balance.It is known that CART neurons are influenced by hormones to regulate energy homeostasis,but whether ghre-lin exerts its pro-appetite function by influencing CART neurons is unknown.Therefore,this study focuses on the role of VMHCART neurons in the regulation of feeding and relative body weight by ghrelin.Firstly,the whole brain expression of CART was determined by immunofluorescence.Then the effect of intraperitoneal injection of ghrelin on the expression of DMHCART neurons was evalua-ted.Finally,the ghrelin was delivered to DMH and the changes of food intake and relative body weight of mice were measured.CART immunoreactive neurons were detected in medial preoptic nucleus(MPA),arcuate nucleus(ARC),dorsomedial hypothalamic nucleus(DMH),thalamic pa-raventricular nucleus(PVT)and raphe nucleus(ROb).Compared with the control group,periph-eral injection of ghrelin significantly increased the expression of DMHC ART immunoreactive neurons(P=0.037 3).DMH long-term injection of ghrelin resulted in an increase in body weight(P=0.004 0)and feed intake(P=0.023 1).The results provide anatomical evidence for the whole brain distribution of CART,which proves that ghrelin affects feed intake and body weight of mice through CART neurons in DMH,suggesting that specific neuron types and regional specificity are involved in ghrelin regulation of feed intake and energy homeostasis.

Vesicular glutamate transporter 2(VGlut2)is expressed in the PVN of the hypothalamic paraventricular nucleus(PVNVG1ut2)as an excitatory neurotransmitter,which regulates food intake and energy metabolism and plays an important role in maintaining homeostasis.However,it is not clear that the upstream and downstream projection network of PVNVGut2 neurons hinders the anal-ysis of glutamatergic neuron circuit function.Anterograde and retrograde tracer viruses were injec-ted into the PVN of VGlut2 mice by stereotactic brain injection technique to find the input and output nuclei of PVNVGlut2 neurons.Anterograde tracing results showed that PVNVGlut2 neurons pro-jected to the downstream medial amygdala(MeAD)and arcuate nucleus(ARC).Retrograde trac-ing results showed that PVNVGlut2 received input from the prefrontal nucleus(Pr),the reticular tegmental nucleus(RtTg),and the hypoglossal nucleus(12N).In addition,VGlut2 was found to be co-expressed with neuronal nitric oxide synthase(nNOS)neurons in the PVN.The anatomical net-work of PVNVG1ut2 neurons was analyzed by virus tracking tool,which laid the anatomical founda-tion for further study on the functional regulation of PVNVGlut2.

This study aims to construct C57/B6-L and A549 cell lines that stably overexpress circu-lar RNA LAMP3(circLAMP3),laying the foundation for further research on the biological func-tions of circLAMP3.Total RNA was extracted and reverse transcripted into cDNA from C57/B6-L and A549 cells to amplify the full-length sequence of circLAMP3.Then,the fragments of cir-cLAMP3 were ligated into pLC5-ciR vector to obtain pLC5-Mouse-circLAMP3 and pLC5-Human-circLAMP3 recombinant plasmids.The lentiviruses expressing circLAMP3 were packaged on tran-sient transfected HEK293T cells.C57/B6-L and A549 cells were infected with lentiviruses to gen-erate cell lines overexpressing circLAMP3 through puromycin screening.To verify the overexpres-sion efficiency of circLAMP3 of cell lines,we performed the fluorescence microscopy,PCR amplifi-cation,quantitative PCR(qPCR),and Sanger sequencing experiments.The results indicated that the overexpression plasmids of pLC5-Mouse-circLAMP3 and pLC5-Human-circLAMP3 were suc-cessfully constructed.Strong green fluorescence was observed under a fluorescence microscopy.C57/B6-L and A549 cell lines showed a significant increase in the expression of circLAMP3 by PCR and qPCR methods.Sanger sequencing results showed that the junction site of circLAMP3 was correct.This study successfully constructed C57/B6-L and A549 cell lines overexpressing circLAMP3,providing biomaterials for further exploration of the biological function of circLAMP3 in influenza virus replication.

The incidence and mortality rates of primary liver cancer remain very high,posing a serious threat to the global public health.In Asia,the guidelines from the Korean Liver Cancer Association-National Cancer Center,the Japan Society of Hepatology,and the Chinese Guidelines for Diagnosis and Treatment of Primary Liver Cancer(2024 edition)have significant influence and provide important guidance for the diagnosis and treatment of primary liver cancer.These guidelines,based on their own national condition,background,evidence and clinical practice,exhibit both commonalities and divergences in the imaging diagnosis of liver cancer.This study aims to provide a more comprehensive and scientific reference for clinicians by comparing the specific contents of three guidelines regarding screening,surveillance,imaging diagnosis and staging of liver cancer,thereby promoting the standardized diagnosis and treatment of clinical practice in the primary liver cancer.

Purpose To construct and validate a machine learning-based diagnostic model for distinguishing between Mycobacterium avium-intracellular complex pulmonary disease(MAC-PD)and pulmonary tuberculosis(PTB)via chest CT images.Materials and Methods Retrospective data from patients diagnosed with MAC-PD and PTB between May 2021 and August 2022 at Beijing Chest Hospital,Capital Medical University,which were collected as the training set.The prospective external validation set was obtained from patients at the First Affiliated Hospital of Henan University of Chinese Medicine between September 2022 and May 2023.Clinical and radiological data were analyzed,and multivariable logistic regression,random forest and support vector machine(SVM)models were established and externally validated using the validation set.The diagnostic performance of models were evaluated using receiver operating characteristic curve and precision-recall curve,and the differences of the areas under the curve of various models were compared via the Delong test.Results There were significant differences in age and hemoptysis rate between the two groups(t=30.414,P<0.001;χ2=6.186,P=0.013).There were statistically significant differences in cavity types and morphology between the two groups(χ2=6.546,P=0.011;χ2=24.113,P<0.001),but there was no significant difference in the distribution and characteristics of cavitary lesions(P>0.05).There were significant differences in the types and distribution of bronchiectasis between the two groups(χ2=4.634,P=0.031;χ2=23.145,P<0.001).Compared with logistic regression and random forest models,the SVM model had better differential diagnostic performance,and the area under the receiver operating characteristic curve,sensitivity,specificity,accuracy,positive predictive value and negative predictive value were 0.960(95%CI 0.935-0.985),85.7%,93.6%,90.5%,93.3%,88.0%and 0.885(95%CI 0.803-0.967),respectively,76.7%,80.0%,78.3%,79.3%,77.4%.The precision-recall curve showed that the SVM model had high precision and low recall,that was,the model performs well.Conclusion The machine learning-based models exhibits excellent diagnostic performance and can assist in differentiating MAC-PD and PTB.

Objective:To investigate the influencing factors of pathologic complete response (pCR) after neoadjuvant chemoradiotherapy (NCRT) in patients with locally advanced esophageal squamous cell carcinoma (ESCC), and to compare the clinical prognosis of ESCC patients with and without pCR after NCRT (40 Gy/ 20F).Methods:Among patients enrolled in a prospective clinical study, 87 ESCC patients treated with NCRT followed by surgery in Tianjin Medical University Cancer Institute & Hospital between June 2015 and October 2019 were selected. They were divided into the pCR ( n=35) and non-pCR groups ( n=52). Clinicopathological characteristics were retrospectively analyzed and subsequent follow-up was performed. Clinical prognosis and influencing factors were compared between two groups by using Kaplan-Meier and Cox regression analyses. Results:After NCRT, 40% of the ESCC patients could achieve pCR. Univariate analysis showed that patients in the pCR group had a disease-free survival (DFS) of 39.3 months and an overall survival (OS) of 64.0 months. In comparison, patients in the non-pCR group had a DFS of only 14.1 months and an OS of only 25.2 months. The differences were statistically significant (DFS: P<0.01, OS: P<0.05). Multivariate analysis revealed that whether pCR or not after NCRT, age, number of primary lesions, evaluation results after NCRT and postoperative pathological outcomes were important prognostic factors. The differences were statistically significant between two groups (all P<0.05). Conclusion:pCR after NCRT is significantly correlated with long-time survival of patients with ESCC, and pCR after NCRT has an important value in predicting clinical prognosis for long-term survival of ESCC patients.

Objective:To investigate the efficacy of 125I seed implantation combined with programmed cell death 1 (PD-1) monoclonal antibody in the treatment of patients with stage Ⅳ non-small cell lung cancer. Methods:A retrospective analysis was performed on 106 patients with stage Ⅳ non-small cell lung cancer treated in Department of Radiology of the Sixth Medical Center of PLA General Hospital from February 2017 to January 2020. All patients were divided into the combination group, radiotherapy group and immunotherapy group according to treatment methods. Besides conventional chemotherapy in all three groups, 125I seed implantation combined with PD-1 monoclonal antibody therapy was given in the combination group, 125I seed implantation therapy was delivered in the radiotherapy, and PD-1 monoclonal antibody therapy was supplemented in the immunotherapy group, respectively. Differences in the general data, maximum tumor diameter, efficacy, progression-free survival (PFS) and safety among the three groups were analyzed. One-way ANOVA was used for comparison between groups, paired sample t-test was used for comparison before and after treatment. Kaplan-Meier method was used to calculate the survival rate, and log-rank test was used to compare the survival curve. Results:After treatment, the maximum tumor diameter in the three groups was decreased, and the data in the combination group was smaller than those in the radiotherapy and immunotherapy groups ( F=4.67, P=0.011). The objective response rate (ORR), disease control rate (DCR) and PFS rate in the combination group were higher than those in the radiotherapy and immunotherapy groups ( χ2=18.93, P<0.001; χ2=11.11, P=0.004; χ2=6.67, P=0.036), and the ORR in the immunoradiotherapy group was higher than that in the radiotherapy group ( χ2=4.23, P=0.040). There was no significant difference in the occurrence of adverse reactions among the three groups ( χ2=1.60, P=0.449). Conclusion:125I seed implantation combined with PD-1 monoclonal antibody may yield high efficacy in patients with stage Ⅳ non-small cell lung cancer, which can alleviate clinical symptoms and improve long-term survival rate with good safety.