In this study, we constructed a GLP-2R-HEK293 cell line and established a method for the determination of the in vitro biological activity of teduglutide based on HTRF, after optimizing experimental conditions and methodological verification. We also carried out relative potency detection of teduglutide pharmaceutical products using this method. The result showed that there was a quantitative-efficient relationship between the teduglutide activity and cAMP contents in GLP-2R-HEK293 cells, which conformed to four-parameter model. Method verification results of five concentrations of teduglutide (64%, 80%, 100%, 125% and 156%) met the requirements of the General Rules of Chinese Pharmacopoeia, 2020 edition, Volume IV (9401). We then analyzed the relative potency of three batches of teduglutide drug substances and three batches of drug products. The linearity, regression and parallelism of the obtained curves all fit the system suitability requirements. The relative potency of six batches of teduglutide was from 83% to 105%. In summary, the biological activity detection method established in this study was accurate, precise, simple and time-saving, which can be used for quality control of teduglutide pharmaceutical products.

Alzheimer’s disease (AD) is a chronic, progressive, and irreversible neurodegenerative disorder that typically begins with a subtle onset and progresses slowly. Pathologically, it is characterized by two hallmark features: the extracellular accumulation of amyloid β-protein (Aβ), forming senile plaques, and the intracellular hyperphosphorylation of tau protein, resulting in neurofibrillary tangles (NFTs). These pathological changes are accompanied by substantial neuronal and synaptic loss, particularly in critical brain regions such as the cerebral cortex and hippocampus. Clinically, AD presents as a gradual decline in memory, language abilities, and spatial orientation, significantly impairing the quality of life of affected individuals. With the aging population steadily increasing in China, the incidence of AD is rising, making it a major public health concern that requires urgent attention. The growing societal and economic burden of AD underscores the pressing need to identify effective diagnostic biomarkers and develop novel therapeutic strategies. Among the various molecular signaling pathways involved in neurological disorders, the Notch signaling pathway is especially noteworthy due to its evolutionary conservation and regulatory roles in cell proliferation, differentiation, development, and apoptosis. In the central nervous system, Notch signaling is essential for neurodevelopment and synaptic plasticity and has been implicated in several neurodegenerative processes. Although some studies suggest that Notch signaling may influence AD-related pathology, its precise role in AD remains poorly understood. In particular, the interaction between Notch signaling and non-coding RNAs (ncRNAs)—key regulators of gene expression—has received limited attention. NcRNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), are known to exert extensive regulatory functions at both transcriptional and post-transcriptional levels. Dysregulation of these molecules has been widely associated with various diseases, including cancers, cardiovascular conditions, and neurodegenerative disorders. Notably, interactions between ncRNAs and major signaling pathways such as Notch can produce widespread biological effects. While such interactions have been increasingly reported in several disease models, comprehensive studies investigating the regulatory relationship between Notch signaling and ncRNAs in the context of AD remain scarce. Given the capacity of ncRNAs to modulate signaling cascades and form complex regulatory networks, a deeper understanding of their crosstalk with the Notch pathway could provide novel insights into AD pathogenesis and reveal potential targets for diagnosis and treatment. In this study, we investigated the regulatory landscape involving the Notch signaling pathway and associated ncRNAs in AD using bioinformatics approaches. By integrating data from multiple public databases, we systematically identified significantly dysregulated Notch pathway-related genes and their interacting ncRNAs in AD. Based on this analysis, we constructed a lncRNA-miRNA-mRNA regulatory network to elucidate the potential mechanisms linking Notch signaling to ncRNA-mediated gene regulation in AD pathogenesis. Furthermore, we explored the internal relationships and molecular mechanisms within this network and assessed the feasibility and clinical relevance of these molecules as early diagnostic biomarkers and potential therapeutic targets for AD. This study aims to deepen our understanding of the molecular basis of AD and offer novel strategies for its diagnosis and treatment.

Objective To investigate the risk factors for recurrence of persistent atrial fibrillation(AF)after radiofrequency ablation with circumferential pulmonary vein isolation+top line+back wall line based on the features of cardiac imaging and serum biomarkers,and then to establish a nomogram risk prediction model.Methods A total of 172 patients with persistent AF admitted to our hospital from June 2022 to September 2023 were enrolled and then according to recurrence or not in 6 months after surgery,they were divided into the recurrence group(51 cases)and the non-ecurrence group(121 cases).Before surgery,routine electrocardiography,and transthoracic and esophageal echocardiography were performed,while blood routine indicators and related biochemi-cal indicators were measured.All patients underwent radiofrequency ablation with circumferential pulmonary vein isolation,top line,and back wall line.They were followed up for 6 months after surgery.Binary logistic analysis was used to analyze the independent risk factors for postoperative recurrence,and then a nomogram risk prediction model was constructed and its diagnostic per-formance was evaluated.Results Lager LAD,higher LAVI,neutrophil count and NLR,and ele-vated BNP and CRP levels,while lower LAAFV,LAAEV and LAAEF were observed in the re-current group than those in the non-recurrent group(P＜0.01).Binary logistic regression analysis showed that elevated LAVI(OR=1.160,95％CI:1.006-1.337),decreased LAAEV(OR=0.740,95％CI:0.583-0.940),decreased LAAEF(OR=0.608,95％CI:0.422-0.877),elevated BNP(OR=1.017,95％CI:1.004-1.030),and higher NLR(OR=10.116,95％CI:1.316-77.755)were independent risk factors for recurrence after radiofrequency ablation of pulmonary vein isolation+top line+posterior wall line in persistent AF patients(P＜0.05,P＜0.01).The AUC value of the nomogram model constructed with LAVI,LAAEV,LAAEF,BNP and NLR in predicting postop-erative recurrence was 0.889(95％CI:0.833-0.932).Conclusion The cardiac imaging parame-ters LAVI,LAAEV and LAAEF,and serum biomarkers BNP and NLR are closely associated with postoperative recurrence of persistent AF in patients after radiofrequency ablation with cir-cumferential pulmonary vein isolation+top line+back wall line,and the relevant nomogram mod-el has better diagnostic value for postoperative recurrence.

【Objection】 To evaluate the effects of aortic valve stenosis on the flow field characteristics of ascending aortic aneurysm by computational fluid dynamics and hemodynamic indexes. MethodsA total of 63 patients aged 18 or over with ascending aortic dilation or aneurysm were retrospectively recruited from Sun Yat-sen Memorial Hospital of Sun Yat-sen University and assigned to aortic valve stenosis （AS） or none aortic valve stenosis （NAS） group. Propensity score matching analysis and ascending aortic division based on centerline were performed to compare the hemodynamic indexes ［pressure， shear stress rate （SSR）， wall shear stress （WSS）， time average shear stress rate （TASSR）， time average wall shear stress （TAWSS）］. ResultsThere were significant differences in SSR （191.70 ［117.22~248.78］ s^-1 vs. 93.23 ［68.59~145.35］ s^-1， Z = 2.167， P = 0.030） and WSS （2.87 ［1.88~4.00］ Pa vs. 1.20 ［1.04~2.18］ Pa， Z = 2.233， P = 0.026） between AS group and NAS group. The differences were mainly found in the proximal and lesser curvature of ascending aorta （P < 0.05）. There was no statistical significance in pressure， TASSR or TAWSS. ConclusionsAortic valve stenosis alters the hemodynamics in ascending aortic dilation or aneurysms， which increases tangential component force （e.g. SSR， WSS） but has no significant effects on perpendicular component force （e.g. pressure）.

Objective:To describe the baseline characteristics of the subjects enrolled in the China Quantitative CT (QCT) big data program in 2018—2019.Methods:Based on baseline data from the Chinese health big data project from January 2018 to December 2019 from the eligible enrolled population, measurements of bone mineral density (BMD) and visceral adipose tissue (VAT) were performed using Mindways′ QCT Pro Model 4 system. The baseline data of age, gender, regional distribution, height, weight, abdominal circumference, blood pressure, blood routine and blood biochemical tests were analyzed. And the single factor analysis of variance (ANOVA) was used to check the age related trend of BMD and VAT in both genders.Results:After screening the inclusion exclusion criteria and outliers of the main indicators, 86 113 people were enrolled in the project. The enrollment rate was 92.47%, including 35 431 (41.1%) women and 50 682 (58.9%) men, and the ratio of men to women was 1.43. The mean age was (50.3±12.7) years in all the subjects, and it was (50.2±12.8) years and (50.4±12.5) years in men and women, respectively, and there was no statistical difference between the two genders ( P>0.05). Total of 43 833 people were enrolled in east China, it was the largest group by region (50.90%), it was followed by central China (16 434 people, 19.08%), and the number of people enrolled in Northeast China was the lowest (2 914 people, 3.38%). The rate of completing of health information indicators related to the main outcome of the study were all above 70%, and there were significant differences between men and women (all P<0.05). The mean BMD was (139.33±46.76) mg/cm 3 in women, (135.90±36.48) mg/cm 3 in men, which showed a decreasing trend with age in both gender (both P<0.001); the mean intra-abdominal fat area was (116.39±56.23) cm 2 in women, (191.67±77.07) cm 2 in men, and there was an increasing trend with age in both men and women (both P<0.001). Conclusions:There are gender differences in BMD and VAT measured by QCT with different age tendency, and there are gender differences in health information index. Regional factors should also be taken into account for regional differences in the inclusion of data.

Objective:To investigate the normal reference values of spinal bone mineral density measured by quantitative computed tomography (QCT) and the differences of bone mineral density (BMD) in different regions of in Chinese adult males.Methods:Men who underwent low-dose CT lung scan for cancer screening in regions of Northeast, North, East, South, Central and Southwest of China from January 2018 to December 2019 were selected. And the lumbar vertebrae BMD values in the male subjects were measured by the QCT system (Mindways Software, Inc.). The mean BMD values and their decline rates were calculated at an age interval of 10 years, and the prevalence of osteoporosis was calculated according to the American College of Radiology spine QCT osteoporosis diagnostic criteria.Results:A total of 50 682 males with a mean age of (50.22±12.79) years (ranged 20 to 98 years) were included in this study. The peak BMD of (173.11±28.56) mg/cm 3 in the healthy Chinese adult male population appeared in the age group of 20 to 29 years and then declined with age. Before the age of 70 years, the BMD was relatively higher in males in South China, and it was lower in Central China and Southwest China, and it was intermediate in Northeast, North and East of China, with statistically significant differences. There was no significant differences in BMD in the males in the two age groups of 70 to 79 years and 80 and older among the regions in China. The overall decline rate of spinal BMD in Chinese males under QCT was about 46.92% over the lifetime, and it declined obviouslyin the 40-49 age group. The overall prevalence of osteoporosis in Chinese male population aged 50 years and above was approximately 11.42%, with the highest prevalence in Southwest China and Central China (14.72% and 13.87%, respectively) and the lowest in North China and South China (8.53% and 7.71%, respectively). Conclusions:A reference of lumbar spine BMD values for healthy males in China based on QCT is established. BMD values were highest in South China and Lowest in Central China.

Objective:To establish the normal reference value of lumbar bone mineral density (BMD) under quantitative CT (QCT) in Chinese healthy adult females and to explore the regional differences.Methods:Total of 35 431 healthy women who met the inclusion criteria of Chinese health quantitative CT big data program were selected in this study. The BMD of the central plane of L 1 and L 2 vertebrae was measured by Mindways′s QCT system, and the mean value was taken. One-way analysis of variance was used to compare the BMD differences of lumbar vertebrae in women of different ages and regions. The subjects were grouped by an age interval of 10 years, and the level of BMD in different regions of the same age group were compaired. Results:The peak BMD of Chinese healthy adult women appeared in the age group of 20-29 years (Northeast China(183.01±24.58) mg/cm 3, North China (188.93±24.80) mg/cm 3, East China (187.54±27.71) mg/cm 3, South China (186.22±33.72) mg/cm 3, Central China (176.33±24.91) mg/cm 3, Southwest China(182.25±28.00) mg/cm 3), and then it decreased with age. The level of BMD in different regions decreased with the age. Before the age of 70 years, BMD in Central and Southwest China was always at a low level((176.23±24.91) to (90.38±28.12) mg/cm 3, 182.25±28.00 to (88.55±25.68) mg/cm 3), lower than those in Northeast China ((183.01±24.58) to (99.69±27.85) mg/cm 3), North China ((188.93±24.80) to (95.89±26.12) mg/cm 3), East China ((187.54±27.71) to (95.65±27.86) mg/cm 3). After 70 years of age, BMD tended to be the same in different regions ( P>0.05). The BMD values in Central China and Southwest China were similar in the age group of 40-60 years ( P>0.05). The BMD values in the health adult femles in the age group of 60 years in different regions of Chinawere all lower than those of bone mass abnormality (all P<0.05). The detection rate of osteoporosis in females over 50 years was the highest in Southwest China (25.65%) and it was the lowest in North China (17.30%). Conclusions:This study establishes reference values of BMD under QCT in healthy Chinese women, which can be used as a reference basis for identifying women with low BMD who are at risk of osteoporosis. The BMD value is the lowest in Southwest China and the highest in South China.

Objective:To use quantitative computed tomography (QCT) technology to measure the bone mineral density of the spine of the Chinese healthy population, and to explore its correlation with hemoglobin and serum albumin.Methods:The data in this study came from the China Health Quantitative CT Big Data Project (China Biobank). The spine bone density was measured by using QCT Pro Image Analysis System and all cooperating centers used the European spine phantom (NO.145) for quality control. Total of 50 053 healthy persons who met the criteria for entry were selected as the research subjects. The subjects were divided into 7 groups according to age. The general data, spine bone density, serum albumin, hemoglobin of the subjects were collected. The single-factor analysis of variance, Pearson correlation analysis and multi-classification logistic regression model were applied to analyze the correlation between bone density and hemoglobin and serum albumin.Results:The bone mineral density of healthy people decreased with age ( P<0.05), and there were significant differences in hemoglobin, serum albumin and body mass index (BMI) among different age groups (all P<0.05). Linear correlation analysis showed that there were positive correlation between bone mineral density and hemoglobin in healthy males in different age groups ( r=0.086, 0.101, 0.076, 0.090, 0.072, 0.123, 0.100, all P<0.01). There were negative correlation between bone mineral density and hemoglobin in certain age groups in women (40-49 years group: r=-0.027; 70-79 yearsgroup: r=-0.077; both P<0.05). And corelation were found between bone mineral density and serum levels of albumin in certain age groups of healthy subjects (among men, 30-39 years group: r=-0.048; 40-49 years group, r=-0.027; 70-79 years group, r=-0.051; among women, 30-39 years group: r=-0.044; 40-49 years group, r=-0.042; 50-59 years group, r=-0.086; 70-79 years group, r=-0.070; all P<0.05). After adjusting for age and BMI, the multi-category logistic regression analysis showed that the hemoglobin level was protective factor of normal bone density ( OR=1.022, 95% CI:1.017-1.027) and decreased bone density ( OR=1.012, 95% CI:1.007-1.016) in healthy males, and the serum albumin was risk factor for normal bone density ( OR=0.926, 95% CI:0.905-0.948) and decreased bone density ( OR=1.006, 95% CI:0.951-1.011) in healthy women. Conclusion:There is a correlation between bone mineral density and hemoglobin and serum albumin in Chinese healthy population. Hemoglobin is a protective factor for bone mineral density in men, and serum albumin is a risk factor for bone mineral densityin women.

Estimation of postmortem interval （PMI） has always been one of the difficult problems for forensic scientists. It is especially hard to estimate the PMI of highly decomposed corpses in the wild or in secluded houses with conventional methods. Therefore, application of insect evidence at the scene is usually required for estimation. Sarcosaprophagous flies of different species have totally different developmental rates. In actual cases, direct measurement of the body length of the larvae, calculation of accumulated temperature and succession stages without species identification, or calculation based on incorrect species identification would often lead to a large deviation between the calculated results and the real PMI. This mistake would also mislead the case investigation. Therefore, accurate species identification should be implemented before any PMI estimation of decomposed corpses with forensic entomological methods. This article reviews the general and ultramicroscopic species identification and molecular biological species identification methods of different stages of sarcosaprophagous flies, in order to provide new ideas and methods for related research and practice, and provide reference for the application and promotion of forensic entomology in the front line of public security.
Animals ; Autopsy ; Cadaver ; Diptera ; Entomology ; Larva ; Postmortem Changes

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.
Microfluidics ; Mutagenesis ; Pichia ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins