ObjectiveThis study proposes a novel single-cell protein localization method based on a class perception graph convolutional network (CP-GCN) to overcome several critical challenges in protein microscopic image analysis, including the scarcity of cell-level annotations, inadequate feature extraction, and the difficulty in achieving precise protein localization within individual cells. The methodology involves multiple innovative components designed to enhance both feature extraction and localization accuracy. MethodsFirst, a class perception module (CPM) is developed to effectively capture and distinguish semantic features across different subcellular categories, enabling more discriminative feature representation. Building upon this, the CP-GCN network is designed to explore global features of subcellular proteins in multicellular environments. This network incorporates a category feature-aware module to extract protein semantic features aligned with label dimensions and establishes a subcellular relationship mining module to model correlations between different subcellular structures. By doing so, it generates co-occurrence embedding features that encode spatial and contextual relationships among subcellular locations, thereby improving feature representation. To further refine localization, a multi-scale feature analysis approach is employed using the K-means clustering algorithm, which classifies multi-scale features within each subcellular category and generates multi-cell class activation maps (CAMs). These CAMs highlight discriminative regions associated with specific subcellular locations, facilitating more accurate protein localization. Additionally, a pseudo-label generation strategy is introduced to address the lack of annotated single-cell data. This strategy segments multicellular images into single-cell images and assigns reliable pseudo-labels based on the CAM-predicted regions, ensuring high-quality training data for single-cell analysis. Under a transfer learning framework, the model is trained to achieve precise single-cell-level protein localization, leveraging both the extracted features and pseudo-labels for robust performance. ResultsExperimental validation on multiple single-cell test datasets demonstrates that the proposed method significantly outperforms existing approaches in terms of robustness and localization accuracy. Specifically, on the Kaggle 2021 dataset, the method achieves superior mean average precision (mAP) metrics across 18 subcellular categories, highlighting its effectiveness in diverse protein localization tasks. Visualization of the generated CAM results further confirms the model’s capability to accurately localize subcellular proteins within individual cells, even in complex multicellular environments. ConclusionThe integration of the CP-GCN network with a pseudo-labeling strategy enables the proposed method to effectively capture heterogeneous cellular features in protein images and achieve precise single-cell protein localization. This advancement not only addresses key limitations in current protein image analysis but also provides a scalable and accurate solution for subcellular protein studies, with potential applications in biomedical research and diagnostic imaging. The success of this method underscores the importance of combining advanced deep learning architectures with innovative training strategies to overcome data scarcity and improve localization performance in biological image analysis. Future work could explore the extension of this framework to other types of microscopic imaging and its application in large-scale protein interaction studies.

H9N2 is a low-pathogenic avian influenza subtype that has a significant impact on the global poultry industry.Since 1994,H9N2 has continuously mutated as a zoonotic pathogen in China,thus posing a severe threat to the poultry indus-try as well as human life and health.In particular,gene rearrangements and recombinations between H9N2 and other influenza viruses have increased the likelihood of avian influenza viruses crossing species barriers and infecting humans and other mam-mals,thereby posing new threats to global public health.Therefore,this article aims to provide a brief discussion of the epide-miology and vaccine research progress related to the H9N2 virus,serving as a valuable resource for safeguarding the economy of the poultry industry and global public health security.

Objective To investigate the effects of ursolic acid(UA)on the TLR4/NF-κB signaling pathway and Th17/Treg cells in type 1 diabetes mellitus(T1DM)rats.Methods T1DM rat models were established by intraperitoneal injection of streptozotocin(STZ)and randomly divided into blank(Control),Model,metformin(MET),and UA groups.General conditions,such as body weight and blood glucose,were recorded,and peripheral blood and pancreatic tissues were collected after 6 weeks of gavage to assess insulin treatment.Immunohistochemistry was used to observe pathological changes in pancreatic tissues.Horseshoe crab reagent was used to assess changes in serum lipopolysaccharide(LPS)content.qRT-PCR was used to measure expression of pancreatic TLR4,MyD88,IκBα,and NF-κB p65 mRNAs,and mRNA expression of transcription factors RORγt and Foxp3.Western blot was used to assess pancreatic TLR4,MyD88,IκBα,NF-κB p65,RORγt,and Foxp3.Flow cytometry was used to assess changes Th17/Treg cell ratio in peripheral blood.ELISA were used to measure serum contents of TNF-α,IL-6,and IL-1 β.Results After STZ-induced diabetic rats were treated by gavage for 6 weeks,compared with the Model group,the fasting blood glucose of rats in MET and UA groups was significantly decreased and their body weights were increased.Inflammatory infiltration of pancreatic islet β-cells was reduced.Expression of TLR4,MyD88,IκBα,NF-κB p65,and RORγt mRNAs and proteins was significantly decreased.LPS content was significantly decreased.IκBα and Foxp3 mRNA and protein expression was significantly increased.The Th17/Treg ratio was significantly decreased,and TNF-α,IL-6,and IL-1 β contents were significantly decreased.Conclusions UA improves the symptoms of rats by reducing the LPS shift,inhibiting the TLR4/NF-κB pathway,down-regulating RORγt expression,and up-regulating Foxp3 expression to correct the imbalance in the Th17/Treg cell ratio in T1DM rats.

Objective:To prepare mesenchymal stem cells with antioxidant capacity (AO-MSC ) from human umbilical cords and evaluate its cell biological properties.Methods:In control group,mesenchymal stem cells (MSC) were isolated by digesting human umbilical cord Wharton's Jelly tissues with 0.2％ collagenase Ⅱ,and the released cells were collected and cultured in an animal serum-free culture medium.In AO-MSC group,incompletely collagenase Ⅱ-digested tissue debris were allowed to adhere to flusk flat bottoms and the AO-MSC was harvested by adherent culture. The conventional digestion and culture method was used as control.MSC colony forming ability was evaluated by fibroblast colony forming assay (CFU-F).MSC proliferative capacity was evaluated by CCK-8 assay.The MSC surface markers were detected by using flow cytometry and immunofluorescence staining.The adipogenic and osteogenic capacity of MSC was evaluated by multi-differentiation in vitro,and the mRNA expression of genes that control adipogenic and osteogenic differentiation was detected by real-time fluorescence quantitative PCR (RT-qPCR );Moreover,the mRNA expression of antioxidant substances such as SOD-1,GSH,GAT,and NQO1 in MSC was also evaluated by RT-qPCR.Results:The AO-MSC isolated by this strategy reached a confluence of 80％-90％ at around 18 days and grew in a swirling pattern.Flow cytometry and immunofluorescence staining assays showed that CD73,CD29,CD105,CD90 were highly expressed and CD31,CD45,HLA-DR were scarcely expressed in AO-MSC.AO-MSC exhibited stronger self-renewal and differentiation ability compared to MSC.However,the in vitro adipogenic-osteogenic capacity of MSC in the control group was stronger than that of AO-MSC.RT-qPCR assay showed that AO-MSC expressed higher mRNA levels of antioxidant substances compared to MSC.Conclusion:Human AO-MSC is successfully prepared from human umbilical cord without animal serum.

Objective To study the diagnostic value of miR-571 for liver fibrosis by detecting miR-571 expression in the peripheral blood of patients with liver fibrosis.Methods From December 2022 to September 2023,40 patients with liver fibrosis,40 patients with chronic hepatitis,and 40 healthy controls were chosen as research subjects.The expression level of miR-571 in peripheral blood was detected using a real-time quantitative polymerase chain reaction,and the relative expression of miR-571 in each group was evaluated.The Spearman correlation method was utilized to examine the relationship between miR-571 and clinical detection indices.To assess the capacity of miR-571 and the multivariate diagnostic model to identify liver fibrosis,binary logistic regression was used to create a multivariate diagnostic model,and ROC curves were generated.Results The expression of miR-571 was significantly higher in the liver fibrosis group than in the healthy control and hepatitis groups,and the difference was statistically significant(P<0.001).The expression level of miR-571 was positively connected with ALT,APRI score,and FIB-4 index(r = 0.23,0.30,0.22,P<0.05)and negatively correlated with PLT(r =-0.19,P<0.05)according to Spearman correlation analysis.Logistic regression research revealed that miR-571 and the FIB-4 index were independent risk factors for liver fibrosis.The AUC for miR-571 to diagnose fibrosis was 0.91(95%CI:0.85～0.96),while the AUC for miR-571 paired with the FIB-4 index was 0.94(95%CI:0.90～0.98).Conclusion MiR-571 expression was shown to be considerably higher in the peripheral blood of hepatic fibrosis patients,and the combined FIB-4 index offers some clinical diagnostic value for hepatic fibrosis.

Female ; Humans ; Breast Neoplasms ; Testosterone ; Prolactin ; Estradiol ; Testosterone Congeners ; China

Objective To perform laboratory detection and molecular traceability analysis on a case of imported kala-azar in Shenzhen to determine the infection strain. Methods Bone marrow puncture fluid and blood samples from a case of kala-azar in Shenzhen were collected for laboratory tests. The patient's bone marrow puncture fluid smears were stained with Giemsa and examined under a microscope. Blood samples were examined for antibodies using the rk39 visceral leishmania rapid diagnostic reagent. Whole blood DNA was extracted, and the ITS-1 sequence was amplified by PCR, sequenced and aligned, and a phylogenetic tree was constructed based on the ITS-1 sequence. Results Microscopic examination of the patient's bone marrow smears revealed a large number of Leishmania amastigotes without flagella, confirming the diagnosis of kala-azar. The patient's blood was tested positive with the rk39 rapid diagnostic reagent, and PCR amplification yielded an ITS-1 gene product sequence that matched the expected size. Sequence alignment with the NCBI database showed 100% sequence similarity with the ITS-1 gene sequence of Leishmania infantum, confirming the infecting strain as Leishmania infantum. Phylogenetic tree construction of the amplified ITS-1 sequence revealed clustering into a clade with Leishmania infantum , and close to KC347299, one of the reference sequence selected. Conclusions The case of kala-azar in Shenzhen was caused by Leishmania infantum. Kala-azar still occurs in China, so the diagnostic technology of medical personnel in non-epidemic areas should be strengthened so that they can actively use new diagnostic technologies to assist in diagnosis, thus improving their prevention and control ability of Leishmania parasites.

Objective To analyze the burden and changing trend of testicular cancer in China from 1990 to 2019.Methods Based on the 2019 Global Burden of Disease Database(GBD 2019),analyze the incidence,mortality,disability-adjusted life years(DALYs),years of life lost(YLLs),years lived with disability(YLDs)and their variation trend of testicular cancer in Chinese population from 1990 to 2019.Evaluating changes in age standardized rate(ASR)by calculating annual estimated percentage change(EAPC).According to the age grouping,analyze the age distribution characteristics of testicular cancer disease burden by age group.Results In 2019,the incident cases,deaths,age-standardized incidence rate,and age-standardized mortality rate of testicular cancer in China were 17.17×103,1.21×103,2.39/105,and 0.16/105,respectively.Compared to 1990,incident cases,deaths,and age-standardized incidence rate increased obviously in China,which was consistent with the global change trend,while the increase was higher than the global level.However,both Chinese and global age-standardized mortality rate showed a downward trend.From 1990 to 2019,DALYs,YLLs and YLDs of testicular cancer increased by 29.66%,9.83%and 720.91%respectively in China.The two age groups,0-15 years group and 30-35 years group,were with highest incidence of testicular cancer,while the highest disease burden of testicular cancer was 30-35 years.Conclusion From 1990 to 2019,the disease burden of testicular cancer in China showed an upward trend.Adolescents and young adults should be the priority population for screening and prevention due to their higher incidence and disease burden.

Objective To culture and expand primary rat aortic vascular stem cells in vitro and evaluate their characteristics as mesenchymal stem cells.Methods The thoracic and abdominal aortas isolated from 2-to 3-week-old Sprague-Dawley rats were cut into vascular segments 2.0 mm in length and cultured in culture flasks till adhesion and solidification of the outer membranes.The primary cells were further cultured to 80%-90%confluence before passaging.The morphology and growth characteristics of the cells were observed under a microscope,and the expressions of surface marker CD molecules on the cells was analyzed using flow cytometry.Adipogenic and osteogenic differentiation assays were performed to assess the capacity of the cells for multilineage differentiation.Results After 3 days of culture,a small number of spindle,star-shaped or polygonal cells migrated out from the peripheral of the vascular segments.At 5-6 days,island-like cell clusters occurred and the cells began to proliferate rapidly.The cell clusters expanded radially and showed signs of cell cloning.At 7-8 days,the cells fused into sheets and displayed a vortex-like distribution.The cells in the third passage presented with a uniform morphology,showing a typical fibroblast-like arrangement.Flow cytometry showed that the cells expressed predominantly CD44(80.3%),CD73(62.2%)and CD90(46.8%)with low expressions of CD34(1.1%),CD45(0.2%)and CD11b/c(0.2%).Adipogenic and osteogenic differentiation experiments demonstrated that the cells were capable of lipogenic and osteogenic differentiation in vitro.Conclusion Rat aortic vascular stem cells with mesenchymal stem cell characteristics can be successfully isolated and cultured by adherent culture of the segmented outer membrane.

Objective:To investigate the effects of targeted silencing of muscle blind-like protein 1 (MBNL1) gene on the proliferation, apoptosis and migration abilities of leukemia cell line K562 and their possible mechanisms.Methods:Single gene analysis was used to search for differences in MBNL1 gene expression between leukemia samples (173 cases) and healthy control samples (70 cases) in The Cancer Genome Atlas (TCGA) database. The data were updated in 2018. The logarithmic growth phase leukemia cell line K562 was taken and divided into sh-MBNL1 group (transfected with shRNA sequence with targeted silencing of MBNL1 gene), sh-NC group (transfected with corresponding negative control shRNA sequence) and blank control group (not transfected with shRNA sequence). Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of MBNL1, transforming growth factor β 1 (TGF-β 1) and Smad7 mRNA in each group of cells; Western blotting was used to detect the relative expression levels of cell migration-related proteins, apoptosis-related proteins, TGF-β 1, and Smad7 proteins; CCK-8 method was used to detect cell proliferation ability; Transwell method was used to detect cell migration ability. Results:In TCGA database, the relative expression level of MBNL1 gene in leukemia samples was higher than that in healthy control samples ( P < 0.05). The relative expression levels of MBNL1 protein in the sh-MBNL1 group, sh-NC group and blank control group were 0.71±0.11, 1.00±0.11 and 1.03±0.10, respectively, and the difference was statistically significant ( F = 7.78, P < 0.05); the relative expression level of MBNL1 protein in the sh-MBNL1 group was lower than that in the sh-NC group and blank control group (both P < 0.05). The results of CCK-8 assay showed that the cell proliferation ability of sh-MBNL1 group at 72 and 96 hours after transfection was higher than that of sh-NC group and blank control group (both P < 0.05). The Transwell method detection results showed that the number of cell membrane penetration in the sh-MBNL1 group, sh-NC group and blank control group were 666±135, 1 072±157 and 1 006±51, respectively, and the difference was statistically significant ( F = 9.40, P = 0.014); the number of cell membrane penetration in the sh-MBNL1 group was less than that in the sh-NC group and blank control group (both P < 0.05). The relative expression level of E-cadherin protein in the sh-MBNL1 group was higher than that in the sh-NC group and blank control group (both P < 0.01); the relative expression levels of Vimentin, Bax, caspase-3, TGF-β 1, and Smad7 proteins in the sh-MBNL1 group were lower than those in the sh-NC group and blank control group (all P < 0.01). The qRT-PCR detection results showed that the relative expression levels of TGF-β 1 mRNA and Smad7 mRNA in the sh-MBNL1 group were lower than those in the sh-NC group and blank control group (both P < 0.05). Conclusions:Silencing of MBNL1 gene can promote the proliferation of leukemia cell line K562, weaken its migration ability, and affect cell apoptosis. The mechanism may be related to the regulatory effect of TGF-β-Smad signaling pathway.