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.

In this study, we explored the pharmacological effects of Siwu Decoction in treating premature ovarian insufficiency(POI) and its molecular mechanism based on the mitophagy pathway modulated and mediated by estrogen receptor(ER) subtypes. Female Balb/c mice were divided into a control group, model group, as well as high-dose and low-dose groups of Siwu Decoction. The POI mice model was constructed by intraperitoneal injection of cisplatin. The high-dose and low-dose groups of Siwu Decoction were administered intragastrically with Siwu Decoction each day for 14 days. During this period, we monitored the estrous cycle and body weight of the mice and calculated the ovarian index. The morphology of the ovaries was detected by hematoxylin-eosin(HE) staining, and the number of primordial follicles was counted. The apoptosis of the ovarian tissue was detected by TUNEL staining. The expression levels of anti-Müllerian hormone(AMH), apoptosis-associated and mitophagy-associated proteins, ER subtypes, and the expression levels of key proteins of its mediated molecular pathways were detected by Western blot and immunohistochemistry. KGN cells were divided into a control group, model group, Siwu Decoction group, and gene silencing group. The apoptosis model was induced by H_2O_2, and PTEN-induced putative kinase 1(PINK1) gene silencing was induced by siRNA transfection. The Siwu Decoction group and gene silencing group were added to the medium containing Siwu Decoction. Cell viability was detected by CCK-8 assay. Cell senescence was detected by senescence-associated-β-galactosidase. The expression levels of apoptosis-associated and mitophagy-associated proteins were detected by Western blot. The results of in vivo experiments showed that compared with the model group, the mice in the high-dose and low-dose groups of Siwu Decoction significantly recovered the rhythm of the estrous cycle, and the levels of ovarian index, number of primordial follicles, and expression of AMH, representative indexes of ovarian function, were significantly higher, suggesting that the level of ovarian function was significantly improved. The expression levels of the apoptosis-related proteins, cytochrome C(Cyt C), cysteinyl aspartate specific proteinase 3(caspase 3), B-cell lymphoma-2(Bcl-2)-associated X(Bax), and mitophagy-associated indicator(Beclin 1) were significantly decreased, and the expression levels of Bcl-2 was significantly elevated. The positive area of TUNEL was significantly reduced, suggesting that the apoptosis level of the ovaries was significantly reduced. The expression levels of PINK1, Parkin, and sequestosome 1(p62) were significantly reduced, suggesting that the level of ovarian mitophagy was significantly down-regulated. The expression levels of ERα and ERβ were significantly elevated, and the ratio of ERα/ERβ was significantly reduced. The expression levels of key proteins in the pathway, phosphoinositide 3-kinase(PI3K) and protein kinase B(Akt), were significantly reduced, suggesting that the regulation of ER subtypes and the mediation of PI3K/Akt pathway were the key mechanisms. In vitro experiments showed that compared with the model group, the proportion of senescent cells in the Siwu Decoction group was significantly reduced. Cyt C, caspase 3, Beclin 1, Parkin, and p62 were significantly reduced, which was in line with in vivo experimental results. The proportion of senescent cells and the expression level of the above proteins were further significantly reduced after PINK1 silencing. It can be seen that Siwu Decoction can regulate the expression level and proportion of ER subtypes in KGN cells, then mediate the PI3K/Akt pathway to inhibit excessive mitophagy and apoptosis, and exert therapeutic effects of POI.
Animals ; Female ; Drugs, Chinese Herbal/administration & dosage* ; Mitophagy/drug effects* ; Primary Ovarian Insufficiency/physiopathology* ; Mice ; Mice, Inbred BALB C ; Humans ; Receptors, Estrogen/genetics* ; Apoptosis/drug effects* ; Ovary/metabolism* ; Signal Transduction/drug effects* ; Anti-Mullerian Hormone/genetics*

Orodispersible films (oral dispersible films), a novel form of oral solid dosage forms, are widely used for patients with dysphagia and those with uncontrollable autonomic behavior. In this study, suvorexant orodispersible film was prepared by hot melt extrusion technology, and the disintegration time, mechanical properties, in vitro dissolution and pharmacokinetics were evaluated, compared with other orodispersible films and commercially available tablets Belsomra. All experiments were approved by the Committee on the Management and Use of Laboratory Animals of Academy of Military Medical Sciences (IACUC-DWZX-2024-504). The results showed that the dissolution rate of suvorexant orodispersible film was faster and the mechanical strength was better than that of other commercially available orodispersible film, which could meet the needs of storage and transportation. Differential scanning calorimetry and X-ray diffraction results showed that suvorexant was dispersed within the orodispersible film in an amorphous state. The in vitro dissolution of the film was observed to be four times faster than that of the commercial tablets, achieving complete dissolution within five minutes. Pharmacokinetic evaluations in Beagle dogs revealed that the self-formulated orodispersible film exhibited no significant differences in the area under the blood concentration-time curve when compared with Belsomra. However, the film showed a faster onset of action, with a peak time that was twice as rapid, and a maximum blood concentration that was twice as high as that of Belsomra. Leveraging hot melt extrusion technology, the suvorexant orodispersible film offers a straightforward, continuous production process with consistent quality. It serves as an excellent platform for the development of solvent-free film preparations tailored for patients with special needs.

The rheological properties of drug and carrier materials have a wide range of guiding significance for the formulation and process development of solid dispersions. In this study, the rheological properties of materials with different drug carrier ratios were systematically studied with suvorexant as the model drug and copovidone as the carrier material, which provided a sufficient basis for determining the formulation and process of solid dispersions. The optimal suvorexant-copovidone ratio obtained by oscillating temperature scanning was 1∶4. If the ratio is greater than 1∶ 4, the glass transformation temperature of the material will increase significantly, and the solubilization effect of the solid dispersion will show a downward trend. The results of oscillation temperature scanning and oscillation temperature sweep can show that when the extrusion temperature is greater than 150 ℃, the viscosity of the material is less than 10 000 Pa·s, and the melt can be extruded smoothly, and the best extrusion temperature of 160-180 ℃ can be obtained by combining the dissolution results. Finally, the dissolution of suvorexant tablets guided by rheological property studies in multiple media is similar to that of the commercially available tablets Belsomra. Therefore, rheological studies can screen and optimize the formulation and process of suvorexant solid dispersions at the mechanism level, which is of great significance to improve the success rate of R&D and shorten the R&D cycle of solid dispersions prepared by hot melt extrusion.

OBJECTIVES: To study new biomarkers for the early diagnosis of retinopathy of prematurity (ROP) by analyzing the differences in blood metabolites based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and metabolomics. METHODS: Dried blood spots were collected from 21 infants with ROP (ROP group) and 21 infants without ROP (non-ROP group) who were hospitalized in the Sixth Affiliated Hospital of Sun Yat-sen University from January 2013 to December 2016. LC-MS/MS was used to measure the metabolites, and orthogonal partial least squares-discriminant analysis was used to search for differentially expressed metabolites and biomarkers. RESULTS: There was a significant difference in blood metabolic profiles between the ROP and non-ROP groups. The pattern recognition analysis, Score-plot, and weight analysis obtained 10 amino acids with a relatively large difference. Further statistical analysis showed that the ROP group had significant increases in blood levels of glutamic acid, leucine, aspartic acid, ornithine, and glycine compared with the non-ROP group (P<0.05). The receiver operating characteristic curve analysis showed that glutamic acid and ornithine had the highest value in diagnosing ROP. CONCLUSIONS Blood metabolites in preterm infants with ROP are different from those without ROP. Glutamic acid and ornithine are the metabolic markers for diagnosing ROP. LC-MS/MS combined with metabolomics analysis has a potential application value in the early identification and diagnosis of ROP.
Infant, Newborn ; Infant ; Humans ; Tandem Mass Spectrometry ; Infant, Premature ; Chromatography, Liquid ; Retinopathy of Prematurity/diagnosis* ; Glutamic Acid ; Ornithine

OBJECTIVES: To investigate the incidence of extrauterine growth retardation (EUGR) and its risk factors in very preterm infants (VPIs) during hospitalization in China. METHODS: A prospective multicenter study was performed on the medical data of 2 514 VPIs who were hospitalized in the department of neonatology in 28 hospitals from 7 areas of China between September 2019 and December 2020. According to the presence or absence of EUGR based on the evaluation of body weight at the corrected gestational age of 36 weeks or at discharge, the VPIs were classified to two groups: EUGR group (n=1 189) and non-EUGR (n=1 325). The clinical features were compared between the two groups, and the incidence of EUGR and risk factors for EUGR were examined. RESULTS: The incidence of EUGR was 47.30% (1 189/2 514) evaluated by weight. The multivariate logistic regression analysis showed that higher weight growth velocity after regaining birth weight and higher cumulative calorie intake during the first week of hospitalization were protective factors against EUGR (P<0.05), while small-for-gestational-age birth, prolonged time to the initiation of total enteral feeding, prolonged cumulative fasting time, lower breast milk intake before starting human milk fortifiers, prolonged time to the initiation of full fortified feeding, and moderate-to-severe bronchopulmonary dysplasia were risk factors for EUGR (P<0.05). CONCLUSIONS It is crucial to reduce the incidence of EUGR by achieving total enteral feeding as early as possible, strengthening breastfeeding, increasing calorie intake in the first week after birth, improving the velocity of weight gain, and preventing moderate-severe bronchopulmonary dysplasia in VPIs.
Female ; Fetal Growth Retardation ; Gestational Age ; Hospitalization ; Humans ; Incidence ; Infant ; Infant, Newborn ; Infant, Premature ; Infant, Very Low Birth Weight ; Prospective Studies ; Risk Factors

Objective: To understand the epidemiological characteristics of two local COVID-19 outbreaks caused by 2019-nCoV Omicron variant in Guangzhou, such as incubation period, serial interval, basic reproductive number (R₀) and the influence of gathering places on R₀, and provide evidence for the prevention and control of Omicron variant infection. Methods: The data of daily confirmed cases of Omicron variant infection from April 8 to May 8, 2022 in two COVID-19 outbreaks in Guangzhou were collected for model fitting. Weibull, Gamma and lognormal distribution were used to estimate incubation period and serial interval. Exponential growth method and the maximum likelihood estimation were used to estimate R₀. Results: The median of incubation period was 2.94 (95%CI: 2.52-3.38) days and median of serial interval was 3.32 (95%CI: 2.89-3.81) days. The estimated R₀ in small-size place was 4.40 (95%CI: 3.95-4.85), while the estimated R₀ at airport was 11.35 (95%CI: 11.02-11.67). Conclusion: The incubation period of Omicron variant in two local COVID-19 outbreaks in Guangzhou is significantly shorter than that of delta variant. The higher the gathering degree in a place, the larger the R₀. Due to its rapid transmission, COVID-19 epidemic is prone to occur. Therefore, the COVID-19 prevention and control strategy should be dynamically adjusted in time.
Humans ; SARS-CoV-2 ; COVID-19/epidemiology* ; Disease Outbreaks ; China/epidemiology*

AIM:To evaluate the macular microstructural changes in patients with rhegmatogenous retinal detachment(RRD)after silicone oil tamponade by spectral-domain optical coherence tomography(SD-OCT).METHODS:From November 2019 to July 2021, 27 patients with 27 eyes in RRD who underwent vitrectomy combined with silicone oil tamponade in Cangzhou Aier Eye Hospital were enrolled in this study as the observation group, other 30 healthy volunteers with 30 eyes were included in the control group. The best corrected visual acuity(BCVA)of patients before and after operation were observed, and quantified evaluation of the postoperative macular microstructural changes were performed by SD-OCT.RESULTS: The BCVA(LogMAR)of the observation group at 1wk and 3mo after operation(0.61±0.23, 0.69±0.34)were improved compared with those before operation(1.43±0.77)(all P<0.01). The cube volume and average cube thickness in the macular area at 3mo after operation in the observation group were lower than those at 1wk and 1mo after operation in the control group(all P<0.05). There were no differences in the average ganglion cell-inner plexiform layer(GCIPL)thickness, minimum GCIPL thickness, average macular retinal nerve fiber layer(mRNFL)thickness and minimum mRNFL thickness at 1wk, 1 and 3mo after operation in the observation group, but all decreased compared with the control group(all P<0.01). There were 9 eyes with subretinal fluid(SRF)in the observation group during postoperative follow-up, SRF had a tendency to be gradually absorbed, but 1 eye had a secondary macular hole; 3 eyes had ellipsoid zone disruption, which had a tendency to be gradually repaired; 2 eyes had submacular perfluorocarbon liquid; 2 eyes had macular edema.CONCLUSION: SD-OCT can show the microstructure and morphological changes very well in macular area in patients with RRD after silicone oil tamponade, and has important clinical value for the preoperative and postoperative follow-up evaluation of RRD.

Aim To study the regulatory effect of FOXA2 on liver function and blood lipid levels in mice with intrahepatic cholestasis and hyperlipidemia. Methods The model was constructed by feeding high cholesterol/cholic acid (CAD), and the FOXA2 plasmid was injected into the liver of mice by tail vein hypertension, so that the hepatocytes overexpressed F0XA2. The automatic blood biochemical analyzer was uses to detect the blood biochemical indicators of the serum, the colorimetric method to detect the cholesterol level in liver, and ELISA method to detect the liver bile acid level. Western blot was used to determine the expression of liver FOXA2, and RT-PCR to assess the mRNA expression of genes related to bile acid metabolism. H&E and oil red staining were employed to observe liver pathology. Results With the extension feeding time of the CAD, the weight of the mice continued to decrease (P < 0.01), the gallbladder increased significantly (P < 0.01), and the level of transaminase increased, and serum cholesterol and low-density lipoprotein cholesterol (P < 0.01) increased significantly. Liver tissue structure was damaged, liver cholesterol was elevated (P <0.01), bile acid level increased (P < 0.01), and lipid accumulation was serious. Overexpression of FOXA2 could significantly improve liver function and dyslipidemia in CAD-fed mice by regulating liver bile acid metabolism genes, and reduce liver bile acid levels (P < 0.01) and liver lipid accumulation. Conclusions FOXA2 improves liver function and blood lipid levels in mice with intrahepatic cholestasis and hyperlipidemia.

OBJECTIVES: To study the effect of glucose metabolism disorders on the short-term prognosis in neonates with asphyxia. METHODS: A retrospective analysis was performed on the medical data of the neonates with asphyxia who were admitted to 52 hospitals in Hubei Province of China from January to December, 2018 and had blood glucose data within 12 hours after birth. Their blood glucose data at 1, 2, 6, and 12 hours after birth (with an allowable time error of 0.5 hour) were recorded. According to the presence or absence of brain injury and/or death during hospitalization, the neonates were divided into a poor prognosis group with 693 neonates and a good prognosis group with 779 neonates. The two groups were compared in the incidence of glucose metabolism disorders within 12 hours after birth and short-term prognosis. RESULTS: Compared with the good prognosis group, the poor prognosis group had a significantly higher proportion of neonates from secondary hospitals (48.5% vs 42.6%, CONCLUSIONS Recurrent hyperglycemia in neonates with asphyxia may suggest poor short-term prognosis, and it is necessary to strengthen the early monitoring and management of the nervous system in such neonates.
Asphyxia ; Asphyxia Neonatorum/epidemiology* ; Humans ; Hyperglycemia ; Infant, Newborn ; Prognosis ; Retrospective Studies