OBJECTIVE: To elucidate the role and mechanism of microRNA-145-5p (miR-145-5p) in hypoxia-induced pyroptosis of human alveolar epithelial cells. METHODS: In vitro, human alveolar epithelial cell line BEAS-2B was cultured. Cells in the logarithmic growth phase were cultured to 80% confluence and then used for the experiment. (1) BEAS-2B cells were cultured under 1% O₂ hypoxic condition, with a normoxic control group. Western blotting was employed to detect the expressions of pyroptosis marker proteins [NOD-like receptor protein 3 (NLRP3), Gasdermin D N-terminal domain (GSDMD-N), and caspase-1] in cells cultured for 24 hours. Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-145-5p in cells cultured for 6 hours and 12 hours. (2) Cells were transfected with 30 nmol/L miR-145-5p mimic to overexpress miR-145-5p expression under normoxic condition or 30 nmol/L miR-145-5p inhibitor to suppress miR-145-5p expression under hypoxic condition. Control group and negative control group were respectively set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of pyroptosis marker proteins and nuclear factor-E2-related factor 2 (Nrf2) in cells. Flow cytometry was applied to detect the level of reactive oxygen species (ROS) in cells. The target genes of miR-145-5p were predicted by miR target gene prediction software miRWalk and verified by Western blotting. (3) Under hypoxic condition, cells were transfected with 6.94 ng/μL silent information regulator 5 (Sirt5) overexpression plasmid or pretreated with 12.5 mmol/L N-acetyl-L-cysteine (NAC) as an ROS inhibitor. The empty plasmid group and control group were set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of Sirt5, Nrf2, and pyroptosis marker proteins in cells. Flow cytometry was used to detect the level of ROS in cells. RESULTS: (1) Compared with the normoxic control group, the expression levels of pyroptosis marker proteins in the 24-hour hypoxia group was significantly increased, indicating that hypoxia could induce pyroptosis in BEAS-2B cells. The expression level of miR-145-5p in cells gradually increased with the extension of hypoxia induction time, indicating that hypoxia could cause the increase of miR-145-5p expression level. (2) The expression levels of pyroptosis marker proteins in cells of miR-145-5p mimic group significantly increased under normoxic condition as compared with the control and negative control groups [NLRP3 protein (NLRP3/β-actin): 1.58±0.07 vs. 1.00±0.01, 0.98±0.07, GSDMD-N protein (GSDMD-N/β-actin): 1.71±0.03 vs. 1.01±0.01, 0.85±0.03, caspase-1 protein (caspase-1/β-actin): 2.33±0.04 vs. 1.01±0.01, 1.05±0.04, all P < 0.05], Nrf2 protein expression level was significantly decreased (Nrf2/β-actin: 0.79±0.03 vs. 1.00±0.01, 1.03±0.04, both P < 0.05), ROS level was significantly up-regulated (fluorescence intensity: 1.74±0.03 vs. 1.00±0.01, 0.92±0.03, both P < 0.05). Under hypoxia condition, compared with control group and negative control group, the expression levels of pyroptosis marker proteins in miR-145-5p inhibitor group were significantly decreased [NLRP3 protein (NLRP3/β-actin): 0.21±0.04 vs. 1.70±0.02, 1.63±0.04; GSDMD-N protein (GSDMD-N/β-actin): 1.32±0.02 vs. 2.51±0.02, 2.72±0.03; caspase-1 protein (caspase-1/β-actin): 0.56±0.01 vs. 2.77±0.02, 3.12±0.03; all P < 0.05], Nrf2 protein expression level was significantly increased (Nrf2/β-actin: 1.57±0.04 vs. 1.22±0.01, 1.28±0.04, both P < 0.05), ROS level was significantly down-regulated (fluorescence intensity: 0.64±0.05 vs. 1.87±0.04, 1.70±0.07, both P < 0.05). The results indicated that miR-145-5p could promote cell pyrodeath. The predictive result of miRWalk showed that the 3' untranslated region (3'UTR) of Sirt5 had complementary base binding sites with miR-145-5p. The expression level of Sirt5 protein in cells of miR-145-5p mimic group was significantly lower than that of control group and negative control group under normoxic condition (Sirt5/β-actin: 0.59±0.03 vs. 1.00±0.01, 1.01±0.03, both P < 0.05), which verified that Sirt5 was the target gene of miR-145-5p. (3) The occurrence of pyrodeath could be partially reversed by transfection with Sirt5 overexpression plasmid or adding ROS inhibitor NAC into cells, and Sirt5 overexpression could also up-regulate Nrf2 expression and eliminate intracellular ROS. CONCLUSION In human alveolar epithelial cells, miR-145-5p can down-regulate Nrf2 by targeting Sirt5, thereby increasing ROS expression and inducing pyrodeath.
Humans ; MicroRNAs ; Pyroptosis ; Cell Hypoxia ; Alveolar Epithelial Cells/cytology* ; Cell Line ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/metabolism* ; Epithelial Cells/metabolism* ; Gasdermins ; Phosphate-Binding Proteins

N⁶-methyladenosine (m⁶A) modification plays a critical role in cell cycle regulation, while the mechanism of m⁶A in regulating mitosis remains underexplored. Here, we found that the total m⁶A modification level in cells increased during mitosis by the liquid chromatography-mass spectrometry/mass spectrometry and m⁶A dot blot assays. Silencing methyltransferase-like 3 (METTL3) or METTL14 results in delayed mitosis, abnormal spindle assembly, and chromosome segregation defects by the immunofluorescence. By analyzing transcriptome-wide m⁶A targets in HeLa cells, we identified polo-like kinase 1 (PLK1) as a key gene modified by m⁶A in regulating mitosis. Specifically, through immunoblotting and RNA pulldown, m⁶A modification inhibits PLK1 translation via YTH N⁶-methyladenosine RNA binding protein 1, thus mediating cell cycle homeostasis. Demethylation of PLK1 mRNA leads to significant mitotic abnormalities. These findings highlight the critical role of m⁶A in regulating mitosis and the potential of m⁶A as a therapeutic target in proliferative diseases such as cancer.
Humans ; Polo-Like Kinase 1 ; Cell Cycle Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Mitosis/physiology* ; HeLa Cells ; Adenosine/genetics* ; Methyltransferases/metabolism* ; RNA, Messenger/metabolism* ; RNA-Binding Proteins/metabolism*

OBJECTIVES: We propose a sparse-view cone-beam CT reconstruction algorithm based on bidirectional flow field guided projection completion (BBC-Recon) to solve the ill-posed inverse problem in sparse-view cone-beam CT imaging. METHODS: The BBC-Recon method consists of two main modules: the projection completion module and the image restoration module. Based on flow field estimation, the projection completion module, through the designed bidirectional and multi-scale correlators, fully calculates the correlation information and redundant information among projections to precisely guide the generation of bidirectional flow fields and missing frames, thus achieving high-precision completion of missing projections and obtaining pseudo complete projections. The image restoration module reconstructs the obtained pseudo complete projections and then refines the image to remove the residual artifacts and further improve the image quality. RESULTS: The experimental results on the public datasets of Mayo Clinic and Guilin Medical University showed that in the case of a 4-fold sparse angle, compared with the suboptimal method, the BBC-Recon method increased the PSNR index by 1.80% and the SSIM index by 0.29%, and reduced the RMSE index by 4.12%; In the case of an 8-fold sparse angle, the BBC-Recon method increased the PSNR index by 1.43% and the SSIM index by 1.49%, and reduced the RMSE index by 0.77%. CONCLUSIONS The BBC-Recon algorithm fully exploits the correlation information between projections to allow effective removal of streak artifacts while preserving image structure information, and demonstrates significant advantages in maintaining inter-slice consistency.
Algorithms ; Cone-Beam Computed Tomography/methods* ; Image Processing, Computer-Assisted/methods* ; Humans

OBJECTIVES: We propose a segmented backprojection tensor degradation feature encoding (SBP-MAC) model for motion artifact correction in dental cone beam computed tomography (CBCT) to improve the quality of the reconstructed images. METHODS: The proposed motion artifact correction model consists of a generator and a degradation encoder. The segmented limited-angle reconstructed sub-images are stacked into the tensors and used as the model input. A degradation encoder is used to extract spatially varying motion information in the tensor, and the generator's skip connection features are adaptively modulated to guide the model for correcting artifacts caused by different motion waveforms. The artifact consistency loss function was designed to simplify the learning task of the generator. RESULTS: The proposed model could effectively remove motion artifacts and improve the quality of the reconstructed images. For simulated data, the proposed model increased the peak signal-to-noise ratio by 8.28%, increased the structural similarity index measurement by 2.29%, and decreased the root mean square error by 23.84%. For real clinical data, the proposed model achieved the highest expert score of 4.4221 (against a 5-point scale), which was significantly higher than those of all the other comparison methods. CONCLUSIONS The SBP-MAC model can effectively extract spatially varying motion information in the tensors and achieve adaptive artifact correction from the tensor domain to the image domain to improve the quality of reconstructed dental CBCT images.
Cone-Beam Computed Tomography/methods* ; Artifacts ; Humans ; Motion ; Image Processing, Computer-Assisted/methods* ; Signal-To-Noise Ratio ; Algorithms

Objective Idiopathic rolandic epilepsy syndrome （IRES） is the most common epilepsy syndrome in childhood， and its lesion site remains undetermined. This article aims to investigate the source of epileptiform discharges in IRES using magnetoencephalography （MEG）.Methods A total of 70 patients with IRES were enrolled in this prospective MEG-based study， among whom there were 53 children with benign epilepsy of childhood with centrotemporal spikes （BECTS）， 12 children with atypical benign partial epilepsy （ABPE）， 3 children with Landau-Kleffner syndrome （LKS）， and 2 children with epileptic encephalopathy with continuous spike-and-waves during slow-wave sleep （CSWS）. Epileptiform discharges were collected independently from each patient 10 times， and an MEG source analysis was performed. Standardized low-resolution brain electromagnetic tomography was used to perform source localization of the distributed source model. The spike source density was quantified into amplitude， and source location was determined according to the Desikan-Killiany atlas. The association between the distribution of spike source in brain and clinical manifestations was analyzed.Results In IRES， there were significant differences in the source locations of epilepsy discharge between BECTS， ABPE， LKS， and CSWS. The current source density of CSWS was stronger in the frontal lobe， the temporal lobe， and the anterior cingulate gyrus， while that of ABPE was stronger in the frontal lobe， and that of BECTS and LKS were stronger in the temporal lobe. The more severe phenotype of epilepsy， such as generalized tonic-clonic seizure， was associated with a stronger current source density in the brain， which was consistent with electroencephalography manifestations.Conclusion This study identifies different sources of epileptiform discharges in IRES. The density distribution of these spike sources may help to explain the discharge， cognitive， and neuropsychological characteristics in different subtypes of IRES.
Magnetoencephalography

OBJECTIVE To investigate the auditory characteristics of patients with congenital unilateral cochlear nerve deficiency(CND).METHODS This study enrolled 21 patients(21 ears)with unilateral auditory nerve dysplasia confirmed by inner ear MRI,including 9 males and 12 females,aged 10 months to 7 years,(1.91±1.38)years.A retrospective analysis was performed on the correlation between behavioral audiometry and auditory steady-state response(ASSR)thresholds,chirp-evoked auditory brainstem response(chirp-ABR),otoacoustic emission(OAE),and other results to analyze the audiological characteristics of unilateral auditory nerve dysplasia.RESULTS In all 21 patients(21 ears)with auditory nerve dysplasia,behavioral audiometry,chirp-ABR,and ASSR results all indicated severe to profound hearing loss in the affected ears.There was a high correlation between behavioral audiometry thresholds and ASSR thresholds,with small differences observed between ASSR and behavioral audiometry results.CONCLUSION The difference between ASSR and behavioral audiometry is small in patients with CND.A-ABR can't be elicited.OAE has a certain false negative rate.The ABR often predominantly shows wave III,with prolonged latency,and a threshold indicative of profound hearing loss.These audiological characteristics can improve the early detection and diagnosis of CND and enhance the effect of intervention.

Objective:To evaluate the sample preparation proficiency and storage proficiency of 11 clinical biobanks in Beijing through simulated experiments, and to establish an assessment method for the quality comparability of biological samples.Methods:An exploratory research design was adopted. In November 2023, artificial composite serum quality control materials containing six recombinant human protein markers—recombinant human alanine aminotransferase (rhALT), recombinant human aspartate aminotransferase (rhAST), recombinant human creatine kinase (rhCK), recombinant human creatine kinase-MB (rhCK-MB), recombinant human B-type natriuretic peptide (rhBNP), and recombinant human troponin I (rhTNI)—were distributed to 11 clinical biobanks in Beijing City. Sample preparation and storage followed the standardized operating procedures. Proficiency differences were assessed through statistical analysis.Results:Three-way repeated measures ANOVA revealed all six protein markers showed a declining trend over storage time in ultra-low-temperature environments ( F values 11.68-4 179.66, all P<0.01). However, neither long-term/temporary refrigerator types ( F values 0.01-1.23, all P>0.05)nor placement locations within refrigerators significantly affected the stability of these six proteins ( F valus 0.03-1.47, all P>0.05). The biases in detection results for rhALT, rhAST, rhTNI, and rhBNP at different storage time points were within the allowable bias limits for each item, supporting their use as markers for protein stability in biobank samples. All 11 institutions passed the storage proficiency assessment. In the preparation proficiency assessment, deviations were observed in post-preparation sample results, with a notably high out-of-control rate for rhCK (36.36%). Conclusion:Sample preparation proficiency can serve as a quality control metric for clinical biobanks. Future external quality assessment systems for biobanks should focus on sample preparation rather than storage processes.

Objective To explore the effect and mechanism of acupuncture at Zusanli(ST36)on inflammatory response in mice with mesenteric lymphadenitis based on the mitogen-activated protein ki-nase(MAPK)/nuclear factor-KB(NF-κB)signaling pathway.Methods Twelve male Kunming strain mice were randomly selected as blank control group,and mice with successfully established mes-enteric lymphadenitis models were randomly divided into model group and acupuncture at Zusanli group,with 12 mice in each group.Mice in the acupuncture at Zusanli group received electroacupunc-ture treatment at Zusanli for 4 days,while those in the blank control group and the model group re-ceived no treatment.Behavioral characteristics of mice in each group were observed.Hematoxylin and eosin(HE)staining was used to evaluate pathological changes in mesenteric lymph node tissues of mice.Enzyme-linked immunosorbent assay(ELISA)was employed to detect the expression levels of pro-inflammatory cytokines[tumor necrosis factor-α(TNF-α),interleukin-1 β(IL-1β),interleu-kin-6(IL-6)]in mouse serum.Western blot was used to detect the expression of autophagy-related proteins(Beclin1,LC3-Ⅱ,LC3-Ⅰ,p62)and MAPK/NF-κB signaling pathway-related proteins(p38 MAPK,p-p38 MAPK,NF-κB p65,p-NF-κB p65)in mesenteric lymph node tissues of mice.Results Mice in the model group exhibited decreased food intake,lethargy,soft and loose stools,and occasional kyphosis.The behavioral characteristics of mice in the acupuncture at Zusanli group were improved compared with those in the model group.HE staining results showed pathological changes in mesenteric lymph node tissues of mice in the model group,while the pathological changes in the acupuncture at Zusanli group were improved compared with those in the model group.ELISA results showed that the levels of TNF-α,IL-1β,and IL-6 in serum of mice in the model group were higher than those in the blank control group,while those in the acupuncture at Zusanli group were lower than those in the model group(P＜0.05).Western blot results showed that the relative ex-pression of Beclin1 protein and the LC3-Ⅱ/LC3-Ⅰ in mesenteric lymph node tissues of mice in the model group were lower than those in the blank control group,while the relative expression of p62 protein and the ratios of p-p38 MAPK/p38 MAPK and p-NF-κB p65/NF-κB p65 were higher than those in the blank control group(P＜0.05).In the acupuncture at Zusanli group,the relative ex-pression of Beclin1 protein and the LC3-Ⅱ/LC3-Ⅰ were higher than those in the model group,while the relative expression of p62 protein and p-p38 MAPK/p38 MAPK and p-NF-κB p65/NF-κB p65 were lower than those in the model group(P＜0.05).Conclusion Acupuncture at Zusanli alleviates the inflammatory response in mice with mesenteric lymphadenitis by inhibiting the p38 MAPK/NF-κB signaling pathway.

Objective To investigate the short-term efficacy of the retrograde semi-retrieval technique(RESET)in the treatment of patients with acute intracranial large vessel occlusion.Methods This retrospective study involved 286 patients with acute intracranial large vessel occlusion who underwent mechanical thrombectomy using RESET at Tianjin Huanhu Hospital from November 2017 to March 2019.The patients were divided into two groups based on the presence or absence of intracranial atherosclerotic stenosis(ICAS):ICAS group(n=186,65％)and non-ICAS group(n=100,35％).Baseline characteristics,procedural outcomes,and 90-day modified Rankin Scale(mRS)scores were compared between the two groups.Results The two groups did not significantly differ in baseline characteristics,including age,gender,past medical history,on-admission National Institutes of Health Stroke Scale(NIHSS)score,and Alberta Stroke Program Early CT(ASPECT)score(all P＞0.05).Successful vascular recanalization was achieved in 272 patients(95.1％),with 209 patients(73.1％)achieving complete recanalization with a single thrombectomy attempt.Rescue therapy was required in 33 patients(11.5％).Two patients in the ICAS group died due to postoperative symptomatic intracranial hemorrhage,and one patient in the non-ICAS group died due to postoperative multiple organ failure.Compared with the non-ICAS group,the ICAS group had a significantly longer puncture-to-recanalization time[141.36(124.11,156.53)min vs.65.17(53.92,83.25)min,P＜0.001]but a significantly smaller number of thrombectomy attempts[1.00(1.00,1.00)vs.1.00(1.00,2.00),P=0.002].However,there were no significant differences between the two groups in terms of final recanalization rate,complete recanalization rate with a single thrombectomy attempt,distal embolism or embolization of new territory,symptomatic intracranial hemorrhage,perioperative death,or 90-day mRS score(all P＞0.05).Conclusion RESET demonstrates satisfactory short-term efficacy in the treatment of acute intracranial large vessel occlusion.The number of thrombectomy attempts required for complete recanalization is significantly lower in the ICAS group than in the non-ICAS group,suggesting that RESET is particularly suitable for patients with ICAS.

Objective:To explore the changes in carcinoembryonic antigen(CEA)during the immune process of programmed death-1(PD-1)inhibitor Sintilimab in human epithelial growth factor receptor 2(HER2)negative advanced gastric cancer patients and its relationship with prognosis.Methods:HER2 negative late stage gastric cancer patients(88 cases)who were treated in North Anhui Coal Power Group General Hospital from May 2020 to April 2022 were selected as study subjects,all of whom received PD-1 inhibitor Sintilimab treatment;according to the prognosis,they were divided into death group(36 cases)and survival group(52 cases).Followed up was conducted every 6 months to collect tumor marker levels before and after treatment.Analyzed relationship between tumor markers(CEA,CA199,CA125)levels and clinical staging,lymph node metastasis and prognosis.Kaplan-Meier sur-vival curve was used to analyze survival time of patients with negative and positive tumor markers.Multivariate Cox regression model and stepwise regression analysis were used to identify risk factors affecting prognosis.Spearman was used for correlation analysis.Results:After two cycles of treatment,72 cases(81.82%)had disease control and 16 cases(18.18%)had progression.Compared with patients before treatment,positive rate of serum tumor markers in patients after treatment was significantly reduced(P<0.05).Positive rates of CEA and CA199 were significantly correlated with clinical staging(P<0.05).When predicting patient death,sensitivity of CEA level was the highest(48.57%),while CA125 had the highest specificity(95.62%)and the lowest sensitivity(25.71%).Kaplan-Meier sur-vival curve analysis showed that the survival time of patients with positive tumor markers were significantly shorter than that of negative patients(P<0.05).Clinical staging,serum CEA and CA199 levels were independent factors for predicting prognosis(P<0.05).Spear-man correlation analysis results showed that the multiple increases in CEA(r=-0.512,P=0.005)and CA199(r=-0.467,P=0.011)were negatively correlated with patient survival time.Conclusion:After treatment with PD-1 inhibitor Sintilimab,serum tumor markers levels in HER2 negative advanced gastric cancer patients have been significantly reduced on average.High serum levels of CEA,CA199 and CA125 can all predict poor prognosis,and clinical staging,serum CEA and CA199 levels are independent factors in pre-dicting prognosis.The higher the increase in CEA and CA199,the shorter the patient's survival time.