Objective This study conducted molecular biological identification of the viruses carried by Culex pipiens pallens specimens collected in Shunyi District, Beijing in 2023, and observed the changes in the infection rate of the viruses carried by Cx. pipiens pallens at different collection times. Methods Cx. pipiens pallens were collected using carbon dioxide mosquito traps. The mosquito samples were ground in batches and analyzed by molecular biology technologies. The virus infection rate at different collection times was analyzed statistically. Results 17 strains of Culex pipiens pallens densovirus(CppDNV)were identified from Cx. pipiens pallens samples collected in Shunyi District, Beijing, in 2023. The nucleotide sequence analysis of the virus genome coding region showed that CppDNV was a single-stranded DNA virus with a total length of 3 335 nt, encoding 2 non-structural proteins(NS1, NS2)and 1 capsid protein(VP). The nucleotide(amino acid)sequence lengths of the three proteins were 2 376 nt(791 aa),1 092 nt(363 aa)and 1 071 nt(356 aa), respectively. Phylogenetic analysis showed that CppDNV was located in genus Brevihamaparvovirus. Statistical analysis showed no significant difference in infection rates across collection times(χ2=4.429, P=0.194). Conclusions CppDNV was identified in Cx. pipiens pallens in Beijing, and it was stably maintained in this natural population.

ObjectiveCell adhesion is a critical process that regulates cellular physiological functions. Quantitative characterization of adhesion dynamics is essential for elucidating the intrinsic mechanical mechanisms underlying cellular activities. Although atomic force microscopy-based single-cell force spectroscopy is widely used for single-cell adhesion measurements, it requires complex chemical modifications for preparation of live-cell probes, leading to limitations such as cumbersome operation, low throughput, and potential impacts on cell viability. Fluidic force microscopy, which combines atomic force microscopy with microfluidic probes, is a technique allowing the operation of force-controlled nanopipettes in aqueous environments. By applying negative or positive pressure via a pressure controller, a single living cell can be captured onto or released from the cantilever under physiological conditions. This procedure offers a simple workflow and high assay throughput for single-cell adhesion measurements without the need for chemical functionalization. In this study, fluidic force microscopy-based single-cell force spectroscopy was adopted to achieve long-term quantitative characterization of single-cell adhesion dynamics in a simpler and more efficient manner, comparing the dynamic differences in adhesion establishment between two cell lines with different differentiation levels. MethodsHEK 293T and hTERT RPE-1 cells were non-invasively captured on the cantilever of a fluidic force microscope via its integrated microfluidic system during 40 h of adhesion culture. Cell-substrate detachment assays were performed, and force-distance curves were recorded to extract key mechanical adhesion parameters, including adhesion force, adhesion energy, and maximum detachment distance. These measurements were combined with real-time monitoring of cell spreading area to systematically characterize the dynamic evolution of single-cell adhesion. ResultshTERT RPE-1 cells rapidly entered a stable adhesion phase within 1 h after seeding, with both area-normalized adhesion force and area-normalized adhesion energy reaching peak values. In contrast, HEK 293T cells required 4 h to achieve stable adhesion. Subsequently, the adhesion force, adhesion energy and maximum detachment distance of hTERT RPE-1 and HEK 293T cells stabilized at approximately 240 nN vs. 30 nN, 2.2 pJ vs. 0.12 pJ and 6 μm vs. 4 μm, respectively. hTERT RPE-1 cells reached the peak of area-normalized adhesion parameters earlier than HEK 293T cells, with their peak area-normalized adhesion force and area-normalized adhesion energy being substantially elevated relative to HEK 293T cells. HEK 293T cells presented stronger linear correlations among adhesion energy, maximum detachment distance and adhesion force compared with hTERT RPE-1 cells. For both cell lines, cell spreading area exhibited a weak correlation with adhesion force. Whereas the area-normalized adhesion parameters of HEK 293T cells remained relatively constant throughout the adhesion process, hTERT RPE-1 cells exhibited elevated values in the early phase, followed by a gradual decline. These results indicated distinct dynamic adhesion patterns between the two cell types, with hTERT RPE-1 cells exhibiting stronger adhesion strength and higher adhesion efficiency. ConclusionIn this study, fluidic force microscopy-based single-cell force spectroscopy was successfully applied to perform long-term in situ quantitative measurement of the adhesion dynamics in single adherent cells. The approach revealed divergent adhesion patterns between HEK 293T and hTERT RPE-1 cells, suggesting a close association between cell differentiation and adhesion behaviors. These findings provide quantitative mechanical evidence for further understanding the underlying mechanisms of cell adhesion.

ObjectiveTreadmill exercise intervention was evaluated for its effects on addictive behavior, learning and memory, and hippocampal neuroinflammation in methamphetamine (MA)-addicted mice, and transcriptomic screening was performed to identify candidate molecules involved in exercise-mediated attenuation of MA-related neuronal injury. MethodsTwo-month-old male C57BL/6 mice were randomly assigned to four groups: control (C), exercise control (CE), methamphetamine (M), and methamphetamine plus exercise (ME). Mice in the M and ME groups received intraperitoneal injections of MA (1 mg/kg) once daily for 7 consecutive days to establish the addiction model, whereas mice in the C and CE groups received equal volumes of saline. Subsequently, mice in the CE and ME groups underwent treadmill exercise at 12 m/min for 60 min/d for 2 weeks. Conditioned place preference (CPP), the Y-maze, and the Morris water maze (MWM) were used to assess addictive behavior, spatial recognition memory, and spatial learning and memory, respectively. Hippocampal neuronal ultrastructure was examined by transmission electron microscopy (TEM), and microglia-related responses were evaluated by immunofluorescence staining for ionized calcium-binding adapter molecule 1 (Iba-1) in the hippocampal CA1 region. Previously generated and publicly available RNA-seq data from brain tissue of the C, M, and ME groups were reanalyzed, with three biological samples per group. Differentially expressed genes (DEGs) were re-screened using the criteria of P<0.05 and fold change>1.5 or <0.67, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. NLR family CARD domain containing 5 (Nlrc5) was further validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western blot was used to determine the protein expression of NLRC5, nuclear factor kappa B (NF‑κB), interleukin-1β (IL-1β), postsynaptic density-95 (PSD-95), and synaptophysin. ResultsTreadmill exercise significantly attenuated MA-induced conditioned place preference and improved learning and memory performance in both the Y-maze and MWM tests. Exercise increased exploration time and distance in the novel arm of the Y-maze, shortened escape latency, and increased the number of platform crossings in the MWM. Average swimming speed during the probe trial did not differ significantly among groups, suggesting that the MWM differences were not attributable to obvious differences in swimming performance. TEM revealed that MA exposure induced mitochondrial swelling, disrupted cristae, reduced organelle abundance, and nuclear envelope abnormalities in hippocampal neurons, whereas these ultrastructural changes were alleviated after exercise. Immunofluorescence staining showed that MA increased Iba-1 fluorescence intensity in the hippocampal CA1 region, while treadmill exercise significantly reduced this change. Transcriptomic analysis identified 160 DEGs associated with MA exposure and 58 DEGs associated with exercise intervention, with 9 overlapping DEGs. KEGG enrichment analysis showed that MA-related DEGs were enriched in the NOD-like receptor signaling pathway. Among the overlapping DEGs, Nlrc5, which was downregulated after MA exposure and restored following exercise intervention, was selected as a candidate gene for further validation. RT-qPCR confirmed that treadmill exercise reversed the MA-induced decrease in Nlrc5 mRNA expression. Western blot analysis further showed that treadmill exercise increased NLRC5, PSD-95, and synaptophysin protein expression while reducing NF-κB and IL-1β expression. ConclusionTwo weeks of treadmill exercise attenuated MA-induced conditioned place preference and ameliorated learning and memory impairment, alleviated hippocampal neuroinflammation and neuronal ultrastructural damage, and restored the expression of synapse-related proteins. Transcriptomic screening combined with molecular validation demonstrated that exercise intervention was accompanied by restoration of NLRC5 expression, reduced NF‑κB and IL-1β expression, and increased PSD-95 and synaptophysin expression. These findings suggest that NLRC5 may represent a candidate molecule involved in exercise-mediated amelioration of MA-related neuronal injury. However, the present results mainly demonstrate coordinated changes in NLRC5 expression, inflammatory indicators, and cognitive outcomes rather than a direct causal relationship, and the specific role of NLRC5 requires further functional validation.

Aim To investigate the mechanism of Guanxinning injection regulating cardiac immune mi-croenvironment to improve myocardial infarction in mice.Methods In this study,MI model was estab-lished by permanent ligation of left anterior descending coronary artery in mice.The mice were divided into five groups:sham operation group,model group,Guanxinning injection low dose group,Guanxinning in-jection high dose group and positive drug captopril group.Hearts were weighed,heart tissues were collect-ed,and Masson staining was used for pathological anal-ysis of heart tissues;immunofluorescence staining was used to detect apoptosis and CCL21 expression in the infarct border zone;flow cytometry was used to detect the proportion of immune cells in myocardial ischemia tissues and lymph nodes;PCR was used to detect CCL21 expression in heart and in vitro human lymphat-ic endothelial cells(HLEC).Results Compared with the model group,the low and high dose groups of Guanxinning injection significantly improved cardiac hypertrophy.Apoptosis in the border zone of myocardi-al infarction was reduced in the low and high dose groups of Guanxinning injection and captopril group.Compared with the model group,the proportion of leu-kocytes in the infarct border zone was dreduced and the proportion of CD4+T cells,Treg cells,and CD8+T cells in the mediastinal lymph nodes and infarct border zone of the heart was regulated in the low and high dose groups of Guanxinning injection;CCL21 secretion by the heart and lymphatic vessels increased.Conclu-sions Guanxinning injection can significantly improve cardiac hypertrophy and fibrosis in MI mice,reduce ap-optosis in the infarct border zone,and play a role in an-ti-myocardial ischemia injury by promoting CCL21 ex-pression in lymphatic vessels to regulate the proportion of mediastinal lymph nodes and cardiac T cells after myocardial infarction.

Electroencephalogram (EEG) is a non-invasive, high temporal-resolution technique for monitoring brain activity. However, affected by the volume conduction effect, EEG has a low spatial resolution and is difficult to locate brain neuronal activity precisely. The surface Laplacian (SL) technique obtains the Laplacian EEG (LEEG) by estimating the second-order spatial derivative of the scalp potential. LEEG can reflect the radial current activity under the scalp, with positive values indicating current flow from the brain to the scalp (“source”) and negative values indicating current flow from the scalp to the brain (“sink”). It attenuates signals from volume conduction, effectively improving the spatial resolution of EEG, and is expected to contribute to breakthroughs in neural engineering. This paper provides a systematic overview of the principles and development of SL technology. Currently, there are two implementation paths for SL technology: current source density algorithms (CSD) and concentric ring electrodes (CRE). CSD performs the Laplace transform of the EEG signals acquired by conventional disc electrodes to indirectly estimate the LEEG. It can be mainly classified into local methods, global methods, and realistic Laplacian methods. The global method is the most commonly used approach in CSD, which can achieve more accurate estimation compared with the local method, and it does not require additional imaging equipment compared with the realistic Laplacian method. CRE employs new concentric ring electrodes instead of the traditional disc electrodes, and measures the LEEG directly by differential acquisition of the multi-ring signals. Depending on the structure, it can be divided into bipolar CRE, quasi-bipolar CRE, tripolar CRE, and multi-pole CRE. The tripolar CRE is widely used due to its optimal detection performance. While ensuring the quality of signal acquisition, the complexity of its preamplifier is relatively acceptable. Here, this paper introduces the study of the SL technique in resting rhythms, visual-related potentials, movement-related potentials, and sensorimotor rhythms. These studies demonstrate that SL technology can improve signal quality and enhance signal characteristics, confirming its potential applications in neuroscientific research, disease diagnosis, visual pathway detection, and brain-computer interfaces. CSD is frequently utilized in applications such as neuroscientific research and disease detection, where high-precision estimation of LEEG is required. And CRE tends to be used in brain-computer interfaces, that have stringent requirements for real-time data processing. Finally, this paper summarizes the strengths and weaknesses of SL technology and envisages its future development. SL technology boasts advantages such as reference independence, high spatial resolution, high temporal resolution, enhanced source connectivity analysis, and noise suppression. However, it also has shortcomings that can be further improved. Theoretically, simulation experiments should be conducted to investigate the theoretical characteristics of SL technology. For CSD methods, the algorithm needs to be optimized to improve the precision of LEEG estimation, reduce dependence on the number of channels, and decrease computational complexity and time consumption. For CRE methods, the electrodes need to be designed with appropriate structures and sizes, and the low-noise, high common-mode rejection ratio preamplifier should be developed. We hope that this paper can promote the in-depth research and wide application of SL technology.

This study investigates the reproductive protective effect and potential mechanism of Cistanches Herba extract(CHE) on a rat model of kidney-Yang deficiency induced by adenine. Rats were randomly divided into five groups: normal, model, low-dose CHE(0.6 g·kg~(-1)·d~(-1)), high-dose CHE(1.2 g·kg~(-1)·d~(-1)), and L-carnitine(100 mg·kg~(-1)·d~(-1)). The rats were administered adenine(200 mg·kg~(-1)·d~(-1)) by gavage for the first 14 days to induce kidney-Yang deficiency, while simultaneously receiving drug treatment. After 14 days, the modeling was discontinued, but drug treatment continued to 49 days. The content of components in CHE was analyzed by high-performance liquid chromatography. The adenine-induced kidney-Yang deficiency model was assessed through symptom characterization and measurement of testosterone(T) levels using an enzyme-linked immunosorbent assay kit. Pathological damage to the testis and epididymis was evaluated based on the wet weight and performing hematoxylin-eosin staining. Sperm density and motility were measured using computer-aided sperm analysis, and sperm viability was assessed using live/dead sperm staining kits, and sperm morphology was evaluated using eosin staining, thereby determining rat sperm quality. Metabolomics was used to analyze changes in serum metabolites, enrich related metabolic pathways, and explore the mechanism of CHE in improving reproductive function damage in rats with kidney-Yang deficiency syndrome. Compared to the normal group, the model group exhibited significant kidney-Yang deficiency symptoms, reduced T levels, decreased testicular and epididymal wet weights, and significant pathological damage to the testis and epididymis. The sperm density, motility, and viability decreased, with an increased rate of sperm abnormalities. In contrast, rats treated with CHE showed marked improvements in kidney-Yang deficiency symptoms, restored T levels, alleviated pathological damage to the testis and epididymis, and improved various sperm parameters. Metabolomics results revealed 286 differential metabolites between the normal and model groups(191 upregulated and 95 downregulated). Seventy-five differential metabolites were identified between the model and low-dose CHE groups(21 upregulated and 54 downregulated). A total of 24 common differential metabolites were identified across the three groups, with 22 of these metabolites exhibiting opposite regulation trends between the two comparison groups. These metabolites were primarily involved in linoleic acid metabolism, ether lipid metabolism, and pantothenic acid and coenzyme A biosynthesis, as well as metabolites including 13-hydroperoxylinoleic acid, lysophosphatidylcholine, and pantethine. CHE can improve kidney-Yang deficiency symptoms in rats, alleviate reproductive organ damage, and enhance sperm quality. The regulation of lipid metabolism may be a potential mechanism through which CHE improves reproductive function in rats with kidney-Yang deficiency. The potential bioactive compounds of CHE include echinacoside, verbascoside, salidroside, betaine, and cistanoside A.
Animals ; Male ; Rats ; Yang Deficiency/physiopathology* ; Metabolomics ; Kidney/physiopathology* ; Rats, Sprague-Dawley ; Drugs, Chinese Herbal/administration & dosage* ; Cistanche/chemistry* ; Kidney Diseases/metabolism* ; Testis/metabolism* ; Humans ; Reproduction/drug effects* ; Testosterone/blood*

Jiuwei Xifeng Granules have become a Chinese patent medicine in the market. Because the formula contains Os Draconis, a top-level protected fossil of ancient organisms, the formula was to be improved by replacing Os Draconis with Ostreae Concha. To evaluate whether the improved formula has the same effectiveness and safety as the original formula, a randomized, double-blind, parallel-controlled, equivalence clinical trial was conducted. This study enrolled 288 tic disorder(TD) of children and assigned them into two groups in 1∶1. The treatment group and control group took the modified formula and original formula, respectively. The treatment lasted for 6 weeks, and follow-up visits were conducted at weeks 2, 4, and 6. The primary efficacy endpoint was the difference in Yale global tic severity scale(YGTSS)-total tic severity(TTS) score from baseline after 6 weeks of treatment. The results showed that after 6 weeks of treatment, the declines in YGTSS-TSS score showed no statistically significant difference between the two groups. The difference in YGTSS-TSS score(treatment group-control group) and the 95%CI of the full analysis set(FAS) were-0.17[-1.42, 1.08] and those of per-protocol set(PPS) were 0.29[-0.97, 1.56], which were within the equivalence boundary [-3, 3]. The equivalence test was therefore concluded. The two groups showed no significant differences in the secondary efficacy endpoints of effective rate for TD, total score and factor scores of YGTSS, clinical global impressions-severity(CGI-S) score, traditional Chinese medicine(TCM) response rate, or symptom disappearance rate, and thus a complete evidence chain with the primary outcome was formed. A total of 6 adverse reactions were reported, including 4(2.82%) cases in the treatment group and 2(1.41%) cases in the control group, which showed no statistically significant difference between the two groups. No serious suspected unexpected adverse reactions were reported, and no laboratory test results indicated serious clinically significant abnormalities. The results support the replacement of Os Draconis by Ostreae Concha in the original formula, and the efficacy and safety of the modified formula are consistent with those of the original formula.
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Male ; Double-Blind Method ; Drugs, Chinese Herbal/therapeutic use* ; Tic Disorders/drug therapy* ; Treatment Outcome

Extensive studies have identified potential adverse effects on semen quality of obesity, based on body mass index, but the association between body fat distribution, a more relevant indicator for obesity, and semen quality remains less clear. We conducted a longitudinal study of 4304 sperm donors from the Guangdong Provincial Human Sperm Bank (Guangzhou, China) during 2017-2021. A body composition analyzer was used to measure total and local body fat percentage for each participant. Generalized estimating equations were employed to assess the association between body fat percentage and sperm count, motility, and morphology. We estimated that each 10% increase in total body fat percentage (estimated change [95% confidence interval, 95% CI]) was significantly associated with a 0.18 × 10 6 (0.09 × 10 6 -0.27 × 10 6 ) ml and 12.21 × 10 6 (4.52 × 10 6 -19.91 × 10 6 ) reduction in semen volume and total sperm count, respectively. Categorical analyses and exposure-response curves showed that the association of body fat distribution with semen volume and total sperm count was stronger at higher body fat percentages. In addition, the association still held among normal weight and overweight participants. We observed similar associations for upper limb, trunk, and lower limb body fact distributions. In conclusion, we found that a higher body fat distribution was significantly associated with lower semen quality (especially semen volume) even in men with a normal weight. These findings provide useful clues in exploring body fat as a risk factor for semen quality decline and add to evidence for improving semen quality for those who are expected to conceive.
Humans ; Male ; Adult ; Semen Analysis ; China ; Body Fat Distribution ; Longitudinal Studies ; Sperm Count ; Sperm Motility ; Body Mass Index ; Tissue Donors ; Obesity/complications* ; Spermatozoa ; Young Adult ; Middle Aged ; East Asian People