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.

Aim To compare the observation results of atomic force microscopy(AFM)and scanning electron microscopy(SEM),and to summarize the main problems and solutions of AFM in observing biological macromolecules,using the observa-tion subjects of protein samples purified by our research group.Methods The protein samples were diluted to 15 nmol·L-1 with PBS,fixed on glass slides,silicon wafers,and mica sheets,dried,and made into solid-phase observation samples.SEM sam-ples were plated with platinum before observation.The surface structures of proteins were observed using AFM and SEM,sample heights were calculated,and differences in results were com-pared.Results Protein samples with positive charges tended to shift to the right during observation due to the repulsion of the AFM probe;mica sheets could effectively eliminate the positive charge of proteins to avoid sample movement;PBS provided a stable environment for protein samples,but the crystallization of PBS salts interfered with probe operation and imaging clarity;SEM samples needed to be plated with platinum before observa-tion and could not achieve the precision of AFM.Conclusions Both AFM and SEM can directly observe protein structures in vitro,with AFM providing higher precision results;when protein sample stability permits,ultrapure water is preferred as the sol-vent carrier,and volatile liquids such as ethanol can also serve as solvent carriers.The application of AFM offers a new approach for pharmacological studies on interactions between biological macromolecules.

The blue fluorescent carbon dots(TMCDs)and cyan fluorescent carbon dots(TMCDs-H2O)were synthesized fromm-phenylenediamine and tricarballylic acid through air-assisted melting polymerization and one-step hydrothermal method,respectively.Air purging could effectively inhibit the side reactions and reduce the derivative structures in the carbon dots product.The structure and morphology of these two materials were systematically characterized using liquid nuclear magnetic resonance spectroscopy(NMR),mass spectrometry(MS),and transmission electron microscopy.Compared to TMCDs-H2O((3.12±0.63)nm),TMCDs showed a smaller average particle size(approximately(1.85±0.02)nm).The NMR and MS analysis revealed that although the main structure of both types of carbon dots was similar,TMCDs exhibited a simpler structure with higher degree of polymerization.These results suggested that supramolecular interactions might introduce numerous small molecule derivatives into TMCDs-H2O particles,resulting in lower polymerization degree,multiple substructures,and larger particle size characteristics for this material.When employed as chemical sensors for metal ion detection,in the linear range of 1×10-5-5×10-4 mol/L,the detection limits of Fe3+by TMCDs and TMCDs-H2O were 3.3×10-6 mol/L and 3.8×10-6 mol/L,respectively.The experimental results demonstrated that the recoveries of CDs and inductively coupled plasma optical emission spectrometer(ICP-OES)were similarity,whereas TMCDs displayed a considerable relative standard deviation.This study demonstrated that endogenously derived structures in CDs could enhance the performance of metal ion sensing.

Recently, male reproductive health has attracted extensive attention, with the adverse effects of circadian disruption on male fertility gradually gaining recognition. However, the mechanism by which circadian disruption leads to damage to male reproductive system remains unclear. In this review, we first summarized the dual regulatory roles of circadian clock genes on the male reproductive system: (1) circadian regulation of testosterone synthesis via the hypothalamic-pituitary-testicular (HPT) and hypothalamic-pituitary-adrenal (HPA) axes; (2) non-circadian regulation of spermatogenesis. Next, we further listed the possible mechanisms by which circadian disruption impairs male fertility, including interference with the oscillatory function of the reproductive system, i.e., synchronization of the HPT axis, crosstalk between the HPT axis and the HPA axis, as well as direct damage to germ cells by disturbing the non-oscillatory function of the reproductive system. Future research using spatiotemporal omics, epigenomic assays, and neural circuit mapping in studying the male reproductive system may provide new clues to systematically unravel the mechanisms by which circadian disruption affects male reproductive system through circadian clock genes.
Male ; Humans ; Animals ; Circadian Clocks/physiology* ; Hypothalamo-Hypophyseal System/physiology* ; Circadian Rhythm/genetics* ; Spermatogenesis/physiology* ; Pituitary-Adrenal System/physiology* ; Testis/physiology* ; Testosterone/biosynthesis* ; CLOCK Proteins ; Infertility, Male/physiopathology*

Most of the life forms on Earth have gradually evolved an endogenous biological clock under the long-term influence of periodic daily light-dark cycles. This biological clock system plays a crucial role in the orderly progression of life activities. In mammals, central circadian clock is located in the suprachiasmatic nucleus of the hypothalamus and the function of the biological clock relies on a transcription-translation negative feedback loop. As a negative regulator in this loop, the function of CHRONO is less known. To deeply explore the role of the Chrono gene in rhythm entrainment and physiology, we constructed a Chrono gene knockout mouse strain using the CRISPR/Cas9 technology and analyzed its entrainment ability under different T cycles. Running wheel tests and glucose tolerance tests were also performed. The results showed that the period of the endogenous biological clock of Chrono knockout mice was prolonged, and the entrainment rate under the T21 cycle was decreased. In addition, metabolic abnormalities, including weight gain and impaired glucose tolerance, were observed in the non-entrained mice. Overall, this study reveals a crucial role of the Chrono gene in maintaining circadian rhythms and metabolic balance, providing a new perspective for understanding the relationship between the biological clock and metabolism. Further research is needed to fully understand the underlying molecular mechanisms.
Animals ; Mice, Knockout ; Mice ; Circadian Rhythm/genetics* ; Metabolic Diseases/physiopathology* ; Photoperiod ; Male ; Period Circadian Proteins/physiology* ; Light ; Circadian Clocks/physiology*

Aim To compare the observation results of atomic force microscopy(AFM)and scanning electron microscopy(SEM),and to summarize the main problems and solutions of AFM in observing biological macromolecules,using the observa-tion subjects of protein samples purified by our research group.Methods The protein samples were diluted to 15 nmol·L-1 with PBS,fixed on glass slides,silicon wafers,and mica sheets,dried,and made into solid-phase observation samples.SEM sam-ples were plated with platinum before observation.The surface structures of proteins were observed using AFM and SEM,sample heights were calculated,and differences in results were com-pared.Results Protein samples with positive charges tended to shift to the right during observation due to the repulsion of the AFM probe;mica sheets could effectively eliminate the positive charge of proteins to avoid sample movement;PBS provided a stable environment for protein samples,but the crystallization of PBS salts interfered with probe operation and imaging clarity;SEM samples needed to be plated with platinum before observa-tion and could not achieve the precision of AFM.Conclusions Both AFM and SEM can directly observe protein structures in vitro,with AFM providing higher precision results;when protein sample stability permits,ultrapure water is preferred as the sol-vent carrier,and volatile liquids such as ethanol can also serve as solvent carriers.The application of AFM offers a new approach for pharmacological studies on interactions between biological macromolecules.

Objective The aim of this study was to explore the acceptance and influencing factors of self-collected urine samples for human papillomavirus(HPV)testing in cervical cancer screening among eligible women,and to provide scientific evidence for promoting this testing in low resource areas.Methods A population-based cross-sectional study was conducted from 2022 to 2023 at the Maternal and Child Health Hospital in Shuangliu district,Chengdu City Sichuan Province.The study subjects were women aged 21 to 69 years old,and a customized questionnaire was used to conduct general information and acceptance surveys on the partic-ipants.Results A total of 2,062 women were included,with an average age of 51.58±9.34 years.Among them,1,501(72.79％)women believed that self-sampling urine was very easy.However,although 1,333(64.65％)women were still willing to accept doctor sampling as a cervical cancer screening method,only 729(35.35％)were more willing to accept self-sampling urine HPV testing.Age,educational level,annual household income,awareness of HPV,HPV vaccination status,and a sense of shame about the doctor's sampling process were all associated with the acceptance of self-collected urine HPV testing among women undergoing cervical cancer screening(P＜0.001).The results of multivariate logistic regression analysis indicated that older women(OR=0.965,95％CI:0.951-0.979)and those who were not familiar with HPV(OR=0.760,95％CI:0.602-0.961)were more likely to undergo self sampling urine HPV testing,while those with junior high school education(OR=1.330,95％CI:1.053-1.682),high school education or a-bove(OR=1.990,95％CI:1.401-2.827),and a sense of shame towards the doctor's sampling process(OR=2.314,95％CI:1.706-3.142)were more likely to undergo self sampling urine for HPV testing.Conclusions Most women believe that self sampling urine for HPV testing is very easy,but compared to doctor sampling,only some women choose to self sample urine for HPV testing.Key health education interventions should be carried out for older and lower educated populations to promote acceptance of urine HPV testing.

Objective The aim of this study was to explore the acceptance and influencing factors of self-collected urine samples for human papillomavirus(HPV)testing in cervical cancer screening among eligible women,and to provide scientific evidence for promoting this testing in low resource areas.Methods A population-based cross-sectional study was conducted from 2022 to 2023 at the Maternal and Child Health Hospital in Shuangliu district,Chengdu City Sichuan Province.The study subjects were women aged 21 to 69 years old,and a customized questionnaire was used to conduct general information and acceptance surveys on the partic-ipants.Results A total of 2,062 women were included,with an average age of 51.58±9.34 years.Among them,1,501(72.79％)women believed that self-sampling urine was very easy.However,although 1,333(64.65％)women were still willing to accept doctor sampling as a cervical cancer screening method,only 729(35.35％)were more willing to accept self-sampling urine HPV testing.Age,educational level,annual household income,awareness of HPV,HPV vaccination status,and a sense of shame about the doctor's sampling process were all associated with the acceptance of self-collected urine HPV testing among women undergoing cervical cancer screening(P＜0.001).The results of multivariate logistic regression analysis indicated that older women(OR=0.965,95％CI:0.951-0.979)and those who were not familiar with HPV(OR=0.760,95％CI:0.602-0.961)were more likely to undergo self sampling urine HPV testing,while those with junior high school education(OR=1.330,95％CI:1.053-1.682),high school education or a-bove(OR=1.990,95％CI:1.401-2.827),and a sense of shame towards the doctor's sampling process(OR=2.314,95％CI:1.706-3.142)were more likely to undergo self sampling urine for HPV testing.Conclusions Most women believe that self sampling urine for HPV testing is very easy,but compared to doctor sampling,only some women choose to self sample urine for HPV testing.Key health education interventions should be carried out for older and lower educated populations to promote acceptance of urine HPV testing.

Objective To investigate the attributable risk(AR)of Acinetobacter baumannii(AB)infection in criti-cally ill patients.Methods A multicenter retrospective cohort study was conducted among adult patients in inten-sive care unit(ICU).Patients with AB isolated from sterile body fluid and confirmed with AB infection in each cen-ter were selected as the infected group.According to the matching criteria that patients should be from the same pe-riod,in the same ICU,as well as with similar APACHE Ⅱ score(±5 points)and primary diagnosis,patients who did not infect with AB were selected as the non-infected group in a 1:2 ratio.The AR was calculated.Results The in-hospital mortality of patients with AB infection in sterile body fluid was 33.3％,and that of non-infected group was 23.1％,with no statistically significant difference between the two groups(P=0.069).The AR was 10.2％(95％CI:-2.3％-22.8％).There is no statistically significant difference in mortality between non-infected pa-tients and infected patients from whose blood,cerebrospinal fluid and other specimen sources AB were isolated(P＞0.05).After infected with AB,critically ill patients with the major diagnosis of pulmonary infection had the high-est AR.There was no statistically significant difference in mortality between patients in the infected and non-infec-ted groups(P＞0.05),or between other diagnostic classifications.Conclusion The prognosis of AB infection in critically ill patients is highly overestimated,but active healthcare-associated infection control for AB in the ICU should still be carried out.