Dissecting positive selection events and immunological drives during the evolution of adeno-associated virus lineages.

Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy.

Multi-omics analysis of renal vein serum with Ischemia-Reperfusion injury.

BACKGROUND: Acute kidney injury (AKI) is frequently caused by renal ischemia-reperfusion injury (IRI). Identifying potential renal IRI disease biomarkers would be useful for evaluating AKI severity. OBJECTIVE: We used proteomics and metabolomics to investigate the differences in renal venous blood between ischemic and healthy kidneys in an animal model by identifying differentially expressed proteins (DEPs) and differentially expressed protein metabolites (DEMs). METHODS: Nine pairs of renal venous blood samples were collected before and at 20, 40, and 60 min post ischemia. The ischemia time of Group A, B and C was 20,40 and 60 min. The proteome and metabolome of renal venous blood were evaluated to establish the differences between renal venous blood before and after ischemia. RESULTS: We identified 79 common DEPs in all samples of Group A, 80 in Group B, and 131 in Group C. Further common DEPs among all three groups were Tyrosineprotein kinase, GPR15LG, KAZALD1, ADH1B. We also identified 81, 64, and 83 common DEMs in each group respectively, in which 30 DEMs were further common to all groups. Bioinformatic analysis of the DEPs and DEMs was conducted. CONCLUSION: This study demonstrated that different pathological processes occur during short- and long-term renal IRI. Tyrosine protein kinase, GPR15LG, Kazal-type serine peptidase inhibitor domain 1, and all-trans-retinol dehydrogenase are potential biomarkers of renal IRI.

Trends and predictors of changes in renal function after radical nephrectomy for renal tumours.

BACKGROUND: Chronic kidney disease (CKD) is a common postoperative complication in patients who undergo radical nephrectomy for renal tumours. However, the factors influencing long-term renal function require further investigation. OBJECTIVE: This study was designed to investigate the trends in renal function changes and risk factors for renal function deterioration in renal tumour patients after radical nephrectomy. METHODS: We monitored changes in renal function before and after surgery for 3 years. The progression of renal function was determined by the progression and degradation of CKD stages. Univariate and multivariate logistic regression analyses were used to analyse the causes of renal function progression. RESULTS: We analysed the data of 329 patients with renal tumours who underwent radical nephrectomies between January 2013 and December 2018. In this study, 43.7% of patients had postoperative acute kidney injury (AKI), and 48.3% had CKD at advanced stages. Further research revealed that patients' renal function stabilized 3 months after surgery. Additionally, renal function changes during these 3 months have a substantial impact on the progression of long-term renal function changes in patients. CONCLUSION: AKI may be an indicator of short-term postoperative changes in renal function. Renal function tests should be performed in patients with AKI after radical nephrectomy to monitor the progression of functional impairment, particularly within the first 3 months after radical nephrectomy.

CPA4 as a biomarker promotes the proliferation, migration and metastasis of clear cell renal cell carcinoma cells.

Clear cell renal cell carcinoma (ccRCC) is a commonly occurring and highly aggressive urological malignancy characterized by a significant mortality rate. Current therapeutic options for advanced ccRCC are limited, necessitating the discovery of novel biomarkers and therapeutic targets. Carboxypeptidase A4 (CPA4) is a zinc-containing metallocarboxypeptidase with implications in various cancer types, but its role in ccRCC remains unexplored. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized in order to investigate the differential expression patterns of CPA4. The expression of CPA4 in ccRCC patients was further verified using immunohistochemical (IHC) examination of 24 clinical specimens. A network of protein-protein interactions (PPI) was established, incorporating CPA4 and its genes that were expressed differentially. Functional enrichment analyses were conducted to anticipate the contribution of CPA4 in the development of ccRCC. To validate our earlier study, we conducted real-time PCR and cell functional tests on ccRCC cell lines. Our findings revealed that CPA4 is overexpressed in ccRCC, and the higher the expression of CPA4, the worse the clinical outcomes such as TNM stage, pathological stage, histological grade, etc. Moreover, patients with high CPA4 expression had worse overall survival, disease-specific survival and progress-free interval than patients with low expression. The PPI network analysis highlighted potential interactions contributing to ccRCC progression. Functional enrichment analysis indicated the involvement of CPA4 in the regulation of key pathways associated with ccRCC development. Additionally, immune infiltration analysis suggested a potential link between CPA4 expression and immune response in the tumour microenvironment. Finally, cell functional studies in ccRCC cell lines shed light on the molecular mechanisms underlying the role of CPA4 in promoting ccRCC formation. Overall, our study unveils CPA4 as a promising biomarker with prognostic potential in ccRCC. The identified interactions and pathways provide valuable insights into its implications in ccRCC development and offer a foundation for future research on targeted therapies. Further investigation of CPA4's involvement in immune responses may contribute to the development of immunotherapeutic strategies for ccRCC treatment.

Fusion expression, purification, and characterization of cytokeratin 19 fragments in E. coli for enhanced stability in diagnostic applications.

Cytokeratin 19 fragment (CYFRA21-1) serves as a crucial tumor marker in the context of lung cancer patients, playing a pivotal role as a calibrator in the realm of in vitro diagnostics. Nevertheless, during practical application, it has come to light that the recombinantly synthesized full-length CYFRA21-1 antigen exhibits suboptimal stability at the requisite concentration, while the utilization of natural antigens incurs a substantial cost. To address this issue, our investigation harnessed a strategic approach whereby the soluble fragment of cytokeratin 19 (Aa244-400) was integrated into the pET32a vector, subsequently being expressed within E. coli through a fusion with the TrxA protein. This process involved induction of protein expression through 0.2 mM IPTG at 16 °C for a duration of 16 h. After induction, the target protein was purified through Ni affinity and ion exchange chromatography. Subsequent characterization of the targeted protein was executed through the SEC-HPLC technique. The attained CYFRA21-1 antigen, as generated within this study, was effectively incorporated into a chemiluminescence-based in vitro diagnostic detection kit. The results indicate that the fusion protein exhibited commendable reactivity and stability, manifesting a deviation of less than 10 % following incubation at 37 °C for 7 days. Importantly, the production yield achieved a notable magnitude of 300 mg/L, thus rendering it a cost-effective and scalable alternative to natural antigens for clinical diagnostic applications.

Phase intensity nanoscope (PINE) opens long-time investigation windows of living matter.

Fundamental to all living organisms and living soft matter are emergent processes in which the reorganization of individual constituents at the nanoscale drives group-level movements and shape changes at the macroscale over time. However, light-induced degradation of fluorophores, photobleaching, is a significant problem in extended bioimaging in life science. Here, we report opening a long-time investigation window by nonbleaching phase intensity nanoscope: PINE. We accomplish phase-intensity separation such that nanoprobe distributions are distinguished by an integrated phase-intensity multilayer thin film (polyvinyl alcohol/liquid crystal). We overcame a physical limit to resolve sub-10 nm cellular architectures, and achieve the first dynamic imaging of nanoscopic reorganization over 250 h using PINE. We discover nanoscopic rearrangements synchronized with the emergence of group-level movements and shape changes at the macroscale according to a set of interaction rules with importance in cellular and soft matter reorganization, self-organization, and pattern formation.

Modulating Zinc Metal Reversibility by Confined Antifluctuator Film for Durable and Dendrite-Free Zinc Ion Batteries.

Aqueous Zn ion batteries are promising systems due to their intrinsic safety, low cost, and non-toxicity, and the Zn corrosion and dendrite growth will cause the poor reversibility of Zn anode. Herein, the porous Zn@C solid, hollow, and yolk-shell microsphere films are developed as Zn anode antifluctuator (ZAAF). The prepared yolk-shell microspheres (Zn@C yolk-shell microsphere [ZCYSM]) film with superior buffering can effectively restrict the deposition of Zn metal in its interior and inhibit the volume expansion during plating/stripping process, thus modulating the Zn2+ flux and enabling stable Zn cycling. As a proof of concept, the ZCYSM@Zn symmetric cells achieve the excellent cyclic stability over 4000 h and cumulative plated capacity of 4 Ah cm-2  at a high current density of 10 mA cm-2 . Concomitantly, the suppressed corrosion reactions and dendrite-free ZAAF significantly improve the durability of full cells (coupled to CaV6 O16 ·3H2 O). Additionally, durable pouch cell and electrochemical neuromorphic inorganic device (ENIDe) are integrated to simulate neural network, providing a strategy for extreme interconnectivity comparable to the human brain.

Wound dehiscence in enhanced recovery after open radical cystectomy: A meta-analysis.

A meta-analysis study to assess the outcome of enhanced recovery (ER) after radical cystectomy (RC) on wound dehiscence was performed. A comprehensive literature examination till January 2023 was implemented and 1457 linked studies were appraised. The picked studies contained 772 open RC subjects in the picked studies' baseline, 436 of them were enhanced recovery after RC, and 336 were open RC. Odds ratio (OR) in addition to 95% confidence intervals (CIs) were used to calculate the consequence of enhanced recovery after RC on wound dehiscence after open RC by the dichotomous styles and a fixed or random model. The ER after RC caused significantly lower wound dehiscence (OR, 0.51; 95% CI, 0.30-0.89, P = .02) with low heterogeneity (I2  = 46%) compared with open RC. The ER after RC caused significantly lower wound dehiscence compared with open RC. Thorough precaution should be taken when commerce with the consequences because a limited number of studies were found and selected for this meta-analysis.

Optical Penetration of Shape-Controlled Metallic Nanosensors across Membrane Barriers.

Precise nanostructure geometry that enables the optical biomolecular delivery of nanosensors to the living intracellular environment is highly desirable for precision biological and clinical therapies. However, the optical delivery through membrane barriers utilizing nanosensors remains difficult due to a lack of design guidelines to avoid inherent conflict between optical force and photothermal heat generation in metallic nanosensors during the process. Here, we present a numerical study reporting significantly enhanced optical penetration of nanosensors by engineering nanostructure geometry with minimized photothermal heating generation for penetrating across membrane barriers. We show that by varying the nanosensor geometry, penetration depths can be maximized while heat generated during the penetration process can be minimized. We demonstrate the effect of lateral stress induced by an angularly rotating nanosensor on a membrane barrier by theoretical analysis. Furthermore, we show that by varying the nanosensor geometry, maximized local stress fields at the nanoparticle-membrane interface enhanced the optical penetration process by four-fold. Owing to the high efficiency and stability, we anticipate that precise optical penetration of nanosensors to specific intracellular locations will be beneficial for biological and therapeutic applications.

Characterizing how size distribution and concentration affect echogenicity of ultrasound contrast agents.

We investigated the effects of UCA gas bubble size distribution and concentration on the generated ultrasound echogenicity signal. Gas bubble size characterization using Coulter Counter and cryogenic-SEM revealed the hollow structure and rare presence of microbubbles >10 µm in a commercial UCA product, Lumason™. Volume-weighed size and concentration were observed to be more sensitive to changes in UCA bubble stability than number-weighted size and concentration. Size distribution measurements showed that the force (e.g., shaking/agitation energy) used to redisperse the sample did not affect the size distribution, concentration, or echogenicity of the UCA sample. The ultrasound backscattering coefficient (BSC) of size fractionated and serial diluted microbubbles showed that the echogenicity signal correlates most with UCA bubble concentration, especially volume-weighted concentration. Findings from this study may be used to support demonstrating the equivalence of a generic UCA product to the reference listed drug.

The association between myopia with overweight and obesity in middle school students in Zhengzhou / 中国学校卫生

Objective@#To understand associated factors of myopia among middle school students in Zhengzhou and to explore the correlation between myopia with overweight and obesity, to provide a scientific basis for myopia prevention.@*Methods@#A total of 3 297 middle school students from 8 middle schools in Erqi District, Zhongmu County and Xingyang County of Zhengzhou City were selected by a cluster random sampling method to participate in vision testing and questionnaire survey. Chi square test, and Logistic regression analysis were used.@*Results@#Overall prevalence of myopia of middle school students was 80.5%, overweight 14.7%, obesity 10.1% in Zhengzhou. The prevalence of myopia differed significantly by schooling stage, parental myopia status and sex ( χ 2= 18.34, 23.55, 8.98, 26.53, 27.46, 47.25, P <0.05). Significant differences in myopia detection rate by after school homework duration were observed in boys and the entire population( χ 2=12.40, 15.25, P <0.01), and significant differences in myopia detection rate by body mass index (BMI) were only observed in boys ( χ 2=6.32, P <0.05). The distribution of myopia severity was statistically significant by sex among junior school students, and by BMI categories among high school students ( χ 2=22.71, 20.37, P <0.01). Multivariate binary Logistic regression analysis showed that the risk for myopia among school students who were overweight and obese was 1.81 times higher than that of students who were not overweight and obese( P <0.01).@*Conclusion@#Overweight and obesity might increase the risk of myopia among middle school students, targeted measures should be taken to maintain the healthy weight of middle school students and reduce the risk of myopia.

Sensitivity of Sniffer Dogs for a Diagnosis of Parkinson's Disease: A Diagnostic Accuracy Study.

BACKGROUND: The diagnostic criteria for Parkinson's disease (PD) remain complex, which is especially problematic for nonmovement disorder experts. A test is required to establish a diagnosis of PD with improved accuracy and reproducibility. OBJECTIVE: The study aimed to investigate the sensitivity and specificity of tests using sniffer dogs to diagnose PD. METHODS: A prospective, diagnostic case-control study was conducted in four tertiary medical centers in China to evaluate the accuracy of sniffer dogs to distinguish between 109 clinically established medicated patients with PD, 654 subjects without PD, 37 drug-naïve patients with PD, and 185 non-PD controls. The primary outcomes were sensitivity and specificity of sniffer dog's identification. RESULTS: In the study with patients who were medicated, when two or all three sniffer dogs yielded positive detection results in a sample tested, the index test sensitivity, specificity, and positive and negative likelihood ratios were 91% (95% CI: 84%-96%), 95% (95% CI: 93%-97%), and 19.16 (95% CI: 13.52-27.16) and 0.10 (95% CI: 0.05-0.17), respectively. The corresponding sensitivity, specificity, and positive and negative likelihood ratios in patients who were drug-naïve were 89% (95% CI: 75%-96%), 86% (95% CI: 81%-91%), and 6.6 (95% CI: 4.51-9.66) and 0.13 (95% CI: 0.05-0.32), respectively. CONCLUSIONS: Tests using sniffer dogs may be a useful, noninvasive, fast, and cost-effective method to identify patients with PD in community screening and health prevention checkups as well as in neurological practice. © 2022 International Parkinson and Movement Disorder Society.

Links between Chinese vocational school students' perception of parents' emotional support and school cooperation climate and their academic performance: The mediating role of school belonging.

The role of social environmental factors on student academic achievement has been conceptualized from the perspective of the ecological system theory. In the present study, a strengths-based approach derived from the theory of positive youth development was adopted to explore the two favorable aspects of proximal social environments, including parents' emotional support and school cooperation climate, and to examine how these two factors influence the academic performance among Chinese senior-secondary vocational school students. Participants were 1,940 students (55.4% male) who took part in the Programme for International Student Assessment (PISA) 2018 test from four regions in China. The students completed the questionnaires to assess parents' emotional support, school cooperation climate, school belonging, and academic performance. By adopting the structural equation model, the results revealed that school belonging fully mediates the association between parents' emotional support and academic scores, and the association between school cooperation climate and academic scores. In addition, multiple group comparison analyses showed there were some gender differences in the relationships between school cooperation climate and academic performance. The practical significance of the influence of parental support and school cooperation climate on student academic achievement was also discussed.

Characteristics of gut microbiota in representative mice strains: Implications for biological research.

BACKGROUND: Experimental animals are used to study physiological phenomena, pathological mechanisms, and disease prevention. The gut microbiome is known as a potential confounding factor for inconsistent data from preclinical studies. Although many gut microbiome studies have been conducted in recent decades, few have focused on gut microbiota fluctuation among representative mouse strains. METHODS: A range of frequently used mouse strains were selected from 34 isolation packages representing disease-related animal (DRA), immunity defect animal (IDA), or gene-editing animal (GEA) from the BALB/c and C57BL/6J backgrounds together with normal mice, and their microbial genomic DNA were isolated from mouse feces to sequence for the exploration of gut microbiota. RESULTS: Mouse background strain, classification, introduced source, introduced year, and reproduction type significantly affected the gut microbiota structure (p < 0.001 for all parameters), with background strain contributing the greatest influence (R2  = 0.237). In normal groups, distinct gut microbiota types existed in different mouse strains. Sixty-four core operational taxonomic units were obtained from normal mice, and 12 belonged to Lactobacillus. Interestingly, the gut microbiota in C57BL/6J was more stable than that in BALB/c mice. Furthermore, the gut microbiota in the IDA, GEA, and DRA groups significantly differed from that in normal groups (p < 0.001 for all). Compared with the normal group, there was a significantly higher Chao1 and Shannon index (p < 0.001 for all) in the IDA, GEA, and DRA groups. Markedly changed classes occurred with Firmicutes and Bacteroidetes. The abundances of Helicobacter, Blautia, Enterobacter, Bacillus, Clostridioides, Paenibacillus, and Clostridiales all significantly decreased in the IDA, GEA, and DRA groups, whereas those of Saccharimonas, Rikenella, and Odoribacter all significantly increased.

Design strategy for an analyte-compensated fluorescent probe to reduce its toxicity.

During biological detection, the toxicity caused by probes to living organisms is neglected. In this study, an analyte-compensated fluorescent probe (NP-SN3) was constructed for the detection of H2S. Through experiments with HepG2 cells and zebrafish embryos and larvae, the NP-SN3 probe showed no significant difference in imaging performance compared with the traditional probe (NP-N3) but exhibited lower detection-induced toxicity in the imaging of liver fibrosis in activated HSC-T6 cells. During the development of zebrafish embryos and continuous administration in rats, NP-SN3 showed a lower death rate, higher hatchability and lower malformation in zebrafish embryos and milder pathological symptoms in stained rat tissues.

Development and characterization of polyurethane-based tissue and blood mimicking materials for high intensity therapeutic ultrasound.

A polyurethane-based tissue mimicking material (TMM) and blood mimicking material (BMM) for the acoustic and thermal characterization of high intensity therapeutic ultrasound (HITU) devices has been developed. Urethane powder and other chemicals were dispersed into either a high temperature hydrogel matrix (gellan gum) or degassed water to form the TMM and BMM, respectively. The ultrasonic properties of both TMM and BMM, including attenuation coefficient, speed of sound, acoustical impedance, and backscatter coefficient, were characterized at room temperature. The thermal conductivity and diffusivity, BMM viscosity, and TMM Young's modulus were also measured. Importantly, the attenuation coefficient has a nearly linear frequency dependence, as is the case for most soft tissues and blood at 37 °C. Their mean values are 0.61f1.2 dB cm-1 (TMM) and 0.2f1.1 dB cm-1 (BMM) based on measurements from 1 to 8 MHz using a time delay spectrometry (TDS) system. Most of the other relevant physical parameters are also close to the reported values of soft tissues and blood. These polyurethane-based TMM and BMM are appropriate for developing standardized dosimetry techniques, validating numerical models, and determining the safety and efficacy of HITU devices.

Benign prostatic hyperplasia wound after surgical removal in subjects on anticoagulant or antiplatelet therapy: A meta-analysis.

We performed a meta-analysis to evaluate the safety of benign prostatic hyperplasia wound after surgical removal in subjects on anticoagulant or antiplatelet therapy. A systematic literature search up to December 2021 was done and 19 studies included 5715 benign prostatic hyperplasia subjects at the start of the study; 1501 of them were on anticoagulant/antiplatelet therapy, and 4214 were control. We calculated the odds ratio (OR) and mean difference (MD) with 95% confidence intervals (CIs) to evaluate the safety of benign prostatic hyperplasia wound after surgical removal in subjects on anticoagulant or antiplatelet therapy by the dichotomous or continuous methods with a random or fixed-influence model. Anticoagulant/antiplatelet therapy had significantly higher bleeding complication (OR, 1.88; 95% CI, 1.36-2.60, P < .001), higher blood transfusion (OR, 2.15; 95% CI, 1.63-2.83, P < .001), lower operation time (MD, -3.53; 95% CI, -6.80-0.27, P = .03), higher catheterization time (MD, 0.30 95% CI, 0.06-0.53, P = .01), longer length of hospital stay (MD, 0.82; 95% CI, 0.37-1.26, P < .001) and higher thromboembolic events (OR, 2.88; 95% CI, 1.26-6.62, P = .01) compared to control in benign prostatic hyperplasia subjects. Anticoagulant/antiplatelet therapy had a significantly higher bleeding complication, higher blood transfusion, lower operation time, higher catheterization time, longer length of hospital stay and higher thromboembolic events compared to control in benign prostatic hyperplasia subjects. Further studies are required.

A Practical Assessment of the Disinfectant Efficacy of UV Light with and without Ozone Using a Novel Transfer Hatch in a Research Animal Facility.

Most in vivo animal research and breeding using mice and rats in China takes place in facilities under barrier conditions. Items being moved across the barrier are typically disinfected using UV radiation in a transfer hatch. However, the time periods necessary for this disinfection technique are inefficient, and disinfection is frequently incomplete, especially if concealed surfaces are present. The current study used a newly developed transfer hatch incorporating both UV and ozone disinfection to examine disinfection efficacy against 4 bacteria species (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii). Disinfection trials used UV and ozone, applied separately and in combination, for up to 30 min. Separate and combined treatments were also tested with a UV barrier. We found that if UV radiation has direct contact with surfaces, it is an efficient disinfection method. However, where surfaces are concealed by a UV barrier, UV radiation performs relatively poorly. The results of this study indicate that a combination of UV and ozone produces the most effective disinfection and is markedly quicker than current disinfection times for UV applied on its own. This novel transfer hatch design therefore allows more complete and efficient disinfection, improves workflow, and reduces barrier breaches by pathogens that may affect animal health and welfare and compromise research outcomes.

Effectiveness of treatment of bedding and feces of laboratory animal with ozone.

BACKGROUND: The incineration and burying of the soiled bedding of laboratory animals, as well as using detergents to treat their feces, is hazardous to the environment. This highlights the need for an alternative, environmentally friendly solution for the treatment of the waste of laboratory animal facilities. This study aims to evaluate the efficacy of ozone disinfection of the soiled bedding and feces of laboratory animals. METHODS: Two grams of soiled beddings were randomly sampled from the cages of mice and rats. These samples were mixed in a beaker with 40ml saline. Ozone was piped into the beaker at a concentration of 500mg/h. Samples were taken from the beaker at time 0min, 30min, 45min and 60min after ozone treatment for microbiological culturing in an incubator for 48h. Colony form unit of each plate (CFU/plate) at each time point were counted, the mean CFU/plate at each time point after ozone treatment were compared with that present at time zero. Feces of rabbits and dogs were treated and pathogens were counted the similar way as that of bedding of the mice and rats; samples being taken at 0min, 15min, 30min, 45min and 60min. RESULTS: Pathogens were observed in beddings of both mice and rats as well as in feces of rabbits and dogs. Ozone treatment for 30min killed more than 93% of pathogens in the bedding of the two rodent species and 60min of treatment killed over 99% of pathogens. Treatment of rabbit and dog feces for 30min killed over 96% pathogens present, and 60min's treatment killed nearly all the pathogens. Both Gram positive and Gram negative pathogens were sensitive to ozone treatment. CONCLUSION: Ozone treatment of bedding and feces is an effective and environment friendly way to deal with the waste of animal facilities, saving energy and potentially enabling their reuse as fertilizer.

Phosphoproteomics of cold stress-responsive mechanisms in Rhododendron chrysanthum.

BACKGROUND: As an alpine plant, Rhododendron chrysanthum (R. chrysanthum) has evolved cold resistance mechanisms and become a valuable plant resource with the responsive mechanism of cold stress. METHODS AND RESULTS: We adopt the phosphoproteomic and proteomic analysis combining with physiological measurement to illustrate the responsive mechanism of R. chrysanthum seedling under cold (4 °C) stress. After chilling for 12 h, 350 significantly changed proteins and 274 significantly changed phosphoproteins were detected. Clusters of Orthologous Groups (COG) analysis showed that significantly changed phosphoproteins and proteins indicated cold changed energy production and conversion and signal transduction. CONCLUSIONS: The results indicated photosynthesis was inhibited under cold stress, but cold induced calcium-mediated signaling, reactive oxygen species (ROS) homeostasis and other transcription regulation factors could protect plants from the destruction caused by cold stress. These data provide the insight to the cold stress response and defense mechanisms of R. chrysanthum leaves at the phosphoproteome level.