PolyQ proteins interfere with nuclear degradation of cytosolic proteins by sequestering the Sis1p chaperone.

Dysfunction of protein quality control contributes to the cellular pathology of polyglutamine (polyQ) expansion diseases and other neurodegenerative disorders associated with aggregate deposition. Here we analyzed how polyQ aggregation interferes with the clearance of misfolded proteins by the ubiquitin-proteasome system (UPS). We show in a yeast model that polyQ-expanded proteins inhibit the UPS-mediated degradation of misfolded cytosolic carboxypeptidase Y(∗) fused to green fluorescent protein (GFP) (CG(∗)) without blocking ubiquitylation or proteasome function. Quantitative proteomic analysis reveals that the polyQ aggregates sequester the low-abundant and essential Hsp40 chaperone Sis1p. Overexpression of Sis1p restores CG(∗) degradation. Surprisingly, we find that Sis1p, and its homolog DnaJB1 in mammalian cells, mediates the delivery of misfolded proteins into the nucleus for proteasomal degradation. Sis1p shuttles between cytosol and nucleus, and its cellular level limits the capacity of this quality control pathway. Upon depletion of Sis1p by polyQ aggregation, misfolded proteins are barred from entering the nucleus and form cytoplasmic inclusions.

The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis.

In eukaryotic cells a molecular chaperone network associates with translating ribosomes, assisting the maturation of emerging nascent polypeptides. Hsp70 is perhaps the major eukaryotic ribosome-associated chaperone and the first reported to bind cotranslationally to nascent chains. However, little is known about the underlying principles and function of this interaction. Here, we use a sensitive and global approach to define the cotranslational substrate specificity of the yeast Hsp70 SSB. We find that SSB binds to a subset of nascent polypeptides whose intrinsic properties and slow translation rates hinder efficient cotranslational folding. The SSB-ribosome cycle and substrate recognition is modulated by its ribosome-bound cochaperone, RAC. Deletion of SSB leads to widespread aggregation of newly synthesized polypeptides. Thus, cotranslationally acting Hsp70 meets the challenge of folding the eukaryotic proteome by stabilizing its longer, more slowly translated, and aggregation-prone nascent polypeptides.

Structural and Functional Characterization of a Fish Type I Subgroup d IFN Reveals Its Binding to Receptors.

Teleost fish type I IFNs and the associated receptors from the cytokine receptor family B (CRFB) are characterized by remarkable diversity and complexity. How the fish type I IFNs bind to their receptors is still not fully understood. In this study, we demonstrate that CRFB1 and CRFB5 constitute the receptor pair through which type I subgroup d IFN (IFNd) from large yellow croaker, Larimichthys crocea, activates the conserved JAK-STAT signaling pathway as a part of the antiviral response. Our data suggest that L. crocea IFNd (LcIFNd) has a higher binding affinity with L. crocea CRFB5 (LcCRFB5) than with LcCRFB1. Furthermore, we report the crystal structure of LcIFNd at a 1.49-Å resolution and construct structural models of LcIFNd in binary complexes with predicted structures of extracellular regions of LcCRFB1 and LcCRFB5, respectively. Despite striking similarities in overall architectures of LcIFNd and its ortholog human IFN-ω, the receptor binding patterns between LcIFNd and its receptors show that teleost and mammalian type I IFNs may have differentially selected helices that bind to their homologous receptors. Correspondingly, key residues mediating binding of LcIFNd to LcCRFB1 and LcCRFB5 are largely distinct from the receptor-interacting residues in other fish and mammalian type I IFNs. Our findings reveal a ligand/receptor complex binding mechanism of IFNd in teleost fish, thus providing new insights into the function and evolution of type I IFNs.

Advances in performance degradation mechanism and safety assessment of LiFePO4for energy storage.

In the context of 'energy shortage', developing a novel energy-based power system is essential for advancing the current power system towards low-carbon solutions. As the usage duration of lithium-ion batteries for energy storage increases, the nonlinear changes in their aging process pose challenges to accurately assess their performance. This paper focuses on the study LiFeO4(LFP), used for energy storage, and explores their performance degradation mechanisms. Furthermore, it introduces common battery models and data structures and algorithms, which used for predicting the correlation between electrode materials and physical parameters, applying to state of health assessment and thermal warning. This paper also discusses the establishment of digital management system. Compared to conventional battery networks, dynamically reconfigurable battery networks can realize real-time monitoring of lithium-ion batteries, and reduce the probability of fault occurrence to an acceptably low level.

Effect of acid-modified biochar coupled with alternate wetting and drying on P leaching, soil P retention and plant P uptake in paddy fields.

H2SO4-modified biochar has been recognized as a means to achieve the advantages of carbon sequestration, and nitrogen loss reduction. However, little information is available on its effect on phosphorus (P) uptake, soil available P, and P leaching under alternate wetting and drying irrigation (IAWD). A split-plot experimental layout was carried out with two irrigation regimes (conventional continuous flooding, ICF, and alternate wetting and drying, IAWD) as main plots and three biochar additions (biochar-free control, B0, non-acidified biochar, B20, and acid-modified biochar, B20A) as subplots. Results indicated that IAWD decreased water percolation by 9.26%-14.74% and P leaching by 50.14%-106.64% and increased surface soil available P by 10.88-29.08%, resulting in 14.21-35.03% apparent phosphorus balance (APB) over the three years as compared with ICF. B20 produced a 6.23% lower grain yield in the 1st year and 5.06% and 11.02% higher yields in the 2nd and 3rd years, while B20A increased or tended to increase it throughout the three years. Both B20 and B20A significantly decreased total water percolation (9.68-28.37%), P leaching (18.26-152.00%), and increased soil available P (9.90-46.24%), dissolved P in surface soil (10.00-62.50%), and P uptake (4.31-49.71%), and thereafter enhanced apparent phosphorus balance (11.06-40.78%). Compared with B20, B20A achieved a better APB due to a 113% lower P leaching and 52.9% lower dissolved P at 60 cm soil profiles. IAWDB20A-M produced the highest APB, surface soil available and dissolved P, and the lowest P leaching, which increased grain yield, APB, surface soil available P, and dissolved P by 9.54%, 129.61%, and 53.19%, and decreased P leaching by 257% over ICFB0, respectively. Therefore, the use of H2SO4-modified biochar could produce higher grain yield with lower P leaching and higher APB for IAWD paddy systems, which is beneficial to enhancing plant P uptake, mitigating P leaching, and ensuring sustainable agricultural production.

FAM3A Ameliorates Brain Impairment Induced by Hypoxia-Ischemia in Neonatal Rat.

Hypoxia-ischemia (HI) during crucial periods of brain formation can lead to changes in brain morphology, propagation of neuronal stimuli, and permanent neurodevelopmental impairment, which can have profound effects on cognitive function later in life. FAM3A, a subgroup of family with sequence similarity 3 (FAM3) gene family, is ubiquitously expressed in almost all cells. Overexpression of FAM3A has been evidenced to reduce hyperglycemia via the PI3K/Akt signaling pathway and protect mitochondrial function in neuronal HT22 cells. This study aims to evaluate the protective role of FAM3A in HI-induced brain impairment. Experimentally, maternal rats underwent uterine artery bilateral ligation to induce neonatal HI on day 14 of gestation. At 6 weeks of age, cognitive development assessments including NSS, wire grip, and water maze were carried out. The animals were then sacrificed to assess cerebral mitochondrial function as well as levels of FAM3A, TNF-α and IFN-γ. Results suggest that HI significantly reduced FAM3A expression in rat brain tissues, and that overexpression of FAM3A through lentiviral transduction effectively improved cognitive and motor functions in HI rats as reflected by improved NSS evaluation, cerebral water content, limb strength, as well as spatial learning and memory. At the molecular level, overexpression of FAM3A was able to promote ATP production, balance mitochondrial membrane potential, and reduce levels of pro-inflammatory cytokines TNF-α and IFN-γ. We conclude that FAM3A overexpression may have a protective effect on neuron morphology, cerebral mitochondrial as well as cognitive function. Created with Biorender.com.

[Integrated management during the perinatal period for total anomalous pulmonary venous connection]. / å®Œå ¨æ€§è‚ºé™è„‰å¼‚ä½è¿žæŽ¥çš„å›´ç”ŸæœŸä¸€ä½“åŒ–ç®¡ç†.

OBJECTIVES: To evaluate the clinical effectiveness of integrated management during the perinatal period for fetuses diagnosed with total anomalous pulmonary venous connection (TAPVC) by prenatal echocardiography. METHODS: Clinical data of 64 cases of TAPVC fetuses diagnosed by prenatal echocardiography and managed with integrated perinatal care in Qingdao Women and Children's Hospital from January 2017 to December 2021 were retrospectively analyzed. Integrated perinatal care included multidisciplinary collaboration among obstetrics, fetal medicine, ultrasound, pediatric cardiology, pediatric anesthesia, and neonatology. RESULTS: Among the 64 TAPVC fetuses, there were 29 cases of supracardiac type, 27 cases of intracardiac type, 2 cases of infracardiac type, and 6 cases of mixed type. Chromosomal analysis was performed in 42 cases, and no obvious abnormalities were found. Among the 64 TAPVC fetuses, 37 were induced labor, and 27 were followed up until term birth. Among the 27 TAPVC cases, 2 cases accepted palliative care, 2 cases were referred to another hospital for treatment and lost to follow-up, while the remaining 23 cases underwent primary repair surgery. One case died within 6 months after the operation due to low cardiac output syndrome, while the other 22 cases were followed up for (2.1±0.3) years with good outcomes (2 cases underwent a second surgery within 1 year after the first operation due to anastomotic stenosis or pulmonary vein stenosis). CONCLUSIONS: TAPVC fetuses can achieve good outcomes with integrated management during the perinatal period.

Intensity-surged and bandwidth-extended terahertz radiation in two-foci cascading plasmas.

The two-color strong-field mixing in gas medium is a widely used approach to generate bright broadband terahertz (THz) radiation. Here, we present a new, to the best of our knowledge, and counterintuitive method to promote THz performance in the two-color scheme. Beyond our knowledge that the maximum THz generation occurs with two-color foci overlapped, we found that, when the foci of two-color beams are noticeably separated along the propagation axis resulting in cascading plasmas, the THz conversion efficiency is surged by one order of magnitude and the bandwidth is stretched by more than two times, achieving 10-3 conversion efficiency and >100 THz bandwidth under the condition of 800/400 nm, ∼35 fs driving lasers. With the help of the pulse propagation equation and photocurrent model, the observations can be partially understood by the compromise between THz generation and absorption due to the spatial redistribution of laser energy in cascading plasmas. The present method can be extended to a mid-infrared driving laser, and new records of THz peak power and conversion efficiency are expected.

Prenatal ultrasound diagnosis of Klippel-Trenaunay-Weber syndrome associated with umbilical cord hemangioma.

We describe a case of prenatal diagnosed Klippel-Trenaunay-Weber syndrome, which mainly manifested as hypertrophy of the left thigh, and was associated with umbilical cord hemangioma and loss of heterozygosity (LOH) for 1q21.2 q44. This case report describes the second reported case associated with umbilical cord hemangioma and the first reported case with LOH for 1q21.2 q44.

Low-Intensity Pulsed Ultrasound Attenuates LPS-Induced Neuroinflammation and Memory Impairment by Modulation of TLR4/NF-κB Signaling and CREB/BDNF Expression.

The purpose of this study was to investigate the restorative role of low-intensity pulsed ultrasound (LIPUS) against lipopolysaccharide (LPS)-induced neuroinflammation and memory impairments in a simulation of Alzheimer's disease. Mice subjected to LPS administration (250 µg/kg, i.p.) were treated with LIPUS daily for 7 days. The levels of brain-derived neurotrophic factor (BDNF) and inflammatory markers were estimated in brain tissue using western blot. After LIPUS treatment, the neuroprotective effects of LIPUS in mice were assessed by behavioral tests. LPS plus LIPUS-treated mice exhibited a significant increase in the average time spent in the target quadrant compared to the LPS-treated group. Compared with the LPS-treated group, LPS plus LIPUS-treated mice revealed a preference for the novel object. LIPUS treatment significantly attenuated LPS-induced increases in the expression of amyloid-beta (Aß) and amyloid precursor protein (APP) in the hippocampus region of LPS-treated mice. Furthermore, LIPUS significantly reduced the protein levels of TNF-α, IL-1ß, and IL-6 in the mice brain induced by LPS. LIPUS treatment induces neuroprotection by inhibiting the LPS-induced activation of TLR4/NF-κB inflammatory signaling and by enhancing the associated CREB/BDNF expression in LPS-treated mice. Our data showed that LIPUS attenuated LPS-induced memory impairment as well as amyloidogenesis via the suppression of neuroinflammatory activity and BDNF decline.

Highly efficient production of functional recombinant human fibroblast growth factor 22 in E. coli and its protective effects on H2O2-lesioned L02 cells.

In the 22 member mammalian FGF family, FGF22 belongs to FGF7 subfamily, and its effects are largely confined to the brain and skin. To explore the functions of FGF22 on other tissues and develop a large-scale production of recombinant human FGF22 (rhFGF22) without a fusion tag, a plasmid encoding human FGF22 (pET3a-rhFGF22) was used to express rhFGF22 in E. coli BL21 (DE3) pLysS. A large amount of rhFGF22 inclusion body protein was obtained. A two-step denaturing method successfully solubilized rhFGF22, and it was refolded and then purified in one step via heparin affinity chromatography. A yield of 105 mg rhFGF22 with a purity of up to 95% was obtained from 100 g wet bacteria. It was found that the rhFGF22 had biological activity, since it effectively attenuated H2O2-induced human hepatic L02 cell death. Analysis by qRT-PCR and Western blot demonstrated that rhFGF22 protects L02 cells from H2O2-induced oxidative damage via suppression of mitochondrial apoptosis pathways. In conclusion, the strategy described in this paper may provide a novel means to solve the production of insoluble rhFGF22 and shine new light on its translational potential.

High-yield of biologically active recombinant human fibroblast growth factor-16 in E. coli and its mechanism of proliferation in NCL-H460 cells.

Fibroblast growth factor-16 (FGF16) is a member of FGF9 subfamily, which plays key role in promoting mitosis and cell survival, and also involved in embryonic development, cell growth, tissue repair, morphogenesis, tumor growth, and invasion. However, the successful high-yield purification of recombinant human fibroblast growth factor-16 (rhFGF16) protein has not been reported. In addition, lung cancer is a major cause of cancer-related deaths, which threats people's lives and its incidence has continued to rise. Learning pathways or proteins, which involved in lung tumor progression will contribute to the development of early diagnosis and targeted therapy. FGF16 promoted proliferation and invasion behavior of SKOV-3 ovarian cancer cells, whose function may be similar in lung cancer. The hFGF16 was cloned into pET-3d and expressed in Escherichia coli BL21 (DE3) pLysS. Finally, obtained two forms of FGF16 that exhibited remarkable biological activity and the purity is over 95%, meanwhile, the yield of soluble 130 mg/100 g and insoluble 240 mg/100 g. Experiments demonstrated FGF16 could promote proliferation of NCL-H460 cells by activating Akt, Erk1/2, and p38 MAPK signaling, whereas JNK had no significant effect. In total, this optimized expression strategy enables significant quantity and activity of rhFGF16, thereby meeting its further pharmacological and clinical usages.

Copper-Mediated Trifluoroacetylation of Arenediazonium Salts with Ethyl Trifluoropyruvate.

A copper-mediated trifluoroacetylation of various arenediazonium salts with ethyl trifluoropyruvate is reported. The reaction proceeded smoothly under mild conditions at room temperature giving trifluoromethyl aryl ketones in moderate to good yields. A variety of functional groups, including methoxy, hydroxy, ester, ketone, trifluoromethyl, and halide groups, were well tolerated. A possible reaction mechanism involving an aryl radical intermediate was proposed and supported by experimental evidence. This reaction provides a new route to trifluoromethyl aryl ketones, notable synthetic targets, from the corresponding anilines.

Research on temperature detection method of liquor distilling pot feeding operation based on a compressed algorithm.

In the process of feeding the distilling bucket after vapor detection, the existing methods can only realize the lag detection after the steam overflow, and can not accurately detect the location of the steam, etc. At the same time, in order to effectively reduce the occupancy of the computational resources and improve the deployment performance, this study established infrared image dataset of fermented grains surface, and fused the YOLO v5n and the knowledge distillation and the model pruning algorithms, and an lightweight method YOLO v5ns-DP was proposed as as a model for detecting temperature changes in the surface layer of fermented grains during the process of feeding the distilling. The experimental results indicated that the improvement makes YOLOv5n improve its performance in all aspects. The number of parameters, GLOPs and model size of YOLO v5ns-DP have been reduced by 28.6%, 16.5%, and 26.4%, respectively, and the mAP has been improved by 0.6. Therefore, the algorithm is able to predict in advance and accurately detect the location of the liquor vapor, which effectively improves the precision and speed of the detection of the temperature of the surface fermented grains , and well completes the real-time detecting task.

Genistin Represses the Proliferation and Angiogenesis While Accelerating the Apoptosis of Glioma Cells by Modulating the FOXC1-Mediated Wnt Signaling Pathway.

BACKGROUND: Glioma is a tumor originating from glial cells and is the most common primary brain tumor. At present, the main treatment methods for glioma include surgical resection and radiotherapy and chemotherapy, but the treatment effect is not very ideal. Genistin (GS) inhibits breast cancer cell growth while promoting apoptosis, but its effect and detailed molecular mechanism on glioma are yet to be defined. In addition, forkhead box C1 (FOXC1) has been found to be involved in the growth, invasion, and angiogenesis processes of glioma cells. METHODS: Human glioma cells in the Control, GS-6.25, GS-12.5, and GS25 (GS) groups were treated with 0, 6.25, 12.5, and 25 µM of Genistin, respectively, for 72 hours, and cells in the GS + NC (negative control) and GS + FOXC1 groups were transfected with negative control or forkhead box C1 (FOXC1) overexpression plasmids, respectively, prior to Genistin (25 µM) treatment for 72 hours. Next, the viability, proliferation, apoptosis, and angiogenesis of treated glioma cells were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'deoxyuridine (EdU) proliferation, flow cytometry, and tube formation assays. Meanwhile, the half-maximal inhibitory concentration (IC50) of Genistin in the treated glioma cells was calculated. Afterwards, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot quantified the levels of FOXC1, Wnt1, Wnt3a, glycogen synthase kinase-3ß (GSK3ß), and phosphorylated GSK3ß (p-GSK3ß). RESULTS: Genistin inhibited viability, proliferation, and angiogenesis while promoting the apoptosis of glioma cells (p < 0.05, p < 0.001). Also, Genistin decreased the levels of FOXC1, Wnt1, and Wnt3a while increasing p-GSK3ß levels in glioma cells (p < 0.05, p < 0.01, p < 0.001). FOXC1 was up-regulated in glioma cells and tissues, and overexpressed FOXC1 overturned the effects of Genistin on the abovementioned factors in glioma cells (p < 0.05, p < 0.001). CONCLUSIONS: Genistin inhibits viability, proliferation, and angiogenesis while accelerating glioma cell apoptosis by modulating the FOXC1-mediated Wnt signaling pathway.

Effects of nitrogen fertilization on the fate of high-risk antibiotic resistance genes in reclaimed water-irrigated soil and plants.

High-risk antibiotic resistance genes (ARGs) in reclaimed water-irrigated soil pose a potential threat to ecosystem and human health. Inorganic fertilization - including with nitrogen, a key ingredient in agricultural production - may affect the ARG profile in soil. However, little is known about nitrogen fertilization's influence on ARGs profiles in the soil-plant system. This study investigated the effects of different nitrogen fertilizer types (CO(NH2)2, NO3--N (NaNO3) and NH4+-N (NH4HCO3)) and different nitrogen fertilizer application rates (low, medium, high) on the distribution of high-risk ARGs in reclaimed water-irrigated soil and plants using quantitative PCR, high-throughput sequencing and metagenomic sequencing. Soil microcosms results revealed that nitrogen fertilization significantly affected the pattern of high-risk ARGs in soil, and also affected high-risk ARGs abundance and transfer capacity in plants. Compared with nitrogen fertilizer application rate, nitrogen fertilizer types significantly contributed to enhancing the soil resistome, with the order of CO(NH2)2 > NO3--N ≈ NH4+-N. The medium application of NO3--N and NH4+-N significantly reduced high-risk ARGs abundance in the leaf endophyte. Bacterial community mainly drove the variation of ARGs in nitrogen-fertilized soil-plant system, and class I integron and metal resistance genes (MRGs) also had direct effects on these high-risk ARGs. A similar high-risk ARGs pattern was also found in field plot experiments, and several dangerous pathogens were observed as the main high-risk ARGs potential hosts in nitrogen-fertilized soil. Based on an economic assessment, application of NH4+-N (NH4HCO3) could reduce costs by $1,312.83 ha-1 compared with NO3--N (NaNO3). These results showed that the more important role of nitrogen type might be an effective and economical way to control high-risk ARGs spread in soil-plant system under reclaimed water irrigation.

The introduction of influent sulfamethoxazole loads induces changes in the removal pathways of sulfamethoxazole in vertical flow constructed wetlands featuring hematite substrate.

High frequent detection of sulfamethoxazole (SMX) in wastewater cannot be effectively removed by constructed wetlands (CWs) with a traditional river sand substrate. The role of emerging substrate of hematite in promoting SMX removal and the effect of influent SMX loads remain unclear. The removal efficiency of SMX in hematite CWs was significantly higher than that in river sand CWs by 12.7-13.8% by improving substrate adsorption capacity, plant uptake and microbial degradation. With increasing influent SMX load, the removal efficiency of SMX in hematite CWs slightly increased, and the removal pathways varied significantly. The contribution of plant uptake was relatively small (< 0.1%) under different influent SMX loads. Substrate adsorption (37.8%) primarily contributed to SMX removal in hematite CWs treated with low-influent SMX. Higher influent SMX loads decreased the contribution of substrate adsorption, and microbial degradation (67.0%) became the main removal pathway. Metagenomic analyses revealed that the rising influent load increased the abundance of SMX-degrading relative bacteria and the activity of key enzymes. Moreover, the abundance of high-risk ARGs and sulfonamide resistance genes in hematite CWs did not increase with the increasing influent load. This study elucidates the potential improvements in CWs with hematite introduction under different influent SMX loads.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation.

Large scale phosphoproteome profiles comprehensive features of mouse embryonic stem cells.

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.