Simultaneous SERS Sensing of Cysteine and Homocysteine in Blood Based on the CBT-Cys Click Reaction: Toward Precisive Diagnosis of Schizophrenia.

At present, there is a lack of sufficiently specific laboratory diagnostic indicators for schizophrenia. Serum homocysteine (Hcy) levels have been found to be related to schizophrenia. Cysteine (Cys) is a demethylation product in the metabolism of Hcy, and they always coexist with highly similar structures in vivo. There are few reports on the use of Cys as a diagnostic biomarker for schizophrenia in collaboration with Hcy, mainly because the rapid, economical, accurate, and high-throughput simultaneous detection of Cys and Hcy in serum is highly challenging. Herein, a click reaction-based surface-enhanced Raman spectroscopy (SERS) sensor was developed for simultaneous and selective detection of Cys and Hcy. Through the efficient and specific CBT-Cys click reaction between the probe containing cyan benzothiazole and Cys/Hcy, the tiny methylene difference between the molecular structures of Cys and Hcy was converted into the difference between the ring skeletons of the corresponding products that could be identified by plasmonic silver nanoparticle enhanced molecular fingerprint spectroscopy to realize discriminative detection. Furthermore, the SERS sensor was successfully applied to the detection in related patient serum samples, and it was found that the combined analysis of Cys and Hcy can improve the diagnostic accuracy of schizophrenia compared to a single indicator.

"Dramatic Growth" of Microbial Aerosols for Visualization and Accurate Counting of Bioaerosols.

While the global COVID-19 pandemic has subsided, microbial aerosol detection has become of high concern. Timely, accurate, and highly sensitive monitoring of microbial aerosols in indoor air is the basis for effective prevention and control of infectious diseases. At present, no commercial equipment or reliable technology can simultaneously control the detection time and limit at 6 h and 102 CFU/mL, respectively. Based on the "safety size range" of particulate matter in the air, we propose a new method of microbial dilation detection, which enables the pathogen to grow rapidly and dramatically into a polymeric microsphere, larger in size than the coexisting aerosol particles. "Like a crane standing among chickens", the microorganism can be easily visualized and counted. Different from routine chemical and biological sensing technologies, this method can achieve absolute counting of microbial particles, and the simple principles can be developed into devices for different life scenarios.

Silent Raman imaging of highly effective anti-bacterial activity synchronous with biofilm breakage using poly(4-cyanostyrene)@silver@polylysine nanocomposites.

Biofilms are known to be a great challenge for their anti-bacterial activity as they obstruct drug action for deeper and more thorough bacteria-killing effects. Therefore, developing highly effective antibacterial agents to destroy biofilms and eradicate bacteria is of great significance. Herein, a new type of nanocomposites (denoted as poly(4-cyanostyrene)@silver@polylysine) is proposed, in which polylysine (PLL) could rapidly capture the biofilms and exhibit excellent antibacterial efficacy together with decorated silver (Ag) nanoparticles (NPs) through the charge effect and Ag+ release. Notably, nearly 100% antibacterial rates against Gram-positive bacterium (Staphylococcus aureus, S. aureus) and Gram-negative bacterium (Escherichia coli, E. coli) were achieved. More importantly, poly(4-cyanostyrene) with biological silent Raman imaging capacity is able to illustrate the relationship between antibacterial efficiency and biofilm breakage. In short, such novel nanocomposites can improve the bioavailability of each component and display tremendous potential in antibacterial applications.

Clinical Significance of Nutrition and Inflammation in Esophageal Cancer Patients with Surgery: A Meta-Analysis.

Many studies have reported that the geriatric nutritional risk index (GNRI) and C-reactive protein to albumin ratio (CAR) may be associated with prognosis of esophageal cancer (EC); however, the results are inconsistent. Therefore, we performed a meta-analysis to evaluate the effect of preoperative GNRI and CAR on the prognosis of EC. PubMed, Embase, Cochrane Library, and Web of Science databases were searched. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were used to analyze the relationship between GNRI/CAR and prognosis. Publication bias was estimated using Begg's funnel plot asymmetry test and Egger's test. A total of 21 studies comprising 5,018 patients were included in the meta-analysis. A decreased GNRI was significantly associated with poorer overall survival (OS) (HR = 1.808, 95% CI: 1.489-2.196, P < 0.001) and cancer-specific survival (CSS) (HR = 1.769, 95% CI: 1.193-2.624, P = 0.005), and an increased CAR was significantly associated with lower OS (HR = 2.179, 95% CI: 1.587-2.992, P < 0.001), CSS (HR = 1.733, 95% CI: 1.333-2.253, P < 0.001), and recurrence-free survival (HR = 2.178, 95% CI: 1.328-3.573, P = 0.002). Thus, preoperative GNRI and CAR may be noninvasive and powerful tools for predicting survival outcomes in patients with EC.

Prognostic Significance of Preoperative Integrated Liver Inflammatory Score in Patients with Hepatocellular Carcinoma.

BACKGROUND The Integrated Liver Inflammatory Score (ILIS), which includes 5 serum indicators (albumin, bilirubin, neutrophil count, alpha-fetoprotein [AFP], and alkaline phosphatase [ALP]), is a novel inflammation-based predictive model associated with poor survival in hepatocellular carcinoma (HCC) patients. Our study aimed to assess the prognostic value of ILIS in HCC patients undergoing radical hepatectomy and establish a nomogram and artificial neural network based on their ILIS scores. MATERIAL AND METHODS This multicenter retrospective study included patients from 2 institutions from 2007 to 2017. Independent risk factors associated with Recurrence-free survival (RFS) and overall survival (OS) were identified through univariate and multifactor analysis in the training and validation groups, respectively. Afterward, column line graphs and artificial neural networks (ANN) were constructed and validated using the validation group. RESULTS A total of 432 patients were included in this study (275 in the training group and 157 in the validation group). In both cohorts, ILIS was correlated with pathological features such as tumor size, degree of differentiation, Child-Pugh class classification, and BCLC staging. Moreover, ILIS was identified as an independent risk factor for OS. ILIS-based nomograms and artificial neural networks also showed the prognostic value of ILIS. CONCLUSIONS Preoperative ILIS is an independent and effective predictor of prognosis in HCC patients treated with radical hepatectomy, as shown by the fact that higher ILIS are associated with worse patient prognosis. We have also established nomograms and ANNs that predict HCC prognosis with high accuracy.

Promoted "Click" SERS Detection for Precise Intracellular Imaging of Caspase-3.

Although homogeneous detection of some biomolecules has been of great significance in clinical assay, it faces great challenges in achieving precise in situ imaging of biomolecules. In addition, nonspecific adsorption between probes and biomolecules and low sensitivity are still unfathomed problems. Herein, we developed a promoted "Click" surface enhanced Raman scattering (SERS) strategy for realizing highly selective homogeneous detection of biomolecules by simultaneous dual enhanced SERS emissions, obtaining mutually confirmed logical judgment. Taking caspase-3 as one of the biotargets, we have realized highly selective homogeneous detection of caspase-3 using this strategy, and precise intracellular imaging of caspase-3 can be in situ monitored in living cells or during cell apoptosis. In detail, polyA-DNA and the Asp-Glu-Val-Asp (DEVD)-containing peptide sequence were modified into alkyne and nitrile-coded Au nanoparticles (NPs). During the cell apoptosis process, the generated caspase-3 would lead to the cleavage of the tetra-peptide sequence DEVD, thereby removing the negative protection part from the peptide on Au NPs. Interestingly, two different triple bond-labeled Au NPs can be connected together through DNA hybridization to form SERS "hotspot", resulting in simultaneously enlarged triple bond Raman signals. Moreover, we found that the SERS intensity was positively related with caspase-3 concentration, which has a wide linear range (0.1 ng/mL to 10 µg/mL) and low detection limit (7.18 × 10-2 ng/mL). Remarkably, these simultaneously enlarged signals by "Click" SERS could be used for more precise imaging of caspase-3, providing mutually confirmed logical judgment based on two spliced SERS emissions, especially for their relative intensity.

Nucleic Acid Hybridization-Based Noise Suppression for Ultraselective Multiplexed Amplification of Mutant Variants.

The analysis of mutant nucleic acid (NA) variants can provide crucial clinical and biological insights for many diseases. Yet, existing analysis techniques are generally constrained by nonspecific "noise" signals from excessive wildtype background sequences, especially under rapid isothermal multiplexed target amplification conditions. Herein, the molecular hybridization chemistry between NA bases is manipulated to suppress noise signals and achieve ultraselective multiplexed detection of cancer gene fusion NA variants. Firstly, modified locked NA (LNA) bases are rationally introduced into oligonucleotide sequences as designed "locker probes" for high affinity hybridization to wildtype sequences, leading to enrichment of mutant variants for multiplexed isothermal amplification. Secondly, locker probes are coupled with a customized "proximity-programmed" (SERS) readout which allows precise control of hybridization-based plasmonic signaling to specifically detect multiple target amplicons within a single reaction. Moreover, the use of triple bond Raman reporters endows NA noise signal-free quantification in the Raman silent region (≈1800-2600 cm-1 ). With this dual molecular hybridization-based strategy, ultraselective multiplexed detection of gene fusion NA variants in cancer cellular models is actualized with successful noise suppression of native wildtype sequences. The distinct benefits of isothermal NA amplification and SERS multiplexing ability are simultaneously harnessed.

Precise Encoding of Triple-Bond Raman Scattering of Single Polymer Nanoparticles for Multiplexed Imaging Application.

Stimulated Raman scattering (SRS) microscopy in combination with innovative tagging strategies offers great potential as a universal high-throughput biomedical imaging tool. Here, we report rationally tailored small molecular monomers containing triple-bond units with large Raman scattering cross-sections, which can be polymerized at the nanoscale for enhancement of SRS contrast with smaller but brighter optical nanotags with artificial fingerprint output. From this, a class of triple-bond rich polymer nanoparticles (NPs) was engineered by regulating the relative dosages of three chemically different triple-bond monomers in co-polymerization. The bonding strategy allowed for 15 spectrally distinguishable triple-bond combinations. These accurately structured nano molecular aggregates, rather than long-chain macromolecules, could establish a universal method for generating small-sized biological SRS imaging tags with high sensitivity for high-throughput multi-color biomedical imaging.

On-Site and Quantitative Detection of Trace Methamphetamine in Urine/Serum Samples with a Surface-Enhanced Raman Scattering-Active Microcavity and Rapid Pretreatment Device.

Here, it reports a high-throughput detection method for reliably quantitative analysis of illegal drugs in complex biological samples by means of a surface-enhanced Raman scattering (SERS) active microcavity and rapid pretreatment device. Based on the well-made hemispherical microcavities that regularly distributed on a glass array, the quality-controllable microcavity device is fabricated by the compact self-assembly of core-shell nanopeanuts (CSNPs) onto the inside surface. Both the CSNPs with a quantifiable internal standard signal of crystal violet acetate anchored inside their gap and the well-made microcavity referred to the physical amplification of the microscale groove surface will do well in trace analysis, which will allow us to realize the accurately quantitative SERS analysis of targeted analytes spread on the bottom area of the microcavity array. As an example, 0.8 nM malachite green and 160 ppb methamphetamine (MATM) have been successively detected in a wide range as standard, while even 0.01 ppm MATM mixed in the urine/serum samples has been efficiently tested by the microcavity device equipped with a rapid pretreatment device (manual monolithic column syringe needle). All of the above suggest that the SERS-active microcavity equipped with a rapid pretreatment device has potential in the on-site quick test of trace amounts of illegal drugs in bodily fluid samples or other field analysis of food sanitation, environmental safety, and public health.

TRAF6-p38/JNK-ATF2 axis promotes microglial inflammatory activation.

Activating transcription factor 2 (ATF2), a member of the alkaline-leucine zipper family, is widely expressed in various tissues, and reportedly involved in inflammatory responses to various irritates, but its role in the central nervous system (CNS) remains unclear. This study aimed to investigate the expression and biological function of ATF2 in CNS inflammation. Utilizing the LPS-induced neuroinflammation model on mice, we first found ATF2 up-regulation and its co-localization with microglia in inflamed mice brain. In vitro, we revealed an increased expression, phosphorylation, and nuclear accumulation of ATF2 in LPS-treated BV2 microglia cells. Inhibiting ATF2 significantly decreased the expression of pro-inflammatory factors in LPS-treated microglia, and alleviated neuronal apoptosis induced by the conditioned medium of activated microglia. Knocking down TRAF6, an important adaptor of the TLR4/MAPK/NF-κB signaling pathway, suppressed the LPS-induced ATF2 expression and phosphorylation, accompanied by the decreased p38/JNK phosphorylation, in microglia. Blocking p38 or JNK signaling pathway by the specific inhibitors reversed the TRAF6-overexpression mediated ATF2 activation. Taken together, our data first proved the pro-inflammatory function of ATF2 in microglia, and suggested that the TRAF6-JNK/p38-ATF2 axis might promote microglial inflammatory activation and thus aggravate neuronal injury in brain, which might become a potential therapeutic target for CNS diseases.

Nanoscale Laser Metallurgy and Patterning in Air Using MOFs.

We report metallurgy on the nanoscale to generate metal nanoparticles and their simultaneous patterning in a single step. This is achieved by the self-reduction of porous metal-organic framework crystals using nanosecond pulsed laser irradiation. Metal nanoparticles of Fe, Co, Ni, Cu, Zn, Cd, In, Bi, and Pb with uniform sizes (controllable between 3 to 200 nm) and gaps (as narrow as 2 nm) are produced by nine different metal-organic frameworks, where atomically dispersed non-noble metal ions are reduced and gathered across the pores. The instant light absorption and cooling at local positions by a laser allows for precise and efficient patterning of metal nanoparticles. This new method is suitable for device fabrication at a speed of 15 mm2 s-1 on glass, consuming only 1.5 W of power. A large variety of metal nanoparticle three-dimensional architectures are demonstrated, among which one architecture exhibits an enhanced plasmonic effect homogeneously across the entire pattern for the detection of molecules at an extremely low concentration (10-12 M). These architectures are extremely stable under air and humidity during production, use, and storage, without altering the oxidation state, for 6 months.

Combined Surface-Enhanced Raman Scattering Emissions for High-Throughput Optical Labels on Micrometer-Scale Objects.

High-throughput optical labeling technologies have become increasingly important with the growing demands for molecular detection, disease diagnosis, and drug discovery. In this thought, a series of CN-bridged coordination polymer encapsulated gold nanoparticles have been developed as a universal and interference-free optical label through a facile and auxiliary agent-free self-assembly route. Moreover, surface-enhanced Raman scattering (SERS) emissions of CN-bridge can be tuned flexibly by simple replacement of Fe2+/Fe3+ with other metal ions relying on the synthesis of three Prussian blue analogues encapsulated gold nanoparticles (Au@PBA NPs). Thus, three distinct Raman frequencies have been acquired, which merely replaced the metal irons. On the basis of the potential supermultiplex optical label, space-confined surface-enhanced Raman scattering (SERS) emissions have been realized. Relying on "Abbe theorem", the focused laser allows the pure and single triple bond-coded SERS emissions to be combined into a unique and independent output, so-called "combined SERS emission" (c-SERS), if the Au@PBA NPs were confined into one micrometer-scale object. This study demonstrated c-SERS may simultaneously provide 2n - 1 optical labels only using n single emissions in the Raman-silent region for micrometer-size objects.

Accurate Clinical Diagnosis of Liver Cancer Based on Simultaneous Detection of Ternary Specific Antigens by Magnetic Induced Mixing Surface-Enhanced Raman Scattering Emissions.

Establishing an accurate, simple, and rapid serodiagnosis method aiming for specific cancer antigens is critically important for the clinical diagnosis, therapy, and prognostication of cancer. Currently, surface-enhanced Raman scattering (SERS) readout techniques challenge fluorescent-based detection methods in terms of both optical stability and more importantly multiple detection capability, which become more desirable for clinical diagnostics. We thus started using an interference-free mixing SERS emission (m-SERS) readout to simultaneously indicate, for the first time, three specific liver cancer antigens, including α-fetoprotein (AFP), carcinoembryonic antigen (CEA), and ferritin (FER), even in one clinical serum sample. Here, three triple bonds (C≡N and C≡C) coded SERS tags contribute separate SERS emissions located at 2105, 2159, and 2227 cm-1, respectively; must have one-to-one correspondence from AFP, to FER, to CEA, In the process of detection, the mature double antibody sandwich allows the formation of microscale core-satellite assembly structure between a magnetic bead (MB) and single SERS tags, and therefore a pure and single SERS emission can be observed under the routine excitation laser spot. Because of the action of magnetic force, the uniform 3D packing of SERS tags absorbed MBs will in contrast generate a so-called m-SERS signals. With the help of enrichment and separation by MBs, the proposed m-SERS immunoassay provides an extremely rapid, sensitive, and accurate solution for multiplex detection of antigens or other biomarkers. Herein, the limit of detection (LOD) for simultaneous m-SERS detection of AFP, CEA, and FER was 0.15, 20, and 4 pg/mL, respectively. As expected for 39 clinical serum samples, simultaneous detection of ternary specific antigens can significantly improve the accuracy of liver cancer diagnosis.

SIRT1 antagonizes liver fibrosis by blocking hepatic stellate cell activation in mice.

Hepatic stellate cells (HSCs) are a major source of fibrogenesis in the liver, contributing to cirrhosis. When activated, HSCs transdifferentiate into myofibroblasts and undergo profound functional alterations paralleling an overhaul of the transcriptome, the mechanism of which remains largely undefined. We investigated the involvement of the class III deacetylase sirtuin [silent information regulator 1 (SIRT1)] in HSC activation and liver fibrosis. SIRT1 levels were down-regulated in the livers in mouse models of liver fibrosis, in patients with cirrhosis, and in activated HSCs as opposed to quiescent HSCs. SIRT1 activation halted, whereas SIRT1 inhibition promoted, HSC transdifferentiation into myofibroblasts. Liver fibrosis was exacerbated in mice with HSC-specific deletion of SIRT1 [conditional knockout (cKO)], receiving CCl4 (1 mg/kg) injection or subjected to bile duct ligation, compared to wild-type littermates. SIRT1 regulated peroxisome proliferator activated receptor γ (PPARγ) transcription by deacetylating enhancer of zeste homolog 2 (EZH2) in quiescent HSCs. Finally, EZH2 inhibition or PPARγ activation ameliorated fibrogenesis in cKO mice. In summary, our data suggest that SIRT1 plays an essential role guiding the transition of HSC phenotypes.-Li, M., Hong, W., Hao, C., Li, L., Wu, D., Shen, A., Lu, J., Zheng, Y., Li, P., Xu, Y. SIRT1 antagonizes liver fibrosis by blocking hepatic stellate cell activation in mice.

Hough Transform-Based Large Dynamic Reflection Coefficient Micro-Motion Target Detection in SAR.

Special phase modulation of SAR echoes resulted from target rotation or vibration, is a phenomenon called the micro-Doppler (m-D) effect. Such an effect offers favorable information for micro-motion (MM) target detection, thereby improving the performance of the synthetic aperture radar (SAR) system. However, when there are MM targets with large differences in reflection coefficient, the weak reflection components will be difficult to be detected. To find a solution to this problem, we propose a novel algorithm. First, we extract and detect the strongest reflection component. By removing the strongest reflection component from the original azimuth echo one by one, we realize the detection of reflection components sequentially, from the strongest to the weakest. Our algorithm applies to detecting MM targets with different reflection coefficients and has high precision of parameter estimation. The results of simulation and field experiments verify the advantages of the algorithm.

Splicing Nanoparticles-Based "Click" SERS Could Aid Multiplex Liquid Biopsy and Accurate Cellular Imaging.

Here, a completely new readout technique, so-called "Click" SERS, has been developed based on Raman scattered light splice derived from nanoparticle (NP) assemblies. The single and narrow (1-2 nm) emission originating from triple bond-containing reporters undergoes dynamic combinatorial output, by means of controllable splice of SERS-active NPs analogous to small molecule units in click chemistry. Entirely different to conventional "sole code related to sole target" readout protocol, the intuitional, predictable and uniquely identifiable "Click" SERS is relies on the number rather than the intensity of combinatorial emissions. By this technique, 10-plex synchronous biomarkers detection under a single scan, and accurate cellular imaging under double exposure have been achieved. "Click" SERS demonstrated multiple single band Raman scattering could be an authentic optical analysis method in biomedicine.

The histone methyltransferase Suv39h2 contributes to nonalcoholic steatohepatitis in mice.

Uncontrolled inflammatory response highlights the central theme of nonalcoholic steatohepatitis (NASH), a growing global pandemic. Hepatocytes and macrophages represent two major sources of hepatic inflammation during NASH pathogenesis, contributing to excessive synthesis of proinflammatory mediators. The epigenetic mechanism that accounts for the activation of hepatocytes and macrophages in this process remains obscure. Here, we report that compared to wild-type littermates, mice with a deficiency in the histone H3K9 methyltransferase suppressor of variegation 39 homolog 2 (Suv39h2, knockout) exhibited a less severe form of NASH induced by feeding with a high-fat, high-carbohydrate diet. Pro-NASH stimuli increased Suv39h2 expression in cell culture, in mice, and in human livers. In hepatocytes, Suv39h2 bound to the Sirt1 gene promoter and repressed Sirt1 transcription. Suv39h2 deficiency normalized Sirt1 expression, allowing nuclear factor kappa B/p65 to become hypoacetylated and thus dampening nuclear factor kappa B-dependent transcription of proinflammatory mediators. In macrophages, Suv39h2-mediated repression of peroxisome proliferator-activated receptor gamma transcription favored a proinflammatory M1 phenotype over an anti-inflammatory M2 phenotype, thereby elevating hepatic inflammation. CONCLUSION: Suv39h2 plays a pivotal role in the regulation of inflammatory response in hepatocytes and macrophages, contributing to NASH pathogenesis. (Hepatology 2017;65:1904-1919).

Heat shock protein 70 protects cardiomyocytes through suppressing SUMOylation and nucleus translocation of phosphorylated eukaryotic elongation factor 2 during myocardial ischemia and reperfusion.

Myocardial ischemia and reperfusion (MIR) results in cardiomyocyte apoptosis with severe outcomes, which blocks cardiac tissue recovering from myocardial ischemia diseases. Heat shock protein 70 (HSP70) is one of protective molecule chaperones which could regulate the nucleus translocation of other proteins. In addition, eukaryotic elongation factor 2 (eEF2), which modulates protein translation process, is vital to the recovery of heart during MIR. However, the relationship between HSP70 and eEF2 and its effects on MIR are unclear. The expression and relationship between HSP70 and eEF2 is confirmed by western blot, immunoprecipitation in vitro using cardiomyocyte cell line H9c2 and in vivo rat MIR model. The further investigation was conducted in H9c2 cells with detection for cell-cycle and apoptosis. It is revealed that eEF2 interacted and be regulated by HSP70, which kept eEF2 as dephosphorylated status and preserved the function of eEF2 during MIR. In addition, HSP70 suppressed the nucleus translocation of phosphorylated eEF2, which inhibited cardiomyocyte apoptosis during myocardial reperfusion stage. Furthermore, HSP70 also interacted with C-terminal fragment of eEF2, which could reverse the nucleus translocation and cardiomyocyte apoptosis caused by N-terminal fragment of eEF2. HSP70 draw on advantage and avoid defect of MIR through regulating phosphorylation and nucleus translocation of eEF2.

Environmentally Safe Mercury(II) Ions Aided Zero-Background and Ultrasensitive SERS Detection of Dipicolinic Acid.

Field, reliable, and ultrasensitive detection of dipicolinic acid (DPA), a general biomarker of bacterial spores and especially Bacillus anthracis, is highly desirable but still challenging in current biometric security emergency response system. Herein we report an environmentally safe mercury(II) ions-mediated and competitive coordination interaction based approach for rationally designed surface-enhanced Raman scattering (SERS)-active gold nanoparticles (AuNPs), enabling rapid, ultrasensitive and zero-background detection of DPA without the pretreatment of samples. By means of competitiveness, these papain-capped gold nanoparticles (P-AuNPs) are induced to undergo controllable aggregation upon the addition of Hg2+ ions and DPA with a concentration range (1 nM∼8 µM), which correspondingly cause quantitative changes of SERS intensity of cresyl violet acetate (CVa) conjugated AuNPs. The decreased Raman intensity obtained by subtracting two cases of additives that contain only Hg2+ and the mixture of Hg2+ and DPA is proportional to the concentration of DPA over a range of 1 nM∼8 µM (R2 = 0.9824), with by far the lowest limit of detection (LOD) of 67.25 pM (0.01 ppb, S/N = 3:1). Of particular significance, mercury(II) ions actually play two roles in the process of measurements: a mediator for two designed competitive ligands (DPA and papain), and also a scavenger for the possibly blended ligands due to the different interaction time between DPA and the interferent with Hg2+ ions, which guarantees the interference-free detection of DPA even under real conditions.

Modification of p27 with O-linked N-acetylglucosamine regulates cell proliferation in hepatocellular carcinoma.

The tumor suppressor p27, which is a member of the Cip/Kip family of Cyclin-dependent kinase inhibitory proteins (CKIs), controls anti-proliferative events. The post-translational addition of O-GlcNAc to p27 occurs in HEK293T and HCC (hepatocellular carcinoma) cell lines, and we identified Ser2, Ser106, Ser110, Thr157, and Thr198 as the glycosylation sites of p27 based on the Q-TOF spectrum. Here, immunoprecipitation analysis showed that Ser2 was O-GlcNAcylated and that this modification was associated with the increased phosphorylation of p27 at Ser10, ultimately resulting in p27 accumulation in the cytoplasm and increased p27 ubiquitination. In addition, O-GlcNAcylation at Ser2 suppressed Cyclin/CDK complex-p27 interactions by promoting the nuclear export of p27, thus facilitating cell cycle progression. Cell proliferation was negatively regulated when Ser2 of p27 was replaced with Ala. Furthermore, western blot and immunohistochemical analyses of HCC tissues and their corresponding nontumorous tissues were performed, and we found that O-GlcNAcylated p27 correlated with cell proliferation in HCC. Together, our results indicate that the dynamic interplay between O-GlcNAcylation and p27 phosphorylation coordinates and regulates cell proliferation in hepatocellular carcinoma. © 2016 Wiley Periodicals, Inc.