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QKI is a vital regulator in RNA splicing and maturation, but its role in cervical cancer (CC) is little known. In this study, we found that QKI is decreased in human CC, and overexpression of QKI inhibits HeLa cell proliferation and promotes the apoptosis of cancer cells. We identified hundreds of endogenous QKI-regulated alternative splicing events (ASEs) and differentially expressed genes (DEGs) in QKI-overexpressed HeLa cells by RNA-seq and selectively validated their expression by quantitative reverse-transcription polymerase chain reaction. The gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that QKI-regulated ASEs and DEGs were closely related to cancer, apoptosis, and transcriptional regulatory functions. In short, QKI may affect the occurrence and development of CC by regulating gene expression through AS.

FMS-like receptor tyrosine kinase 3 (Flt3) expression was reported to increase in the heart in response to pathological stress, but the role of Flt3 activation and its underlying mechanisms remain poorly elucidated. This study was designed to investigate the role of Flt3 activation in sympathetic hyperactivity-induced cardiac hypertrophy and its mechanisms through autophagy and mitochondrial dynamics. In vivo, cardiac hypertrophy was established by subcutaneous injection of isoprenaline (6 mg/kg·day) in C57BL/6 mice for 7 consecutive days. The Flt3-ligand intervention was launched 2 h prior to isoprenaline each day. In vitro, experiments of cardiomyocyte hypertrophy, autophagy, and mitochondrial dynamics were performed in neonatal rat cardiomyocytes (NRCMs). Our results revealed that the expression level of Flt3 protein was significantly increased in the hypertrophic myocardium provoked by isoprenaline administration. Flt3-ligand intervention alleviated isoprenaline-induced cardiac oxidative stress, hypertrophy, fibrosis, and contractile dysfunction. Isoprenaline stimulation impaired autophagic flux in hypertrophic mouse hearts, supported by the accumulation of LC3II and P62 proteins, while Flt3-ligand restored the impairment of autophagic flux. Flt3 activation normalized the imbalance of mitochondrial fission and fusion in the hearts of mice evoked by isoprenaline as evidenced by the neutralization of elevated mitochondrial fission markers and reduced mitochondrial fusion markers. In NRCMs, Flt3-ligand treatment attenuated isoprenaline-stimulated hypertrophy, which was abolished by a Flt3-specific blocker AC220. Activating Flt3 reversed isoprenaline-induced autophagosome accumulation and impairment of autophagic flux probably by enhancing SIRT1 expression and consequently TFEB nuclear translocation. Flt3 activation improved the imbalance of mitochondrial dynamics induced by isoprenaline in NRCMs through the SIRT1/P53 pathway. Activation of Flt3 mitigated ISO-stimulated hypertrophy probably involves the restoration of autophagic flux and balance of mitochondrial dynamics. Therefore, activation of Flt3 attenuates isoprenaline-induced cardiac hypertrophy in vivo and in vitro, the potential mechanism probably attributes to SIRT1/TFEB-mediated autophagy promotion and SIRT1/P53-mediated mitochondrial dynamics balance. These findings suggest that activation of Flt3 may be a novel target for protection against cardiac remodeling and heart failure during sympathetic hyperactivity.

Sorafenib (Sor), a novel multi-target anticancer drug also induces severe toxicity in heart, while the mechanism of its cardiotoxicity remains to be fully elucidated. Dysregulation of autophagy and mitochondrial dynamics imbalance have been implicated in cardiomyocyte death. The aim of this study is to test the hypothesis that Sor disrupts autophagy and mitochondrial dynamics, thereby aggravating Sor-induced oxidative stress damage to cardiomyocytes. Our results revealed that Sor (≥ 5 μM) concentration- and time-dependently reduced cell viability and induced apoptosis in H9c2 myoblasts. Sor treatment promoted intracellular reactive oxygen species (ROS) generation, and subsequent Ca

Neointimal hyperplasia is the major cause of carotid stenosis after vascular injury, which restricts the long-term efficacy of endovascular treatment and endarterectomy in preventing stenosis. Ginsenoside Re (Re) is a major active ingredient of ginseng having multifaceted pharmacological effects on the cardiovascular system, and is a potential treatment for restenosis. In this study, we demonstrated that Re treatment significantly inhibited vascular injury-induced neointimal thickening, reduced the intimal area and intima/media (I/M) ratio, increased the lumen area, and inhibited pro-inflammatory cytokines. In cultured A7R5 cells, Re inhibited LPS-induced proliferation and migration as evidenced by suppressed colony formation and shortened migration distance, accompanied by the downregulated expression of pro-inflammatory cytokines. Re promoted VSMC apoptosis induced by balloon injury in vivo and LPS challenge in vitro. Moreover, Re inhibited autophagy in VSMCs evoked by balloon injury and LPS as supported by reduced LC3II and increased p62 expressions. Suppression of autophagy with the specific autophagy inhibitor spautin-1 efficiently inhibited LPS-induced cell proliferation and inflammation and promoted caspase-3/7 activities. Mechanistically, we found that Re attenuated Ras/ERK1/2 expression in VSMCs in vivo and in vitro. The MEK1/2 inhibitor PD98059 showed similar effects to Re on cell proliferation, migration, apoptosis, and the levels of autophagy and cytokines. In conclusion, we provided significant evidence that Re inhibited vascular injury-induced neointimal thickening probably by promoting VSMC apoptosis and inhibiting autophagy via suppression of the Ras/MEK/ERK1/2 signaling pathway.

Background Ovarian cancer is the most lethal gynecological cancer worldwide. Since the early clinical symptoms are not obvious, most patients with ovarian cancer are already in an advanced clinical stage when they are diagnosed. HGSOC accounts for the greatest number of ovarian cancer deaths and shows an association between an enhanced immune response and prolonged patient survival. Methods 88 normal ovarian samples from TCGA and 149 ovarian cancer patients from GTEx were used to analyze gene expression, proteins and pathways associated with INPP4B in HGSOC, LGSOC and ovarian cancer. The correlation between INPP4B gene and immune factors was analyzed by TIMER website. The expression of INPP4B was verified by immunohistochemistry and Western Blotting. MTT assay, flow cytometry, and scratch assay were used to verify the effects of INPP4B on proliferation, apoptosis and migration. Tumor formation experiment to verify the effect of INPP4B on tumor formation. Results In this study, we performed data mining analysis of HGSOC and LGSOC samples. We first identified 25 tumor suppressor genes associated with poor prognosis and survival in HGSOC. Further immune infiltration analysis of these 25 genes with the TIMER web tool showed that among the 25 genes, INPP4B had the most pronounced effect on CD8 + T-cell infiltration. In addition, detection of INPP4B expression by IHC staining in clinical samples indicated that the INPP4B level was significantly lower in ovarian cancer tissues and negatively related to patient survival. Moreover, knockdown of INPP4B accelerated ovarian cancer cell growth and migration in vitro and vivo. Finally, the genes related to INPP4B in ovarian cancer were mainly enriched in the immune response and inflammatory response. Conclusions INPP4B plays an important role in cell proliferation, cell growth, cell migration, apoptosis and immune infiltration in OC. The INPP4B gene, as a potential tumor suppressor gene, may become a biomarker and therapeutic target for ovarian cancer.

A hydrophilic metal-organic network based on Ti4+ and dual natural ligand, tannic acid (TA) and phytic acid (PA), has been developed to enrich phosphopeptides from complex bio-samples prior to liquid chromatography-mass spectrometric analysis. Due to the strong chelation ability of TA and PA, abundant Ti4+ can be immobilized in the material, forming hydrophilic network by one-step coordination-driven self-assembly approach. The sorbent, TA-Ti-PA@Fe3O4, exhibited satisfactory selectivity for the phosphopeptides in the tryptic digest of β-casein, and can eliminate the interference components in 1000-fold excess of bovine serum albumin. The adsorption capacity of the sorbents for phosphopeptides was up to 35.2 mg g−1 and the adsorbing equilibrium can be reached in 5 min. The adsorbing mechanism has been investigated and the results indicated that the Ti4+ in forms of [Ti(f-TA)(H2O)4]2+, [Ti(f-PA)(H2O)4]2+ and Ti(f-PA)2(H2O)2 may play an important role in the adsorption process. The sorbent of the TA-Ti-PA@Fe3O4 has been applied to enrichment of the phosphopeptides in tryptic digest of rat liver lysate, and 3,408 phosphopeptides have been identified, while the numbers of the identified phosphopeptides were 2730 and 1217 when the sample was enriched by the commercial TiO2 and Fe3+-IMAC kit, respectively. This work provides a strategy to enrich phosphopeptides from complex samples and shows great potential application in phosphoproteome research.

Recent studies reported that miR-128 was differentially expressed in cardiomyocytes in response to pathologic stress. However, its function and mechanism remain to be fully elucidated. The aim of the present study was to investigate the role of miR-128 in chronic angiotensin II (Ang II) infusion-induced cardiac remodeling and its underlying mechanism. The cardiac remodeling and heart failure in vivo were established in C57BL/6 mice by chronic subcutaneous Ang II delivery. Knocking down miR-128 was conducted in the hearts of the mice by intravenous injection of HBAAV2/9-miR-128-GFP sponge (miR-128 inhibitor). In vitro experiments of cardiac hypertrophy, apoptosis, and aberrant autophagy were performed in cultured cells after Ang II treatment or transfection of miR-128 antagomir. Our results showed that chronic Ang II delivery for 28 days induced cardiac dysfunction, hypertrophy, fibrosis, apoptosis, and oxidative stress in the mice, while the miR-128 expression was notably enhanced in the left ventricle. Silencing miR-128 in the hearts of mice ameliorated Ang II-induced cardiac dysfunction, hypertrophy, fibrosis apoptosis, and oxidative stress injury. Moreover, Ang II induced excessive autophagy in the mouse hearts, which was suppressed by miR-128 knockdown. In cultured cells, Ang II treatment induced a marked elevation in the miR-128 expression. Downregulation of miR-128 in the cells by transfection with miR-128 antagomir attenuated Ang II-induced apoptosis and oxidative injury probably via directly targeting on the SIRT1/p53 pathway. Intriguingly, we found that miR-128 inhibition activated PIK3R1/Akt/mTOR pathway and thereby significantly damped Ang II-stimulated pathological autophagy in cardiomyocytes, which consequently mitigated cell oxidative stress and apoptosis. In conclusion, downregulation of miR-128 ameliorates Ang II-provoked cardiac oxidative stress, hypertrophy, fibrosis, apoptosis, and dysfunction in mice, likely through targeting on PIK3R1/Akt/mTORC1 and/or SIRT1/p53 pathways. These results indicate that miR-128 inhibition might be a potent therapeutic strategy for maladaptive cardiac remodeling and heart failure.

The abuse of cephalosporins poses a serious threat to human health and the ecological environment. In this work, cephalosporinase (AmpC enzyme) and Prussian blue (PB) crystals were encapsulated into ZIF-8 metal-organic frameworks (MOFs), and a photothermal AmpC/PB@ZIF-8 MOFs (APZ) nanocatalyst was prepared for the catalytic degradation of cephalosporin. The temperature of the APZ catalytic degradation system can be regulated by irradiation with near infrared light due to the photothermal effect of PB, and then, the activity of the APZ biocatalyst is significantly enhanced. Thereby, the degradation efficiency of cefuroxime can reach to 96%, and the degradation kinetic rate of cefuroxime augmented 4.5-fold comparing with that catalyzed by free enzyme. Moreover, encapsulation of the enzyme and PB can increase the affinity and charge transfer efficiency between APZ and substrate molecules, which can also improve the degradation efficiency of cephalosporins. Catalytic degradation pathways for three generations of cephalosporins were proposed based on their degradation products. The dual-enhancer biocatalyst based on the photothermal effect and immobilization of the PB and enzyme can significantly enhance the activity and stability of the enzyme, and it can also be recycled. Therefore, the biocatalyst has potential applications for the effective degradation of cephalosporins in the environment.

AIM FMS-like receptor tyrosine kinase 3 (Flt3) has been reported to be increased in cardiomyocytes responding to ischaemic stress. This study was to determine whether Flt3 activation could ameliorate pressure overload-induced heart hypertrophy and fibrosis, and to elucidate the mechanisms of action. METHODS In vivo cardiac hypertrophy and remodelling experiments were conducted by infusing angiotensin II (Ang II) chronically in male C57BL/6 mice. Flt3-specific ligand (FL) was administered intraperitoneally every two days (5 µg/mouse). In vitro experiments on hypertrophy, apoptosis and autophagy mechanism were performed in neonatal rat cardiomyocytes (NRCMs) and H9c2 cells with adenovirus vector-mediated overexpression of Flt3. RESULTS Our results demonstrated that following chronic Ang II infusion for 4 weeks, the mice exhibited heart hypertrophy, fibrosis, apoptosis and contractile dysfunction. Meanwhile, Ang II induced autophagic responses in mouse hearts, as evidenced by increased LC3 II and decreased P62 expression. These pathological alterations in Ang II-treated mice were significantly ameliorated by Flt3 activation with FL administration. In NRCMs and Flt3-overexpressed H9c2 cells, FL attenuated Ang II-induced pathological autophagy and inactivated AMPK/mTORC1/FoxO3a signalling, thereby efficiently mitigating cell hypertrophy and apoptosis. Conversely, the AMPK activator metformin or the mTORC1 inhibitor rapamycin reversed the effects of FL on the alterations of autophagy, hypertrophy and apoptosis in cardiomyocytes induced by Ang II. CONCLUSION Flt3 activation ameliorates cardiac hypertrophy, fibrosis and contractile dysfunction in the mouse model of chronic pressure overload, most likely via suppressing AMPK/mTORC1/FoxO3a-mediated autophagy. These results provide new evidence supporting Flt3 as a novel therapeutic target in maladaptive cardiac remodelling.

Severe microplastic pollution from anthropogenic activities in coastal zones presents an imminent risk to marine ecosystems. In this study, abundant microplastics (15–12,852 items kg−1) with sizes ranging between 0.16 and 5.0 mm were extracted from 17 sediment samples collected in sandy beaches and mangrove wetlands of the Qinzhou Bay, Guangxi Province, Southwest China. Three types of microplastics (i.e. polystyrene, polypropylene, and polyethylene) were identified with Fourier transform infrared (FTIR) spectroscopy analysis. These detected microplastics were characterized by different colors (white, transparent, yellow, green, red, and blue) and shapes (fragment, fiber, and sphere). Microplastics were concentrated on supratidal beaches and wetlands outside of mangrove, and less abundant on intertidal beaches and inside of mangrove wetlands. Meanwhile, high microplastic concentrations were observed near mollusk farms. The spatial distribution and chemical speciation indicated that microplastics were derived from disintegration of large plastic debris (e.g., Styrofoam buoys used to support mollusk rafts) abandoned by aquaculture industry. Further, coastal vegetation (e.g. mangrove) could trap microplastic particles.

s The tyrosine phosphorylation of proteins and peptides plays a vital role in cell signal transduction pathways, and it is very important to assay them for understanding their action mechanism. Due to the low levels of the tyrosine phosphopeptides (pTyr) in cells, it is a challenge to enrich them with traditional sorbents, therefore, development of specific and selective sorbents is urgent and necessary. In this work, the phosphate-imprinted magnetic nanoparticles (PMNPs) to enrich the pTyr with high efficiency and selectivity have been fabricated using the phenylphosphonic acid as a template for the “epitope” of pTyr. The magnetic nanoparticles have been functionalized with TiO2 and then the imprinting silica shells have been coated on the surface of the functional core to obtain the PMNPs sorbents. The PMNPs can obviously shorten the enrichment time and improve the adsorption efficiency for pTyr, and the epitope imprinting films provide an excellent selective recognition ability to target. The recognition capability of PMNPs for pTyr is 90.3 μg/mg and the imprinting factor of the sorbents can reach 24.4. The results indicate that the PMNPs can enrich the pTyr from the tryptic digest of β-casein samples with high specificity, and the spiking recoveries of the pTyr range from 85.1% to 93.8% with the RSD from 0.04 to 3.73. With the high adsorption capacity, rapid separation, excellent specificity and recyclability, the PMNPs sorbents show great potential for analysis of the phosphorylation of peptides in biological and medical fields.

Fluorescent hydroxyapatite nanoparticles (HAP-NPs) were synthesized by introducing trisodium citrate during the HAP-NP synthesis process. The HAP-NPs display strong emission at around 440 nm when excited at 340 nm. In the presence of low levels of biothiols, such as cysteine (Cys) and homocysteine (hCys), the fluorescence intensity of HAP-NPs was found to be enhanced, while for the same concentration of glutathione (GSH), a smaller fluorescence intensity increase was observed. So a selective and sensitive fluorescence turn-on assay for detection of low levels of biothiols is reported. The linear range of the assay for Cys is from 2 to 100 nM, and for hCys is from 2 to 200 nM. The assay was successfully applied to the detection of Cys in human serum samples. The interesting fluorescence properties of HAP-NPs, plus their good biocompatibility, mean that HAP-NPs may find wide application as fluorescence probes in different assays and bio-imaging areas.

Accurate detection of the biological biomarkers can provide valuable information for early diagnosis and treatment of many diseases. In this work, an enzyme-free immunosensor based on nanobrushes and the fluorescent dye (Rhodamine-6G) for detection of the biomarker procalcitonin (PCT) in human serum albumin has been successfully developed. The immuno nanobrush has been prepared by conjugating the poly-glutamic acid (PGA) and the detection antibody of PCT on the surface of the polystyrene (PS) nanospheres sequentially. The abundant functional groups (-COO-) on the PGA chain of the nanobrushes have the potential ability to bind tightly with a lot of the fluorescent dye (Rhodamine-6G) by electrostatic interaction, which can significantly augment the fluorescence signal intensity of the dyed nanobrushes, and then acted as the signal amplification system of the immunosensor to improve the sensitivity for determination of the PCT. Meanwhile, the immunomagnetic beads can selectively capture the PCT from the samples. The results showed that the immunoassay exhibited high sensitivity and selectivity with satisfactory reproducibility and stability, and successfully applied to determine the PCT levels in real human serum samples. The enzyme-free immunoassay overcomes the defects of traditional Enzyme-linked immunosorbent assay and provides a novel approach to detect the biomarker PCT for clinical diagnosis of the inflammation.

The authors describe a carbon dot (CD) based dual-emission ratiometric optical probe for the on-site visual and fluorometric determination of mercury(II) ions. The nanoparticle (NP) probe was obtained by covalently linking the blue emissive carbon dots to the surface of silica nanoparticles containing red-emissive quantum dots (QDs). The red emitting QDs in the silica matrix are inert to Hg(II) and provide a reliable and constant reference signal. They also reduce their toxicity and improve the optical and chemical stabilities, while the blue emission CDs are very sensitive to Hg(II). With increasing concentration of Hg(II), a solution containing the NP probe undergoes a continuous color change from light purple to red. This can be seen with bare eyes or detected instrumentally by measurement of fluorescence intensity under excitation/emission wavelengths of 350/453 and 658 nm. The probe exhibits high sensitivity to Hg(II), with a detection limit of 0.47 nM (at an S/N ratio of 3). This is much lower than the allowable level of mercury (10 nM, ~10 ppb) in drinking water set by the U.S. Environmental Protection Agency. For practical use, the probe was used to quantify Hg(II) in (spiked) tap water where it gave recoveries between 95 and 106% and relative standard deviations between 1.9 and 3.2%. The probe can also be applied in filter paper-based assays, and this paves the way to point-of-care pollution control. This ratiometric probe is nontoxic and easily operated, and therefore shows potential applications for rapid and low-cost visual identification of Hg(II).

Near space target is of high velocity and high acceleration, which will result in the velocity ambiguity, range migration (RM), and Doppler frequency migration (DFM). To achieve good performance of coherent integration, the velocity ambiguity, RM and DFM should be mitigated. Due to the high acceleration of the target, the velocity ambiguity numbers may vary in the coherent integration time, which leads to the decrease of the signal-to-noise ratio (SNR). A variable velocity ambiguity numbers compensation method is proposed in this paper. The signal is divided into two segments according to the position where the velocity ambiguity number changes, which is obtained by searching. To correct the RM and DFM, the second-order Keystone transform (SKT) and the fractional Fourier transform (FRFT) are combined. The initial phases of the two segments are different. Therefore, the initial range and velocity of the second segment are compensated based on the target parameters. The proposed method solves the problem of variable velocity ambiguity numbers in the coherent integration time, which improves the performance of target detection. Finally, the effectiveness of the proposed method is verified through simulation results.

The traditional enzyme-linked immunosorbent assay (ELISA) has some disadvantages, such as insufficient sensitivity and low stability of the labeled enzyme, which limit its further applications. In this study, a more stable enzyme, Amp cephalosporinase (AmpC), was selected as the labeled enzyme, and its substrate was designed and synthesized. This substrate contained the cephalosporin ring core as the enzymatic recognition section and the structural motif of the 3-hydroxyflavone (3-HF) as the reporter molecule. AmpC can specifically catalyze the substrate and release 3-HF, which can enter the cavity of β-cyclodextrin (β-CD) on the surface of ZnS quantum dots and form a fluorescence resonance energy transfer (FRET) signal amplification system. An AmpC-catalyzed, FRET-mediated ultrasensitive immunosensor (ACF immunosensor) for procalcitonin (PCT) was developed by combining the signal amplification system of the polystyrene microspheres and effective immune-based magnetic separation. The ACF immunosensor has high sensitivity and specificity for the detection of PCT: its linear range is from 0.1 ng mL

A novel magnetic nanomaterial for use in metal ion based affinity chromatography is described. It is based on the chelation between the phosphate groups of phytic acid (PA) and Ti(IV) ions. Due to the large number (6) of phosphate groups of PA, it has a large capacity for Ti(IV) ions. PA was first immobilized on magnetite nanoparticles (PA-MNPs) and then loaded with Ti(IV) ions to obtain the sorbent (Ti-PA-MNPs). The fraction of Ti(IV) ions on the surface of PA-MNPs that is exposed to the solution binds the phosphate groups of phosphopeptides. The bound phosphopeptides can then be magnetically separated. The method was applied to the enrichment of the phosphopeptides in a β-casein tryptic digest. A tryptic digest of bovine serum albumin (BSA) was added at a molar ratio (β-casein to BSA) of 1:2000 to study selectivity. The phosphopeptides were quantified by mass spectrometry. The limit of detection can be as low as 8 × 10−10 mol L−1. This sorbent has a high absorption capacity (53.5 μg mg-1) and shows good recoveries (90%). As many as 2145 phosphopeptides were isolated from 500 μg tryptic digest of a rat liver lysate after enrichment by Ti-PA-MNPs. This is superior to that (1568 phosphopeptides) of commercial TiO2 kit.

A sensitive and specific multiplex immunoassay for the biomarkers of hepatocellular carcinoma (HCC), Alpha-fetoprotein (AFP) and Golgi protein 73 (GP73), has been developed. The enzymes, Horse radish peroxidase (HRP) and Alkaline phosphatase (ALP) were modified with active reactive groups of click chemistry, and then self-assembled to form enzyme aggregates, HRPn and ALPn, respectively. The carbon nanotubes (CNT) were conjugated with the enzyme aggregates and the detection antibody of biomarkers by bioorthogonal reaction, forming the immuno nanosensor for AFP and GP73 individually. The enzymes on the nanosensor can catalyze its substrate to emit chemiluminescences, which can be read out in different time windows. Owing to the high reaction efficiency of the bioorthogonal reaction, large capacity of the catalytic enzymes has been conjugated to the surface of CNT, and then the signal intensity of chemiluminiscence has been greatly amplified. The multiplex immunoassay has been successfully applied to detect the biomarkers in real serum samples, which can improve the accuracy and preciseness for the clinical diagnosis of HCC. The nanosensors have high specifity for the biomarkers of HCC, and their sensitivities can be adjustable according to practical demand. This work provides a novel clue to develope immunoassay for clinical diagnosis of HCC.

Protein kinase (PKA) and alkaline phosphatase (ALP) are clinically relevant enzymes for a number of diseases. In this work, we developed a new simple electrochemical biosensor for the detection of the activity and inhibition of both PKA and ALP. One common feature of the PKA and ALP catalyzing process is that PKA can hydrolysis adenosine-5'-triphosphate (ATP) and ALP can hydrolysis pyrophosphate, both reactions produce phosphate ions, and the amount of phosphate ion produced is proportional to enzyme activity. Our assay is based on the principle that phosphate ions react with molybdate to form redox molybdophosphate precipitates on the electrode surface, thus generating electrochemical current. The detection limit for PKA and ALP were much lower than existing assays. The biosensor has good specificity and was used to measure drug-stimulated PKA from lysates of HeLa cells. We also evaluated the use of the biosensor as a screening tool for enzyme inhibitors. To the best of our knowledge, this is the first report of a biosensor capable of detecting the activity of both PKA and ALP. This tool has the potential to simplify PKA and ALP clinical measurement, thereby improving diagnostics of relevant diseases. It also may serve as the basis for a simple screening method for new enzyme inhibitors for disease treatment.

The authors describe a method for highly sensitive and selective determination of the activity of protein kinase (PKA). It is based on the finding that silver nanoclusters (AgNCs) can act as a nucleus to catalyze further deposition of silver nanoparticles. This causes the color of a solution to change from pale yellow to black. In the detection scheme presented here, the substrate peptide is phosphorylated by PKA in the presence of ATP. The resulting phosphopeptides bind to oligonucleotide-stabilized AgNCs in the presence of Zr(IV) ions due to electrostatic interactions between Zr(IV) and the phosphate groups, thereby capping the AgNCs. The silver enhancement process (leading to a color change to black) does not work if the AgNCs are capped. The degree of inhibition is proportional to the activity of the kinase. The color change can be detected visually or photographically in a microplate format by exploiting the changes in the grey values of the digital photos. In addition, the DNA-AgNCs display fluorescence emission at 635 nm when excited at 565 nm. Electrochemical assays were performed (at a working voltage as low as 38 mV vs. Ag/AgCl) by using a glassy carbon electrode modified with a solution containing AgNCs, Zr(IV) ions and the peptide, and immersing it into the silver enhancement solution. The assay is highly sensitive and selective. It was applied to the determination of PKA in lysates of HeLa cells. The detection limits typically are between 32 and 37 U⋅ L-1 based on a signal-to-noise ratio of 3.

A novel magnetic nanomaterial for use in metal ion based affinity chromatography is described. It is based on the chelation between the phosphate groups of phytic acid (PA) and Ti(IV) ions. Due to the large number (6) of phosphate groups of PA, it has a large capacity for Ti(IV) ions. PA was first immobilized on magnetite nanoparticles (PA-MNPs) and then loaded with Ti(IV) ions to obtain the sorbent (Ti-PA-MNPs). The fraction of Ti(IV) ions on the surface of PA-MNPs that is exposed to the solution binds the phosphate groups of phosphopeptides. The bound phosphopeptides can then be magnetically separated. The method was applied to the enrichment of the phosphopeptides in a β-casein tryptic digest. A tryptic digest of bovine serum albumin (BSA) was added at a molar ratio (β-casein to BSA) of 1:2000 to study selectivity. The phosphopeptides were quantified by mass spectrometry. The limit of detection can be as low as 8 × 10

The authors describe a fluorometric method for the determination of the activity and inhibition of protein kinase A (PKA). In the presence of ATP, PKA catalyzes the transfer of phosphate groups from ATP to a peptide, and the generated phosphorylated peptide quenches the fluorescence (measured at excitation/emission peaks of 340/440 nm) of the hydroxyapatite nanoparticles (HAP-NPs). A linear logarithmic relationship of PKA concentrations with fluorescence intensity in the range from 1 to 50 U·L−1 was obtained, and the lower limit of detection (LOD) is 0.5 U·L−1. This is much lower than LODs reported in the literature. The PKA inhibitor H-89 was studied, and the inhibition plot has a sigmoidal shape with a half-maximal inhibitory concentration of around 750 nM of H-89. At a 4.5 nM level of H-89, fluorescence of HAP-NPs fell to levels of no PKA controls, demonstrating that the assay is a viable tool to screen for kinase inhibitors. An assay with Hela cell lysates in combination with forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor used to activate the cellular activity of PKA) resulted in decreased fluorescence of HAP-NPs. This suggests that the assay can be applied for testing in vitro cell kinase activity. In our perception, this method will enable high-throughput screening for kinase-related drugs and fluorometric enzymatic detection in various areas.

// Ya Zhou 1, 2, * , Yuanyuan Zhu 1, 2, * , Xiaoyan Fan 1, 2, * , Chundong Zhang 1, 2 , Yitao Wang 1, 2 , Lian Zhang 1, 2 , Huan Zhang 3 , Tao Wen 4 , Kaina Zhang 3 , Xiao Huo 5 , Xue Jiang 1, 2 , Youquan Bu 1, 2 and Ying Zhang 1, 2 1 Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016, China 2 Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China 3 College of Pharmacy, Chongqing Medical University, Chongqing 400016, China 4 First Clinical College, Chongqing Medical University, Chongqing 400016, China 5 College of Biomedical Engineering, Chongqing Medical University, Chongqing 400016, China * These authors contributed equally to this work Correspondence to: Ying Zhang, email: zhangying078@aliyun.com Keywords: ovarian cancer, NID1, EMT, metastasis, chemoresistance Abbreviations: ECM: extracellular matrix; EMT: epithelial-mesenchymal transition; FAK: focal adhesion kinase; MET: mesenchymal-epithelial transition; NID1: nidogen-1 Received: January 09, 2017 Accepted: February 08, 2017 Published: March 13, 2017 ABSTRACT Nidogen-1 (NID1) has been identified as a novel candidate diagnostic biomarker of ovarian cancer in our previous study. Nevertheless, the role of NID1 in the pathogenesis of ovarian cancer is unclear. In the present study, we demonstrated that NID1 was a mesenchymal associated gene and its high expression was significantly correlated with shorter overall survival of ovarian cancer patients. The ectopic expression of NID1 in OVCAR-3 cells revealed a epithelial-mesenchymal transition (EMT) phenotype accompanied by enhancement of motility, invasiveness and cisplatin resistance, whereas the knockdown of NID1 was sufficient to convert HEY cells into epithelial phenotype with decreased capability of motility, invasiveness and cisplatin resistance. Mechanistic studies disclosed that NID1 activated ERK/MAPK signaling pathway to promote EMT. Collectively, our findings have uncovered the molecular mechanisms of NID1 in promoting ovarian cancer metastasis and chemoresistance, and provide a rationale for the therapeutic potential of NID1 suppression in ovarian cancer.