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At present, the occurrence and development of inflammatory diseases are closely related to the abnormal changes of the content and function of many cytokines. At the same time, targeting related cytokines to prevent and treat diseases has also achieved good results. Therefore, it is very important to explore the role of various cytokines in inflammatory diseases. As an inflammation related protein, Tumor necrosis factor alpha stimulating gene-6 (TSG-6) has attracted more and more attention. In the process of disease, it's like a double-edged sword, showing different responses. It is constitutively expressed in some tissues with high metabolic activity and barrier protection. The diversity of its functions depends on the binding of TSG-6 with a variety of ligands, including matrix molecules, autoimmune regulatory factors and growth factors, participating in extracellular matrix remodeling and regulating protease network. This paper reviews the structure, biological function and research progress of TSG-6 in inflammatory diseases, in order to provide reference for drug development in the future.

Monoclonal antibodies (mAbs) are one of the most widely used types of protein therapeutics. Charge variants are important quality attributes for evaluating developability, activity, and safety for mAb therapeutics. Here, we report a novel online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for mAb charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. Key factors that enable online CIEF-MS include effective capillary electrophoresis-MS (CE-MS) interface with enhanced sensitivity, utilization of MS-friendly electrolytes, beneficial effects of glycerol that reduces non-CIEF electrophoretic mobility and limits band broadening, appropriate ampholyte type and concentration selection for balanced separation resolution and MS detection sensitivity, optimized sheath liquid composition to realize high-resolution CIEF separation and effective MS electrospray ionization, as well as judiciously selected CIEF running parameters. The fundamental premise of CIEF has been verified by the linear correlation between isoelectric point (pI) values and migration time using a mixture of pI markers. By achieving high separation resolutions that are similar as those obtained from imaged CIEF (iCIEF), this method successfully provides highly sensitive MS identification for intact mAb charge variants. Furthermore, a middle-up sample treatment workflow can be adopted to provide in-depth charge variant analysis at subunit level for mAbs with complex charge heterogeneity. The mAb subunit CIEF-MS reveals the source of charge variant with enhanced resolution on both CIEF separation and MS spectra. This novel CIEF-MS method is a valuable tool with distinct advantage for objective and accurate assessment of charge heterogeneity of protein therapeutics.

Background Thrombosis is the pathological basis of cardiovascular and cerebrovascular diseases, which seriously threaten human life and health. Among them, nearly half of cardiovascular disease patients suffer from severe hypertension complications. Hypertension is thought to cause abnormal platelet activation and increases the risk of thrombosis, but the related mechanism is still vague. Objectives This study hypothesized that the abnormal hemodynamics of blood under hypertension might affect platelet function and accelerate thrombosis by activating mechanoreceptor Piezo1. Methods To assess the activation effect of hypertension on mechanoreceptor Piezo1, we injected Piezo1 agonist Yoda1 and antagonist GsMTx-4 through the tail vein, then examined the platelet activation status and thrombosis. Results Our results displayed that antagonist GsMTx-4 effectively inhibited calcium influx caused by hypertension and agonist Yoda1. Anti-thrombotic studies proved that the inhibition of Piezo1 effectively inhibited arterial thrombosis and reduced the infarct size of stroke in hypertensive mice. Conclusions Our study explains the activation of mechanoreceptor Piezo1 under hypertension is the key to abnormal platelet activation and thrombosis while providing novel platelet intervention strategies to prevent thrombosis.

Background: Our previous studies have demonstrated that diosgenin and diosgenin derivatives exhibit excellent antithrombotic activity via regulating platelet function and coagulation factor level. Platelets and blood coagulation system are highly associated with tumor hematogenous metastasis. Therefore, the purpose of this study was to evaluate whether dihydrodiosgenin (dydio) mediated-platelet inhibition or coagulation factor level modulation is involved in hepatocellular carcinoma cell (HCC) metastasis. Methods: Cell viability was examined by MTT and colony formation assays. Platelet aggregation text and morphology were used to assess dydio's role on tumor cell-induced platelet activation (TCIPA). Scratch assay, adhesion assay and Western blot were used to evaluate dydio's role on platelet-mediated metastasis. Western blot and fluorescence detection were performed to clarify dydio's role on endothelial cell (EC) function. The mice lung metastasis model was constructed to investigated dydio's function on coagulation factor and platelet-mediated metastasis. Results: This study found that pretreatment with dydio caused a significant inhibition of TCIPA. Platelets exposed to dydio significantly inhibited their adhesion to tumor cells, meanwhile, releasates of platelets that pretreated with dydio led to diminished cancer cell proliferation and migration along with the increase of epithelial markers E-cadherin and loss of mesenchymal phenotype. Additionally, ECs pretreated with dydio suppressed factor VIII (FVIII) level which in turn restrained the activation of platelets and the adhesion of cancer cells or platelets to ECs. Interestingly, our study demonstrated that FVIII could promote HCC proliferation. In vivo study revealed that mice intragastrical (i.g.) administration with dydio significantly inhibited the lung metastasis of hepal-6 cells which is highly correlated with the altered platelet function and coagulation level. Conclusion: Taken together, these results demonstrated that dydio altered platelet function and coagulation FVIII level, resulting in decreased metastatic potential of HCC. Thus, our study reveals that dydio exerts novel mechanisms of antitumor action beside its direct antitumor activity.

Background There are limited data on vitamin D status of Sichuan province, and no investigation has been carried out on the correlations of 25(OH)D and BTMs between healthy Hans and Tibetans of Sichuan province. This study aimed to examine 25(OH)D levels around Sichuan province and to assess differences by ethnicity, age, gender, sunlight exposure, geographic location, and seasons. Methods Blood samples from 2317 healthy adults aged of 18 to 75 years and of Han and Tibetan ethnicities were collected in six regions and during four seasons. Serum 25(OH)D2 and 25(OH)D3 levels were measured by LC‐MS/MS method. Serum total P1NP and β‐CTX were measured by immunoassay. Results Participants aged 18‐40 years showed significantly lower 25(OH)D levels than participants aged 41‐75 years old (P, This multicenter cross‐sectional study showed that serum 25(OH)D levels of adults in Sichuan province among four seasons and different districts varied significantly (P

MucA and MucB are critical negative modulators of sigma factor AlgU and regulate the mucoid conversion of Pseudomonas aeruginosa. Previous studies have revealed that lipid signals antagonize MucA-MucB binding. Here we report the crystal structure of MucB in complex with the periplasmic domain of MucA and polyethylene glycol (PEG), which unveiled an intermediate state preceding the MucA-MucB dissociation. Based on the biochemical experiments, the aliphatic side chain with a polar group was found to be of primary importance for inducing MucA cleavage. These results provide evidence that the hydrophobic cavity of MucB is a primary site for sensing lipid molecules and illustrates the detailed control of conformational switching within MucA-MucB in response to lipophilic effectors., Tao Li et al. report crystal structure of MucA-MucB complex and identify a cavity region of MucB that binds PEG molecule. They further show that this hydrophobic cavity of MucB is a primary site for sensing lipid molecules and that lipid A stimulates MucB to release MucA for AlgW cleavage, helping to understand the regulation mechanism of alginate biosynthesis.

Thrombosis is the leading cause of death in patients with cardiovascular disease in the world. Current antithrombotic agent aspirin has serious side effects such as higher bleeding risk and serious gastrointestinal ulcers. Diosgenin reported in clinical research could prevent thrombosis without side effects. However, poor bioavailability and low knowledge on its molecular targets limit its clinical application. A novel prodrug with antithrombotic effect was prepared based on conjugating diosgenin derivatives to PEG with Schiff-base bond. The prodrug with long blood circulation time and satisfying safety could self-assemble into micelles in water. The prodrug micelles with pH-responsibility could targetedly release diosgenin in position of thrombus in vivo. The results indicate that the prodrug micelles without bleeding risk and histological damages prevent thrombosis by inhibiting platelet activation and apoptosis. Our studies demonstrate that the prodrug micelles could obviously enhance the efficacy in the prevention of arterial thrombus and venous thrombus than aspirin.

Aim As the global population has reached 7 billion and the baby boom generation reaches old age, thrombosis has become the major contributor to the global disease burden. It has been reported that, in moderate doses, beer may protect against thrombosis. Xanthohumol (XN), an antioxidant, is found at high concentrations in hop cones ( Humulus lupulus L.) and is a common ingredient of beer. Here, the aim of the present work was to investigate the effects of XN on antithrombotic and antiplatelet activities, and study its mechanism. Approach and Results Using ferric chloride-induced carotid artery injury, inferior vena cava ligation model, and platelet function tests, we demonstrated that XN uniquely prevents both venous and arterial thrombosis by inhibiting platelet activation. Interestingly, in tail bleeding time studies, XN did not increase bleeding risk, which is recognized as a major limitation of current antithrombotic therapies. We also demonstrated that XN induces Sirt1 expression and thereby decreases reactive oxygen species (ROS) overload, prevents mitochondrial dysfunction, and reduces activated platelet-induced mitochondrial hyperpolarization, respiratory disorders, and associated membrane damage at low concentrations. In mitochondrial function assays designed to detect amounts of extracellular mitochondrial DNA (mtDNA), we found that XN prevents mtDNA release, which induces platelet activation in a DC-SIGN-dependent manner. Conclusions XN exemplifies a promising new class of antiplatelet agents that are highly effective at inhibiting platelet activation by decreasing ROS accumulation and platelet mtDNA release without incurring a bleeding risk. This study has also provided novel insights into mechanisms of thrombotic diseases with possible therapeutic implications.

Current therapy for blood vessel thrombosis has the risk of leading to gastrointestinal bleeding and thrombocytopenia. We previously reported that a new derivative of diosgenin, compound 5, had significant anti-inflammatory activity superior to that of aspirin, prolonged bleeding time, and inhibited platelet aggregation in vitro. In the present study, we investigated the in vivo efficacy and safety of compound 5 using the ferric chloride (FeCl3)-induced arterial and venous thrombosis models in rats as well as its toxicity in mice. Compared with the control rats, those treated with compound 5 showed significantly less adenosine diphosphate (ADP)-induced platelet aggregation and a prolonged activated partial thromboplastin time mediated by the specific regulation of factor VIII. Furthermore, compound 5 significantly reduced the average length and weight of thrombi in both arteries and veins. These findings were similar to those of aspirin at the same dose. The safety evaluation revealed a much lower risk of bleeding and lesser gastric mucosal damage with compound 5 than with the same dose of aspirin. An oral dose of up to 575.5mg/kg showed no toxicity in mice. In conclusion, consistent with our in vitro findings, compound 5 exhibited an in vivo antithrombotic activity that was comparable to aspirin mainly by reducing platelet aggregation and regulating factor VIII, but with fewer side effects.

Background Monoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family. It functions as a regulator of cell apoptosis and a potential tumor suppressor, playing a vital role in the processes of immune cell function and apoptosis. Methods The present study constructed GFP-pMNSFβ swine umbilical vein endothelial cell (SUVEC) lines and investigated the function of porcine MNSFβ (pMNSFβ) in apoptosis, as well as its interactions with pBCL-G. Results revealed that stably expressed pMNSFβ protein in SUVEC lines significantly enhanced staurosporine (STS)-induced apoptosis. pMNSFβ proteins interacted with pBCL-G proteins and the expression of these interacting proteins synergized to further enhance STS-induced apoptosis. Results GFP-pMNSFβ stably expressed SUVEC lines through transient transfection and neomycin screening methods. Over 90% of the SUVEC cultures expressed GFP signals, and 41.5 kDa GFP-pMNSFβ proteins were detected with western blotting methods. Annexin V-PE/PI staining and flow cytometry analyses showed that overexpression of pMNSFβ proteins significantly elevated STS-induced apoptosis rates. Co-immunoprecipitation methods revealed an interaction between pMNSFβ and pBCL-G proteins. BCL-G is a proapoptotic member of the BCL-2 family that has been shown to be misexpressed in human systemic lupus erythematosus, as well as mammary and prostate cancers. Here, we demonstrated that the co-expression and potential conjugation of pMNSFβ and pBCL-G proteins synergistically enhanced STS-induced apoptosis. Conclusions The present study was the first to characterize the function of MNSFβ in porcine cells, and to clarify the function of MNSFβ in apoptosis. These results reveal that pMNSFβ is a potential molecular model for future investigations of diseases related to human MNSFβ dysfunction.

Peripheral blood, menstrual blood, semen, saliva and vaginal secretions are the five most common body fluids found at crime scenes, and the identification of these five body fluids is of great significance to the reconstruction of a crime scene and resolution of the case. However, accurate identification of these five body fluids is still a challenge. To address this problem, a mathematical model for differentiating five types of forensic body fluids based on the differential expression characteristics of multiple miRNAs in five body fluids (peripheral blood, menstrual blood, semen, saliva and vaginal secretions) was developed. A total of 350 forensic body fluids (70 of each type) were collected and tested, and relative expression of 10 miRNAs (miR-451a, miR-205-5p, miR-203-3p, miR-214-3p, miR-144-3p, miR-144-5p, miR-654-5p, miR-888-5p, miR-891a-5p, miR-124a-3p) in all samples was detected by SYBR Green real-time qPCR. Three hundred samples (60 samples of each body fluid) were used as the training set to screen meaningful identification markers by stepwise discriminant analysis, and a discriminant function was established. Fifty samples (10 samples of each body fluid) were used as a validation set to examine the accuracy of the model, and 25 samples (the types of samples were unknown to the experimenter) were used for a blind test. Except for miR-144-3p, the other miRNAs were selected to construct discriminant analysis models. The self-validation accuracy of the model was 99.7 %, cross-validation accuracy was 99.3 %, accuracy of the identification validation set was 100 %, and accuracy of the blind test result was 100 %. This study provides a reliable and accurate identification strategy for five common body fluids (peripheral blood, menstrual blood, semen, saliva, and vaginal secretions) in forensic medicine.

Platelet thrombosis is the main pathogeny resulting in the low curability of ischemic stroke, a leading cause of mortality and disability worldwide. Metformin, a biguanide derivative that is the first-line oral medicine for type 2 diabetes, alleviates the severity of ischemic stroke in diabetic patients and suppresses platelet activation in experimental animal model. However, the clinical implementation of commercial biguanide analogs for stroke related to platelet thrombosis remains challenging due to its weak potency, poor pharmacokinetic characteristics and possible hypoglycemia. Here, twenty-three biguanide derivatives were designed and synthesized based on the principles of bioisosteres. These derivatives were evaluated for the activity of antiplatelet thrombosis in vivo. We found that N-trifluoromethanesulfonyl biguanide derivative, compound b10, uniquely prevented cerebral infarction as well as neuronal function injury, and significantly decrease the mortality rate of ischemic stroke in the middle cerebral artery occlusion mice without significant side effects. We verified that b10 directly inhibited platelets thrombus formation and decreased the compactness of stroke thrombi. Particularly, b10 exhibited good potency to inhibit human platelet activation including platelet aggregation, adhesion, pseudopodia formation, integrin GPIIb/IIIa activation, CD62P expression and clot retraction. Meanwhile, the pharmacokinetics assessment showed that b10 had satisfying pharmacological characteristics including a longer duration and a higher oral absorption ratio than its parent compound. In addition, b10 remarkably ameliorated not only stroke related to platelet thrombosis but also carotid artery thrombus formation. It is concluded that the novel potent antiplatelet thrombotic agent derived from biguanide is a promising candidate for stroke treatment.

BACKGROUND: Novel haloemodin (HEI2) synthesized by modifying emodin, a traditional Chinese medicine component, possesses remarkable antibacterial activity, being much more effective than its parent nucleus, emodin. METHODS: Firstly, we discovered that HEI2 increases bacterial cell membrane permeability to potassium ions more drastically than emodin. We thus further investigated the interaction of haloemodin and protein using a fluorescence quenching and circular dichroism (CD) study based on bovine serum albumin (BSA). RESULTS: HEI2 spontaneously bound to BSA at Trp 212 residue (subdomain IIA) by hydrogen bonds and van der Waals interactions to forms HEI2-BSA complexes, and this binding decreased the α-helical content of BSA. We also confirmed that emodin bound to BSA by hydrophobic interaction alone. CONCLUSIONS: These results suggest that the main responses for the substantial antibacterial activities of HEI2 are a disruption of the bacterial plasma membrane function and the interaction with biological functional proteins. Furthermore, the study of the interaction of drugs with BSA, which has a fluorescent group tryptophan residue similar to many bio-functional proteins, will be a simple and inexpensive scope-reducing method in screening new drugs.

Cabbage extract containing chlorogenic acid has notable scavenging activity against hydroxyl radicals, superoxide anion radicals, DPPH radicals as well as potent reducing power. Oral administration of the extract (240 mg/kg BW/day) to hypercholesterolaemic rats reduced by high–fat diet for 30 days lowered their blood TC, TG, and LDL–C levels by 36.3, 43.5 and 45.8%, respectively. However, the blood HDL–C levels in the same treated rats increased by 17.1%. Treatment of hypercholesterolaemic rats with the extract significantly increased the GSH level along with enhanced SOD, CAT activities in liver tissues. Furthermore, the extract significantly decreased hepatic MDA as well as GPx and GR activities in extract–treated rats. It can therefore be concluded that the extract has a high hypolipidaemic activity and this may be attributed to its antioxidative potential.

Thrombosis and its complications are the leading cause of death in patients with diabetes. Metformin, a first-line therapy for type 2 diabetes, is the only drug demonstrated to reduce cardiovascular complications in diabetic patients. However, whether metformin can effectively prevent thrombosis and its potential mechanism of action is unknown. Here we show, metformin prevents both venous and arterial thrombosis with no significant prolonged bleeding time by inhibiting platelet activation and extracellular mitochondrial DNA (mtDNA) release. Specifically, metformin inhibits mitochondrial complex I and thereby protects mitochondrial function, reduces activated platelet-induced mitochondrial hyperpolarization, reactive oxygen species overload and associated membrane damage. In mitochondrial function assays designed to detect amounts of extracellular mtDNA, we found that metformin prevents mtDNA release. This study also demonstrated that mtDNA induces platelet activation through a DC-SIGN dependent pathway. Metformin exemplifies a promising new class of antiplatelet agents that are highly effective at inhibiting platelet activation by decreasing the release of free mtDNA, which induces platelet activation in a DC-SIGN-dependent manner. This study has established a novel therapeutic strategy and molecular target for thrombotic diseases, especially for thrombotic complications of diabetes mellitus.

RATIONALE N-Linked glycosylation plays important roles in modulating protein structure and function. The direct impact of the modification on protein conformation is not yet well understood. METHODS Here we probed the dynamic changes following Endo H trimming of high mannose glycans in α-amylase by means of amide hydrogen/deuterium exchange mass spectrometry. RESULTS The results revealed that deglycosylation elicited extensive alterations in backbone dynamics, affecting regions both adjacent to and distal from the glycosylation site. CONCLUSIONS The overall exchange rate is reduced in the glycosylated state, which indicates rigidity enhancement due to the attached carbohydrates. Copyright © 2013 John Wiley & Sons, Ltd.

Background The establishment of a proper reference range is necessary for both the prognosis and diagnosis of clinical disease. Our study aimed to establish the hemoglobin reference interval for the population of Southwest of China and to compare this with published data for other races. Methods A total of 21085 healthy individuals (11561 men and 9524 women, age range 18 - 100 years) living in Southwest China were selected. CBC was performed on an XE-2100 hematology analyzer. The medians were calculated and reference values were determined at the 2.5th and 95th percentiles. Results There was a statistically significant gender difference for RBC count, Hb, and MCV (p = 0.000). The comparison of Hb by gender showed higher values among males, with lower values in females and this difference was similar to the values for RBC count and MCV. For males, mean values for Hb and RBC decreased with age. However, mean values for Hb in females increased after middle age. Conclusions Reference intervals for major red blood cell parameters established in this study showed significant gender differences. For males, mean values for Hb and RBC decreased with age. This is the first region-wide population study on hematologic reference values among people in Southwest China.

To assess the feasibility of using sandwich-cultured human hepatocytes (SCHHs) as a model to characterize transport kinetics for in vivo pharmacokinetic prediction, the expression of organic anion-transporting polypeptide (OATP) proteins in SCHHs, along with biliary efflux transporters, was confirmed quantitatively by liquid chromatography-tandem mass spectrometry. Rifamycin SV (Rif SV), which was shown to completely block the function of OATP transporters, was selected as an inhibitor to assess the initial rates of active uptake. The optimized SCHH model was applied in a retrospective investigation of compounds with known clinically significant OATP-mediated uptake and was applied further to explore drug-drug interactions (DDIs). Greater than 50% inhibition of active uptake by Rif SV was found to be associated with clinically significant OATP-mediated DDIs. We propose that the in vitro active uptake value therefore could serve as a cutoff for class 3 and 4 compounds of the Biopharmaceutics Drug Disposition Classification System, which could be integrated into the International Transporter Consortium decision tree recommendations to trigger clinical evaluations for potential DDI risks. Furthermore, the kinetics of in vitro hepatobiliary transport obtained from SCHHs, along with protein expression scaling factors, offer an opportunity to predict complex in vivo processes using mathematical models, such as physiologically based pharmacokinetics models.

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2 μg mL(-1) (0.040 μg mg(-1)) to 20 μg mL(-1) (4.0 μg mg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites produced by cytochrome P450 epoxygenases which are highly expressed in hepatocytes. The functions of EETs in hepatocytes are not well understood. In this study, we investigated the effects of 14,15-EETs treatment on the insulin signal transduction pathway in hepatocytes. We report that chronic treatment, not acute treatment, with 30 μM 14,15-EETs prevents palmitate induced insulin resistance and potentiates insulin action in cultured HepG2 hepatocytes. 14,15-EETs increase Akt phosphorylation at S473, activating Akt, in an insulin dependent manner in HepG2 cells. Under insulin resistant conditions induced by palmitate, 14,15-EETs restore the insulin response by increasing S473-phosphorylated Akt. 8,9-EETs and 11,12-EETs demonstrated similar effects to 14,15-EETs. Furthermore, 14,15-EETs potentiate insulin-suppression of gluconeogenesis in cultured H4IIE hepatocytes. To elucidate the mechanism of EETs function, we analyzed the insulin signaling factors upstream of Akt. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 attenuated the 14,15-EETs-induced activating phosphorylation of Akt. 14,15-EETs reduced palmitate-stimulated phosphorylation of IRS-1 on S312 and phosphorylation of c-Jun N-terminal kinase (JNK) at threonine 183 and tyrosine 185 residues. The regulation of insulin sensitivity in cultured hepatocytes by chronic 14,15-EETs treatment appears to involve the JNK-IRS-PI3K pathway. The requirement of chronic treatment with EETs suggests that the effects of EETs on insulin response may be indirect.

The primary challenge associated with the development of an LC/MS/MS-based assay for simultaneous determination of biogenic monoamine neurotransmitters such as norepinephrine (NE), dopamine (DA), serotonin (5-HT), and normetanephrine (NM) in rat brain microdialysates is to improve detection sensitivity. In this work, a UPLC/ MS/MS-based method combined with a diethyl labeling technique was developed for simultaneous determination of a panel of monoamines in rat prefrontal cortex microdialysates. The chromatographic run time is 3.5 min/ sample. The limits of detection of the UPLC/MS/MS-based method for NE, DA, 5-HT/ and NM, with/without diethyl labeling of monoamines, are 0.005/0.4 (30/2367 pM), 0.005/0.1 (33/653 pM), 0.005/0.2 (28/1136 pM), and 0.002/0.2 ng/mL (11/1092 pM), respectively. Diethyl labeling of amino groups of monoamines affords 20-100 times increased detection sensitivity of corresponding native monoamines during the UPLC/MS/MS analysis. This could result from the following: (1) improved fragmentation patterns; (2) increased hydrophobicity and concomitantly increased ionization efficiency in ESI MS and MS/MS analysis; (3) reduced matrix interference. This labeling reaction employs a commercially available reagent, acetaldehyde-d4, to label the amine groups on the monoamines via reductive amination. It is also simple, fast (approximately 25-min reaction time), specific, and quantitative under mild reaction conditions. Data are also presented from the application of this assay to monitor the drug-induced changes of monoamine concentrations in rat prefrontal cortex microdialysate samples followed by administration of SKF 81297, a selective D1 dopamine receptor agonist known to elevate the extracellular level of the neurotransmitters DA and NE in the central nervous system.

One of the challenges associated with metabolome profiling in complex biological samples is to generate quantitative information on the metabolites of interest. In this work, a targeted metabolome analysis strategy is presented for the quantification of amine-containing metabolites. A dimethylation reaction is used to introduce a stable isotopic tag onto amine-containing metabolites followed by LC-ESI MS analysis. This labeling reaction employs a common reagent, formaldehyde, to label globally the amine groups through reductive amination. The performance of this strategy was investigated in the analysis of 20 amino acids and 15 amines by LC-ESI MS. It is shown that the labeling chemistry is simple, fast (10-min reaction time), specific, and provides high yields under mild reaction conditions. The issue of isotopic effects of the labeled amines on reversed-phase (RP) and hydrophilic interaction (HILIC) LC separations was examined. It was found that deuterium labeling causes an isotope effect on the elution of labeled amines on RPLC but has no effect on HILIC LC. However, 13C-dimethylation does not show any isotope effect on either RPLC or HILIC LC, indicating that 13C-labeling is a preferred approach for relative quantification of amine-containing metabolites in different samples. The isotopically labeled 35 amine-containing analogues were found to be stable and proved to be effective in overcoming matrix effects in both relative and absolute quantification of these analytes present in a complicated sample, human urine. Finally, the characteristic mass difference provides additional structural information that reveals the existence of primary or secondary amine functional groups in amine-containing metabolites. As an example, for a human urine sample, a total of 438 pairs of different amine-containing metabolites were detected, at signal-to-noise ratios of greater than 10, by using the labeling strategy in conjunction with RP LC-ESI Fourier-transform ion cyclotron resonance MS.

A method for the characterization of modifications of low molecular weight proteins (

An enhancement of the room temperature magnetoresistance is induced by the substitution of Fe or Ni for Mn with appropriate content x in La0.65Sr0.35Mn1−xTxO3 (T=Fe and Ni) manganites, since the temperature of metal–insulator transition of La0.65Sr0.35Mn1−xTxO3 can be shifted from a high temperature to near room temperature through the substitution.

Drug-resistant bacterial infections and lack of available antibacterial agents in clinical practice are becoming serious risks to public health. We synthesized a new class of haloemodins by modifying a traditional Chinese medicine component, emodin. The novel haloemodin exerts strong inhibitory activity on bacterial topoisomerase I and DNA gyrase, and not on the topoisomerases of human origin. In principle, it shows remarkable antibacterial activities against laboratory and clinically isolated Gram-positive bacteria, including vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus. We further expanded its antibacterial spectrum into against Gram-negative bacteria with the assistance of polymyxin B nonapeptide, which helps haloemodin to penetrate through the bacterial outer membrane. Finally, the therapeutic effect of haloemodin in vivo was confirmed in curing S. aureus-induced keratitis on rabbit model. With distinctive structural difference from the antibiotics we used, the haloemodins are of value as promising antibacterial pharmacophore, especially for combat the infections caused by drug-resistant pathogens.

N-Linked glycosylation plays important roles in modulating protein structure and function. The direct impact of the modification on protein conformation is not yet well understood.Here we probed the dynamic changes following Endo H trimming of high mannose glycans in α-amylase by means of amide hydrogen/deuterium exchange mass spectrometry.The results revealed that deglycosylation elicited extensive alterations in backbone dynamics, affecting regions both adjacent to and distal from the glycosylation site.The overall exchange rate is reduced in the glycosylated state, which indicates rigidity enhancement due to the attached carbohydrates.

Contemporary drug discovery leverages quantitative modeling and simulation with increasing emphasis, both to gain deeper knowledge of drug targets and mechanisms as well as improve predictions between preclinical models and clinical applications, such as first-in-human dose projections. Proliferation of novel biotherapeutic modalities increases the need for applied PK/PD modeling as a quantitative tool to advance new therapies. Of particular relevance is the understanding of exposure, target binding and associated pharmacology at the target site of interest. Bioanalytical methods are key to informing PK/PD models and require assessment of both PK and PD end points. Where targets are sequestered in tissues (noncirculating), the ability to quantitatively measure drug or biomarker in tissue compartments becomes particularly important. This perspective provides an overview of contemporary applications of quantitative bioanalysis in tissue compartments as applied to PK and PD assessments associated with novel biotherapeutics. Case studies and key references are provided.

Measurement of endogenous epinephrine (E) and norepinephrine (NE) in human plasma is very challenging due to lower endogenous concentrations as compared with animal plasma. An LC-MS/MS in combination with alumina-based SPE and derivatization procedure was validated for the measurement of E and NE in human plasma with acceptable intra-day and inter-day accuracy and precision. Sample was extracted with semi-automated alumina 96-well solid phase extraction (SPE) cartridge. The resulting eluent was dried and derivatized using d4-acetaldehyde. The analytes were separated on a monolithic C(18) column. Extraction efficiencies were >66% for E and NE. The lower limit of quantitation (LLOQ) was 5.00 pg/mL for E and 20.0 pg/mL for NE.

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) is a widely utilized analytical tool for quantifying small molecules in complex biological matrices. In certain situations the mass-selection capabilities of the tandem mass spectrometer may be insufficient to discriminate between the analyte of interest and its metabolites, particularly those metabolites that are isobaric with the analyte. One scenario by which isobaric interference may occur is the metabolism of a chlorine- or bromine-containing small molecule to a metabolite with the concomitant loss of 2 Da. This report describes the detection and characterization of two distinct dehydrogenation [M-2] metabolites during LC/MS/MS quantification of a chlorinated small molecule in rat plasma samples derived from a toxicokinetic study. The potential isotope-related impact of these metabolites on quantification of the parent compound was assessed. Several alternate precursor ion and product ion combinations were evaluated and shown to minimize the quantitative impact of the interfering metabolites without having to rely on their stringent chromatographic resolution from the parent compound. These results indicate that when quantifying chlorine- or bromine-containing small molecules from in vivo samples or in vitro metabolic incubations: (1) efforts to detect potential dehydrogenation metabolites should be undertaken and (2) if such metabolites are detected, the judicious choice of alternate multiple-reaction monitoring (MRM) transitions can limit their impact on quantification of the parent molecule without the need for robust chromatographic resolution.

A novel, automated, simple, sensitive, specific, accurate, precise and high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method has been developed for simultaneous determination of epinephrine (E) and norepinephrine (NE) in plasma by using the combination of a fully automated protein precipitation technique for plasma sample preparation, reductive ethylation labeling with UPLC-MS/MS. A simple protein precipitation procedure was used to clean up 50 microL calibration samples prepared in stripped human plasma and 50 microL quality control plasma samples containing 25 microL plasma and 25 microL stabilizing additives. The supernatants were subsequently dried down and then reconstituted with commercially available and cost-effective reductive ethylation labeling reagents, followed by UPLC-MS/MS analysis. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and quality control samples, shaking of 96-well plates, adding and transferring liquids. Processing time, which included the preparation of standards and quality control samples, protein precipitation and reductive ethylation labeling, is less than 2 h per 96-well plate. The chromatographic run time is 3.5 min per sample. The limits of detection of UPLC-MS/MS-based methods for E and NE, with/without reductive ethylation labeling, are 0.025/0.40 and 0.025/2.00 ng mL(-1), respectively. Reductive ethylation labeling of amino groups of E and NE affords 16 and 80 times increased detection sensitivity of corresponding native counterparts during the UPLC-MS/MS analysis. The linearity of this method was established from 0.05 to 25 ng mL(-1) for E and NE with accuracy and precision within 15% at all concentrations. The intra-run and inter-run assay accuracy (%RE) and coefficient of variations (CV%) are all within 15% for all QC samples prepared in commercially purchased plasma samples.

Although the strategic use of enzymatic digestion combined with isotope dilution mass spectrometry has been increasingly developed and used for the absolute quantification of therapeutic and endogenous proteins in the biopharmaceutical industry over the past several years, the lack of an appropriate internal standard has become the rate-limiting step in the development of a standardized analytical approach to provide bioanalytical support for both preclinical and clinical studies. In this study, we present a universal strategy for fast development and validation (within 1-2 weeks) of a method for absolute quantification of a therapeutic monoclonal antibody in biological matrices using differential dimethyl labeling coupled with UPLC-MS/MS. Differential dimethyl labeling of tryptic peptides generated from the purified therapeutic monoclonal antibody and those derived from proteins in cynomolgus monkey serum with either d(2)- or d(0)-formaldehyde provided a fast, cost-effective, and standardized approach to generate internal standards for any surrogate peptides that are used to quantify the therapeutic monoclonal antibody in biological matrices. This labeling reaction employs inexpensive and commercially available reagents, d(0)- or d(2)-formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys in a peptide via reductive amination. Moreover, the process is simple, relatively fast (2 h reaction time), specific, and quantitative under mild reaction conditions. The chromatographic run time is 6 min per sample. The linearity of the assay for the selected monoclonal antibody was established from 1.00 to 1000 mug/mL with accuracy and precision within 15% at all concentrations. The intraday and interday assay accuracy (%RE) and coefficient of variations (CV%) are all within 15% for all QCs (2.00, 4.00, 20.0, 100, 750 mug/mL) prepared in three different serum pools from male and female cynomolgus monkeys.

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25pM with the linearity of the standard curve spanned to 2500pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.

The combination of dimethylation after guanidination (2MEGA) isotope labeling with microbore liquid chromatography (LC)-matrix-assisted laser desorption ionization (MALDI) MS and MS/MS [C. Ji, N. Guo, L. Li, J. Proteome Res. 4 (2005) 2099] has been reported as a promising strategy for abundance ratio-dependent quantitative proteome analysis. A critical step in using this integrated strategy is to set up the abundance ratio threshold of peptide pairs, above which the peptide pairs are used for quantifying and identifying the protein that is considered to be differentially expressed between two different samples. The threshold is determined by technical variation (i.e., the overall abundance ratio variation caused by the experimental process including sample workup, MS analysis and data processing) as well as biological variation (i.e., the abundance ratio variation caused by the biological process including cell growth), which can be defined and assessed by a coefficient of variation (CV). We have designed experiments and measured three different levels of variations, starting with the same membrane protein preparation, the same batch of cells and three batches of cells from the same cell line grown under the same conditions, respectively. It is shown that technical variation from the experimental processes involved in 2MEGA labeling LC-MALDI MS has a CV of

One of the challenges associated with large-scale proteome analysis using tandem mass spectrometry (MS/MS) and automated database searching is to reduce the number of false positive identifications without sacrificing the number of true positives found. In this work, a systematic investigation of the effect of 2MEGA labeling (N-terminal dimethylation after lysine guanidination) on the proteome analysis of a membrane fraction of an Escherichia coli cell extract by 2-dimensional liquid chromatography MS/MS is presented. By a large-scale comparison of MS/MS spectra of native peptides with those from the 2MEGA-labeled peptides, the labeled peptides were found to undergo facile fragmentation with enhanced a1 or a1-related (a(1)-17 and a(1)-45) ions derived from all N-terminal amino acids in the MS/MS spectra; these ions are usually difficult to detect in the MS/MS spectra of nonderivatized peptides. The 2MEGA labeling alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI and ESI MS experiments. 2MEGA labeling was found not only to increase the number of peptides and proteins identified but also to generate enhanced a1 or a1-related ions as a constraint to reduce the number of false positive identifications. In total, 640 proteins were identified from the E. coli membrane fraction, with each protein identified based on peptide mass and sequence match of one or more peptides using MASCOT database search algorithm from the MS/MS spectra generated by a quadrupole time-of-flight mass spectrometer. Among them, the subcellular locations of 336 proteins are presently known, including 258 membrane and membrane-associated proteins (76.8%). Among the classified proteins, there was a dramatic increase in the total number of integral membrane proteins identified in the 2MEGA-labeled sample (153 proteins) versus the unlabeled sample (77 proteins).

We describe an enabling technique for proteome analysis based on isotope-differential dimethyl labeling of N-termini of tryptic peptides followed by microbore liquid chromatography (LC) matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS). In this method, lysine side chains are blocked by guanidination to prevent the incorporation of multiple labels, followed by N-terminal labeling via reductive amination using d(0),(12)C-formaldehyde or d(2),(13)C-formaldehyde. Relative quantification of peptide mixtures is achieved by examining the MALDI mass spectra of the peptide pairs labeled with different isotope tags. A nominal mass difference of 6 Da between the peptide pair allows negligible interference between the two isotopic clusters for quantification of peptides of up to 3000 Da. Since only the N-termini of tryptic peptides are differentially labeled and the a(1) ions are also enhanced in the MALDI MS/MS spectra, interpretation of the fragment ion spectra to obtain sequence information is greatly simplified. It is demonstrated that this technique of N-terminal dimethylation (2ME) after lysine guanidination (GA) or 2MEGA offers several desirable features, including simple experimental procedure, stable products, using inexpensive and commercially available reagents, and negligible isotope effect on reversed-phase separation. LC-MALDI MS combined with this 2MEGA labeling technique was successfully used to identify proteins that included polymorphic variants and low abundance proteins in bovine milk. In addition, by analyzing a mixture of two equal amounts of milk whey fraction as a control, it is shown that the measured average ratio for 56 peptide pairs from 14 different proteins is 1.02, which is very close to the theoretical ratio of 1.00. The calculated percentage error is 2.0% and relative standard deviation is 4.6%.

A strategy based on isotope labeling of peptides and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry (LC-MALDI MS) has been employed to accurately quantify and confidently identify differentially expressed proteins between an E-cadherin-deficient human carcinoma cell line (SCC9) and its transfectants expressing E-cadherin (SCC9-E). Proteins extracted from each cell line were tryptically digested and the resultant peptides were labeled individually with either d(0)- or d(2)-formaldehyde. The labeled peptides were combined and the peptide mixture was separated and fractionated by a strong cation exchange (SCX) column. Peptides from each SCX fraction were further separated by a microbore reversed-phase (RP) LC column. The effluents were then directly spotted onto a MALDI target using a heated droplet LC-MALDI interface. After mixing with a MALDI matrix, individual sample spots were analyzed by MALDI quadrupole time-of-flight MS, using an initial MS scan to quantify the dimethyl labeled peptide pairs. MS/MS analysis was then carried out on the peptide pairs having relative peak intensity changes of greater than 2-fold. The MS/MS spectra were subjected to database searching for protein identification. The search results were further confirmed by comparing the MS/MS spectra of the peptide pairs. Using this strategy, we detected and compared relative peak intensity changes of 5480 peptide pairs. Among them, 320 peptide pairs showed changes of greater than 2-fold. MS/MS analysis of these changing pairs led to the identification of 49 differentially expressed proteins between the parental SCC9 cells and SCC9-E transfectants. These proteins were determined to be involved in different pathways regulating cytoskeletal organization, cell adhesion, epithelial polarity, and cell proliferation. The changes in protein expression were consistent with increased cell-cell and cell-matrix adhesion and decreased proliferation in SCC9-E cells, in line with E-cadherin tumor suppressor activity. Finally, the accuracy of the MS quantification and subcellular localization for 6 differentially expressed proteins were validated by immunoblotting and immunofluorescence assays.

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.