Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics.

RATIONALE: Mass spectrometry (MS)-based protein identification depends mainly on protein extraction and digestion. Although sodium dodecyl sulfate (SDS) can preclude enzymatic digestion and interfere with MS analysis, it is still the most widely used surfactant in these steps. To overcome these disadvantages, a SDS-compatible proteomic technique for SDS removal prior to MS-based analyses was developed, namely filter-aided sample preparation (FASP). METHODS: Herein, based on the effectiveness of sodium deoxycholate and a detergent removal spin column, we developed a modified FASP (mFASP) method and compared its overall performance, total number of peptides and proteins identified for shotgun proteomic experiments with that of the FASP method. RESULTS: Identification of 4570 ± 392 and 9139 ± 317 peptides and description of 862 ± 46 and 1377 ± 33 protein groups with two or more peptides from the ovarian cancer cell line A2780 was accomplished by FASP and mFASP methods, respectively. The mFASP method (21.2 ± 0.2%) had higher average peptide to protein coverage than FASP method (13.2 ± 0.5%). More hydrophobic peptides were identified by mFASP than by FASP, as indicated by the GRAVY score distribution. CONCLUSIONS: The reported method enables reliable and efficient identification of proteins and peptides in whole-cell extracts containing SDS. The new approach allows for higher throughput (the simultaneous identification of more proteins), a more comprehensive investigation of proteins, and potentially the discovery of new biomarkers. Copyright © 2016 John Wiley & Sons, Ltd.

Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins.

Fluorescent staining of protein in sodium dodecyl sulfate polyacrylamide gels by salicylaldehyde azine.

As a non-covalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence-based dye for detecting proteins both in 1-D and 2-D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which similars to that of glutaraldehyde (GA)-silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.

Improved staining of phosphoproteins with high sensitivity in polyacrylamide gels using Stains-All.

An improved Stains-All (ISA) staining method for phosphoproteins in SDS-PAGE was described. Down to 0.5-1 ng phosphoproteins (α-casein, ß-casein, or phosvitin) can be successfully selectively detected by ISA stain, which is approximately 120-fold higher than that of original Stains-All stain, but is similar to that of commonly used Pro-Q Diamond stain. Furthermore, unlike the original Stains-All protocol that was time consuming and light unstable, ISA stain could be completed within 60 min without resorting to protect the gels from light during the whole staining procedure. According to the results, it is concluded that ISA stain is a rapid, sensitive, specific, and economic staining method for a broad application to the research of phosphoproteins.

Quantification method of tubing defects based on machine learning algorithm and magnetic flux leakage signals.

Tubing is the pipeline that transports crude oil and natural gas from the oil and gas layer to the surface of the earth. Due to the harsh operating environment, the tubing will suffer from etch pits, scratches, cracks, perforations, and even direct fractures of different degrees of defective conditions. If tubing defects are not detected and quantified in a timely manner, the continued use of tubing will result in tubing leakage and failure. Magnetic flux leakage (MFL) testing as a nondestructive testing method enables the identification and quantitative analysis of defects in metal tubing. To improve the quantification accuracy of defects in the wellhead MFL testing of tubing defects during workover operations, this paper proposes a multi-output least-squares support vector regression machine (MLSSVR) model optimized based on the simulated annealing algorithm. The size of tubing defects can be quantified by establishing the mapping between the characteristic quantity of MFL signals and the defect size. The experimental results of MFL testing of tubing defects show that the root mean square error (RMSE) of the diameter of tubing defects of the simulated annealing algorithm optimized multi-output least-squares support vector regression (SA-MLSSVR) machine model proposed in this paper is 0.4562 mm, and the RMSE of the depth of tubing defects is 0.1504 mm. Compared with the non-optimized MLSSVR model, the overall RMSE of tubing defects is reduced by 36.48%. The SA-MLSSVR model only needs one-ninth of the time to achieve the same quantification accuracy as the particle swarm optimized multi-output least-squares support vector regression machine model.

Microstructure and Mechanical Properties of Gradient Interfaces in Wire Arc Additive Remanufacturing of Hot Forging Die Steel.

Hot forging dies are subjected to periodic thermal stress and often fail in the forms of thermal fatigue, wear, plastic deformation, and fracture. A gradient multi-material wire arc additive remanufacturing method for hot forging dies was proposed to extend the service life of hot forging dies and reduce total production costs. The properties of multi-material gradient interfaces play a critical role in determining the overall performance of the final products. In this study, the remanufacturing zone of a hot forging die was divided into three deposition layers: the transition layer, the intermediate layer, and the strengthening layer. Experiments of wire arc additive manufacturing with gradient material were conducted on a 5CrNiMo hot forging die steel. The microstructure, microhardness, bonding strength, and impact property of gradient interfaces were characterized and analyzed. The results revealed that the gradient additive layers and their interfaces were defect-free and that the gradient interfaces had obtained a high-strength metallurgical bonding. The microstructure of the gradient additive layers presented a gradient transformation process of bainite-to-martensite from the bottom to the top layer. The microhardness gradually increased from the substrate layer to the surface-strengthening layer, forming a three-level gradient in the range of 100 HV. The impact toughness values of the three interfaces were 46.15 J/cm2, 54.96 J/cm2, and 22.53 J/cm2, and the impact fracture morphology ranged from ductile fracture to quasi-cleavage fracture. The mechanical properties of the gradient interfaces showed a gradient increase in hardness and strength, and a gradient decrease in toughness. The practical application of hot forging die remanufactured by the proposed method had an increase of 37.5% in average lifespan, which provided scientific support for the engineering application of the gradient multi-material wire arc additive remanufacturing of hot forging dies.

Effect of ultrasonic intensity on microstructure and mechanical properties of steel alloy in direct energy deposition-Arc.

To study the effect of ultrasonic intensity on the microstructure and mechanical properties during the direct energy deposition-Arc (DED-Arc) of ER70S-6 steel alloy, an ultrasound assisted DED-Arc system was developed by coupling ultrasonic energy with the electric arc deposition process. The propagation and vibration distribution of ultrasound in the substrate were analyzed by numerical simulation method. Deposition layers were fabricated using different ultrasonic amplitudes, and the microstructure, microhardness and tensile properties of the fabricated parts were systematically investigated. The results show that as the ultrasonic intensity increased, the grain refinement area expanded from the center of the molten pool to the surrounding area, and the grain morphology transforms from coarse columnar grains to fine equiaxed grains. When the ultrasonic amplitude was 15 µm, the grain refinement area of the cross-section was 94.6%, the average grain size was significantly increased to about grade 6. The microhardness increased by 10.6%. Thousands of ultrasonic cavitation events not only enhance the supercooling and wettability of the melt pool to promote nucleation, but also break the columnar grains into small grains by intense shock waves, which significantly improve the microstructure homogeneity and mechanical properties. The research provides an alternative approach to overcoming the long-standing problem of coarse columnar grains in the field of DED-Arc.

Fecal microbiota transplantation in obesity metabolism: A meta analysis and systematic review.

OBJECTIVE: The effect of fecal microbiota transplantation (FMT) on microbiota engraftment in patients with metabolic syndrome remains unclear. This systematic review employed a meta-analysis of RCTs for assessment on the role of FMT in treating obesity and metabolic syndrome, and its impact on clinically relevant parameters. METHOD: Major databases and grey literatures were searched identifying RCTs comparing FMT of lean donors with placebo in obese/metabolic syndrome patients. Studies using any form of placebo were included. Variations in the parameters before and after treatment were calculated followed by meta-analyses. RESULT: Ten studies met the inclusion criteria and a total of 334 patients were included for further analysis. Clinically significant parameters associated with obesity and metabolic syndrome were explored and FMT was identified significantly and negatively associated with most indices of abdominal adiposity including caloric intake, fasting glucose, HOMA-IR, systolic blood pressure, diastolic blood pressure, total cholesterol, HDL, LDL, triglycerides and CRP, Obesity parameters including fasting glucose and acetic acid were increased following FMT. CONCLUSION: FMT is more advantageous for obese patients with elevated blood pressure, disordered glucose and insulin metabolism, and elevated blood lipids. The study of metabolic factors in obese patients will be our starting point in the future.

Integrative proteomic characterization of adenocarcinoma of esophagogastric junction.

The incidence of adenocarcinoma of the esophagogastric junction (AEG) has been rapidly increasing in recent decades, but its molecular alterations and subtypes are still obscure. Here, we conduct proteomics and phosphoproteomics profiling of 103 AEG tumors with paired normal adjacent tissues (NATs), whole exome sequencing of 94 tumor-NAT pairs, and RNA sequencing in 83 tumor-NAT pairs. Our analysis reveals an extensively altered proteome and 252 potential druggable proteins in AEG tumors. We identify three proteomic subtypes with significant clinical and molecular differences. The S-II subtype signature protein, FBXO44, is demonstrated to promote tumor progression and metastasis in vitro and in vivo. Our comparative analyses reveal distinct genomic features in AEG subtypes. We find a specific decrease of fibroblasts in the S-III subtype. Further phosphoproteomic comparisons reveal different kinase-phosphosubstrate regulatory networks among AEG subtypes. Our proteogenomics dataset provides valuable resources for understanding molecular mechanisms and developing precision treatment strategies of AEG.

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels.

A sensitive, brief, and user-friendly silver stain to meet the needs in high-efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde-based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user-friendly method could be a good choice for LPS visualization on polyacrylamide gels.

[Dissolved Organic Matter Component and Source Characteristics of the Metropolitan Lakes and Reservoirs in a Typical Karst Region].

In order to explore dissolved organic matter (DOM) components and their origins in metropolitan lakes and reservoirs in the karst region, the typical Hongfeng Lake, Baihua Lake, Songbaishan Reservoir, and Aha Reservoir were investigated in Guiyang City. Surface water parameters, including dissolved organic carbon (DOC), chlorophyll a (Chla), and optical parameters (a254, a280, a350, E2:E3, S275-295, FI, ß:α, BIX, and HIX) were analyzed. Fluorescence peaks (B, T, A, M, C, D, and N) and three-dimensional matrix fluorescence with parallel factor analysis (EEM-PARAFAC) were employed to explain distinct DOM abundances and proportions. Meanwhile, Spearman's correlation coefficients and principal component analysis (PCA) were used to decipher parameter types and primary environmental processes. The results showed that aquatic ρ(DOC) and ρ(Chla) ranged between 4.24-11.9 mg·L-1 and 0.32-19.7 µg·L-1, respectively. High humic-like (a254) and protein DOM (a280) were observed in the Songbaishan Reservoir, resulting in higher DOM molecular weight when compared to that in other lakes and reservoirs. Surface water DOM mainly contained visible-light humic-like (23.8%-46.9%) and terrestrial fulvic-like components (17.6%-28.4%). High FI, ß:α, and BIX but low HIX values in this study suggested that endogenous inputs largely contributed to aquatic DOM. Aquatic DOM component and source characteristics were significantly correlated with each other. Furthermore, inputs of humic-like DOM and microbial metabolism, as well as coupled carbonate dissolution and photosynthesis, drove dynamic DOM behaviors in the karst lakes and reservoirs.

[Water-air Carbon Dioxide Exchange and Nutritional Controls in a Typical Karst River].

To explore water-air carbon dioxide (CO2) exchange and its nutritional controls in karst rivers, water sampling and analyses were conducted in the Furong River as a representative karst river system. Regional hydrometeorological characteristics, carbonate system parameters, total organic carbon (TOC), nutrients and their stoichiometric ratios, and the riverine pressure of aqueous CO2 (pCO2) and its couplings to trophic status were analyzed. The results showed that gas transfer velocity of CO2 (k) varied between 2.71 and 13.0 m·d-1. Riverine pCO2 varied from 78.5 Pa to 21491.2 Pa and was significantly higher in the tributaries than the main stem. Riverine TOC, total nitrogen (TN), and total phosphorus (TP) concentrations were (302.8±50.1), (128.9±67.9), and (0.65±0.98) µmol·L-1, respectively, demonstrating the trophic status of P-limitation. Riverine pCO2 was tightly linked to P-related parameters, suggesting that in-situ metabolism triggered aquatic CO2 supersaturation. Water-air CO2 flux (F) averaged (534.5±801.4) mmol·(m2·d)-1 in the Furong River, which is higher than most of the world's rivers, and thus indicates high potential CO2 emissions.

[Water-Air Interface CO2 Exchange Flux of Typical Lakes in a Mountainous Area of the Western Chongqing and Their Influencing Factors].

To examine the mountainous lake CO2 evasion in Southwest China, partial pressures of carbon dioxide[p(CO2)] and the CO2 exchange flux[F(CO2)] via the water-air interface of nine mountainous lakes in Chongqing, China, have been studied in summer using the thin boundary layer model (TBL) and floating chambers. Key water quality parameters were concomitantly measured. The results indicate that the pCO2 in the mountainous lakes in western Chongqing ranges from 2.1 to 45.0 Pa, with a mean value of (18.1±12.1) Pa. The mean CO2 fluxes calculated by the TBL model and chamber method are (-8.0±2.9), (-3.4±3.6), and (-7.1±22.3) mmol·(m2·d)-1, respectively. The p(CO2) and F(CO2) have positive correlations with the wind speed and ORP but negative correlations with the pH. Our study indicates that mountainous lakes are atmospheric sinks of CO2 and the TBL model should be cautiously adopted.

Downregulation of LINC00515 in high-grade serous ovarian cancer and its relationship with platinum resistance.

Aim: To investigate the expression of long intergenic noncoding RNA 00515 (LINC00515) in high-grade serous ovarian cancer (HGSOC) and its potential correlation with platinum resistance. Patients & methods: Expression of LINC00515 in HGSOC (n = 115) and normal (n = 19) tissues was detected via quantitative real-time PCR (qRT-PCR). We further explored the statistical significance of the relationship between LINC00515 expression and platinum resistance in HGSOC. Results: LINC00515 was gradually downregulated in the order of normal > platinum-sensitive > platinum-resistant tissue (p < 0.05). Results demonstrated that LINC00515 downregulation was correlated with platinum resistance and relapse-free survival (RFS) of HGSOC (p < 0.05). Conclusion: LINC00515 downregulation is correlated with HGSOC development, platinum resistance and RFS, supporting its utility as a potential biomarker to predict platinum resistance and prognosis of RFS.

FMRFamide modulates outward potassium currents in mouse olfactory sensory neurons.

1. The olfactory system can detect the presence of low concentrations of odourant molecules and discriminate even slight differences among molecules with a very similar chemical structure. The detection and discrimination of odourants are correlated with the electrophysiology of the olfactory sensory neurons. To get a better understanding of the molecular mechanisms of olfactory transduction, it is therefore of considerable importance to obtain electrophysiological recordings of olfactory sensory neurons. FMRFamide (Phe-Met-Arg-Phe-NH(2)), secreted from the nerve terminals of the nasal cavity, has been suggested to act as a neurotransmitter or neuromodulator, playing an important role in modulating the activity of olfactory receptor neurons. Its effects on voltage-dependent potassium currents in the mouse olfactory sensory neurons were investigated in the present study using the whole-cell patch-clamp technique. 2. Olfactory sensory neurons were isolated from the Kunming Mouse (KM) mouse olfactory epithelium. Different protocols were applied to obtain delayed-rectifier potassium current (I(K)) and fast transient potassium current (I(A)). The effects of FMRFamide on the outward potassium currents, including I(K) and I(A), in mouse olfactory sensory neurons were investigated. 3. We found that FMRFamide (5 micromol/L) increased the magnitude of I(K). However no effect on I(A) was observed. The activation dynamics of both currents were not changed by FMRFamide. 4. In conclusion, FMRFamide may play a role in the modulation of peripheral olfactory signals by regulating I(K). This modulation may shorten the phase of the fast repolarization of the action potential in mouse olfactory sensory neurons and increase the excitability of the neuronal membrane.

Simultaneous determination of six tyrosine kinase inhibitors in human plasma using HPLC-Q-Orbitrap mass spectrometry.

AIM: Gefitinib, erlotinib, icotinib, crizotinib, lapatinib and apatinib are targeted cancer therapy agents acting through inhibition of tyrosine kinase. Method for quantifying these six drugs in human plasma of patients was required. MATERIALS & METHODS: An HPLC-Q-Orbitrap method (based on HPLC-MS/MS) was developed and validated for the simultaneous detection and quantitation of six tyrosine kinase inhibitors in human plasma. Sample was extracted by liquid-liquid extraction (ethyl acetate: tert-Butyl methyl ether, 1:1 v/v). The method shows a high level of accuracy and reproducibility. The lower limit of quantification was 0.02 ng/ml for apatinib, 0.1 ng/ml for crizotinib, 2.0 ng/ml for lapatinib and 0.05 ng/ml for erlotinib, gefitinib and icotinib. This method was successfully used for apatinib monitoring in plasma of patients with NSCLC. CONCLUSION: This simple and reproducible method has potential for monitoring of tyrosine kinase inhibitors in patients' plasma.

The Essential Role of H19 Contributing to Cisplatin Resistance by Regulating Glutathione Metabolism in High-Grade Serous Ovarian Cancer.

Primary and acquired drug resistance is one of the main obstacles encountered in high-grade serous ovarian cancer (HGSC) chemotherapy. Cisplatin induces DNA damage through cross-linking and long integrated non-coding RNAs (lincRNAs) play an important role in chemical induced DNA-damage response, which suggests that lincRNAs may be also associated with cisplatin resistance. However, the mechanism of long integrated non-coding RNAs (lincRNAs) acting on cisplatin resistance is not well understood. Here, we showed that expression of lin-RECK-3, H19, LUCAT1, LINC00961, and linc-CARS2-2 was enhanced in cisplatin-resistant A2780-DR cells, while transcriptome sequencing showed decreased Linc-TNFRSF19-1 and LINC00515 expression. Additionally, we verified that different H19 expression levels in HGSC tissues showed strong correlation with cancer recurrence. H19 knockdown in A2780-DR cells resulted in recovery of cisplatin sensitivity in vitro and in vivo. Quantitative proteomics analysis indicated that six NRF2-targeted proteins, including NQO1, GSR, G6PD, GCLC, GCLM and GSTP1 involved in the glutathione metabolism pathway, were reduced in H19-knockdown cells. Furthermore, H19-knockdown cells were markedly more sensitive to hydrogen-peroxide treatment and exhibited lower glutathione levels. Our results reveal a previously unknown link between H19 and glutathione metabolism in the regulation of cancer-drug resistance.

The inhibitory effect of Shaoyao Ruangan formula on mice with transplanted H22 hepatocarcinoma and its mechanism research.

BACKGROUND: The incidence of hepatocellular carcinoma (HCC) is very high in the world. However, a safe and effective strategy is still under research. AIMS: Our aim was to demonstrate the inhibitory effect of Shaoyao Ruangan Formmula (SRF) on the tumor of H22-bearing mice and explore its antitumor mechanisms. SETTINGS AND DESIGN: Corresponding physiological indexes of H22-bearing mice treated with SRF were compared with that of saline treated mice, which could reflect the tumor-suppressing effect of SRF. MATERIALS AND METHODS: After treatment, tumor weight, survival time, related gene expression levels etc., were recorded or detected. STATISTICAL ANALYSIS: Data analyzed using a computer SPSS program. RESULTS AND CONCLUSIONS: Comparing with blank control group, the tumor inhibitor rate (IR) of low, middle and high dose group of SRF was 17.72%, 33.99% and 23.73%, respectively. IR of CTX was 43.95%. The results also showed that each group of SRF could prolong the life span of H22-bearing mice to some extent. In addition, reverse transcription polymerase chain reaction (RT-PCR) results revealed that SRF was able to influence related genes expression in the tumor tissues of H22-bearing mice. The expression of TGF-ß receptor type II (TBRII) gene was significantly upregulated in each SRF group comparing with normal saline group. On the contrary, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was significantly downregulated in each SRF group comparing with normal saline group. In summary, SRF showed tumor-suppressing effect on mice with transplanted H22 hepatocarcinoma. The mechanism of antitumor effect may induced by upregulating TBRII expression and down-regulating NF-κB expression.

A method for sensitive staining of DNA in polyacrylamide gels using basic fuchsin.

BACKGROUND: PAGE is a widely used analytical method to resolve components of a DNA mixture based on their size. Various DNA visualization methods including fluorescence, visible dye and silver have been used for the detection of gel-separated DNA, with each having different advantages and disadvantages in terms of sensitivity, safety and simplicity. RESULTS: A fast and sensitive visible dye-based staining method for DNA in polyacrylamide gels using basic fuchsin (BF) is described. As low as 10-20 pg of DNA can be visualized within 10 min; the sensitivity is fourfold more sensitive than that of SYBR® Gold stain, the most sensitive commercial fluorescent probe, but similar to silver staining kit from GE Healthcare. In addition, the mechanism studies suggest that the interaction of BF with DNA is mainly contributed by non-intercalative binding mode. CONCLUSION: By comprehensive studies of this visible dye-based protocol, we concluded that BF stain is a fast and sensitive method currently available for detecting DNA in polyacrylamide gels.