Effect of boiling on texture of scallop adductor muscles and its mechanism based on label-free quantitative proteomic technique.

The mechanism behind textural changes in scallop adductor muscle during boiling was investigated through proteomic analysis, determination of water holding capacity (WHC) and oxidative indices, as well as observation with scanning electron microscopy and multiphoton nonlinear optical microscopy. The hardness and shear force showed the trend of first rising and then falling in 45 min-boiling time. The results suggested that short-time boiling caused the oxidation, denaturation and aggregation of proteins, resulting in the transverse contraction of myofibers and lateral cross-linked aggregation of muscle fibers and a rise in WHC, which led to the increase in hardness and shear force. While long-time boiling caused the progressive degradation of structural proteins such as fibrillin-1, collagen alpha-2(I) chain, myosin heavy chain, basement membrane-specific heparan sulfate proteoglycan core protein, and paramyosin, resulting in a loose myofibril network and the decrease in WHC, which led to the decrease in hardness and shear force.

Analysis of texture properties and water-soluble fraction proteome of sea cucumber body wall with different boiling heating treatment.

Label-free quantitative proteomic analysis was utilized to determine the key proteins that affect texture properties of sea cucumber body wall (SCBW) with different boiling heating treatment. 862, 363, 315, and 258 proteins were confirmed in water-soluble fractions from fresh group, 0.5 h-, 2 h- and 4 h-heat treatment group, respectively. During boiling heating treatment, proteins with an increased abundance in water-soluble fraction primarily belong to structural proteins, such as collagens, microfibril-associated proteins, glycoproteins, and muscle proteins. It was speculated that the degradation of these structural proteins caused the progressive disintegration of network skeleton of collagen fibres and FMs as well as the gelatinization, thus resulted in the decrease of hardness and shear force. Besides, the degradation of FMs was occurred layer by layer during boiling heating treatment, and the fibrilin-1 outer layer degraded first, followed by the fibrilin-2 core component.

Identification of Phosphorus Stress Related Proteins in the Seedlings of Dongxiang Wild Rice (Oryza Rufipogon Griff.) Using Label-Free Quantitative Proteomic Analysis.

Phosphorus (P) deficiency tolerance in rice is a complex character controlled by polygenes. Through proteomics analysis, we could find more low P tolerance related proteins in unique P-deficiency tolerance germplasm Dongxiang wild rice (Oryza Rufipogon, DXWR), which will provide the basis for the research of its regulation mechanism. In this study, a proteomic approach as well as joint analysis with transcriptome data were conducted to identify potential unique low P response genes in DXWR during seedlings. The results showed that 3589 significant differential accumulation proteins were identified between the low P and the normal P treated root samples of DXWR. The degree of change was more than 1.5 times, including 60 up-regulated and 15 downregulated proteins, 24 of which also detected expression changes of more than 1.5-fold in the transcriptome data. Through quantitative trait locus (QTLs) matching analysis, seven genes corresponding to the significantly different expression proteins identified in this study were found to be uncharacterized and distributed in the QTLs interval related to low P tolerance, two of which (LOC_Os12g09620 and LOC_Os03g40670) were detected at both transcriptome and proteome levels. Based on the comprehensive analysis, it was found that DXWR could increase the expression of purple acid phosphatases (PAPs), membrane location of P transporters (PTs), rhizosphere area, and alternative splicing, and it could decrease reactive oxygen species (ROS) activity to deal with low P stress. This study would provide some useful insights in cloning the P-deficiency tolerance genes from wild rice, as well as elucidating the molecular mechanism of low P resistance in DXWR.

Simplified ARCHITECT microfluidic chip through a dual-flip strategy enables stable and versatile tumoroid formation combined with label-free quantitative proteomic analysis.

Recent years, microfluidic three-dimensional (3D) tumor culture technique has made great progress in tumor microenvironment simulation and drug screening. Meanwhile, as their functionality and complexity increase, it is more difficult for current chip models to selectively collect specific-layer cells from tumoroids for further analysis. Moreover, a simplified and robust method for tumoroid formation with highly consistent size and repeatable 3D morphology is relatively ncessary. Here, we report an ARCHITECT (ARtificial CHIp for Tumor Enables Confocal Topography observation) chip, through a dual-flip strategy to implement straightforward tumoroid establishment. This platform guarantees stable batch-to-batch tumoroids formation and allows high resolution confocal imaging. Moreover, an initial cell density as low as 65 cells per chamber is efficient to deliver a tumoroid. With this ARCHITECT chip, different-layer cells of interest could be collected from tumoroid for label-free quantitative (LFQ) proteomic analysis. For application demonstration, we mainly verified this platform for lung carcinoma (A549) tumoroid construction and proteomic analysis at out layer. Our data indicate that the out-layer cells of A549 tumoroid show extensively distinct proteomic expressions compared to two-dimensional cultured A549 cells. The up-regulated proteins are mainly related to tumorigenicity, proliferation and metastasis. And the differentially expressed proteins are mainly relevant to lipid metabolism pathway which is essential to tumor progression and proliferation. This platform provides a simplified yet robust technique to connect microfluidic tumoroid construction and LFQ proteomic analysis. The simplicity of this technique should open the way to numerous applications such as discovering the innovative targets for cancer treatment, and studying the mophological and proteomic heterogeneity of different-layer cells across the tumoroid.

TAB1——Potential Biomarker of Paclitaxel Resistance in Ovarian Cancer Based on Label-Free Quantitative Proteomics / 中国实验方剂学杂志

Objective:To explore the potential targets and related mechanism involved in the paclitaxel resistance to ovarian cancer. Method:Ovarian cancer A2780 cells and A2780 paclitaxel-resistant cells （A2780/T） were treated by 2， 4， 8， 16， 32， 64， 128， 256 μmol·L^-1 paclitaxel （PTX） for 24 h or 48 h respectively <italic>in vitro</italic>. The proliferation rate of A2780 cells and A2780/T cells treated with paclitaxel was determined by methyl thiazolyl tetrazolium （MTT） colorimetric method assay. A2780 and A2780/T cells were analyzed by LC-MS/MS Label-Free quantitative proteomics to identify and screen differentially expressed proteins in the two groups of cells. Gene ontology （GO） annotation and Kyoto encyclopedia of genes and genomes （KEGG） pathway analysis were used to determine the potential biomarkers of paclitaxel resistance in ovarian cancer. Conventionally cultured A2780 cells were used as a control group， and A2780/T cells were treated with 0， 1， 4 μmol·L^-1 PTX. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot methods were used to detect and verify the mRNA and protein expression levels of potential target transforming growth factor-<italic>β</italic>-activated kinase 1 binding protein 1 （TAB1） and its downstream related molecules transforming growth factor-<italic>β</italic>-activated kinase （TAK1） and p38. Result:After PTX treatment for 24 h and 48 h， the cell viability of A2780 and A2780/T cells decreased. The inhibitory rate of PTX on A2780 cells was significantly higher than that of A2780/T cells. In A2780 cells， the IC₅₀ of PTX treatment for 48 h was 0.002 μmol·L^-1， while in A2780/T cells， the IC₅₀of PTX was greater than the maximum concentration of 128 μmol·L^-1， indicating that A2780/T cells were resistant to PTX compared with A2780 cells. 441 differentially expressed proteins and 421 special differentially expressed proteins between A2780/T and A2780 cells were screened by label-free quantitative proteomic analysis. GO function enrichment analysis showed that the binding proteins accounted for the majority （80%） among the differentially expressed proteins. According to the results of KEGG pathway analysis and expression site analysis， TAB1 might be a potential biomarker in paclitaxel-resistant ovarian cancer. Compared with A2780 cells， mRNA and protein expression levels of TAB1 in A2780/T cells were significantly reduced （<italic>P</italic><0.01）. mRNA expression of TAK1 and p38 that interacted with TAB1 were also significantly reduced （<italic>P</italic><0.05， <italic>P</italic><0.01）， while there was no significant change in protein expression. Conclusion:TAB1 may be a potential biomarker of paclitaxel resistance to ovarian cancer ， and its mechanism may be related to the TAB1/TAK1/p38 MAPK pathway.

Microbial insights of enhanced anaerobic conversion of syngas into volatile fatty acids by co-fermentation with carbohydrate-rich synthetic wastewater.

BACKGROUND: The co-fermentation of syngas (mainly CO, H2 and CO2) and different concentrations of carbohydrate/protein synthetic wastewater to produce volatile fatty acids (VFAs) was conducted in the present study. RESULTS: It was found that co-fermentation of syngas with carbohydrate-rich synthetic wastewater could enhance the conversion efficiency of syngas and the most efficient conversion of syngas was obtained by co-fermentation of syngas with 5 g/L glucose, which resulted in 25% and 43% increased conversion efficiencies of CO and H2, compared to syngas alone. The protein-rich synthetic wastewater as co-substrate, however, had inhibition on syngas conversion due to the presence of high concentration of NH4 +-N (> 900 mg/L) produced from protein degradation. qPCR analysis found higher concentration of acetogens, which could use CO and H2, was present in syngas and glucose co-fermentation system, compared to glucose solo-fermentation or syngas solo-fermentation. In addition, the known acetogen Clostridium formicoaceticum, which could utilize both carbohydrate and CO/H2 was enriched in syngas solo-fermentation and syngas with glucose co-fermentation. In addition, butyrate was detected in syngas and glucose co-fermentation system, compared to glucose solo-fermentation. The detected n-butyrate could be converted from acetate and lactate/ethanol which produced from glucose in syngas and glucose co-fermentation system supported by label-free quantitative proteomic analysis. CONCLUSIONS: These results demonstrated that the co-fermentation with syngas and carbohydrate-rich wastewater could be a promising technology to increase the conversion of syngas to VFAs. In addition, the syngas and glucose co-fermentation system could change the degradation pathway of glucose in co-fermentation and produce fatty acids with longer carbon chain supported by microbial community and label-free quantitative proteomic analysis. The above results are innovative and lead to achieve effective conversion of syngas into VFAs/longer chain fatty acids, which would for sure have a great interest for the scientific and engineering community. Furthermore, the present study also used the combination of high-throughput sequencing of 16S rRNA genes, qPCR analysis and label-free quantitative proteomic analysis to provide deep insights of the co-fermentation process from the taxonomic and proteomic aspects, which should be applied for future studies relating with anaerobic fermentation.

Label-free quantitative proteomic analysis reveals the lifestyle of Lactobacillus hordei in the presence of Sacchromyces cerevisiae.

Water kefir is a fermented beverage, which is traditionally prepared from sucrose, kefir grains, dried or fresh fruits, and water. L. hordei and S. cerevisiae are isolated as predominant and stable species of lactic acid bacteria and yeasts, respectively. In this study we demonstrate that label free quantitative proteomics is useful to study microbial interaction along the response of co-cultivated L. hordei TMW 1.1822 in the presence of S. cerevisiae TMW 3.221 as compared with their single cultures in a water kefir model. It is shown and L. hordei responds to S. cerevisiae in many respects revealing a mutualistic relationship. The data suggest that L. hordei responds to the presence of S. cerevisiae with adjustment of intracellular redox reactions controlled of proteins, which are part of Rex regulons and proteins involved in the glycolytic pathway and energy fermentation. An NADH, H+-driven metabolic switch to preferential production of butanediol instead of acetate or lactate, and up-regulation of arginine deiminase, alleviated acid stress and concomitantly protected S. cerevisiae against an acidic environment, which L. hordei generated in single culture. Moreover, the data suggest that the presence of S. cerevisiae in the nitrogen and fatty acids limited environment of the water kefir facilitated and improved the growth of L. hordei by delivering gluconate, fructose, amino acids, fatty acids or substrates for their biosynthesis. Up-regulation of the OppABCDF peptide transport and enzymes involved in amino acid metabolism indicates enhanced peptide uptake, as well as cross-feeding of L. hordei by glutamine, glutamate, histidine, tryptophan, methionine, proline, tryptophan delivered by S. cerevisiae.

Label-free quantitative proteomic analysis identifies CTNNB1 as a direct target of FOXP3 in gastric cancer cells.

Forkhead box protein 3 (FOXP3) is expressed in numerous types of tumor cell and is associated with tumor progression and prognosis. A previous study reported that FOXP3 inhibited cellular proliferation and induced apoptosis of gastric cancer (GC) cells by activating the apoptosis signaling pathway. In the present study, label-free quantitative proteomic analysis and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) was performed to investigate the mechanism by which the anticancer role of FOXP3 was mediated and the proteins that with which it may interact. Label-free quantitative proteomic analysis was used to screen for proteins differentially expressed between FOXP3-overexpressing GC (AF) and vector (ANC) cells. Catenin ß1 (CTNNB1) was one of the proteins that exhibited the greatest difference between AF and ANC among 3,313 proteins identified by liquid chromatography with tandem mass spectrometry analysis. The expression of CTNNB1 was evaluated by reverse transcription-quantitative PCR and western blotting. The association between FOXP3 and CTNNB1 was confirmed by ChIP-PCR in AGS cells. The changes in expression of epithelial-mesenchymal transition-associated proteins were analyzed by western blotting. The level of FOXP3 expression was positively associated with CTNNB1 and E-cadherin expression, but not with vimentin and N-cadherin expression. FOXP3 positively regulates CTNNB1 and binds to it directly. Along with the upregulation of glycogen synthase kinase 3ß (GSK3ß), which was also a protein whose expression was found to change significantly in proteomic analysis and has a key role in the Wnt pathway. This association is an attractive and novel hypothesis for the mechanism by which FOXP3 inhibits the invasion and metastasis of GC cells.

Label-free quantitative proteomic profiling of colon cancer cells identifies acetyl-CoA carboxylase alpha as antitumor target of Citrus limon-derived nanovesicles.

We have previously isolated exosome-like nanoparticles from Citrus-limon juice, able to inhibit in vitro and in vivo tumor cell growth. In order to deeply understand the mechanism underlying nanovesicle effects, we performed a proteomic profile of treated colorectal cancer cells. Among the proteins differentially expressed after nanovesicle treatment, we found a significant downregulation of the Acetyl-CoA Carboxylase 1 (ACACA) and we demonstrated that silencing ACACA in cancer cells leads to a reduction of cell growth. Our study proved that the anti-tumor effects of Citrus-limon nanovesicles is partly mediated by lipid metabolism inhibition, in particular via ACACA downregulation. SIGNIFICANCE: This study represents the attempt to achieve, by a proteomic approach, a better understanding of the role of lemon nanovesicles in affecting colorectal cancer cell growth.

Differential label-free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property.

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label-free MS-based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.