Comparison of Pairwise Venous and Fingertip Plasma Using Quantitative Proteomics Based on Data-Independent Acquisition.

Blood contains a great deal of health-related information and can be used to monitor human health status. Clinically, venous or fingertip blood is usually used for blood tests. However, the clinical application settings of the two sources of blood are unclear. In this study, the proteomes of pairwise venous plasma (VP) and fingertip plasma (FP) were analyzed, and the levels of 3797 proteins were compared between VP and FP. The Spearman's correlation coefficient for the relationship between protein levels of VP and FP ranges from 0.64 to 0.78 (p < 0.0001). The common pathways of VP and FP are related to cell-cell adhesion, protein stabilization, innate immune response, and complement activation, the classical pathway. The VP-overrepresented pathway is related to actin filament organization, while the FP-overrepresented pathway is related to the hydrogen peroxide catabolic process. ADAMTSL4, ADIPOQ, HIBADH, and XPO5 both in VP and FP are potential gender-related proteins. Notably, the VP proteome has a higher interpretation on age than the FP proteome, and CD14 is a potential age-related protein in VP but not in FP. Our study mapped the different proteomes between VP and FP, which can provide value for the standardization of clinical blood tests.

Expression of a thermo- and alkali-philic fungal laccase in Pichia pastoris and its application.

The fungal laccase Lcc9 from Coprinopsis cinerea is a promising candidate for biotechnological applications due to its distinct biochemical properties. In the present work, Lcc9 cDNA was cloned from C. cinerea using reverse transcription polymerase chain reaction and heterologously expressed in Pichia pastoris GS115. The recombinant laccase was found to be a heavily hyperglycoprotein, with the molecular weight of 60.2 kDa as determined by MALDI-TOF. Laccase activity in the culture supernatant was 1750 ±â€¯83 U/L and reached 3138 ±â€¯62 U/L after expression condition optimization using orthogonal experiment. The biochemical property of the purified recombinant Lcc9 (rLcc9) was compared to that of wild-type Lcc9. rLcc9 shows a higher specific activity (315.3 U/mg) than Lcc9 (92.9 U/mg) when using ABTS (2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate)) as the substrate. Although rLcc9 and Lcc9 showed comparable optimal pH (6.5) and temperature (70 °C) toward syringaldazine, rLcc9 displayed higher activity and stability in the pH range of 6.5-8.5. rLcc9 showed improved ability to oxidize indigo carmine and 5 azo dyes when methyl syringate was used as the mediator, with the decolorization rate range from 71.9 ±â€¯3.2% to 99.1 ±â€¯1.6% for different dyes in a wide pH (4.5-9.0) and temperature (4-70 °C) ranges. In comparison, Lcc9 decolorized 50.3 ±â€¯2.1% to 98.2 ±â€¯2.0% of the dyes used. The improved activity and stability in alkaline pH of rLcc9 relative to Lcc9, and improved dye decolorization ability towards 6 dyes suggested greater application potential of rLcc9 in biotechnologies such as wastewater treatment.

[Effect of sodium houttuyfonate in combination with erythromycin on luxS, agr/RNAâ ¢ of Staphylococus epidermidis].

Quorum sensing of bacteria and its specific gene expression regulation have a very important role in bacterial biofilm formation. LuxS and agr are the key regulatory genes in quorum sensing of Staphylococcus epidermidis, and RNA Ⅲ is the effector molecule of agr system. In order to evaluate the effects of sodium houttuyfonate in combination with erythromycin on the transcription level of S. epidermidis, serial dilution method was used to determine the MIC of sodium houttuyfonate, erythromycin and vancomycin on S. epidermidis, and fluorescent quantitative PCR method was used to detect the transcription levels of luxS, agr/RNAⅢ in different time periods after treatment on S. epidermidis by sodium houttuyfonate in combination with erythromycin, vancomycin, and erythromycin alone. Our results showed that in treatment by 1/2MIC, 1/4MIC sodium houttuyfonate, 1/2MIC sodium houttuyfonate +1/2MIC erythromycin, 1/4MIC sodium houttuyfonate+1/4MIC erythromycin, and 1/8MIC sodium houttuyfonate+1/8MIC erythromycin for ATCC 35984, they could rapidly up-regulate the expression of luxS of S. epidermidis from the beginning as compared with negative control, with significant differences (P<0.05); furthermore, sodium houttuyfonate can still up-regulate the expression of luxS even after treatment for 6, 12 and 48 h. Sodium houttuyfonate in MIC and 1/2MIC concentration can significantly down-regulate the expression of agr (P<0.05); 1/2MIC sodium houttuyfonate+1/2MIC erythromycin, 1/4MIC sodium houttuyfonate+1/4MIC erythromycin, can also significantly down-regulate the expression of agr in 6 h, 12 h and 24 h(P<0.05). Sodium houttuyfonate in MIC, can significantly down-regulate the expression of RNA Ⅲ (P<0.05), and 1/2MIC sodium houttuyfonate+1/2MIC erythromycin can also significantly down-regulate the expression of RNAⅢ(P<0.05). Therefore, our presented results showed that sodium houttuyfonate in combination with erythromycin can rapidly up-regulate the transcription of luxS of S. epidermidis, and can down-regulate the expression of agr/RNA Ⅲ in certain concentrations, and suggested that sodium houttuyfonate in combination of erythromycin could inhibit mutual aggregation between S. epidermidis and biofilm bacteria, inhibit membrane nutrition and formation of water transport channels, prevent separation of bacterial cells in biofilm, and inhibit the formation of bacterial exotoxin of S. epidermidis.

Proteomic Profiling of Serum Extracellular Vesicles Identifies Diagnostic Signatures and Therapeutic Targets in Breast Cancer.

Analysis of extracellular vesicles (EVs) is a promising noninvasive liquid biopsy approach for breast cancer (BC) detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of BC-specific EVs from human samples is required to realize the potential of this strategy. In this study, we applied a mass spectrometry-based, data-independent acquisition (DIA) proteomic approach to characterize human serum EVs derived from patients with BC (n = 126) and healthy donors (HDs, n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled construction of specific EV protein classifiers for diagnosing BC and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of BC. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating BC invasion and metastasis. Finally, high-throughput molecular docking and virtual screening of a library consisting of 271,380 small molecules identified a potent TALDO1 allosteric inhibitor, AO-022, which could inhibit BC migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of BC patients to guide development of improved diagnosis, monitoring, and treatment strategies.

Proteomic characterization of the colorectal cancer response to chemoradiation and targeted therapies reveals potential therapeutic strategies.

Chemoradiation and targeted therapies are the major treatments for colorectal cancer (CRC); however, molecular properties associated with therapy resistance are incompletely characterized. Here, we profile the proteome of 254 tumor tissues from patients with CRC undergoing chemotherapy, chemoradiation, or chemotherapy combined with targeted therapy. Proteome-based classification reveals four subtypes featured with distinct biological and therapeutic characteristics. The integrative analysis of CRC cell lines and clinical samples indicates that immune regulation is significantly associated with drug sensitivity. HSF1 can increase DNA damage repair and cell cycle, thus inducing resistance to radiation, while high expression of HDAC6 is negatively associated with response of cetuximab. Furthermore, we develop prognostic models with high accuracy to predict the therapeutic response, further validated by parallel reaction monitoring (PRM) assay in an independent validation cohort. This study provides a rich resource for investigating the mechanisms and indicators of chemoradiation and targeted therapy in CRC.

Role of N-glycosylation on the specific activity of a Coprinopsis cinerea laccase Lcc9 expressed in Pichia pastoris.

Laccase Lcc9 from Coprinopsis cinerea heterologously expressed in Pichia pastoris (rLcc9) displayed different molecular weight and specific activity from the native laccase (nLcc9). Glycosylation may play a role in regulating the Lcc9 specific activity. To elucidate this hypothesis, in this study, firstly we demonstrated that rLcc9 and nLcc9 were glycoproteins, and then enzymatically deglycosylated them. The obtained drLcc9 and dnLcc9 showed an apparent decrease in their specific activities. Three putative N-glycosylation sites (N293, N313, and N454) were then predicted in Lcc9 and substituted to evaluate their roles in its specific activity. Molecular weight analysis on those mutants suggested that glycosylation should have occurred on N313 and N454 whereas not on N293 in rLcc9. Comparison of catalytic properties of those mutants revealed that glycosylation at N313 and N454 in rLcc9 could affect the binding affinity to substrates and the catalytic rate, respectively. In addition, the glycosylation could also affect the thermal stability of rLcc9 and nLcc9 since deglycosylation of those Lcc9s resulted in decreases in their thermal stability to some extent. These results will help us to understand the effect of glycosylation on biochemical characteristics of fungal laccases, and provide us support for the improvement of fungal laccase activity based on N-linked glycosylation modification.