Strategy for Microscale Extraction and Proteome Profiling of Peripheral Blood Mononuclear Cells.

Peripheral blood mononuclear cells (PBMCs) play vital roles in physiological and pathological processes and represent a rich source for disease monitoring. Typical molecular profiling on PBMCs involves the sorting of cell subsets and thus requires a large volume of peripheral blood (PB), which impedes the clinical practicability of omics tools in PBMC measurements. It would be clinically invaluable to develop a convenient approach for preparing PBMCs from small volumes of PB and for deep proteome profiling of PBMCs. To this end, here, we designed an apparatus (PBMC-mCap) for microscale enrichment and proteome analysis of PBMCs, which pushed the needed PB volume from the normal 2 mL or higher to 100 µL or lower, comparable to the volume of a drop of finger blood. A PBMC-specific mass spectra library containing 8869 proteins and 121,956 peptides was further built, which, in combination with the optimized data-independent acquisition strategy, helped to identify 6000 and 6500 proteins from PBMCs with 100 µL and 1 mL of PB as initial materials, respectively. Further application of the strategy for PBMC proteomes revealed a steady difference between gender (male vs female) and upon stimulus (COVID-19 vaccination). For the latter, we observed differentially expressed genes and pathways involving the activation of immune cells, including the NF-κB pathway, inflammation response, and antiviral response. Our strategy for the proteome analysis of microscale PBMCs may provide a convenient clinical toolkit for disease diagnosis and healthy state monitoring.

[Efficacy of Chinese Herbs in Multiple Paths for Tubal Factor Infertility Patients and Their Effects on Serum Inflammatory Factors].

Objective To evaluate the clinical efficacy of Chinese herbs in multiple paths for tub- al factor infertility (TFI) patients, and to observe their effects on serum inflammatory factor. Methods Totally 100 TFl patients were assigned to the observation group and the control group according to grouping sequence, 50 in each group. All patients received laparoscopy. Patients in the observation group were additionally combined with traditional Chinese herbal treatment program in multiple paths (oral administration of Chinese herbs + retention enema of Chinese herbs + iontophoresis). All treatment lasted for 3 successive months. Scores of Chinese medicine (CM) symptoms, clinical efficacy, pregnancy rate, and levels of interleukin-6 ( IL-6) and tumor necrosis factor-α ( TNF-α) were compared between the two groups after 6 months of treatment. Results Scores of CM symptoms were significantly lower in the two groups after treatment ( P <0. 05). They were lower in the observation group than in the control group (P <0. 05). Serum levels of TNF-a and IL-6 were significantly lower in the observation group than in the control group, with statistical difference (P <0. 05). The effective rate was 96. 0% (48/50) in the observation group and 82. 0% (41/50) in the control group, with statistical difference (x² =5. 005, P <0. 05). After one year of postoperative follow-up, the intrauterine pregnancy rate was 58. 0% (29/50) in the observation group and 34. 0% (17/50) in the control group, with statistical difference (x² =5.797, P <0. 05). The ectopic gestation occurred in one patient of the control group, and none in the observation group, with no statistical difference in the ectopic gestation rate between the two groups (x² =1. 010, P >0. 05). Conclusion Chinese herbs in multiple paths for treating TFI could significantly improve clinical symptoms, reduce expressions of serum inflammatory factors, and elevate efficacy and pregnancy rate, which showed superiority when compared with laparoscopy alone.

The Frictional Vibration Attenuation of Rubber Utilizing a Groove on the Body.

Frictionally induced vibrations in rubber are readily triggered due to their lower stiffness and higher elasticity. This study developed a numerical model to investigate the frictional vibration of a rubber block with a groove on its side surface against an aluminum disc. The results indicate that a backside groove (GB) on the block significantly enhances vibration attenuation, with a decay time 0.6 s faster than a non-grooved (NG) block, despite a potentially higher initial vibrational amplitude. In contrast, a frontside groove (GF) results in persistent frictional oscillations, with the steady-state time being similar for both GB and GF configurations. The underlying mechanism is attributed to the GB's effectiveness in reducing the maximum energy imparted to the block initially, dissipating vibrational energy more swiftly, and distributing the contact stress more uniformly. The discrepancies in frictional forces between the conducted experiment and the simulation for the NG, GB and GF cases were 11.3%, 9.3% and 12.1%, respectively, quantitatively indicating the moderate precision of the results from the simulation. The insights gained from this study hold promise for enriching methods of mitigating vibrations arising from rubber friction.

Autophagy and biotransformation affect sorafenib resistance in hepatocellular carcinoma.

As sorafenib is a first-line drug for treating advanced hepatocellular carcinoma, sorafenib resistance has historically attracted attention. However, most of this attention has been focused on a series of mechanisms related to drug resistance arising after sorafenib treatment. In this study, we used proteomic techniques to explore the potential mechanisms by which pretreatment factors affect sorafenib resistance. The degree of redundant pathway PI3K/AKT activation, biotransformation capacity, and autophagy level in hepatocellular carcinoma patients prior to sorafenib treatment might affect their sensitivity to sorafenib, in which ADH1A and STING1 are key molecules. These three factors could interact mechanistically to promote tumor cell survival, might be malignant features of tumor cells, and are associated with hepatocellular carcinoma prognosis. Our study suggests possible avenues of therapeutic intervention for patients with sorafenib-resistance and the potential application of immunotherapy with the aim of improving the survival of such patients.

Proteomic overview of hepatocellular carcinoma cell lines and generation of the spectral library.

Cell lines are extensively used tools, therefore a comprehensive proteomic overview of hepatocellular carcinoma (HCC) cell lines and an extensive spectral library for data independent acquisition (DIA) quantification are necessary. Here, we present the proteome of nine commonly used HCC cell lines covering 9,208 protein groups, and the HCC spectral library containing 253,921 precursors, 168,811 peptides and 10,098 protein groups. The proteomic overview reveals the heterogeneity between different cell lines, and the similarity in proliferation and metastasis characteristics and drug targets-expression with tumour tissues. The HCC spectral library generating consumed 108 hours' runtime for data dependent acquisition (DDA) of 48 runs, 24 hours' runtime for database searching by MaxQuant version 2.0.3.0, and 1 hour' runtime for processing by SpectronautTM version 15.2. The HCC spectral library supports quantification of 7,637 protein groups of triples 2-hour DIA analysis of HepG2 and discovering biological alteration. This study provides valuable resources for HCC cell lines and efficient DIA quantification on LC-Orbitrap platform, further help to explore the molecular mechanism and candidate therapeutic targets.

YTHDF2 Inhibits the Migration and Invasion of Lung Adenocarcinoma by Negatively Regulating the FAM83D-TGFß1-SMAD2/3 Pathway.

OBJECTIVE: YTH domain family 2 (YTHDF2) is an important N6-methyladenosine (m6A) reader, but its role in lung adenocarcinoma remains elusive. This study assessed its function in lung adenocarcinoma. METHODS: YTHDF2 expression in lung adenocarcinoma was explored using public databases, such as The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumour Analysis Consortium (CPTAC). The effect of YTHDF2 on a lung adenocarcinoma cell line was explored by performing cytological and molecular experiments. Molecules downstream of YTHDF2 were identified using proteomics, and the related pathways were verified through cytological and molecular biology experiments. RESULTS: YTHDF2 expression was upregulated in lung adenocarcinoma, and patients with high YTHDF2 expression experienced prolonged overall survival. In two lung cancer cell lines, YTHDF2 knockdown inhibited proliferation but promoted migration, invasion, and the epithelial-mesenchymal transition. The proteomic analysis identified 142 molecules downstream of YTHDF2, and 11 were closely related to survival. Further experiments revealed that YTHDF2 inhibited expression of the family with sequence similarity 83D (FAM83D)-TGFß1-SMAD2/3 pathway components. This study is the first to show that YTHDF2 regulated the downstream TGFß1-SMAD2/3 pathway through FAM83D in lung adenocarcinoma. CONCLUSION: YTHDF2 inhibits the migration and invasion of lung adenocarcinoma cells by regulating the FAM83D-TGFß1-pSMAD2/3 pathway, which may play an important role in lung cancer metastasis.

Optimised data-independent acquisition strategy recaptures the classification of early-stage hepatocellular carcinoma based on data-dependent acquisition.

Proteomics is increasingly used for exploring disease biomarkers and therapeutic targets. The data-independent acquisition (DIA) method collects all peptide signals in a sample, and provides a convenient way to archive disease-related molecular features for further exploration. In this study, we first established a high-coverage human hepatocellular carcinoma (HCC) spectral library containing 9393 protein groups, 119,903 peptides. Furthermore, we optimised the DIA method with respect to four key parameters: settings for mass spectrometry acquisition, gradient length, amount of sample loading, and length of analytical column. More than 6000 proteins from HepG2 cells could be stably quantified using the optimised one-shot DIA approach with a 2 h gradient time. One-shot DIA identified a similar number of proteins as did multi-fraction data-dependent acquisition (DDA) from the same group of HCC samples, but at a quarter of the total acquisition time. DIA data could recapture the classification results obtained from DDA data, thus paving the way for large-scale, multi-centre proteomics analysis of clinical samples. SIGNIFICANCE: The organ-specific spectral library for HCC and the optimised 2 h DIA approach met the urgent demands for large-scale quantitative proteomics analysis of HCC clinical samples. Compared with multi-fraction-DDA, the optimised one-shot DIA could reach a similar identification while consuming shorter acquisition time, thus making it possible to analyse thousands of clinical samples.