[Applications of high performance liquid chromatography-mass spectrometry in proteomics].

Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the "bottom-up" approach and the "top-down" approach. The "shotgun" method is a typical "bottom-up" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. "Top-down" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the "bottom-up" "top-down" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.

DEPDC1 as a metabolic target regulates glycolysis in renal cell carcinoma through AKT/mTOR/HIF1α pathway.

Renal cell carcinoma (RCC) is considered a "metabolic disease" characterized by elevated glycolysis in patients with advanced RCC. Tyrosine kinase inhibitor (TKI) therapy is currently an important treatment option for advanced RCC, but drug resistance may develop in some patients. Combining TKI with targeted metabolic therapy may provide a more effective approach for patients with advanced RCC. An analysis of 14 RCC patients (including three needle biopsy samples with TKI resistance) revealed by sing-cell RNA sequencing (scRNA-seq) that glycolysis played a crucial role in poor prognosis and drug resistance in RCC. TCGA-KIRC and glycolysis gene set analysis identified DEPDC1 as a target associated with malignant progression and drug resistance in KIRC. Subsequent experiments demonstrated that DEPDC1 promoted malignant progression and glycolysis of RCC, and knockdown DEPDC1 could reverse TKI resistance in RCC cell lines. Bulk RNA sequencing (RNA-seq) and non-targeted metabolomics sequencing suggested that DEPDC1 may regulate RCC glycolysis via AKT/mTOR/HIF1α pathway, a finding supported by protein-level analysis. Clinical tissue samples from 98 RCC patients demonstrated that DEPDC1 was associated with poor prognosis and predicted RCC metastasis. In conclusion, this multi-omics analysis suggests that DEPDC1 could serve as a novel target for TKI combined with targeted metabolic therapy in advanced RCC patients with TKI resistance.

Current progression in application of extracellular vesicles in central nervous system diseases.

Early diagnosis and pharmacological treatment of central nervous system (CNS) diseases has been a long-standing challenge for clinical research due to the presence of the blood-brain barrier. Specific proteins and RNAs in brain-derived extracellular vesicles (EVs) usually reflect the corresponding state of brain disease, and therefore, EVs can be used as diagnostic biomarkers for CNS diseases. In addition, EVs can be engineered and fused to target cells for delivery of cargo, demonstrating the great potential of EVs as a nanocarrier platform. We review the progress of EVs as markers and drug carriers in the diagnosis and treatment of neurological diseases. The main areas include visual imaging, biomarker diagnosis and drug loading therapy for different types of CNS diseases. It is hoped that increased knowledge of EVs will facilitate their clinical translation in CNS diseases.

ETNK2 Low-Expression Predicts Poor Prognosis in Renal Cell Carcinoma with Immunosuppressive Tumor Microenvironment.

Background: The ethanolamine kinase 2 (ETNK2) gene is implicated in carcinogenesis, but its expression and involvement in kidney renal clear cell carcinoma (KIRC) remain unknown. Methods: Initially, we conducted a pan-cancer study in which we searched the Gene Expression Profiling Interactive Analysis, the UALCAN, and the Human Protein Atlas databases to determine the expression level of the ETNK2 gene in KIRC. The Kaplan-Meier curve was then used to calculate the overall survival (OS) of KIRC patients. We then used the differentially expressed genes (DEGs) and enrichment analysis to explain the mechanism of the ETNK2 gene. Finally, the immune cell infiltration analysis was performed. Results: Although the ETNK2 gene expression was lower in KIRC tissues, the findings illustrated a link between the ETNK2 gene expression and a shorter OS time for KIRC patients. DEGs and enrichment analysis revealed that the ETNK2 gene in KIRC involved multiple metabolic pathways. Finally, the ETNK2 gene expression has been linked to several immune cell infiltrations. Conclusions: According to the findings, the ETNK2 gene plays a crucial role in tumor growth. It can potentially serve as a negative prognostic biological marker for KIRC by modifying immune infiltrating cells.

"Zero ischemia" laparoscopic partial nephrectomy by high-power GreenLight laser enucleation for renal carcinoma: A single-center experience.

BACKGROUND: Laparoscopic partial nephrectomy has been widely used in renal cell carcinoma treatment. The efficacy of GreenLight laser on Laparoscopic partial nephrectomy is still unknown. AIM: To present the first series of laparoscopic partial nephrectomy (LPN) by GreenLight laser enucleation without renal artery clamping. Due to the excellent coagulation and hemostatic properties of the laser, laser-assisted LPN (LLPN) makes it possible to perform a "zero ischemia" resection. METHODS: Fifteen patients with T1a exogenous renal tumors who received high-power GreenLight laser non-ischemic LPN in our hospital were retrospectively analyzed. All clinical information, surgical and post-operative data, complications, pathological and functional outcomes were analyzed. RESULTS: Surgery was successfully completed in all patients, and no open or radical nephrectomy was performed. The renal artery was not clamped, leading to no ischemic time. No blood transfusions were required, the average hemoglobin level ranged from 96.0 to 132.0 g/L and no postoperative complications occurred. The mean operation time was 104.3 ± 8.2 min. The postoperative removal of negative pressure drainage time ranged from 5.0 to 7.0 d, and the mean postoperative hospital stay was 6.5 ± 0.7 d. No serious complications occurred. Postoperative pathological results showed clear cell carcinoma in 12 patients, papillary renal cell carcinoma in 2 patients, and hamartoma in 1 patient. The mean creatinine level was 75.0 ± 0.8 µmol/L (range 61.0-90.4 µmol/L) at 1 mo after surgery, and there were no statistically significant differences compared with pre-operation (P > 0.05). The glomerular filtration rate ranged from 45.1 to 60.8 mL/min, with an average of 54.0 ± 5.0 mL/min, and these levels were not significantly different from those before surgery (P > 0.05). CONCLUSION: GreenLight laser has extraordinary cutting and sealing advantages when used for small renal tumors (exogenous tumors of stage T1a) during LPN. However, use of this technique can lead to the generation of excessive smoke.

Short-Term Impact of Sleep Apnea/Hypopnea on the Interaction Between Various Cortical Areas.

INTRODUCTION: Sleep apnea/hypopnea syndrome (SAHS) can change brain structure and function. These alterations are related to respiratory event-induced abnormal sleep, however, how brain activity changes during these events is less well understood. METHODS: To study information content and interaction among various cortical regions, we analyzed the variations of permutation entropy (PeEn) and symbolic transfer entropy (STE) of electroencephalography (EEG) activity during respiratory events. In this study, 57 patients with moderate SAHS were enrolled, including 2804 respiratory events. The events terminated with cortical arousal were independently researched. RESULTS: PeEn and STE were lower during apnea/hypopnea, and most of the brain interaction was higher after apnea/hypopnea termination than that before apnea in N2 stage. As indicated by STE, the respiratory events also affected the stability of information transmission mode. In N1, N2, and rapid eye movement (REM) stages, the information flow direction was posterior-to-anterior, but the anterior-to-posterior increased relatively during apnea/hypopnea. The above EEG activity trends maintained in events with cortical arousal. CONCLUSIONS: These results may be related to the intermittent hypoxia during apnea and the cortical response. Furthermore, increased frontal information outflow, which was related to the compensatory activation of frontal neurons, may associate with cognitive function.

Appropriate locations of fixed bearings of continuous beams considering rail-bridge thermal interaction.

Due to the rail-bridge thermal interaction, the high additional axial force in continuously welded rails on continuous bridges may lead to rail buckling or breaking. However, there is little research on the influence of the location of the fixed bearing of continuous beam on the additional force of rail. In order to study the influence of bridge bearing arrangement on the additional longitudinal force of CWR, the thermal interaction model is established for rail, and simple and continuous beams considering nonlinear stiffness and the methods are proposed to determine the locations of fixed bearings of continuous beams corresponding to the maximum additional forces in rail reaching minimum values. Multiple continuous beams with several different lengths and simple beams with three types of bearing arrangements are taken into account to find the effect laws of the locations of the fixed bearings of continuous beams on the maximum additional forces in rail. The results show that as long as the same number of continuous beams, the ratios of the distances of adjacent two fixed bearings to the distance between the two fixed bearings of the simple beams neighbour to the first and last continuous beams respectively are approximately equal to each other. Furthermore the appropriate locations of the fixed bearings of continuous beams are recommended. The results can guide designing the location of the fixed bearing of continuous railway bridge while reducing the additional axial force in continuously welded rails due to bridge thermal effect.

Gankyrin is a novel biomarker for disease progression and prognosis of patients with renal cell carcinoma.

BACKGROUND: In the clinic, how to stratify renal cell carcinoma (RCC) patients with different risks and to accurately predict their prognostic outcome remains a crucial issue. In this study, we assessed the expression and prognostic value of gankyrin in RCC patients. METHODS: The expression of gankyrin was examined in public databases and validated in specimens from two independent centers. The clinical practice and disease correlation of gankyrin in RCC were evaluated in RCC patients, various cell lines and an orthotopic RCC model. FINDINGS: Upregulation of gankyrin expression in RCC was corroborated in two independent cohorts. High gankyrin expression positively associated with disease progression and metastasis of RCC patients. A positive correlation between gankyrin and sunitinib-resistance was also observed in RCC cell lines and in an orthotopic RCC model. Kaplan-Meier analysis revealed that patients with higher gankyrin expression presented worse prognosis of RCC patients in the two cohorts. Gankyrin served as an independent prognostic factor for RCC patients even after multivariable adjustment by clinical variables. Time-dependent AUC and Harrell's c-index analysis presented that the incorporation of the gankyrin classifier into the current clinical prognostic parameters such as TNM stage, Fuhrman nuclear grade or SSIGN score achieved a greater accuracy than without it in predicting prognosis of RCC patients. All results were confirmed in randomized training and validation sets from the two patient cohorts. INTERPRETATION: Gankyrin can serve as a reliable biomarker for disease progression and for prognosis of RCC patients. Combining gankyrin with the current clinical parameters may help patient management. FUND: National Natural Science Foundation of China (No. 81773154, 81772747 and 81301861), Medical Discipline Construction Project of Pudong New Area Commission of Health and Family Planning (PWYgf2018-03), the Shanghai Medical Guidance (Chinese and Western Medicine) Science and Technology Support Project (No. 17411960200), Outstanding Leaders Training Program of Pudong Health Bureau of Shanghai (No. PWR12016-05).

[The development of techniques served to widen the lateral velopharyngeal space for treating OSAHS].

Since Fujita first described uvulopalatopharyngoplasty(UPPP)in 1981,UPPP and its modified procedures have been widely used to treat obstructive sleep apnea and hyponea syndrome(OSAHS).However,despite of its wide application,the success rates was uncertain,ranging from 20% to 80%,with patients of varing Friedman stages.It is well known that the principle of UPPP is to remove the redundant tissue of palate,elongated uvula and hypertrophic tonsils in order to widen the anteroposterior space at the level of palate.But recently,surgeons have found that not only the collapse of soft palate but also the collapse of lateral wall at the palate level can contribute to the obstruction of upper airway at the level of palate.As a result,many surgeries which can widen the lateral velopharyneal space have sprung up in these years.This review focuses on the development of techniques that emphasize the enlargement of lateral velopharyneal space in patients with OSAHS.

[Femoral supracondylar osteotomy for the treatment of valgus knee osteoarthritis in young and middle-aged patients].

OBJECTIVE: To evaluate the clinical efficacy of open supracondylar osteotomy of the femoral condyle for the treatment of valgus knee osteoarthritis. METHODS: From April 2008 to June 2015, 21 patients with valgus knee osteoarthritis underwent an open wedge femoral supracondylar osteotomy using the distal femur dissection plates combined with autologous iliac bone graft for the bone defect. There were 8 males (8 knees) and 13 females (15 knees), ranging in age from 30 to 54 years old, with a mean age of 41.2 years old. All the patients had valgus deformity and knee joint pain in the lateral compartment. The average tibiofemoral angle was (162.0±2.6)° which was measured on the image of preoperative lower extremity weight-bearing X-ray. Clinical outcomes were comprehensively assessed according to the bone healing time, postoperative complications, progress of knee osteoarthritis after operation, the Hospital for Special Surgery rating system (HSS), and tibiofemoral angle before and after operation. RESULTS: All 21 patients were followed up, the valgus deformity of knee joint was corrected in all patients after operation. No obvious delayed union or nonunion were found, and no serious complications were found. The HSS knee score was improved from the preoperative 57.3±3.1 to the final follow-up time 88.6±2.7. Tibiofemoral angle was improved to the postoperative(176.0±1.4)°. CONCLUSIONS: Open wedge femoral supracondylar osteotomy has a clear surgical approach, and it is easy to control the bone mass of osteotomy and can effectively correct the valgus deformity and improve the function of knee joint using this method. It is an effective method for the treatment of valgus knee osteoarthritis in young and middle-aged patients.