Protein Quantification Research Articles

Abstract 7111: Homoharringtonine is effective in treating acute lymphoblastic leukaemia via WEE1 downregulation and inducing DNA damage

Abstract Acute lymphoblastic leukaemia (ALL) is an aggressive haematolymphoid malignancy that originated from clonal mutations in lymphoid precursors. The prognosis is poor in adult patients and relapse/refractory disease is not uncommon which carries dismal prognosis. There is an urgent need to develop novel therapeutics. Homoharringtonine (HHT) is a plant alkaloid which can bind to ribosomal A-site and thus inhibit protein synthesis. It was used to treat acute myeloid leukaemia in China during 1980s. Herein, we conducted a study to explore the therapeutic efficacy and mechanism of action in ALL. T-ALL (DND-41, PEER) and B-ALL (PALL-2, SUP-B15) cell lines were treated with HHT for 24 and 72 hours. Cell viability was reduced at a time and dose-dependent manner in all cell lines we tested, with increased in percentage of apoptosis. Western blot showed upregulation of p53, with PARP and caspase 3 cleavages upon treating with HHT for 24 hours. Moreover, we observed WEE1 downregulation which allowed cell cycle progression through G2/M with damaged DNA. Comet assay showed evidence of DNA damage. Proteomics study by label-free protein quantitation showed downregulation of proteins involving in homologous recombination-mediated DNA repairing machinery, including BRCA1 and RAD51. The findings not only showed therapeutic efficacy of HHT in ALL, but also demonstrated the first evidence of DNA damage induced by HHT which might account for its therapeutic effects. Further studies will be conducted for delineating the mechanisms of DNA damage and its relationship with therapeutic efficacy. Citation Format: Chun-fung Sin, Kei-ching Yuen, Anan Jiao, Bai-lin Li, Hau-wai Yu. Homoharringtonine is effective in treating acute lymphoblastic leukaemia via WEE1 downregulation and inducing DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7111.

Less is more: Validating a single method for comprehensive rh-insulin analysis

The objective of this current study is to establish a single method for potency and related proteins analysis of human insulin formulations using reverse-phase high performance liquid (RP-HPLC) chromatography technique which was validated and verified for the potency analysis in insulin formulations. Chromatographic separation was achieved using an octadecylsilane (C-18) stationary phase and a mobile phase composed of 55% (v/v) buffer (0.2 M sodium sulfate in water, {pH 2.3}) and 45% (v/v) acetonitrile. Detection was performed by UV detector at 214 nm with a flow rate of 1 ml/min and an injection volume of 20 µL, at 40°C. Currently there are separate methods available in Indian Pharmacopoeia for analysis of Potency and Related proteins in human insulin. We have validated a single method where quantitation of potency and related proteins can be performed in the same run. The method validation exhibited linearity over the concentration range of 0.08–4.5 mg/ml (r2=0.999) with limit of detection of 0.094 mg/ml The accuracy of the method was 99–102.8%. Thus, it is proposed that both potency and related proteins in insulin formulations can be precisely evaluated using a single run thus saving the time and cost for quality analysis of insulin preparations both at manufacturing and regulatory laboratories which in turn will increase the market availability of such standard quality insulin preparations for public health use.

Platycodi Radix Extract Prevents Hepatic Steatosis by Enhancing Bile Acid Synthesis in a High-Fat Diet-Induced Fatty Liver Mouse Model.

We aimed to identify the mechanism underlying the preventive effects of non-alcoholic fatty liver disease (NAFLD) through Platycodi Radix consumption using liver proteomic and bioinformatic analysis. C57BL/6J mice were categorized into three groups: those receiving a standard chow diet (NCD), those on a high-fat diet (HFD), and those on an HFD supplemented with 5% Platycodi Radix extract (PRE). After a 12-week period, PRE-fed mice exhibited a noteworthy prevention of hepatic steatosis. Protein identification and quantification in liver samples were conducted using LC-MS/MS. The identified proteins were analyzed through Ingenuity Pathway Analysis software, revealing a decrease in proteins associated with FXR/RXR activation and a concurrent increase in cholesterol biosynthesis proteins in the PRE-treated mouse liver. Subsequent network analysis predicted enhanced bile acid synthesis from these proteins. Indeed, the quantity of bile acids, which was reduced in HFD conditions, increased in the PRE group, accompanied by an elevation in the expression of synthesis-related proteins. Our findings suggest that the beneficial effects of PRE in preventing hepatic steatosis may be mediated, at least in part, through the modulation of FXR/RXR activation, cholesterol biosynthesis, and bile acid synthesis pathways.

A universal molecular control for DNA, mRNA and protein expression

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Quantifying Bound Proteins on Pegylated Gold Nanoparticles Using Infrared Spectroscopy.

Protein-nanoparticle (NP) complexes are nanomaterials that have numerous potential uses ranging from biosensing to biomedical applications such as drug delivery and nanomedicine. Despite their extensive use quantifying the number of bound proteins per NP remains a challenging characterization step that is crucial for further developments of the conjugate, particularly for metal NPs that often interfere with standard protein quantification techniques. In this work, we present a method for quantifying the number of proteins bound to pegylated thiol-capped gold nanoparticles (AuNPs) using an infrared (IR) spectrometer, a readily available instrument. This method takes advantage of the strong IR bands present in proteins and the capping ligands to quantify protein-NP ratios and circumvents the need to degrade the NPs prior to analysis. We show that this method is generalizable where calibration curves made using inexpensive and commercially available proteins such as bovine serum albumin (BSA) can be used to quantify protein-NP ratios for proteins of different sizes and structures.

Advancing High-Throughput MS-Based Protein Quantification: A Case Study on Quantifying 10 Major Food Allergens by LC-MS/MS Using a One-Sample Multipoint External Calibration Curve.

The LC-MS-based method has emerged as the preferred approach for quantifying food allergens. However, the preparation of a traditional calibration curve (MSCC) is labor-intensive and error-prone. Here, a sensitive and robust LC-MS/MS method for quantifying 10 major food allergens was developed and validated, where the one-sample multipoint external calibration curve (OSCC) was employed instead of MSCC. By employing the multiple isotopologue reaction monitoring (MIRM) technique with only one spiked level in the blank, OSCC can be effectively established. Results demonstrate that the proposed method exhibits excellent performance in selectivity, sensitivity, accuracy, and precision, comparable to that of the traditional MSCC. Additionally, this strategy allows for isotope sample dilution by monitoring the less abundant MIRM channel. Moreover, the developed method was successfully applied to investigate the contamination of 10 food allergens in commercial food products. With its high throughput and robustness, the MIRM-OSCC-LC-MS/MS methodology has many potential applications, especially in the MS-based protein quantification analysis.

Analysis of metabolic compounds and antitumorigenic effects of Albizia niopoides and Senegalia polyphylla leaves

The objective of this study was to quantify metabolic compounds in leaves of A. niopoides and S. polyphylla and to evaluate the antitumor potential of extracts from both species in cervical tumour cells. The physiological analyses performed were quantification of starch, sucrose, phenolic compounds and proteins. An aqueous extract was prepared and added to the SiHa cell line at concentrations of 10, 100 and 1000 μg/mL at 4h, 24h, 48h and 72h. Cell morphology, proliferation and viability were analysed. The species showed a large amount of starch and phenolic compounds. Treatment with the extract of both species caused morphological changes in SiHa cells and exhibited antiproliferative effects at a concentration of 1000 µg/ml. In cell viability test, only A. niopoides showed a significant reduction. The study presented the effects of the species against a cervical cancer cell line, where A. niopoides has already shown to be a promising plant drug.

Prospective Multicenter Study on Early Proximal Tubular Injury in COVID-19–Related Acute Respiratory Distress Syndrome

IntroductionDuring COVID-19, renal impairment is the most frequent after lung impairment and is associated with poor prognosis particularly in intensive care unit (ICU). We aimed to assess the existence and incidence of early renal dysfunction and its prognostic value in patients with COVID-19-related acute respiratory distress syndrome (ARDS). MethodsIn this prospective multicenter study, patients aged over 18 years with invasive mechanical ventilation for ARDS were enrolled in 3 ICUs. Precise evaluation of renal dysfunction markers including urinary proteins electrophoresis (UPE) and quantification, was performed within 24 hours after mechanical ventilation onset. ResultsFrom March 2020 to December 2021, 135 patients were enrolled: 100 COVID-19 ARDS and 35 non-COVID-19 ARDS. UPE found more tubular dysfunction in COVID-19 patients (68% vs. 21.4%, p<0.0001) and more normal profiles in non-COVID-19 patients (65.0% vs. 11.2%, p=0.0003). COVID-19 patients significantly displayed early urinary leakage of tubular proteins like beta-2-microglobulin and free-light chains, tended to display more frequently acute kidney injury (AKI) (51.0% vs 34.3%, p=0.088), had longer mechanical ventilation (20 vs. 9 days, p<0.0001) and longer ICU stay (26 vs. 15 days, p<0.0001). In COVID-19 ARDS, leakage of free lambda light chain was associated with the onset of KDIGO ≥2 AKI (OR: 1.014, 95%CI [1.003-1.025], p=0.011). ConclusionsPatients with COVID-19-related ARDS display a proximal tubular dysfunction, prior to the onset of AKI, which predicts AKI. Proximal tubular damage seems an important mechanism of COVID-19-induced nephropathy. Analysis of urinary proteins is a reliable non-invasive tool to assess proximal tubular dysfunction in the ICU.

Estimation and Association of Total Protein Concentration with Early Childhood Caries in 3-6-year-old Children: A Randomized Clinical Trial.

Caries is a common problem witnessed in children, early childhood caries (ECC) is the most predominant chronic disease which not only leads to distress and pain but also poor quality of life, thus affecting the overall well-being of children. Salivary protein plays a vital part in monitoring health status or disease. It was stated that the salivary proteins could regulate the equilibrium of oral health, preserve a stable ecosystem, and constrain the growth of cariogenic bacteria. The aim of this study is to estimate the total protein concentration in saliva and its correlation to ECC. A total of 20 patients with ECC in the age-group of 3-6 years were selected as the experimental group and 20 patients without caries for the control group. Unstimulated saliva samples were collected and subjected to spectrophotometry. The data obtained was subjected to statistical analysis. Independent student's t-test was used for the comparison of mean salivary pH between the caries group and the control group. Mann-Whitney test was used for a comparison of salivary total protein concentrations between the two groups. The mean pH of the carious group showed a statistically significant slightly lower value than that of the noncarious group. On the contrary, the mean total protein concentration of the carious group presented a statistically significant higher value than that of the noncarious group. Age-wise comparison of mean salivary proteins in the carious group and the noncarious group showed an increase in the protein concentration in the children aged 4 years or younger. Based on the results of this study, it can be concluded that there is a strong association between the total protein concentration in saliva and ECC. There exists a significant increase in the total protein concentration in children with ECC. As age increases, total protein concentration decreases with age. Total protein concentration and particular protein estimation and quantification help us in assessing the risk of caries in children at the earliest and prevention of caries through preventive measures. Estimation of total salivary protein concentration in children can be a marker for ECC in children. Thimmegowda U, Pai S, Chikkanarasaiah N, et al. Estimation and Association of Total Protein Concentration with Early Childhood Caries in 3-6-year-old Children: A Randomized Clinical Trial. Int J Clin Pediatr Dent 2024;17(1):36-40.

Neat plasma proteomics: getting the best out of the worst

Plasma proteomics holds immense potential for clinical research and biomarker discovery, serving as a non-invasive "liquid biopsy" for tissue sampling. Mass spectrometry (MS)-based proteomics, thanks to improvement in speed and robustness, emerges as an ideal technology for exploring the plasma proteome for its unbiased and highly specific protein identification and quantification. Despite its potential, plasma proteomics is still a challenge due to the vast dynamic range of protein abundance, hindering the detection of less abundant proteins. Different approaches can help overcome this challenge. Conventional depletion methods face limitations in cost, throughput, accuracy, and off-target depletion. Nanoparticle-based enrichment shows promise in compressing dynamic range, but cost remains a constraint. Enrichment strategies for extracellular vesicles (EVs) can enhance plasma proteome coverage dramatically, but current methods are still too laborious for large series. Neat plasma remains popular for its cost-effectiveness, time efficiency, and low volume requirement. We used a test set of 33 plasma samples for all evaluations. Samples were digested using S-Trap and analyzed on Evosep One and nanoElute coupled to a timsTOF Pro using different elution gradients and ion mobility ranges. Data were mainly analyzed using library-free searches using DIA-NN. This study explores ways to improve proteome coverage in neat plasma both in MS data acquisition and MS data analysis. We demonstrate the value of sampling smaller hydrophilic peptides, increasing chromatographic separation, and using library-free searches. Additionally, we introduce the EV boost approach, that leverages on the extracellular vesicle fraction to enhance protein identification in neat plasma samples. Globally, our optimized analysis workflow allows the quantification of over 1000 proteins in neat plasma with a 24SPD throughput. We believe that these considerations can be of help independently of the LC–MS platform used.

Cyanobacterial Proteomics: Diversity and Dynamics.

Cyanobacteria (oxygenic photoautrophs) comprise a diverse group holding significance both environmentally and for biotechnological applications. The utilization of proteomic techniques has significantly influenced investigations concerning cyanobacteria. Application of proteomics allows for large-scale analysis of protein expression and function within cyanobacterial systems. The cyanobacterial proteome exhibits tremendous functional, spatial, and temporal diversity regulated by multiple factors that continuously modify protein abundance, post-translational modifications, interactions, localization, and activity to meet the dynamic needs of these tiny blue greens. Modern mass spectrometry-based proteomics techniques enable system-wide examination of proteome complexity through global identification and high-throughput quantification of proteins. These powerful approaches have revolutionized our understanding of proteome dynamics and promise to provide novel insights into integrated cellular behavior at an unprecedented scale. In this Review, we present modern methods and cutting-edge technologies employed for unraveling the spatiotemporal diversity and dynamics of cyanobacterial proteomics with a specific focus on the methods used to analyze post-translational modifications (PTMs) and examples of dynamic changes in the cyanobacterial proteome investigated by proteomic approaches.

Orosomucoid 2 upregulation mediates liver injury-induced colorectal cancer liver metastasis by promoting EMT and cell migration.

The relationship between drug-induced liver injury and liver metastasis of colorectal cancer and the underlying mechanisms are not well understood. In this study, we used carbon tetrachloride to construct a classic mouse liver injury model and injected CT26 colorectal cancer cells into the mouse spleen to simulate the natural route of colorectal cancer liver metastasis. Liver injury significantly increased the number of colorectal cancer liver metastases. Transcriptome sequencing and data-independent acquisition protein quantification identified proteins that were significantly differentially expressed in injured livers, and orosomucoid (ORM) 2 was identified as a target protein for tumor liver metastasis. Invitro experiments showed that exogenous ORM2 protein increased the expression of EMT markers such as Twist, Zeb1, Vim, Snail1 and Snail2 and chemokine ligands to promote CT26 cell migration. In addition, liver-specific overexpression of the ORM2 protein in the mouse model significantly promoted tumor cell liver metastasis without inducing liver injury. Our results indicate that drug-induced liver injury can promote colorectal cancer liver metastasis and that ORM2 can promote cell migration by inducing EMT in tumor cells.

VPBrowse: Genome-based representation of MS/MS spectra to quantify 10,000 bovine proteins.

SWATH is a data acquisition strategy acclaimed for generating quantitatively accurate and consistent measurements of proteins across multiple samples. Its utility for proteomics studies in nonlaboratory animals, however, is currently compromised by the lack of sufficiently comprehensive and reliable public libraries, either experimental or predicted, and relevant platforms that support their sharing and utilization in an intuitive manner. Here we describe the development of the Veterinary Proteome Browser, VPBrowse (http://browser.proteo.cloud/), an on-line platform for genome-based representation of the Bos taurus proteome, which is equipped with an interactive database and tools for searching, visualization, and building quantitative mass spectrometry assays. In its current version (VPBrowse 1.0), it contains high-quality fragmentation spectra acquired on QToF instrument for over 36,000 proteotypic peptides, the experimental evidence for over 10,000 proteins. Data can be downloaded in different formats to enable analysis using popular software packages for SWATH data processing whilst normalization to iRT scale ensures compatibility with diverse chromatography systems. When applied to published blood plasma dataset from the biomarker discovery study, the resource supported label-free quantification of additional proteins not reported by the authors previously including PSMA4, a tissue leakage protein and a promising candidate biomarker of animal's response to dehorning-related injury.

AlphaPept: a modern and open framework for MS-based proteomics

In common with other omics technologies, mass spectrometry (MS)-based proteomics produces ever-increasing amounts of raw data, making efficient analysis a principal challenge. A plethora of different computational tools can process the MS data to derive peptide and protein identification and quantification. However, during the last years there has been dramatic progress in computer science, including collaboration tools that have transformed research and industry. To leverage these advances, we develop AlphaPept, a Python-based open-source framework for efficient processing of large high-resolution MS data sets. Numba for just-in-time compilation on CPU and GPU achieves hundred-fold speed improvements. AlphaPept uses the Python scientific stack of highly optimized packages, reducing the code base to domain-specific tasks while accessing the latest advances. We provide an easy on-ramp for community contributions through the concept of literate programming, implemented in Jupyter Notebooks. Large datasets can rapidly be processed as shown by the analysis of hundreds of proteomes in minutes per file, many-fold faster than acquisition. AlphaPept can be used to build automated processing pipelines with web-serving functionality and compatibility with downstream analysis tools. It provides easy access via one-click installation, a modular Python library for advanced users, and via an open GitHub repository for developers.

Evaluation of cell disruption methods for protein and coenzyme Q10 quantification in purple non-sulfur bacteria.

A recent focus has been on the recovery of single-cell protein and other nutritionally valuable bioproducts, such as Coenzyme Q10 (CoQ10) from purple non-sulfur bacteria (PNSB) biomass following wastewater treatment. However, due to PNSB's peculiar cell envelope (e.g., increased membrane cross-section for energy transduction) and relatively smaller cell size compared to well-studied microbial protein sources like yeast and microalgae, the effectiveness of common cell disruption methods for protein quantification from PNSB may differ. Thus, this study examines the efficiency of selected chemical (NaOH and EDTA), mechanical (homogenization and bead milling), physical (thermal and bath/probe sonication), and combined chemical-mechanical/physical treatment techniques on the PNSB cell lysis. PNSB biomass was recovered from the treatment of gas-to-liquid process water. Biomass protein and CoQ10 contents were quantified based on extraction efficiency. Considering single-treatment techniques, bead milling resulted in the best protein yields (p < 0.001), with the other techniques resulting in poor yields. However, the NaOH-assisted sonication (combined chemical/physical treatment technique) resulted in similar protein recovery (p = 1.00) with bead milling, with the former having a better amino acid profile. For example, close to 50% of the amino acids, such as sensitive ones like tryptophan, threonine, cystine, and methionine, were detected in higher concentrations in NaOH-assisted sonication (>10% relative difference) compared to bead-milling due to its less disruptive nature and improved solubility of amino acids in alkaline conditions. Overall, PNSB required more intensive protein extraction techniques than were reported to be effective on other single-cell organisms. NaOH was the preferred chemical for chemical-aided mechanical/physical extraction as EDTA was observed to interfere with the Lowry protein kit, resulting in significantly lower concentrations. However, EDTA was the preferred chemical agent for CoQ10 extraction and quantification. CoQ10 extraction efficiency was also suspected to be adversely influenced by pH and temperature.

ProTInSeq: transposon insertion tracking by ultra-deep DNA sequencing to identify translated large and small ORFs

Identifying open reading frames (ORFs) being translated is not a trivial task. ProTInSeq is a technique designed to characterize proteomes by sequencing transposon insertions engineered to express a selection marker when they occur in-frame within a protein-coding gene. In the bacterium Mycoplasma pneumoniae, ProTInSeq identifies 83% of its annotated proteins, along with 5 proteins and 153 small ORF-encoded proteins (SEPs; ≤100 aa) that were not previously annotated. Moreover, ProTInSeq can be utilized for detecting translational noise, as well as for relative quantification and transmembrane topology estimation of fitness and non-essential proteins. By integrating various identification approaches, the number of initially annotated SEPs in this bacterium increases from 27 to 329, with a quarter of them predicted to possess antimicrobial potential. Herein, we describe a methodology complementary to Ribo-Seq and mass spectroscopy that can identify SEPs while providing other insights in a proteome with a flexible and cost-effective DNA ultra-deep sequencing approach.

Single Isotopologue for In-Sample Calibration and Absolute Quantitation by LC-MS/MS.

Targeted mass spectrometry (MS)-based absolute quantitative analysis has been increasingly used in biomarker discovery. The ability to accurately measure the masses by MS enabled the use of isotope-incorporated surrogates having virtually identical physiochemical properties with the target analytes as calibrators. Such a unique capacity allowed for accurate in-sample calibration. Current in-sample calibration uses multiple isotopologues or structural analogues for both the surrogate and the internal standard. Here, we simplified this common practice by using endogenous light peptides as the internal standards and used a mathematical deduction of "heavy matching light, HML" to directly quantify an endogenous analyte. This method provides all necessary assay performance parameters in the authentic matrix, including the lower limit of quantitation (LLOQ) and intercept of the calibration curve, by using only a single isotopologue of the analyte. This method can be applied to the quantitation of proteins, peptides, and small molecules. Using this method, we quantified the efficiency of heart tissue digestion and recovery using sodium deoxycholate as a detergent and two spiked exogenous proteins as mimics of heart proteins. The results demonstrated the robustness of the assay.

Evaluation of serum and urine biomarkers for severe COVID-19.

The new coronavirus disease, COVID-19, poses complex challenges exacerbated by several factors, with respiratory tissue lesions being notably significant among them. Consequently, there is a pressing need to identify informative biological markers that can indicate the severity of the disease. Several studies have highlighted the involvement of proteins such as APOA1, XPNPEP2, ORP150, CUBN, HCII, and CREB3L3 in these respiratory tissue lesions. However, there is a lack of information regarding antibodies to these proteins in the human body, which could potentially serve as valuable diagnostic markers for COVID-19. Simultaneously, it is relevant to select biological fluids that can be obtained without invasive procedures. Urine is one such fluid, but its effect on clinical laboratory analysis is not yet fully understood due to lack of study on its composition. Methods used in this study are as follows: total serum protein analysis; ELISA on moderate and severe COVID-19 patients' serum and urine; bioinformatic methods: ROC analysis, PCA, SVM. The levels of antiAPOA1, antiXPNPEP2, antiORP150, antiCUBN, antiHCII, and antiCREB3L3 exhibit gradual fluctuations ranging from moderate to severe in both the serum and urine of COVID-19 patients. However, the diagnostic value of individual anti-protein antibodies is low, in both blood serum and urine. On the contrary, joint detection of these antibodies in patients' serum significantly increases the diagnostic value as demonstrated by the results of principal component analysis (PCA) and support vector machine (SVM). The non-linear regression model achieved an accuracy of 0.833. Furthermore, PCA aided in identifying serum protein markers that have the greatest impact on patient group discrimination. The study revealed that serum serves as a superior analyte for describing protein quantification due to its consistent composition and lack of organic salts and drug residues, which can otherwise affect protein stability.

BAP31 Promotes Angiogenesis via Galectin-3 Upregulation in Neuroblastoma.

Neuroblastoma (NB) is one of the highly vascularized childhood solid tumors, and understanding the molecular mechanisms underlying angiogenesis in NB is crucial for developing effective therapeutic strategies. B-cell receptor-associated protein 31 (BAP31) has been implicated in tumor progression, but its role in angiogenesis remains unexplored. This study investigated BAP31 modulation of pro-angiogenic factors in SH-SY5Y NB cells. Through protein overexpression, knockdown, antibody blocking, and quantification experiments, we demonstrated that overexpression of BAP31 led to increased levels of vascular endothelial growth factor A (VEGFA) and Galectin-3 (GAL-3), which are known to promote angiogenesis. Conditioned medium derived from BAP31-overexpressing neuroblastoma cells stimulated migration and tube formation in endothelial cells, indicating its pro-angiogenic properties. Also, we demonstrated that BAP31 enhances capillary tube formation by regulating hypoxia-inducible factor 1 alpha (HIF-1α) and its downstream target, GAL-3. Furthermore, GAL-3 downstream proteins, Jagged 1 and VEGF receptor 2 (VEGFR2), were up-regulated, and blocking GAL-3 partially inhibited the BAP31-induced tube formation. These findings suggest that BAP31 promotes angiogenesis in NB by modulating GAL-3 and VEGF signaling, thereby shaping the tumor microenvironment. This study provides novel insights into the pro-angiogenic role of BAP31 in NB.

Urinary protein and coagulation-fibrinolysis indicators in preeclampsia: Expression and significance.

This study investigates the expression and significance of urinary protein and coagulation-fibrinolysis indicators in preeclampsia, categorized into mild preeclampsia (109 cases) and severe preeclampsia (97 cases) based on disease severity. Additionally, 110 patients with gestational hypertension (gestational hypertension group) were included for comparative analysis. General information, laboratory indicators, urinary protein, and coagulation-fibrinolysis indicator levels were collected for each group. Significant differences were observed in blood pressure among groups (P<.05), while uric acid, serum creatinine, aspartate transaminase, alanine transaminase, and triglycerides showed no significant differences (P>.05). Total cholesterol, triglycerides, and low-density Lipoprotein levels in severe preeclampsia were higher than those in mild preeclampsia and gestational hypertension groups, whereas high-density lipoprotein, albumin, and platelet levels were lower in severe preeclampsia. No significant differences were observed in prothrombin time or D-dimer levels among groups (P>.05). Urinary protein, urinary protein quantification, activated partial thromboplastin time, thrombin time, and fibrinogen were identified as influencing factors for adverse maternal and infant outcomes in severe preeclampsia patients. The study concludes that urinary protein and coagulation-fibrinolysis indicators are elevated in preeclampsia, particularly in severe preeclampsia cases, suggesting their potential use as diagnostic influencing factors for severe preeclampsia.