Protein Extracts Research Articles

Preparation and extraction of chorion proteins from Salmo salar embryos at the pigmented eye stage for electrophoresis with SDS-polyacrylamide gel

The chorion fulfills important functions in fish embryos, including protecting the embryo during development. The characterization of the protein profile of this envelope could be used as a bioindicator in the evaluation of the quality of embryonic development. The object of this work was to validate a standardized protocol for protein extraction from chorion of Salmo salar embryos at 280 accumulated thermal units (ATU) by comparing and combining existing methods. The protocol consists of consecutive washing of the chorion samples followed by protein extraction with the solution that was named SDS solution (Tris HCl 100 mM (pH 8), Urea 8 M, 1% SDS, β-mercaptoethanol 300 mM and EGTA 10 Mm, and 1% protease inhibitor cocktail) and mechanical methods. Protein extraction is enhanced by a working temperature of 75°C and use of a disperser. The protein concentration was quantified by Bradford Assay. After extraction, the samples were diluted (dilution factor 10) before reading against the calibration curve. After gel electrophoresis with a load of 3 µg of protein, staining showed more than 4 bands, with molecular weights between 25 kDa and 180 kDa.• The protein profile of fish chorion was between 25 kDa and 180 kDa.• Solution containing 1% SDS allows a higher extraction of proteins from the chorion of Atlantic salmon embryos with 280 ATU.

Technology Prospection of Ora-pro-Nóbis (Pereskia aculeata Mill.): A Non-conventional Food Plant.

The ora-pro-nóbis (Pereskia aculeata Mill.) is a plant from Brazilian biodiversity used for food and medicinal purposes. It has ample technological potential, however, it is still underutilized, being classified as a Non-Conventional Food Plant (PANC). Prospective studies in intellectual property banks make it possible to expand perspectives for scientific research, enhancing the generation of new products. Evaluate the patents of products containing Pereskia aculeata Mill. for the areas of food and health in intellectual property databases. The study was conducted through structured prospective investigation (collection, processing and analysis) in 4 patent databases: National Institute of Intellectual Property (INPI) - Brazil, United States Patent and Trademark Office, World Trade Organization Intellectual Property (WIPO) and Espacenet. The evaluation showed a reduced number of registered patents. In general, 8 patent applications were examined, of which 7 are directly associated with the species (and its derivatives) and 1 is related to a device specially designed for harvesting leaves/fruits and removing thorns. The focus of the patents was the use of the species in the food, pharmaceutical and biotechnological areas, with emphasis on the use of the leaves in the extraction of mucilage and proteins. This study showed that Pereskia aculeata Mill. is a technologically promising plant, because of its nutritional and medicinal composition, and it is important to encourage innovation and the development of new products with the species.

Determination of vancomycin and meropenem in serum and synovial fluid of patients with prosthetic joint infections using UPLC-MS/MS.

Numerous studies have suggested that intra-articular administration of antibiotics following primary revision surgery may be one of the methods for treating prosthetic joint infection (PJI). Vancomycin and meropenem are the two most commonly used antibiotics for local application. Determining the concentrations of vancomycin and meropenem in the serum and synovial fluid of patients with PJI plays a significant role in further optimizing local medication schemes and effectively eradicating biofilm infections. This study aimed to establish a rapid, sensitive, and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining the concentrations of vancomycin and meropenem in human serum and synovial fluid. Serum samples were processed using acetonitrile precipitation of proteins and dichloromethane extraction, while synovial fluid samples were diluted before analysis. Chromatographic separation was achieved in 6min on a Waters Acquity UPLC BEH C18 column, with the mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Quantification was carried out using a Waters XEVO TQD triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) mode was employed to detect the following quantifier ion transitions: 717.95-99.97 (norvancomycin), 725.90-100.04 (vancomycin), 384.16-67.99 (meropenem). The method validation conformed to the guidelines of the FDA and the Chinese Pharmacopoeia. The method demonstrated good linearity within the range of 0.5-50 μg/ml for serum and 0.5-100 μg/ml for synovial fluid. Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, matrix effect, and stability validation results all met the required standards. This method has been successfully applied in the pharmacokinetic/pharmacodynamic (PK/PD) studies of patients with PJI.

Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

Modified xanthan gum (natural polymeric material) and its derivative for wastewater treatment: A review

AbstractNaturally occurring xanthan gum (XG) has much commercial importance because of its excellent physicochemical, eco‐friendly, and non‐toxic properties. It is used in various applications like protein extraction, wastewater treatment, tissue engineering, drug delivery, food packaging, and so forth. XG is a natural material, and it has some limitations related to the mechanical stress, thermal stability, and hydration. To overcome the limitation, XG can be modified by adding third component or modification of operation for specific applications. XG can be modified by physical, genetically, enzymatically, or chemical processes. Revamped XG also enhances the efficiency toward adsorbing toxic metal ions and organics from synthetic or industrial effluents. The emphasis of present review article is to address the structural characteristics along with the focus on the developing chemically modified XG like grafted, cross‐linked, nanocomposites, and functionally modified biopolymer. However, this review commences thorough discussion on numerous ways of modifications, which can be attempted in XG structure, expanding its applications for heavy metal ion along with dye removal.

Anaerobically mineralized nitrogen as a potential indicator of the activity and abundance of mycorrhizal fungi in Mollisols

Anaerobically mineralized nitrogen (AN) is a suitable soil health indicator. The AN is sensitive to soil use changes and is related to soil and particulate organic carbon and aggregate stability. This work aims to evaluate the relationship between AN and 1) easily extractable glomalin-related soil proteins; 2) abundance of arbuscular mycorrhizal fungi measured by the number of arbuscular mycorrhizal fungi spores, and 3) arbuscular mycorrhizal fungi activity (root colonization). Soil samples were taken at depths of 0-5 and 5-20 cm from cultivated and uncultivated plots throughout the southeastern province of Buenos Aires. Anaerobically mineralized nitrogen, soil organic carbon, particulate organic carbon, aggregate stability, easily extracted glomalin-related soil proteins and the logarithm of the number of arbuscular mycorrhizal fungi spores (log spores) at 0-5, 5-20 and 0-20 cm depths were determined. In wheat roots, the percentages of total infection and arbuscules at 0-20 cm were measured. At all depths, AN was positively correlated to easily extractable glomalin-related soil proteins (r=0.34-0.65), which is an indicator of arbuscular mycorrhizal fungi activity and abundance. Likewise, AN was positively related to log-spores (r=0.58-0.78), which is an indicator of arbuscular mycorrhizal fungi abundance. However, AN was not related to root colonization (the percentages of total infection and arbuscules) that manifests the activity of arbuscular mycorrhizal fungi at a specific moment. Thus, anaerobically mineralized nitrogen would be an indicator of mid- to long-term changes in arbuscular mycorrhizal fungi abundance and activity (easily extractable glomalin-related soil proteins and log-spores) resulting from soil use. Consequently, the AN would allow monitoring an important aspect of soil microbiological health associated with arbuscular mycorrhizal fungi. However, it is necessary to evaluate the relationships studied in this work in a wider range of soil situations.

Investigating the Influence of Spinach Sauce in Modulating Serum Calcium in Osteoporotic Females

Background: The modulation of serum calcium levels through diet is a critical aspect of managing osteoporosis, particularly in postmenopausal women. Spinach, known for its high calcium content, offers potential benefits for bone health due to its rich nutrient profile. This study aimed to develop a spinach sauce, evaluate its consumer acceptability, and investigate its impact on serum calcium levels in osteoporotic females. Objective: To develop a nutritionally rich spinach sauce, assess its sensory acceptability, and evaluate its effectiveness in modulating serum calcium levels among osteoporotic females. Methods: The spinach sauce was formulated using various recipes to maximize calcium bioavailability. The preparation process involved controlled cooking techniques to preserve nutrients, followed by proximate composition analysis. A sensory evaluation was conducted with osteoporotic females and a control group using standard hedonic scales. Participants were instructed to incorporate the spinach sauce into their daily diet for a specified period. Serum calcium levels were measured before and after the intervention. Statistical analysis was conducted using SPSS version 25, controlling for covariates such as age, baseline dietary habits, and baseline serum calcium levels. Ethical approval was obtained, and informed consent was provided by all participants. Results: Treatment T2, containing 40% spinach and 60% mint, scored highest in sensory evaluation with color (8.94 ± 0.26), flavor (8.92 ± 0.30), taste (8.98 ± 0.27), texture (8.22 ± 0.29), and overall acceptability (8.94 ± 0.31). The proximate composition of the spinach sauce showed moisture (79.84%), crude protein (6.27%), crude fat (1.91%), fiber (0.57%), ash (4.21%), and nitrogen-free extract (10.27%). Serum calcium levels increased significantly in the spinach sauce group from 0.84 ± 0.01 to 1.03 ± 0.04, and in the combined spinach sauce and calcium supplement group from 0.69 ± 0.01 to 0.91 ± 0.07. ANOVA revealed significant effects of treatments (F = 1.99, p ≤ 0.01) and days (F = 41.96, p ≤ 0.01) on bone mineral density. Conclusion: The developed spinach sauce was nutritionally rich and well-received, particularly in treatment T2. It demonstrated potential in improving serum calcium levels among osteoporotic females, suggesting that dietary interventions with spinach could be a viable strategy for managing osteoporosis. Further research with larger sample sizes and longer durations is recommended to substantiate these findings.

Composition and functionality differences of oat protein concentrates: Potential of less refined concentrates obtained by wet milling

The objective of this study was to determine differences in composition and functionality for oat protein concentrates. This was achieved by comparing preparations extracted at different pH levels using either a twin screw press or alkaline extraction followed by isoelectric precipitation. Extraction by twin screw technology provided a higher protein yield compared to isoelectric precipitation. Analysis by SDS-PAGE revealed similar protein composition regardless of the extraction method or extraction pH except for extraction at pH 6. Extraction at a higher pH led to a higher protein purity and yield, while extraction closer to the isoelectric point of the protein resulted in less pure concentrates. All extracts were freeze-dried, and their composition and functionality were compared. Thermal analysis of the protein concentrates extracted by twin screw pressing showed a higher denaturation enthalpy compared to protein isolates prepared by isoelectric precipitation, suggesting that the native structure of the proteins is preserved to a higher extent. All protein fractions exhibited the typical U-shaped solubility profile, except for the concentrate extracted at pH 6, which showed an increase in solubility with increasing pH. Oat protein concentrates prepared by twin screw pressing had a higher affinity for binding to oil than to water, and this was also higher than for isoelectric precipitated protein extracts. Overall results demonstrate a potential for twin screw technology as a viable alternative to conventional wet extraction methods for producing less refined fractions from oats with tunable functional properties.

The feasibility and applicability of sequential extraction of high value-added biogenic materials from sewage sludge

ABSTRACT The sequential extraction routes of biogenic materials from sewage sludge (SS) were investigated. Physical methods (ultrasound, heating) and chemical methods (sodium hydroxide, sodium carbonate) were used to extract extracellular polymeric substances (EPS) and alginate-like extracellular polymers (ALEs) from SS. The residues after extraction were further subjected to physical methods (heating) and chemical methods (sulfuric acid, sodium hydroxide) for protein extraction. A comparison was made between sequential extraction routes and direct extraction of biomaterials from sludge in terms of extraction quantity, material properties, and applicability. The results showed that sequential extraction of biomaterials is feasible. The highest extraction quantities were obtained when using sodium carbonate for EPS and ALE extraction and sodium hydroxide for protein, reaching 449.80 mg/gVSS, 109.78 mg/gVSS, and 5447.08 mg/L, respectively. Sequential extraction procedures facilitate the extraction of biomaterials. Finally, suitable extraction methods for different application scenarios were analyzed.

GPSFun: geometry-aware protein sequence function predictions with language models.

Knowledge of protein function is essential for elucidating disease mechanisms and discovering new drug targets. However, there is a widening gap between the exponential growth of protein sequences and their limited function annotations. In our prior studies, we have developed a series of methods including GraphPPIS, GraphSite, LMetalSiteand SPROF-GO for protein function annotations at residue or protein level. To further enhance their applicability and performance, we now present GPSFun, a versatile web server for Geometry-aware Protein Sequence Function annotations, which equips our previous tools with language models and geometric deep learning. Specifically, GPSFun employs large language models to efficiently predict 3D conformations of the input protein sequences and extract informative sequence embeddings. Subsequently, geometric graph neural networks are utilized to capture the sequence and structure patterns in the protein graphs, facilitating various downstream predictions including protein-ligand binding sites, gene ontologies, subcellular locationsand protein solubility. Notably, GPSFun achieves superior performance to state-of-the-art methods across diverse tasks without requiring multiple sequence alignments or experimental protein structures. GPSFun is freely available to all users at https://bio-web1.nscc-gz.cn/app/GPSFun with user-friendly interfaces and rich visualizations.

Bio-based composite active film/coating from deccan hemp seed protein, taro starch and leaf extract: Characterizations and application in grapes

This study aimed to create a novel, cost-effective, and environmentally friendly food packaging film or coating using agricultural waste materials such as deccan hemp seeds and leaves. The process involved blending deccan hemp seed protein and leaf extract at an optimal ratio through a casting technique. Additionally, starch isolated from taro root was added into the protein-based film to enhance its structural, barrier, and mechanical properties. The introduction of bioactive compounds, like deccan hemp leaf extract, led increase in antioxidant activity and enriching the film with beneficial phenolic compounds.The combination of leaf extract and starch with the protein film resulted in a homogeneous, dense, and smooth film matrix, as evidenced by scanning microscopy images. FTIR spectroscopy confirmed interlinkage among the various constituents in the film. Rheological analysis of the film-forming solution showed its shear-thinning behavior, with the storage modulus exceeding the loss modulus in the angular frequency range of 0.1 rad/s to 100 rad/s, indicating the film's elastic nature. Furthermore, the physical and optical attributes of the films were enhanced through the addition of starch and leaf extract.In a practical application, the film-forming solution was applied to grapes, leading to improved quality and extended shelf life for grapes stored under ambient conditions for ten days. Overall, this study's findings suggest that the use of deccan hemp seed protein-based packaging film holds promise as an emerging solution for enhancing the shelf life of vegetables and fruits, offering a sustainable and effective alternative for food packaging.

Surface activity of Lupinus angustifolius (blue lupine) seed extracts

Surface tension (γeq) of the seed extracts of four lupine cultivars showed values in the range 44.9–46.4 mN/m. The surface compression elasticity (E') of the adsorbed layers and foaming capacity (FC) also showed similar values (E' ∼ 30 mN/m, FC ∼ 100%). The effect of defatting prior to extraction at pH 8.5 depends on the solvent employed – hexane and dichloromethane improved the subsequent protein extraction yield, while ethanol reduced it. The effect of defatting on surface tension could be positive (for hexane and ethanol) or negative (for dichloromethane). Generally, defatting improved the surface compression rheological and foaming parameters. On the other hand, fractionation of the extracts obtained at pH 8.5 from hexane-defatted seeds did not improve significantly the surface activity parameters. Some improvement with respect to the unfractionated extracts was observed only for the extracts of undefatted seeds. γeq, E', E" and FC isotherms confirm the surfactant-like behavior of the lupine seed extracts.

Apparent digestibility and nutritional status of capuchin monkeys (Sapajus sp.) under human care.

Studing the nutritional needs and dietary habits of primates is essential to ensure their health and well-being. This includes the understanding of the use of nutrients and its correlation with health parameters. Diet and nutritional parameters of 13 captive capuchin monkeys (Sapajus sp.) were assesed. Apparent digestibility of dry matter and nutrients were evaluated. Nutritional status was established based on body condition score (BCS) and muscle mass score (MMS). High apparent digestibility coefficients (ADC) were observed for crude protein and ether extract, but low for minerals and crude fiber. The ADC of EE and CF were related to MMS, and the biochemical parameters did not correlate with the AD coefficients obtained in the group. The nutritional status of the animals remained inadequate in 84.6% of the individuals, suggesting a reformulation of the diet and nutritional management.

Perspective: could Ethiopian potatoes contribute to environmental sustainability, the Ethiopian economy, and human health?

Environmental data are rapidly accruing on the unsustainability of diets based on animal products, such as dairy and meats. Shifting to alternative sources of protein is inevitable given an increase in the projected global population and protein demand. Left unchecked, a collision between food security and sustainability is imminent. Potatoes could be the strategic food and cash crop to harmonize food security and sustainability worldwide. Recently, there has been a growing interest in extracting proteins from the byproduct of the potato starch industry known as potato fruit juice. These proteins are garnering attention due to their nutritional value, characterized by a well-balanced amino acid profile, as well as their functional properties including emulsifying, foaming, and gelling capabilities. Moreover, these proteins are considered to be less allergenic than some other protein sources. Extracting potato protein, which is sourced as a byproduct, reduces food loss and waste, thereby eliminating pathogenic microorganisms from the environment and mitigating greenhouse gas emissions. Ethiopia is a major potato producer in East Africa. Potatoes help the country increase household income, ensure food security and revenue generation, and produce starch. However, Ethiopia’s potato starch industry has not yet begun protein extraction, despite the vital role of the proteins and the country’s huge cultivation potential. Furthermore, the global potato protein market is experiencing significant growth. This information urgently calls for innovative approaches to assess the impact of extracting protein from potatoes produced in Ethiopia. Therefore, this perspective article has two main objectives. First, to scan the extent of potato production in Ethiopia in relation to environmental sustainability and the economy. Second, to provide prospects on the impact of extracting protein isolate from potatoes produced in Ethiopia on environmental sustainability, Ethiopia’s economy, and human health.

A Review of Background and Application of ATPSs in Protein and Enzyme Extraction

A Review of Background and Application of ATPSs in Protein and Enzyme Extraction

A methodological exploration of distinguishing hair quality based on hair proteomics

Hair is an advantageous biological sample due to its recordable, collectable, and storable nature. Hair's primary components are keratin and keratin-associated proteins. Owing to its abundance of cystine, keratin possesses impressive mechanical strength and chemical stability, formed by creating disulfide bonds as crosslinks within the protein peptide chain. Furthermore, keratin is cross-linked with keratin-associated proteins to create a complex network structure that provides the hair with strength and rigidity. Protein extraction serves as the foundation for hair analysis research. Bleaching hair causes damage to the structure between keratin and keratin-associated proteins, resulting in texture issues and hair breakage. This article outlines various physical treatment methods and lysate analysis that enhance the efficiency of hair protein extraction. The PLEE method achieves a three-fold increase in hair protein extraction efficiency when using a lysis solution containing SDS and combining high temperatures with intense shaking, compared to previous methods found in literature. We utilized the PLEE method to extract hair from both normal and damaged groups. Normal samples identified 156–157 proteins, including 51 keratin and keratin-associated proteins. The damaged group consisted of 155–158 identified proteins, of which 48–50 were keratin and keratin-associated proteins. Bleaching did not cause any notable difference in the protein identification of hair. However, it did reduce coverage of keratin and keratin-associated proteins significantly. Our hair protein extraction method provides extensive coverage of the hair proteome. Our findings indicate that bleaching damage results in subpar hair quality due to reduced coverage of protein primary sequences in keratin and keratin-associated proteins.

Abstract PO5-13-07: Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics

Abstract Background Early-stage triple-negative breast cancer (TNBC) is usually treated with neoadjuvant chemotherapy (NADC), but prognosis remains poor compared with other BC subtypes. Unfortunately, less than 30% of BC patients achieve pathological complete response (pCR) to NADC and the overall survival of TNBC when cancer spreads is estimated at 10-13 months. Complete response is associated with improved progression-free and overall survival rates, but more accurate prognostic markers are needed. In this study, we assessed the prognostic value of blood circulating metabolites by using targeted-based metabolomics. Methods Patients received standard neoadjuvant chemotherapy with epirubicin plus cyclophosphamide followed by paclitaxel. Blood samples were obtained upon completion of chemotherapy and before surgery. Pathological response to treatment was recorded according to the Miller & Payne system, and categorized as partial (MP 1, 2, & 3) or complete response (MP 4 & 5). Blood samples were collected in EDTA tubes, centrifuged and plasma was stored at -80°C. After QC standard addition and methanol extraction of proteins, four fractions of the resulting extract were analysed: two by reverse-phase/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one by reverse phase/UPLC-MS/MS with negative ion mode ESI, and one by hydrophilic interaction liquid chromatography/UPLC-MS/MS) by Metabolon using a Thermo Scientific Q-Exactive mass spectrometer with a heated electrospray ionization source and an Orbitrap mass analyser. Using Metabolon's hardware and software, raw data were extracted, identified, QC processed and quantified using the area under the curve (AUC). Identified compounds were compared with library entries of purified standards or recurrent unknown entities. The prognostic capacity of each metabolite was assessed using BRB ArrayTools. Selected metabolites were validated using a targeted approach using compound standards as follows. Plasma samples were re-analysed by UPLC/MS using a Vanquish LC coupled to an Orbitrap Exploris 240 MS (Thermo Fisher Scientific) with positive and negative ionization mode ESI under polarity switching. Identification of compounds were performed by comparison of retention times (against in-house authentic standards), accurate mass (with an accepted deviation of 3ppm), and MS/MS spectra. These analyses were carried out by MS-Omics (Denmark). Results Twenty patients treated in the Medical Oncology Unit of the University Hospital of Jaén were recruited. Median age was 50 (31-76). Twelve patients obtained a pathological complete response. Median follow-up after surgery was 25 months. Seven patients eventually had distant metastases (2 in the lungs, 1 in the brain, 1 in the bones and 3 multi-site). Metabolon untargeted analysis identified and quantified 985 metabolites, 789 after xenobiotics exclusion. Survival analysis identified 16 metabolites related with distant relapse (p< 0.05). The ten metabolites with lower p-value were selected for targeted validation. Ms-Omics pipeline allowed absolute quantification of 5 metabolites, 3 of them showing high correlation (R >0.7) between both measurements (targeted and untargeted). Validation of the prognostic value of the candidate metabolites is ongoing in a larger TNBC cohort. Conclusions Metabolomics is a useful tool for the detection of simply assessable and cost-effective prognostic biomarkers in TNBC. Easily monitoring the presence of minimal residual disease is worth to be settled up in the clinical practice for the most aggressive molecular subtype of breast cancer. In this work we have defined a set of metabolites that could predict distant relapse events in TNBC patients treated with neoadjuvant chemotherapy. Further analyses are being carried out to validate the prognostic value of the proposed candidate metabolites. Citation Format: Pedro Sánchez-Rovira, Rocio Urbano-Cubero, Angelo Gamez Pozo, Carmen González-Olmedo, Alicia Cano-Jimenez, Andrea Zapater- Moros, Leticia Díaz-Beltrán, Lucia Trilla-Fuertes, Mariana Díaz-Almirón, Enrique Espinosa, Pilar Zamora, Juan Angel Fresno Vara. Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-07.

Phosphorylation of cell cycle and apoptosis regulatory protein-1 by stress activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy.

CARP-1, a perinuclear phospho-protein, regulates cell survival and apoptosis signaling induced by genotoxic drugs. However, kinase(s) phosphorylating CARP-1 and down-stream signal transduction events remain unclear. Here we find that CARP-1 Serine (S)626 and Threonine (T)627 substitution to Alanines (AA) inhibits genotoxic drug-induced apoptosis. CARP-1 T627 is followed by a Proline (P), and this TP motif is conserved in vertebrates. Based on these findings, we generated affinity-purified, anti-phospho-CARP-1 T627 rabbit polyclonal antibodies, and utilized them to elucidate chemotherapy-activated, CARP-1-dependent cell growth signaling mechanisms. Our kinase profiling studies revealed that MAPKs/SAPKs phosphorylated CARP-1 T627. We then UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed SAPK p38γ interaction with CARP-1 (614-638) peptide. Our studies further established that SAPK p38γ, but not other MAPKs, phosphorylates CARP-1 T627 in cancer cells treated with genotoxic drugs. Loss of p38γ abrogates CARP-1 T627 phosphorylation, and results in enhanced survival of breast cancer cells by genotoxic drugs. CARP-1 T627 phosphorylation was also noted in breast tumors from patients treated with radiation or endocrine therapies. We conclude that genotoxic drugs activate p38γ-dependent CARP-1 T627 phosphorylation to inhibit cell growth.

SYNTHESIS OF NEW BODIPY HYDRAZIDE FLUORESCENT PROBES FOR THE DETECTION OF CARBONYLATED PROTEINS GENERATED BY OXIDATIVE STRESS.

Oxidative stress is a cellular disorder implicated in various severe diseases and redox biology and represents an important field of research for the last decades. One of the major consequences of oxidative stress is the carbonylation of proteins, which is also a reliable marker to assess protein oxidative modifications. Accumulation of carbonylated proteins has been associated with aging and age-related diseases and can ultimately causes cell death. Detection of these oxidative modifications is essential to understand and discover new treatments against oxidative stress. We describe the design and the synthetic pathway of new BODIPY fluorescent probes functionalized with hydrazide function for protein carbonyl labeling to improve existing methodologies such as 2D-Oxi electrophoresis. Hydrazide BODIPY analogues show very good fluorescent properties such as NIR emission up to 633 nm and quantum yield up to 0.88. These new probes were validated for the detection and quantification of carbonylated proteins with 2D-Oxi electrophoresis using mouse muscle protein extracts, as well as both flow cytometry and microscopy using oxidant stressed C2C12 cells.

Alkaline solubilization of microalgal protein and its impact on the functional properties of protein extract

Microalgal alternative proteins are increasingly recognized as a sustainable and promising source for future food production. Despite the widespread use of alkaline solubilization in protein extraction, its influence on the functional properties of microalgal protein extracts has received limited attention. This study marks the pioneering effort to assess the impact of alkaline (NaOH) solubilization on both the extraction process and the functional properties of Chlorella vulgaris protein extract (CVPE) obtained through isoelectric precipitation. With the gradual escalation of alkaline concentration from 0 to 0.7 M, there was a notable improvement in protein extraction efficiency, increasing significantly from 6.22% to 56.23%. However, elevated NaOH concentrations resulted in protein degradation, as evidenced in SDS-PAGE studies. Alkaline solubilization exhibited a positive impact on various functional properties of CVPE. It enhanced solubility, water absorption capacity, foaming capacity and stability, as well as emulsifying activity and stability. However, there was a slight decrease in oil absorption capacity with increasing NaOH concentration. Notably, the maximum solubility, oil adsorption capacity, foaming capacity, and emulsion activity of CVPE were 2.3-, 1.8-, 2-, and 1.3-fold higher, respectively, than those of commercial soy protein isolate (SPI). This highlights the effectiveness of alkaline solubilization in in modifying C. vulgaris protein and emphasizes the high potential of CVPE as a functional food ingredient.