Bicinchoninic Acid Protein Research Articles

The Impact of Acute Myeloblastic Leukemia-Derived Small Extracellular Vesicles on the Bone Marrow Mesenchymal Stem Cells: Expression of G1 Cell Cycle Progression Genes

Background: Leukemia progression is intricately linked to interactions with neighboring cells within the bone marrow microenvironment (BMM), and small extracellular vesicles (sEVs) emerge as vital mediators in facilitating these interactions. Objectives: This study examined the proliferation effects of sEVs-derived from acute myeloid leukemia (AML) in the HL60 cell line on the cell cycle progression of bone marrow mesenchymal stem cells (BM-MSCs), a key element of the BMM. Methods: Small extracellular vesicles were isolated from the HL60 cell line supernatant, using the ExoCib kit, and characterized through flow cytometry, transmission electron microscopy (TEM), dynamic light scattering (DLS), and bicinchoninic acid (BCA) protein assay. bone marrow mesenchymal stem cells were cultured and treated with various concentrations (20 μg/mL, 50 μg/mL, and 80 μg/mL) of AML-derived sEVs for 24, 48, and 72 hours. The effects on cell proliferation and viability were assessed, using methylthiazole tetrazolium test (MTT) and Ki-67 assays, while cell cycle progression and apoptosis were analyzed via flow cytometry. RT-qPCR was performed to evaluate the expression levels of CCND1, CDK4, CDK6, and AKT1 genes. Results: The proliferation effects of AML-derived sEVs on BM-MSCs were both dose- and time-dependent, with optimal effects observed at 50 μg/mL after 48 hours. Flow cytometry analysis revealed a significant increase in the G1 phase, showing a 1.8-fold change compared to the control group (P < 0.0001). RT-qPCR results demonstrated a significant upregulation of CCND1 (3.5-fold, P < 0.0001), CDK4 (3.2-fold, P < 0.0001), CDK6 (2.5-fold, P < 0.0001), and AKT1 (3.2-fold, P < 0.0001) expression levels, along with increased Ki-67 (2.3-fold, P < 0.0001) levels. Moreover, treatment with 50 μg/mL of AML-derived sEVs resulted in a notable reduction in BM-MSC apoptosis (0.57-fold decrease, P < 0.0001). Conclusions: Our findings revealed that AML extracellular vesicles could alter the gene expression associated with the proliferation of BMSCs to increase their proliferation by stimulating the cell cycle of MSCs through the G1 phase.

Effect of AML-exosomes on the cellular and molecular properties of bone marrow mesenchymal stromal cells: Expression of JAK/STAT signaling genes

Purpose of studyDespite the various therapeutic options introduced for AML treatment, therapy resistance and relapse are still the main obstacles. It is well known that alterations in the bone marrow microenvironment (BMM) play a crucial role in leukemia growth and the treatment failure of AML. Evidence shows that exosomes alter the components of BMM in a way that support leukemia survival, leading to chemoresistance. In this study, we evaluated the effect of AML exosomes on the biological functions of human bone marrow mesenchymal stromal cells (h BM-MSCs), especially alteration in the expression of the JAK/STAT signaling genes, as a leukemia-favoring pathway. MethodExosomes were isolated from the HL-60 cell line and characterized using flow cytometry, Transmission Electron Microscopy (TEM), and Dynamic Light Scattering (DLS) technique. The exosome protein content was assessed using a bicinchoninic acid (BCA) protein assay kit in order to determine the concentration of exosomes. Subsequently, MSCs were treated with varying concentrations of AML exosomes, and data was obtained using MTT, cell cycle, apoptosis, and ki67 assays. Additionally, gene expression analysis was conducted through qRT-PCR. ResultAML exosomes regulated the viability and survival of MSCs in a concentration-dependent manner. The qRT-PCR data revealed that treatment with AML exosomes at a concentration of 50 μg/mL led to a significant upregulation of JAK2, STAT3, and STAT5 genes in MSCs. ConclusionBecause the JAK/STAT signaling pathway has been shown to play a role in the proliferation and survival of leukemic cells, our results suggest that AML exosomes stimulate MSCs to activate this pathway. This activation may impede AML cell apoptosis, potentially leading to chemoresistance and relapse.

A novel intralamellar semi‐bioresorbable keratoprosthesis—Part B: Surface functionalization and physico‐chemical characterization toward site‐specific cellular activity

AbstractIn this article, site‐specific functionalized keratoprosthesis (KPro) was accomplished to meet epithelial on‐growth on the anterior optical surface, inhibit cell adhesion on the posterior optic surface, and promote cell ingrowth at the peripheral tissue‐haptic‐flange interface toward tissue integration. Gelatin immobilization on PC4 hydrogel surfaces was achieved through acid hydrolysis followed by EDC/NHS chemistry, and polyethylene glycol‐diacrylate (PEGDA)‐modified inter‐penetrating network (IPN) surfaces were prepared using photo‐polymerization of ethylene glycol‐diacrylate (EGDA) for enhancing and inhibiting cellular growth, respectively. The surface chemical structures of modified hydrogels were characterized using attenuated total reflectance‐infrared (ATR‐IR) spectroscopy and a bicinchoninic acid (BCA) protein adsorption assay. Further, suitable differentially functionalized surfaces were prepared and characterized using the universal testing machine (UTM), refractometer, atomic force microscopy (AFM), and contact angle measurements to understand mechanical, optical, surface morphological, and wettability properties, respectively. Finally, in vitro site‐specific cell adhesion, cell inhibition, and cell ingrowth functionalities were tested using isolated goat corneal limbal epithelial stem cells and corneal stromal fibroblast cells, respectively. Overall, the two connective articles represent a successful endeavor to develop a more esthetically pleasing, mechanically stable, tissue‐free hydrogel KPro with site‐specific surface functionality nearly mimicking the cornea's functionality. A further pre‐clinical investigation is necessary to advance this functional novel KPro to reality, especially the tissue integration aspect.

Comprehensive Characterization of Surface-Bound Proteins and Measurement of Fibrin Fiber Thickness on Extracorporeal Membrane Oxygenation Circuits Collected From Patients.

To characterize surface-bound proteins and to measure the thickness of fibrin fibers bound to extracorporeal membrane oxygenation (ECMO) circuits used in children. Single-center observational prospective study, April to November 2021. PICU, Royal Children's Hospital, Melbourne, Australia. Patients aged less than 18 years on venoarterial ECMO and without preexisting disorder. None. ECMO circuits were collected from six patients. Circuit samples were collected from five different sites, and subsequently processed for proteomic and scanning electron microscopy (SEM) studies. The concentration of proteins bound to ECMO circuit samples was measured using a bicinchoninic acid protein assay, whereas characterization of the bound proteome was performed using data-independent acquisition mass spectrometry. The Reactome Over-representation Pathway Analyses tool was used to identify functional pathways related to bound proteins. For the SEM studies, ECMO circuit samples were prepared and imaged, and the thickness of bound fibrin fibers was measured using the Fiji ImageJ software, version 1.53c (https://imagej.net/software/fiji/). Protein binding to ECMO circuit samples and fibrin networks showed significant intra-circuit and interpatient variation. The median (range) total protein concentration was 19.0 (0-76.9) μg/mL, and the median total number of proteins was 2011 (1435-2777). A total of 933 proteins were commonly bound to ECMO circuit samples from all patients and were functionally involved in 212 pathways, with signal transduction, cell cycle, and metabolism of proteins being the top three pathway categories. The median intra-circuit fibrin fiber thickness was 0.20 (0.15-0.24) μm, whereas the median interpatient fibrin fiber thickness was 0.18 (0.15-0.21) μm. In this report, we have characterized proteins and fiber fibrin thickness bound to ECMO circuits in six children. The techniques and approaches may be useful for investigating interactions between blood, coagulation, and the ECMO circuit and have the potential for circuit design.

Investigating the proliferative inhibition of HepG2 cells by exosome-like nanovesicles derived from Centella asiatica extract through metabolomics

Nano-particles demonstrating excellent anticancer properties have gradually found application in cancer therapy. However, their widespread use is impeded by their potential toxicity, high cost, and the complexity of the preparation process. In this study, we achieved exosome-like Centella asiatica-derived nanovesicles (ADNVs) through a straightforward juicing and high-speed centrifugation process. We employed transmission electron microscopy and nanoparticle flow cytometry to characterize the morphology, diameter, and stability of the ADNVs. We evaluated the in vitro anticancer effects of ADNVs using Cell Counting Kit-8 and apoptosis assays. Through sequencing and bicinchoninic acid protein analysis, we discovered the abundant presence of proteins and microRNAs in ADNVs. These microRNAs can target various diseases such as cancer and infection. Furthermore, we demonstrated the effective internalization of ADNVs by HepG2 cells, resulting in an increase in reactive oxygen species levels, mitochondrial damage, cell cycle arrest at the G1 phase, and apoptosis. Finally, we analyzed changes in cellular metabolites post-treatment using cell metabolomics techniques. Our findings indicated that ADNVs primarily influence metabolic pathways such as amino acid metabolism and lipid biosynthesis, which are closely associated with HepG2 treatment. Our results demonstrate the potential utility of ADNVs as anticancer agents.

Evaluating smartphone-based optical readouts for immunoassays in human and veterinary healthcare: A comparative study

Recent advances have significantly enhanced the use of smartphone devices for medical diagnostics. This study uses high-resolution cameras in mobile devices to capture and process bioassay images, enabling the quantification of diverse biomarkers across a range of diagnostic tests conducted on 96-well microplates. The study evaluates the effectiveness of this technology through protein quantification techniques and immunoassays that generate colorimetric responses at specific wavelengths. It includes the assessment of bicinchoninic acid and Bradford protein quantification methods, alongside a conventional immunoassay for detecting mare antibodies in colostrum to monitor foal immunodeficiencies. Further application involves the readout of magneto-actuated immunoassays aimed at quantifying bacteria. The results obtained from benchtop spectrophotometry at 595, 562, and 450 nm are compared with those acquired using a smartphone, which identified color intensities in shades of blue, purple, and yellow. This comparison yields promising correlations for the samples tested, suggesting a high degree of accuracy in the smartphone capability to analyze bioassay outcomes. The analysis via smartphone is facilitated by a specific app, which processes the images captured by the phone camera to quantify color intensities corresponding to different biomarker concentrations. Detection limits of 12.3 and 22.8 μg mL−1 for the bicinchoninic acid assay and 36.7 and 45.4 μg mL−1 for the Bradford are obtained for protein quantification using the spectrophotometer and the smartphone app, respectively. For mare's antibodies in colostrum, the values are 1.14 and 1.72 ng mL−1, while the detection of E. coli is performed at 2.0 x 104 and 2.9 × 104 CFU mL−1, respectively. This approach offers further advantages, including wide availability, cost-effectiveness, portability, compared to traditional and expensive benchtop instruments.

Influence of chirality on protein corona formation of low-fouling chiral poly(2-oxazoline) coated nanoparticles

Low-fouling polymers play a crucial role in the development of therapeutic nanoparticles (NPs), which are commonly described as nanomedicines. These hydrophilic polymers are typically designed to favourably alter the protein corona that forms around a NP by reducing the binding of opsonins, resulting in reduced immune recognition and clearance. The polymer specific proteome of the corona is mainly dictated by the physicochemical properties of the polymer and several parameters such as hydrophilicity and charge have been explored to regulate its composition. To date, there has been little attention given to the influence of the chirality of the hydrophilic polymer on the composition of the corona. Water-soluble poly(2,4-dialkyl-2-oxazoline)s (PdOx) are a unique synthetic polymer class to explore the effect of chirality as they can be rendered optically active if enantiopure monomers are used for their synthesis. Herein we prepared two water-soluble poly(2,4-dimethyl-2-oxazoline)s (PdMeOx) of different handedness, alongside racemic PdMeOx and the well-established low-fouling structural isomer poly(2-ethyl-2-oxazoline) (PEtOx). All polymers were equipped with sulphur-containing xanthate moieties with high end-group fidelity, which enabled anchoring to gold nanoparticles (AuNPs) as a useful handle for evaluation of the polymer-specific fouling. It was revealed that a hard corona formed after exposing the coated AuNP to human plasma proteins as investigated by dynamic light scattering, SDS-PAGE and PierceTM bicinchoninic acid protein assay (BCA) revealing a similar protein adsorption between PEtOx and all three PdMeOx. Moreover, a detailed proteomics study using liquid chromatography coupled to mass spectrometry (LC–MS/MS) was performed and analysed by label-free quantitation (LFQ). Overall, the PdMeOx systems showed similar human plasma protein corona profiles compared to the PEtOx control. However, between the R- and S-PdMeOx enantiomers differences in protein population abundances were observed, suggesting the propensity for specific interaction of chiral PdOx with different elements of the proteome. Overall, the present study introduces PdMeOx as a chiral, low fouling polymer system, which can reduce protein fouling in a similar magnitude to the PEtOx control. Importantly, we provide a first insight into how changes in the chirality of PdMeOx can influence the components of the protein corona, which will inform the future design of PdOx nanotherapeutics.

The effects of tumor-derived exosomes enriched with miRNA-211a on B16F10 cells.

Exosomes have emerged as a novel alternative delivery system for transporting small molecules. Tumor-derived exosomes (TEXs) possess anti-cancer properties and serve as natural carriers of microRNAs. Using this knowledge as a foundation, the current study evaluated the efficacy of delivering a miR-211 mimic via B16F10 cell-derived exosomes to block the growth and development of a melanoma cell line. After exposing B16F10 cultured cells to serum-free media for 24 hours, we collected the supernatant. Subsequently, we purified the exosomes from the supernatant using a commercial kit. Scanning electron microscopy, transmission electron microscopy, dynamic light scattering, Western blot, and bicinchoninic acid protein assay were used to characterize exosomes. Following that, miR-211 mimic was loaded into exosomes (termed TEXomiR) via a modified calcium chloride procedure. The assessment of miR-211a loading efficiency into exosomes was conducted by analyzing its relative expression. MTT, annexin V/PI, and quantitative real-time polymerase chain reaction were used to measure the proliferation, apoptosis and relative expression of miR-211 target genes, respectively. Our study showed that the exosomes can deliver miR-211 mimic efficiently. The treatment of B16F10 cells with miR-211-enriched TEX downregulated miR-211 target genes, including brain-specific homeobox, vascular endothelial growth factor, and transforming growth factor-β receptor. The results indicated the antiproliferative effect of TEXomiR as time-dose-dependent. The flow cytometry evaluation showed that TEXomiR could induce the apoptosis of B16F10 cells. Our data indicated that exosomes can be suitable carriers for miR-211 mimic. Moreover, TEXomiR via anti-cancer effects could inhibit the progression of melanoma cancer.

Tear Samples for Protein Extraction: Comparative Analysis of Schirmer's Test Strip and Microcapillary Tube Methods.

Tear sampling is an attractive option for collecting biological samples in ophthalmology clinics, as it offers a non-invasive alternative to other invasive techniques. However, there are many tear sampling methods still in consideration. This study explores the suitability of Schirmer's test strip and microcapillary tube as reliable and satisfactory methods for tear sampling. Tear samples were collected from eight healthy volunteers using the standard Schirmer's test strip method with or without anesthesia and microcapillary tubes. The total tear protein concentrations were analyzed via spectrophotometry and bicinchoninic acid (BCA) protein assay. The protein profile was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal wetting length of Schirmer's strip and suitable buffer solutions were compared. Discomfort levels reported by participants and the ease of execution for ophthalmologists were also evaluated. Tear samples exhibited typical protein profiles as shown by SDS-PAGE.The mean total protein obtained from an optimum wetting length of 20 mm using Schirmer's strip without anesthesia in phosphate-buffered saline (PBS) yielded substantial quantities of protein as measured by nanophotometer (220.20 ± 67.43 µg) and the BCA protein assay (210.34 ± 59.46 µg). This method collected a significantly higher quantity of protein compared to the microcapillary tube method (p=0.004) which was much more difficult to standardize. The clinician found it harder to utilize microcapillary tubes, while participants experienced higher insecurity and less discomfort with the microcapillary tube method.PBS used during the tear protein extraction process eluted higher tear protein concentration than ammonium bicarbonate, although the difference was not statistically significant. Using anaesthesia did not ease the sampling procedure substantially and protein quantity was maintained. Good quality and quantity of protein from tear samples were extracted with the optimized procedure. Schirmer's strip test in the absence of local anesthesia provided a standard, convenient, and non-invasive method for tear collection.

Factors affecting total protein and lactoferrin in human milk

The aim of this study was to investigate factors affecting total, true protein and lactoferrin (Lf) concentrations in human milk (HM) and to evaluate the changes in protein concentrations over the course of lactation (first to sixth month postpartum). HM samples were collected from exclusively breastfeeding mothers during six time periods (1–6 months postpartum); 198 breast milk samples were collected in total. The concentrations of total and true protein in HM were determined using the MIRIS human milk analyzer (HMA). The assessment of HM protein content was also performed in skim HM samples and quantified by bicinchoninic methods with the Bicinchoninic Acid Protein Assay Kit. In turn, Lf content in skim HM samples was determined by the enzyme-linked immunosorbent assay (ELISA) in accordance with a slightly modified procedure. In the first month of lactation total protein concentration was negatively correlated with maternal pre-pregnancy BMI (r = − 0.397; p = 0.022), whereas in the third month postpartum, positive correlation with maternal age was found (r = 0.399; p = 0.021). Considering Lf concentration, in the first month of lactation, it was positively correlated with baby’s birth weight (r = 0.514; p = 0.002). In the next months (from second to sixth) no relationships between Lf concentration and maternal and infants’ factors were observed. The concentration of protein and Lf in HM changes dynamically throughout lactation. Maternal and infant characteristics may impact the HM protein and Lf content, especially in the first month postpartum.

Assessment of DMFT Score, Salivary Protein, and Flow Rate in UiTM’s Year One Dental Students

Objectives: The aim of the study was to evaluate the year one dental students for their score of decayed, missing, filled teeth (DMFT), total salivary protein, and unstimulated and stimulated flow rate of salivary secretions. In addition, correlations between DMFT index with their salivary parameters were also elucidated in our study. Methods: Fifty-seven of dental students were selected in the study, and informed consents were obtained prior to study. Intraoral examination specifically the DMFT scoring was performed. Both salivary proteins in unstimulated and stimulated saliva were measured using Bicinchoninic acid protein assay, and finally flow rates of unstimulated and stimulated saliva were calculated in the study. Data obtained for DMFT score and salivary parameters were analyzed by descriptive test and Spearman’s correlation test using SPSS version 26.0. Software Program (IBM, New York), Statistical significance was set at p < 0.05. Results: Majority subjects showed DMFT index of 40.6% in DMFT 1-3 and 36.9% in DMFT 4-10 respectively. Statistically significant inverse correlations were observed between salivary total protein and unstimulated salivary flow rate (r=-0.314, p=0.017), and DFMT score with stimulated salivary flow rate (r=-0.244, p=0.067). Conclusion: In our study, majority of first year dental students evidenced DMFT scores of 1-10, with slightly significant associated dental caries prevalence with salivary flow rate. The findings obtained may serve as reference values for the growing interest in saliva as a diagnostic tool in predicting dental caries risk.

Small Extracellular Vesicles Secreted by Peri-urethral Tissues Regulate Fibroblast Function and Contribute to the Pathogenesis of Female Stress Urinary Incontinence.

This study aimed to explore the existence of small extracellular vesicles (sEVs) in peri-urethral tissues and the role of abnormal expression of sEVs in the pathogenesis of female stress urinary incontinence (SUI). sEVs were extracted from peri-urethral vaginal wall tissues using differential centrifugation and were observed by transmission electron microscopy (TEM). The number of sEVs and their protein contents were compared between SUI and control groups using nanoparticle tracking analysis (NTA) and bicinchoninic acid (BCA) protein assay. Fibroblasts were cultured separately with SUI (SsEVs group) and normal tissue sEVs (NsEVs group). Proliferation and migration of fibroblasts were compared between groups using CCK-8 and wound healing assays, respectively. Expression levels of collagen I and III were compared among blank control (BC), NsEVs, and SsEVs groups using real-time PCR. Protein mass spectrometry was used to test the differentially expressed proteins contained in sEVs between groups. sEVs were extracted and found under the electron microscope. There were significantly more sEVs extracted from the SUI group compared to the normal group. Fibroblasts showed increased proliferative and decreased migratory abilities, and expressed more collagen in the SsEVs group compared to the NsEVs and BC groups. Protein spectrum analysis demonstrated several differentially expressed targets, including components of microfibrils, elastin polymer, and anti-inflammatory factors. sEVs were detected in the peri-urethral tissues. SUI tissues expressed more sEVs than control. The abnormal expression of sEVs and their protein contents may contribute to the pathogenesis and progression of SUI.

Adsorption characteristics of bovine serum albumin on the surface of 316L stainless steel coated with diethoxydimethylsilane/chitosan composite films

In this study, diethoxydimethylsilane (DEDMS) was blended with chitosan (CS) to fabricate DEDMS/CS composite films, which were deposited on the surface of 316L stainless steel to assess their anticoagulant properties. A neat CS film exhibits a high concentration of bovine serum albumin (BSA) protein adhesion of 458.3 ± 6.2 μg/L in a bicinchoninic acid protein assay because the carbonyl and amide functional groups on the CS surface easily form hydrogen bonds with the carboxylic acid functional groups of the BSA protein. The DEDMS/CS composite films exhibited lower BSA adhesion concentration than neat CS films because some of the carbonyl and amide functional groups on the surface of CS were replaced by the –Si-O-Si and –Si-(CH3)2 functional groups. Increasing the DEDMS content in the DEDMS/CS composite films led to a higher concentration of –Si-O-Si and –Si-(CH3)2 functional groups on the surface and lower BSA adhesion concentration. Blending excess amounts of DEDMS caused an undesirable rougher surface morphology because of the hydrolysis and self-condensation reactions of DEDMS; this deteriorated the anticoagulant properties of the composite films. The study confirmed that a DEDMS/CS composite film with an appropriate DEDMS/CS content ratio of 8/92 mL/mL possesses hydrophobic characteristics and the lowest BSA protein adhesion of 155.5 ± 1.0 μg/L and has potential for biomedical coating applications.

Development of Novel Paper-Based Assay for Direct Serum Separation.

Background: Many conventional laboratory tests require serum separation using a clot activator/gel tube, followed by centrifugation in an equipped laboratory. The aim of this study is development of novel, equipment-free, paper-based assay for direct and efficient serum separation. Methods: Fresh blood was directly applied to wax-channeled filter paper treated with clotting activator/s and then observed for serum separation. The purity, efficiency, recovery, reproducibility, and applicability of the assay were validated after optimization. Results: Serum was successfully separated using activated partial thromboplastin time (APTT) reagent and calcium chloride-treated wax-channeled filter paper within 2 min. The assay was optimized using different coagulation activators, paper types, blood collection methods, and incubation conditions. Confirmation of serum separation from cellular components was achieved by direct visualization of the yellow serum band, microscopic imaging of the pure serum band, and absence of blood cells in recovered serum samples. Successful clotting was evaluated by the absence of clotting of recovered serum by prolonged prothrombin time and APTT, absence of fibrin degradation products, and absence of Staphylococcus aureus-induced coagulation. Absence of hemolysis was confirmed by undetectable hemoglobin from recovered serum bands. The applicability of serum separated in paper was tested directly by positive color change on paper using bicinchoninic acid protein reagent, on recovered serum samples treated with Biuret and Bradford reagents in tubes, or measurement of thyroid-stimulating hormone and urea compared to standard serum samples. Serum was separated using the paper-based assay from 40 voluntary donors and from the same donor for 15 days to confirm reproducibility. Dryness of coagulants in paper prevents serum separation that can be re-stored by a re-wetting step. Conclusions: Paper-based serum separation allows for development of sample-to-answer paper-based point-of-care tests or simple and direct blood sampling for routine diagnostic tests.

Pea and Soy Protein Stabilized Emulsions: Formulation, Structure, and Stability Studies

During the last decades, there has been a huge consumer concern about animal proteins that has led to their replacement with plant proteins. Most of those proteins exhibit emulsifying properties; thus, the food industry begins their extensive use in various food matrices. In the present study, pea and soy protein isolates (PPI and SPI) were tested as potential candidates for stabilizing food emulsions to encapsulate α-tocopherol and squalene. More specifically, PPI and SPI particles were formulated using the pH modification method. Following, emulsions were prepared using high-shear homogenization and were observed at both a microscopic and macroscopic level. Furthermore, the adsorption of the proteins was measured using the bicinchoninic acid protein assay. The emulsions’ droplet size as well as their antioxidant capacity were also evaluated. It was found that the droplet diameter of the SPI-based emulsions was 60.0 μm, while the PPI ones had a relatively smaller diameter of approximately 57.9 μm. In the presence of the bioactives, both emulsions showed scavenging activity of the 2,20-Azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTS·+) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, with the ones loaded with α-tocopherol having the greatest antioxidant capacity. Overall, the proposed systems are very good candidates in different food matrices, with applications ranging from vegan milks and soups to meat alternative products.

Identification of transient receptor potential melastatin 3 proteotypic peptides employing an efficient membrane protein extraction method for natural killer cells.

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer’s recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150–200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.

Innovative process for facile dextran-bovine serum albumin conjugate synthesis: Mechanism, kinetics, and characterization

Three process variations are developed to synthesize dextran-bovine serum albumin (BSA) model conjugate through the Maillard reaction and a Schiff base-Amadori solid phase rearrangement mechanism at 90 °C. The influences of lyophilization pH and dextran:BSA molar ratio on the reaction are investigated, with the highest yields achieved by absorbing heat evenly and continuously removing the water by-product under vacuum. Lyophilizing at pH 7.8 provides higher yields than materials lyophilized at pH 5.9, a result attributed to higher reactivity of deprotonated amine at pH 7.8. The purified dextran-BSA conjugate solution is heated to above the BSA denaturation temperature (80 °C) at pH 5.2 to form nanogels with a hydrodynamic diameter of 195–400 nm, with the size dependent on the conjugate composition estimated by bicinchoninic acid protein and glycoprotein carbohydrate assays.

Arginine Vasopressin is not elevated in Early Pregnancy Loss

BackgroundAdministration of arginine vasopressin (AVP) has been previously shown to influence myometrial contractility during the first trimester of pregnancy in women who were undergoing a planned therapeutic abortion. Consequently, it was hypothesized that AVP may play a role in early spontaneous pregnancy loss. AVP is made in a 1:1 molar ratio with copeptin; thus, copeptin can be used as a surrogate measure of AVP. While early pregnancy loss is likely a multifactorial problem, we sought to fill the gap in knowledge of the causes of using pre‐collected samples from pregnancies that subsequently underwent spontaneous abortion and matched controls.MethodsA retrospective case control study was performed using plasma samples from the Maternal Fetal Tissue Bank (IRB# 200910784) at the University of Iowa Hospitals &amp; Clinics. Whole blood samples were collected (ACD‐A tubes, Becton Dickinson) at confirmation of pregnancy appointments in which a viable pregnancy was detected by ultrasound (6‐10 weeks gestation). Samples were processed and plasma aliquots were snap frozen and stored at ‐80oC until analysis. Control samples (N=67) from viable, healthy pregnancies were matched by gestational age, race, ethnicity, and body mass index to cases (N=123). Copeptin was measured by an automated immunoassay (Brahms KRYPTOR) and total protein concentration via a bicinchoninic acid (BCA) protein assay (Pierce). Copeptin concentrations were normalized to total protein in samples. Continuous variables were analyzed by T test.ResultsNo significant differences were detected in plasma samples that were collected while the pregnancies appeared viable. Copeptin was 7.2 +/‐ 6.0 mg/dL in case plasma samples and 6.0 +/‐ 3.4 mg/dL in control plasma samples (P=0.066). It is unclear if vasopressin increases acutely prior to the occurrence of spontaneous abortion.ConclusionThese results suggest that vasopressin cannot be used to identify pregnancies at risk of early loss at the time of pregnancy confirmation.

Elevated Urinary Arginine Vasopressin Concentrations during Preeclamptic Pregnancies do not Persist Postpartum

BackgroundPreeclampsia (PreE) is a common hypertensive disorder during the second and third trimesters of pregnancy. It is estimated to affect 5‐7% of all U.S. pregnancies; about 400,000 pregnancies per year. PreE leads to increased risks of fetal abnormalities, pregnancy complications, chronic hypertension, and cardiovascular disease. Arginine vasopressin (AVP) plays an important role in controlling osmotic balance, regulating blood pressure, maintaining sodium homeostasis, and functioning of the kidney. AVP is secreted by the hypothalamus as a proprotein with copeptin. Because copeptin and vasopressin are secreted in a 1:1 molar ratio, copeptin measurements can be used a surrogate measure of vasopressin. Our lab has previously demonstrated that vasopressin, as measured by copeptin, is elevated in plasma and urine throughout preeclamptic pregnancies; even before PreE can be clinically diagnosed. However, it is unknown whether urine vasopressin is elevated chronically postpartum. Our objective was to determine whether there is a difference in copeptin concentration between women with and without a history of preeclampsia at 1‐4 years postpartum. We hypothesized that AVP concentrations of subjects who had received a PreE diagnosis would continue to be elevated postpartum.MethodsInclusion criteria for this study (IRB# 201808705) included being 1‐4 years postpartum, not currently pregnant, non‐prisoner, and able to provide informed consent. Women without (N=26; Age 34 +/‐ 3.4 yrs, BMI 25.99 +/‐ 6.34 kg/m2) and with a history (N=25; age 33 +/‐ 4.5 yrs, BMI 30.32 +/‐ 9.12 kg/m2) of preeclampsia in their most recent pregnancy were enrolled. Women fasted, did not smoke, and did not have caffeine for 4 hours prior to their study visit, nor did they have alcohol or exercise for the prior 24 hours. Urine samples were assayed for copeptin concentrations via an automated immunoassay (Brahms KRYPTOR), creatinine concentrations via a QuantiChrom assay, total protein concentrations via a bicinchoninic acid (BCA) protein assay, osmolality by freezing point depression, and specific gravity using a hydrometer.ResultsThere were no differences between the cohorts in race or ethnicity. There was no statistically significant difference in urinary copeptin, as a marker of AVP, in the postpartum period between women with vs. without a history of preeclampsia (4.5 vs. 3.2 mg/dL, P=0.4). No significant differences in copeptin were detected in women without a current hypertension diagnosis (5.49 mg/dL, N=43) and those being treated for hypertension (4.79 mg/dL, N=7, P=0.35).ConclusionsThis study suggests that copeptin, a surrogate measure for AVP, is not chronically elevated in urine after a preeclamptic pregnancy.

D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter beta-casein, proteins and metabolites linked in milk protein synthesis in bovine mammary epithelial cells.

This study aims to determine the effects of D-methionine (D-Met) isomer and the methionine precursor 2-hydroxy-4-methylthiobutanoic acid i (HMBi) supplementation on milk protein synthesis on immortalized bovine mammary epithelial cell (MAC-T). MAC-T cells were seeded using 10-cm dishes and cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) basic medium. The basic medium of DMEM/F12 was replaced with the lactogenic DMEM/F12 differentiation medium when 90% of MAC-T cells reached confluency. The best dosage at 0.6 mM of D-Met and HMBi and incubation time at 72 h were used uniformly for all treatments. Each treatment was replicated six times wherein treatments were randomly assigned in a 6-well plate. Cell, medium, and total protein were determined using a bicinchoninic acid protein assay kit. Genes, proteomics and metabolomics analyses were also done to determine the mechanism of the milk protein synthesis pathway. Data were analyzed by two-way analysis of variance (ANOVA) with supplement type and plate as fixed effects. The least significant difference test was used to evaluate the differences among treatments. The HMBi treatment group had the highest beta-casein and S6 kinase beta-1 (S6K1) mRNA gene expression levels. HMBi and D-Met treatments have higher gene expressions compared to the control group. In terms of medium protein content, HMBi had a higher medium protein quantity than the control although not significantly different from the D-Met group. HMBi supplementation stimulated the production of eukaryotic translation initiation factor 3 subunit protein essential for protein translation initiation resulting in higher medium protein synthesis in the HMBi group than in the control group. The protein pathway analysis results showed that the D-Met group stimulated fructose–galactose metabolism, glycolysis pathway, phosphoinositide 3 kinase, and pyruvate metabolism. The HMBi group stimulated the pentose phosphate and glycolysis pathways. Metabolite analysis revealed that the D-Met treatment group increased seven metabolites and decreased uridine monophosphate (UMP) production. HMBi supplementation increased the production of three metabolites and decreased UMP and N-acetyl-L-glutamate production. Taken together, D-Met and HMBi supplementation are effective in stimulating milk protein synthesis in MAC-T cells by genes, proteins, and metabolites stimulation linked to milk protein synthesis.