Bicinchoninic Acid Assay Research Articles

Creation of Single Cell Protein-Producing Mutants of Phaffia Rhodozyma

Recently, there has been an increasing demand for sustainable protein sources for aquaculture. In aquaculture, sustainable protein includes sources that can be produced with a minimal environmental footprint, such as single-cell proteins, proteins from algae, insects, and by-products from the agricultural industry. Single-cell proteins that are derived from microorganisms show a sustainable alternative to traditional protein sources due to their high protein content and rapid growth. This research focuses on creating mutants of the yeast Phaffia rhodozyma to increase single-cell protein production. This yeast has significant industrial potential and biological features. The yeast Phaffia rhodozyma is already known for its unique ability to synthesize astaxanthin, so this case will focus on its ability to produce protein. In the study, we used wild strain yeast and previously obtained white mutants on inorganic nitrogen media for further random mutagenesis methods to introduce specific mutations into the genome aimed at improving their ability to biosynthesize protein. Amino acid inhibitor was used to select potential protein-producing mutants. The protein content of the biomass obtained from the experiments was analyzed in detail using BCA (Protein Assay Reagent) analysis, which confirmed their potential as a source of nutrients for aquaculture. This method is based on the reaction between bicinchoninic acid and the peptide bonds of proteins, which results in the formation of a color whose intensity is proportional to the amount of protein in the sample. The results obtained allow further research into the development of cost-effective and environmentally friendly alternatives for feed in aquaculture.

Platelet microparticles influence gene expression and modulate biological activities of chronic myeloid leukemia cells (K562).

The current understanding emphasizes the intricate interplay between the Leukemic cell and its environment. Platelet-derived microparticles play a crucial role in facilitating intercellular communication and contribute to the complex landscape of cancer pathology. This study aimed to investigate the influence of platelet-derived microparticles on cell proliferation, apoptosis, and the expression of key genes, including P53, P21, Cyclin D1, Bax, and Bcl-2, within the context of a chronic myeloid leukemia cell line (K562). Platelet-derived microparticles were obtained through centrifugation at various speeds, and their concentration was quantified using the BCA assay. To determine the size and immunophenotypic characteristics of the PMPs, both the DLS technique and flow cytometry were employed. Cell proliferation was assessed using the MTT assay and hemocytometer, and cell cycle analysis was conducted through DNA content evaluation. Real-time PCR was utilized for gene expression analysis of Bax, Bcl-2, Cyclin D1, P53, and P21. Flow cytometry was employed to examine cell apoptosis. The findings revealed that platelet-derived microparticles have the ability to decrease proliferation of the K562 cell line, while not exerting an impact on apoptosis and cell cycle progression. Analysis through real-time PCR indicated an upregulation in the gene expression of P53, P21, and Bcl-2, accompanied by a downregulation in Bax and Cyclin D1. This investigation sheds light on the intricate relationship between chronic myeloid leukemia and its microenvironment, particularly the involvement of platelet-derived microparticles. The study underscores the potential of platelet-derived microparticles to influence cell behavior and gene expression, providing a deeper understanding of their role in CML and its therapeutic implications.

High-Moisture Extrusion of a Dietary Protein Blend Impairs In Vitro Digestion and Delays In Vivo Postprandial Plasma Amino Acid Availability in Humans

BackgroundIndustrial processing can alter the structural complexity of dietary proteins and, potentially, their digestion and absorption upon ingestion. High-moisture extrusion (HME), a common processing method used to produce meat alternative products, affects in vitro digestion, but human data are lacking. We hypothesized that HME of a mycoprotein/pea protein blend would impair in vitro digestion and in vivo postprandial plasma amino acid availability. MethodsIn Study A, 9 healthy volunteers completed 2 experimental trials in a randomized, double-blind, crossover design. Participants consumed a beverage containing 25 g protein from a “dry” blend (CON) of mycoprotein/pea protein (39%/61%) or an HME content-matched blend (EXT). Arterialized venous blood samples were collected in the postabsorptive state and regularly over a 5-h postprandial period to assess plasma amino acid concentrations. In Study B, in vitro digestibility of the 2 beverages were assessed using bicinchoninic acid assay and optical fluorescence microscopy at baseline and during and following gastric and intestinal digestion using the INFOGEST model of digestion. ResultsProtein ingestion increased plasma total, essential (EAA), and branched-chain amino acid (BCAA) concentrations (time effect, P < 0.0001) but more rapidly and to a greater magnitude in the CON compared with the EXT condition (condition × time interaction, P < 0.0001). This resulted in greater plasma availability of EAA and BCAA concentrations during the early postprandial period (0–150 min). These data were corroborated by the in vitro approach, which showed greater protein availability in the CON (2150 ± 129 mg/mL) compared with the EXT (590 ± 41 mg/mL) condition during the gastric phase. Fluorescence microscopy revealed clear structural differences between the 2 conditions. ConclusionsThese data demonstrate that HME delays in vivo plasma amino acid availability following ingestion of a mycoprotein/pea protein blend. This is likely due to impaired gastric phase digestion as a result of HME-induced aggregate formation in the pea protein.This trial was registered at clinicaltrials.gov as NCT05584358.

Exosomal ROR1 in peritoneal fluid identifies peritoneal disseminated PDAC and is associated with poor survival.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and peritoneal dissemination is one major cause for this poor prognosis. Exosomes have emerged as promising biomarkers for gastrointestinal cancers and can be found in all kinds of bodily fluids, also in peritoneal fluid (PF). This is a unique sample due to its closeness to gastrointestinal malignancies. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been identified as a potential biomarker in human cancers and represents a promising target for an immunotherapy approach, which could be considered for future treatment strategies. Here we prospectively analyzed the exosomal surface protein ROR1 (exo-ROR1) in PF in localized PDAC patients (PER-) on the one hand and peritoneal disseminated tumor stages (PER+) on the other hand followed by the correlation of exo-ROR1 with clinical-pathological parameters. Exosomes were isolated from PF and plasma samples of non-cancerous (NC) (n = 15), chronic pancreatitis (CP) (n = 4), localized PDAC (PER-) (n = 18) and peritoneal disseminated PDAC (PER+) (n = 9) patients and the surface protein ROR1 was detected via FACS analysis. Additionally, soluble ROR1 in PF was analyzed. ROR1 expression in tissue was investigated using western blots (WB), qPCR, and immunohistochemistry (IHC). Exosome isolation was proven by Nano Tracking Analysis (NTA), WB, Transmission electron microscopy (TEM), and BCA protein assay. The results were correlated with clinical data and survival analysis was performed. PDAC (PER+) patients have the highest exo-ROR1 values in PF and can be discriminated from NC (p <0.0001), PDAC (PER-) (p <0.0001), and CP (p = 0.0112). PDAC (PER-) can be discriminated from NC (p = 0.0003). In plasma, exo-ROR1 is not able to distinguish between the groups. While there is no expression of ROR1 in the exocrine pancreatic tissue, PDAC and peritoneal metastasis show expression of ROR1. High exo-ROR1 expression in PF is associated with lower overall survival (p = 0.0482). With exo-ROR1 in PF we found a promising diagnostic and prognostic biomarker possibly discriminating between NC, PDAC (PER-) and PDAC (PER+) and might shed light on future diagnostic and therapeutic concepts in PDAC.

#1841 Trimethylamine N-oxide (TMAO) induces the formation of endothelial extracellular vesicles

Abstract Background and Aims Endothelial dysfunction caused by the accumulation of uremic toxins is an important contributor to the development of cardiovascular disease in patients with chronic kidney disease (CKD). The uremic toxin trimethylamine n-oxide (TMAO) is associated with oxidative stress, vascular calcification, and inflammation, triggering the release of several proinflammatory molecules and Extracellular Vesicles (EVs). Clinical studies have shown that patients with CKD have high levels of circulating EVs, however, their role is still poorly understood. In this dysfunctional scenario, EVs can be used as damage biomarkers for CKD, as they carry information from their cells of origin. Here, we aim to elucidate the formation of EVs in endothelial cells exposed to TMAO. Method Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed for 24 h with TMAO at uremic concentrations (7.49 mg/L). EVs were isolated by differential ultracentrifugation. Protein concentration was analyzed by Pierce™ BCA Protein Assay Kit, and particle size and particle concentration were analyzed by NanoParticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and Scanning Electron Microscopy (SEM). EVs were stained with calcein and their internalization in endothelial cells was analyzed with Fluorescence Confocal Microscopy. Results TMAO treatment at uremic concentration induced the formation of EVs with a size of 202.8 ± 3.1 nm (image 1B) and 1.09 × 109 ± 2.68 × 107 particles/mL and a total protein concentration of 88.57 µg/mL. The morphological features of EVs are shown in images 1C-1F. TMAO-induced EVs were incorporated by endothelial cells, being dispersed in the cytoplasm or perinuclear regions (images 1G and end 1H). Conclusion TMAO was able to induce the formation of EVs in endothelial cells, with characteristics morphological features. When compared to control EVs (image 1A), TMAO-EVs have a more heterogeneous size, which indicate a change in the size pattern, and thus the nature of the EVs induced by TMAO. TMAO-EVs were able to be internalized by human endothelial cells. This uptake can be closely related to the effects of EVs in endothelial cells, but more experiments need to be performed. Taken together, these data could contribute to understanding how the formation and uptake of TMAO-EVs by the endothelium occurs, relating their effects to the cardiovascular complications of CKD.

Human gingival mesenchymal stem cells-lyosecretome attenuates adverse effect of hydrogen peroxide-induced oxidative stress on osteoblast cells

ObjectiveTo determine total protein content, antioxidant activity, and protective ability of lyophilized human gingival mesenchymal stem cells (hGMSCs)-secretome in hydrogen peroxide (H2O2) induced oxidative stress model. MethodsHuman GMSCs were cultured to obtain a conditioned medium (secretome), then lyophilized to produce lyosecretome. Total protein was determined by bicinchoninic acid assay (BCA) and SDS-PAGE to improve protein measurements. Antioxidant concentration was measured by ABTS assay, while the protective ability of lyosecretome against oxidative stress was determined by the metabolic activity of osteoblast cells. The study group was divided into a control group (culture medium) and a lyosecretome treatment group (0.0; 0.157, 0.313, 0.625, 1.25, 2.5, 5, and 10 mg/mL + H2O2). ResultsLyosecretome had a protein concentration of 2086.00 ± 0.20 μg/ml, with a molecular weight of 174, 74, 61, 55, and 26 kDa, which are thought to facilitate cell migration, as well as bind cytokines and growth factors. Lyosecretome also provided the highest antioxidant activity of 93.51% at a concentration of 4.8 mg/ml, with an IC50 value of 2.08 mg/ml. The highest cell metabolic activity (79.53 ± 2.41%) was shown in the 1.25 mg/ml lyosecretome treatment group. All concentrations of hGMSC-lyosecretome attenuate the adverse effect of H2O2-induced oxidative stress. ConclusionLyosecretome obtained from hGMSCs can maintain metabolic activity in osteoblast cells as protection against H2O2 oxidative stress.

Effective delivery of anti-PD-L1 siRNA with human heavy chain ferritin (HFn) in acute myeloid leukemia cell lines.

Because of the high biocompatibility, self-assembly capability, and CD71-mediated endocytosis, using human heavy chain ferritin (HFn) as a nanocarrier would greatly increase therapeutic effectiveness and reduce possible adverse events. Anti-PD-L1 siRNA can downregulate the level of PD-L1 on tumor cells, resulting in the activation of effector T cells against leukemia. Therefore, this study aimed to produce the tumor-targeting siPD-L1/HFn nanocarrier. Briefly, the HFn coding sequence was cloned into a pET-28a, and the constructed expression plasmid was subsequently transformed into E. coli BL21. After induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG), HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The protein characteristics were analyzed using SDS-PAGE, Western Blot, and Dynamic light scattering (DLS). The final concentration was assessed using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The RT-PCR was used to determine the mRNA expression of PD-L1. The biocompatibility and excretion of siPD-L1/HFn have also been evaluated. The expression and purity of HFn were well verified through SDS-PAGE, WB, and DLS. RT-PCR analyses also showed significant siRNA-mediated PD-L1 silencing in both HL-60 and K-562 cells. Our study suggested a promising approach for siRNA delivery. This efficient delivery system can pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy.

PLGA nanocapsules as a delivery system for a recombinant LRP-based therapeutic.

Telomerase activity is directly affected by the laminin receptor precursor (LRP) protein, a highly conserved nonintegrin transmembrane receptor, which has been shown to have therapeutic effects in ageing, and age-related diseases. Recently, it has been found that overexpression of LRP-FLAG, by plasmid transfection, leads to a significant increase in telomerase activity in cell culture models. This may indicate that upregulation of LRP can be used to treat various age-related diseases. However, transfection is not a viable treatment strategy for patients. Therefore, we present a nanoencapsulated protein-based drug synthesised using poly(lactic-co-glycolic acid) (PLGA) nanocapsules for delivery of the 37 kDa LRP protein therapeutic. PLGA nanocapsules were synthesised using the double emulsification-solvent evaporation technique. Different purification methods, including filtration and centrifugation, were tested to ensure that the nanocapsules were within the optimal size range, and the BCA assay was used to determine encapsulation efficiency. The completed drug was tested in a HEK-293 cell culture model, to investigate the effect on cell viability, LRP protein levels and telomerase activity. A significant increase in total LRP protein levels with a concomitant increase in cell viability and telomerase activity was observed. Due to the observed increase in telomerase activity, this approach could represent a safer alternative to plasmid transfection for the treatment of age-related diseases.

Analytical Methods and Effects of Bioactive Peptides Derived from Animal Products: A Mini-Review.

Peptides with bioactive effects are being researched for various purposes. However, there is a lack of overall research on pork-derived peptides. In this study, we reviewed the process of obtaining bioactive peptides, available analytical methods, and the study of bioactive peptides derived from pork. Pepsin and trypsin, two representative protein digestive enzymes in the body, are hydrolyzed by other cofactors to produce peptides. Bicinchoninic acid assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chromatography, and in vitro digestion simulation systems are utilized to analyze bioactive peptides for protein digestibility and molecular weight distribution. Pork-derived peptides mainly exhibit antioxidant and antihypertensive activities. The antioxidant activity of bioactive peptides increases the accessibility of amino acid residues by disrupting the three-dimensional structure of proteins, affecting free radical scavenging, reactive oxygen species inactivation, and metal ion chelating. In addition, the antihypertensive activity decreases angiotensin II production by inhibiting angiotensin converting enzyme and suppresses blood pressure by blocking the AT1 receptor. Pork-derived bioactive peptides, primarily obtained using papain and pepsin, exhibit significant antioxidant and antihypertensive activities, with most having low molecular weights below 1 kDa. This study may aid in the future development of bioactive peptides and serve as a valuable reference for pork-derived peptides.

Liver Androgen Receptor Knockout (LivARKO) Male Mice on 3-months of High Fat Diet (HFD) Did Not Experience Insulin Resistance at the Level of p-AKT in White Adipose Tissue

Introduction: Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome (PCOS) affects up to 10% of reproductive-age adult women. Andrisse et al 2021 (PMCID: PMC9097557) showed that deleting the liver androgen receptor (LivARKO) prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. Here, we asked the question, does LivARKO prevent high fat diet (HFD) induced insulin resistance? Hypothesis: It was hypothesized that (unlike HA-LivARKO) HFD LivARKO male mice would display impaired insulin action (lowered insulin-stimulated p-AKT) in white adipose tissue in comparison to the Control diet fed LivARKO male mice, suggesting that AR does not play a significant role in regulating HFD-induced insulin resistance. Methods: Male LivARKO mice were placed on two diets: Control (Con, Research Diets Inc, RDI D12450J, Protein (20%), Carbs (70%, corn starch-50%, sucrose-maldextrin-20%, fructose-0%), Fat 10% (lard)) and High Fat (HFD, RDI D12492, Protein (20%), Carbs (20%, corn starch-0%, sucrose-maldextrin-20%, fructose-0%), Fat 60% (lard)). The mice were sacrificed after 3 months on the diet. Half of the mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of the diets on insulin signaling. Western blots were used to determine protein expression in white adipose tissue (WAT). BCA assays were used to standardize the protein concentration in each sample. Insulin action can be measured molecularly by examining p-AKT Serine 473 (positive regulator, Santa Cruz sc-514032). If p-AKT S473 levels increase in the presence of insulin, this indicates that insulin is likely functioning properly in the sample. Results: Male LivARKO fed a control diet (LivARKO-Con) and administered with 0.5 U/kg insulin displayed significantly lowered p-AKT protein levels compared to male LivARKO-Con that did not get administered an i.p. injection of insulin (Basal), suggesting that male LivARKO-Con mice were experiencing insulin resistance. Additionally, male LivARKO fed a high fructose diet (LivARKO-HFrD) and administered with 0.5 U/kg insulin displayed slightly increased p-AKT protein levels compared to male LivARKO-HFrD-Basal mice. Conclusion: In conclusion, LivARKO male mice on 3-months of HFD were not experiencing insulin resistance at the level of p-AKT in WAT. We are not certain why insulin negatively regulated (or lowered) p-AKT in WAT of LivARKO-Con male mice. This could be an aspect of needing a higher n-value. Further studies in different energy storage tissues and higher n-values are warranted. We would like to thank the NSF for award # 1931045 and the NIH for award # 1R01DK126892-01A1 for supporting this work. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Effect of High Fructose Diet on Insulin Signaling in Pituitary Tissue of Liver Androgen Receptor Knockout in Female Mice

Intro: Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome affects up to 10% of reproductive-age adult women. Hyperandrogenism in females can increase predisposition to insulin resistance. Andrisse et al 2021 (PMCID: PMC9097557) showed that deleting the liver androgen receptor (LivARKO) prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. This study placed female LivARKO mice on a high fructose diet (HFrD) to determine if LivARKO mice demonstrate blunted insulin resistance on HFrD compared to control diet. Hypothesize: It was hypothesized that (unlike HA-LivARKO) HFrD LivARKO female mice would display impaired insulin action (lowered insulin-stimulated p-AKT) in PIT tissue in comparison to the Control diet fed LivARKO female mice, suggesting that liver AR does not play a significant role in regulating HFrD-induced insulin resistance in the pituitary. Methods: Female LivARKO mice were placed on two diets: Control (Con, Research Diets Inc, RDI D12450J) and High Fructose (HFrD, RDI D02022704). After 3 months on the diet, the mice were sacrificed. Half of the mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of the diets on insulin signaling. Western blots were used to determine protein expression in tissue from the pituitary tissue (PIT). BCA assays were used to standardize the protein concentration in each sample. Insulin action can be measured molecularly by examining p-AKT Serine 473 (positive regulator, Santa Cruz sc-514032). If p-AKT S473 levels increase in the presence of insulin, this indicates that insulin is likely functioning properly in the sample. Results:LivARKO female mice on a control diet (Con) for 3-months administered with insulin displayed a 3-fold significant increase in p-AKT levels in the pituitary compared to LivARKO-Con female mice not injected with insulin (basal), suggesting that LivARKO-Con mice pituitaries were insulin sensitive. However, LivARKO female mice fed a high fructose diet (HFrD) for 3-months and then administered with insulin displayed significantly lowered p-AKT levels in the pituitary compared to basal LivARKO-HFrD female mice and compared to insulin LivARKO-Con, indicating that LivARKO-HFrD female mice were experiencing insulin resistance (lowered insulin sensitivity compared to LivARKO-Con). Interestingly, p-AKT was significantly increased by 2.5 fold in LivARKO-HFrD female mice compared to LivARKO-Con, suggesting that HFrD increased insulin sensitivity in the pituitary. Conclusion: In conclusion, these data suggest that LivARKO indirectly disrupted insulin-stimulated p-AKT in PIT possibly via altering metabolites in the blood. Additionally, HFrD is known to cause insulin resistance, however, it is not known to alter p-AKT levels. Thus, the change in p-AKT levels is presumably associated with the LivARKO. Further research is required on what components of the Control Diet are prompting this difference in insulin action and if it only takes place in the LivARKO mouse model. We would like to thank the National Science Foundation for award # NSF 1931045 and the National Institutes of Health 1R01DK126892-01A1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

DTT protein equalization and Tryptophan protein quantification as a powerful tool in analytical proteomics.

Assessing total protein levels in biological samples is a common procedure in biochemistry and molecular biology. In this study, we compare tryptophan fluorescence (WF) with Bradford and BCA assays to determine total protein in serum samples. Our results indicate that tryptophan fluorescence spectrometry is an efficient, sensitive, and straightforward technique for quantifying proteins in serum. We observed minimal variation between the three methods: BCA de one with the lowers LOD and LOQ. The tryptophan method offers the possibility of reusing the intact sample that does not need colourimetric reagents for quantification. Consequently, free tryptophan serves as a reliable universal standard. This assay can be performed using a conventional fluorescence spectrometer with cuvettes or in a 96-well plate format with a plate reader. The method was successfully used as proof of concept, using serum from patients diagnosed with myeloma and serum from healthy donors.

Characterization of Antimicrobial Activities of Bifidobacterium lactis BB-12 and Their Inhibitory Effect Against Some Foodborne Pathogens.

Bifidobacterium animalis subsp. lactis BB-12, a probiotic, has shown potential to promote health benefits and control pathogens. This study aimed to investigate the effectiveness of BB-12 and its cell-free supernatant (CFS) in inhibiting the growth of Listeria monocytogenes and Salmonella enterica serovar Typhimurium. To assess the antimicrobial activity of BB-12, agar well diffusion, disk diffusion, and minimum inhibitory concentration (MIC) tests were conducted. The bicinchoninic acid (BCA) assay was performed to measure the protein concentration in CFS. The study's results indicated that the BB-12 strain inhibited the pathogens' growth. The disk diffusion test using BB-12 showed inhibitory results ranging from 11 to 14 mm for both bacteria. The agar well diffusion test reported the zone of inhibition ranging from 11.6 to 16 mm for both bacteria. The MIC test was conducted as a confirmatory test, which demonstrated the highest inhibitory zone using 2 McFarland (6 × 108 CFU/mL) concentrations of probiotics on L. monocytogenes (44.98%) and S. Typhimurium (66.41%). The disk diffusion test revealed that the probiotic CFS had a significant inhibitory impact on S. Typhimurium with a 16.6 mm zone of inhibition. The BCA test findings indicated that the 24- and 48-h CFSs exhibited inhibitory properties against infections. Notably, the 24-h CFS, including a protein level of 78.47 μg/mL, demonstrated a more pronounced inhibitory impact on both pathogens. The findings highlight that utilizing the BB-12 strain and its CFS can serve as a viable approach to battle infections, enhancing food safety and public health.

Skin-derived precursor conditioned medium alleviated photoaging via early activation of TGF-β/Smad signaling pathway by thrombospondin1: In vitro and in vivo studies

Photoaging is one major exogenous factor of skin aging. Efficacy and safety of current anti-photoaging therapies remained to be improved. Our previous studies indicated that skin-derived precursors (SKPs) alleviated photodamage by early activation of TGF-β/Smad signaling pathway via thrombospondin1 (TSP1). However, the research concerning SKP conditioned medium (SKP-CM) has never been reported. In the current study, we aimed to explore the anti-photoaging effects of SKP-CM both in vitro and in vivo, and to elucidate the possible mechanisms. Mouse SKP-CM (mSKP-CM) collection was optimized by a comparative method. The concentration of protein and growth factors in mSKP-CM was detected using BCA protein assay kit and growth factor protein chip. The anti-photoaging effects of mSKP-CM and its regulation of key factors in the TGF-β/Smad signaling pathway were explored using UVA + UVB photoaged mouse fibroblasts (mFBs) and nude mice dorsal skin. The research revealed that mSKP-CM contained significantly higher-concentration of protein and growth factors than mouse mesenchymal stem cell conditioned medium (mDMSC-CM). mSKP-CM alleviated mFBs photoaging by restoring cell viability and relieving senescence and death. ELISA, qRT-PCR, and western blot results implied the potential mechanisms were associated with the early activation of TGF-β/Smad signaling pathway by TSP1. In vivo experiments demonstrated that compared with the topical intradermal mDMSC-CM injection and retinoic acid cream application, the photodamaged mice dorsal skin intradermally injected with mSKP-CM showed significantly better improvement. Consistent with the in vitro results, both western blot and immunohistochemistry results confirmed that protein expression of TSP1, smad2/3, p-smad2/3, TGF-β1, and collagen I increased, and matrix metalloproteinases decreased. In summary, both in vitro and in vivo experiments demonstrated that mSKP-CM alleviated photoaging through an early activation of TGF-β/Smad signaling pathway via TSP1. SKP-CM may serve as a novel and promising cell-free therapeutical approach for anti-photoaging treatment and regenerative medicine.

Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 β, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 β were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 β, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

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.

Abstract 1588: Identification of new signaling pathways between fibroblasts &amp; tumor cells in pancreatic cancer

Abstract We explore the unstudied role of nanoparticles (NPs), recently discovered secreted non-vesicular nanoparticles, in signaling between pre-cancer associated fibroblasts (preCAF), CAFs, and tumor cells. We generated two novel primary lines preCAF PFUM1 from a patient with high-grade intraductal papillary mucinous neoplasms and CAF FUM2 from a patient with pancreatic ductal adenocarcinoma, all tests performed between passages 2 and 3, fibroblast purity validated by RT-PCR and ACTA2 immunofluorescence. Small extracellular vesicles (sEVs), exomeres, and supermeres (the two main forms of NPs) were isolated by the ultracentrifugation method from PF-UM1 and F-UM2 with n=2-3 experiments. To validate the isolation, we used nanoparticle tracking, electron, and fluid-phase atomic force microscopy. Proteins were quantified by BCA assay. Liquid chromatography-mass spectrometry characterized the cargo from each fraction. Human cancer lines MIAPACA2 and SU8686 were treated with 10 μg of each fraction and cell proliferation was tested by phase-contrast images (Cytation 5), analyzed by Ilastik-based 1.4.0 machine-learning. We performed fluorescent Ki-67 and vimentin staining and quantified in QuPath 0.4.3. Non-parametric statistics were performed in Prism GraphPad. NPs constitute the major fraction of small particles released by fibroblasts, containing 80.5% (PFUM1) and 77.5% (FUM2) of the protein load by quantification. Mass spectrometry reveals enrichment of growth factors, epithelial-to-mesenchymal (EMT) transition, and metabolism-associated proteins in the NPs compared to sEVs. At 72 hours, cell proliferation for Su8686 reveals a proliferative effect for all fractions from both PFUM1 and FUM2 (19-63% increase compared to controls, p=0.049 to &amp;lt;0.0001). For MIAPACA2, only supermeres from PFUM1 and FUM2 consistently increased proliferation (9.5-35% increase, p=0.0051 to &amp;lt;0.0001). Ki-67 expression in MIAPACA2, increased by 9.6-23.0% following treatment by NPs (all p&amp;lt;0.0001) and by 2.9-9.2% with sEVs (p=0.011 to &amp;lt;0.0001). EMT was estimated by vimentin in MIAPACA2, as all preCAF fractions from PFUM1 reduced vimentin expression (p&amp;lt;0.001), while CAF FUM2 NPs increased vimentin expression (p&amp;lt;0.0001) while sEVs had no effect (p=0.075). In conclusion, the majority of pancreatic tumor-associated fibroblast small secretome is composed of NPs, exhibiting significant functional pro-tumoral effects. Citation Format: Ayman Al Shoukari, Maelle Batardiere, Nooshin Movahed, Camille beaussier, Jumanah Baig, Melissa gonzalez, Patricia Moraille, Eric Bonneil, Louise Rousseau, Simon turcotte, Kathleen Delgiorno, Marcus Tan, Anna Means, Elham Dianati Ajibisheh, Quoc-huy Trinh. Identification of new signaling pathways between fibroblasts &amp; tumor cells in pancreatic cancer [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 1588.

Abstract 3897: Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood-brain barrier

Abstract Glioblastoma (GBM) is a highly aggressive tumor of the brain. Current techniques of detection like magnetic resonance imaging and tissue biopsy are inaccurate or highly invasive, requiring alternatives like liquid biopsy. Exosomes are nanosized vesicles that contain cargo, reflective of their cellular origin and have been used for early diagnosis of cancer. However, in GBM, the blood-brain barrier (BBB) leads to a paucity of pathological information in blood. This study investigates the effect of opening the BBB on circulating exosomes and their potential for early detection of GBM using a microfluidic platform (GlioExoChip). In 20 patients enrolled in a clinical trial NCT03744026, a pulsed ultrasound emitter- Sonocloud-9 was implanted into their brains to open the BBB. We show that GlioExoChip isolates GBM-specific exosomes with a high capture efficiency using phosphatidylserine and Annexin-V chemistry. Further, the amount of circulating EVs increases twofold after opening the BBB. Finally, the correlation between tumor growth rate and the exosome concentrations is evaluated longitudinally. Methods: GlioExoChip is a microfluidic platform with a polydimethylsiloxane top and a glass bottom, functionalized with neutravidin-biotinylated annexin-V. Experiments are performed with GBM cell-line derived exosomes, followed by patient plasma. Isolated exosomes are characterized using Nanoparticle tracking analysis (NTA), scanning electron microscopy, Bicinchoninic acid (BCA) assay and western blot. Statistical analyses are performed using Student’s t-test and Pearson correlation. Results: The microfluidic device GlioExoChip has a high capture efficiency of exosomes derived from GBM cell lines, i.e., 93.03±3.31% and 96.77±0.72% for GBM6 and U87, respectively. NTA and BCA showed that 15 times more exosomes were captured from patient plasma when compared with healthy. Further, western blot proved the presence of a glial protein and Flotillin-1 in exosomes isolated from patients. Exosome concentrations varied with different time points after sonication and had an average twofold increase from the BBB opening. In our longitudinal study with 8 patients, an association between the volumetric enhancement of the tumor and exosome concentrations after sonication shows that the average Pearson correlation among short progression-free survival (PFS) patients was 0.73 (avg. p=0.39), while no correlation was seen among long PFS patients (avg. r value=0.10; avg. p-value=0.54). Conclusions: A microfluidic approach was used to investigate the role of circulating exosomes using phosphatidylserine-annexin V chemistry in GBM. BBB opening via sonication leads to an increase in the average GBM-specific exosome concentrations in plasma. Thus, exosomes are promising biomarkers for early detection in GBM. Citation Format: Abha Kumari, Mark Youngblood, Andrew Gould, Yoon-Tae Kang, Li Chen, Karl Habashy, Chris Amidei, Rachel Ward, Cristal Gomez, Guillaume Bouchoux, Michael Canney, Roger Stupp, Adam M. Sonabend, Sunitha Nagrath. Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood-brain barrier [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 3897.

Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis

Determination of protein concentration in Hymenoptera venoms requires an accurate and reproducible assay as the results will be used to support subsequent proteomic techniques employed in their analyses. However, all protein assay techniques have inherent strengths and weaknesses, demanding their assessment before selecting the most suitable platform for sample analysis. In this study, protein profiles of ant, honeybee, and wasp venoms, and bovine serum albumin (BSA) and hyaluronidase standards were qualitatively assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid and protein concentration were quantitatively determined via Amino Acid Analysis (AAA). Amino acid concentration was determined via hydrolysis, derivatization, and chromatographic quantification. Protein concentration was estimated using four different protein concentration assays. The ratios of protein concentration in venom samples to protein standards were calculated, and the accuracy of the protein concentration assays was analysed relative to the concentration determined from AAA. SDS-PAGE analysis showed that BSA contained several protein bands, while hyaluronidase contained a mixture of peptide and protein bands. Ant and honeybee venoms contained a higher proportion of peptide bands, while wasp venom contained more protein bands. As determined by AAA, the ratio of protein concentration in Hymenoptera venoms varied between 1.01 and 1.11 to BSA, and between 0.96 and 1.06 to hyaluronidase. Overall, the Bradford assay was found to be the least accurate and the BCA assay was the most accurate in estimating protein concentration in Hymenoptera venoms. There was no significant advantage in using hyaluronidase as a standard or increasing incubation temperature of BCA assay when analysing Hymenoptera venoms. Diluent solutions containing phenol and human serum albumin interfered with Lowry-based assays.