Metabolically healthy/unhealthy obesity and breast cancer: A possible role of plasma-derived extracellular vesicles on the cancerous behavior of triple-negative breast cancer.

PURPOSE: Obesity has known detrimental effects on breast cancer (BC) development and progression. However, it's essential to consider the obesity phenotype based on metabolic health. This study aims to evaluate the impact of circulating extracellular vesicles (EVs) from women with metabolically healthy or unhealthy normal weight, overweight, and obesity on MDA-MB-231 cell migration, invasion, and apoptosis. METHODS: Plasma EVs were isolated from different obesity phenotypes in women. EVs were characterized and EVs uptake by MDA-MB-231 cells was assessed. MDA-MB-231 cell lines were treated with EVs obtained from various studied groups, and migration, invasion, MMP-2 and MMP-9 activity, Bax and Bcl-2 mRNA expression, p-53 and Thr55 p-p53 protein expression, and apoptosis were assessed. RESULTS: EVs from obese individuals, regardless of phenotype, increased invasion and MMP-2 activity compared to healthy normal-weight EVs. Normal-weight EVs led to higher invasion under unhealthy conditions. BC cell migration was enhanced by EVs from healthy obese individuals compared to healthy normal-weight EVs. EVs from unhealthy obese women exhibited significantly lower p53/p-p53 levels and reduced apoptosis compared to healthy obese groups. CONCLUSION: It appears that EVs from both normal-weight women with unhealthy conditions and those with obesity or overweight, irrespective of metabolic status, worsened the cancerous behavior of TNBC cells. Therefore, considering metabolic health, in addition to BMI, is crucial for understanding obesity-related disorders.

The obeticholic acid can positively regulate the cancerous behavior of MCF7 breast cancer cell line.

BACKGROUND: The diagnosis and treatment processes of cancer are among the main challenges of medical science in recent decades. The use of different therapeutic agents is one of the most common methods frequently utilized for cancer treatment. Accumulating evidence points to a potential effect of Obeticholic acid (OCA), a specific ligand for farnesoid X receptor, on the regulation of cancer-associated pathways. In spite of tremendous efforts to introduce OCA into the clinical setting, there is a great deal of uncertainty about its impact on breast cancer treatment. This study was performed to evaluate the effects of OCA on breast cancer. METHODS AND RESULTS: In this experiment, the MCF-7 (Michigan Cancer Foundation-7) cell line was treated with 0.1 µM OCA, and cancerous characteristics of the MCF-7 cell line was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, gelatin zymography, western blot, Real-time PCR, flow cytometry, and ELISA techniques. The results indicated that OCA increased the rate of apoptosis and the expression levels of PPARα (Peroxisome proliferator-activated receptor alpha) and TIMP-1 (tissue inhibitor of metalloproteinase-1) genes in this cell line, while it reduced the mRNA levels of MMP7 (matrix metalloproteinase 7) and Bcl-2 (B-cell lymphoma 2) genes, as well as the protein levels of the active form of AKT (protein kinase B), Erk1/2 (extracellular signal-regulated kinase 1/2) and STAT3 (Signal transducers and activators of transcription-3). Also, OCA decreased the activity of MMP9, while it increased the secretion of VEGF-A (vascular endothelial growth factor-A). CONCLUSIONS: It seems that OCA can exert anti-cancer effects on the MCF-7 cells by reducing growth, proliferation, migration, invasion, and regulation of the expression of genes involved in cancer-associated pathways. However, it should be noted that further studies are warranted to establish this concept, especially the increase of VEGF-A can be considered a challenge for the results of this study.

Paper based analytical devices for blood grouping: a comprehensive review.

The clinical importance of blood group (BG) antigens is related to their ability to induce immune antibodies that can cause hemolysis. Yet, ABO and D (Rh) are still considered to be the key antigens for healthy blood transfusion and secondary antigens are the next priority. Serological typing is the most widely used typing method. Rapid and accurate blood grouping plays an important role in some clinical conditions, rather than conventional techniques. Hence, developing a simple and economical model for rapid blood grouping would facilitate these tests. In recent decades, paper-based microfluidics such as µPADs has gained much interest in wide application areas such as point-of-care diagnostic. In this study, we evaluated µPADs that are performed for blood grouping and its recent progress. A comprehensive literature search was performed using databases including PUBMED, SCOPUS, Web of Science and Google Scholar. Keywords were blood grouping or typing, paper analytical device, rapid test, etc. After investigation of search results, 16 papers from 2010 to 2020 were included. Further information in detail was classified in Table 1. Generally, two principles for blood typing µPADs are introduced. The lateral chromatographic flow method and the vertical flow-through method that detects BG in a visual-based manner. To detect results with acceptable clarity many factors and challenges like paper, blood sample, buffer, Ab and RBC interaction and also µPADs stability need to be considered, which are discussed. In conclusion, the simplicity, stability, cheapness, portability and biocompatibility of µPADs for blood grouping confirming its utility and also they have the capability to robust, universal blood-grouping platform. Table 1 Summary of blood grouping tests using paper-based analytical devices Antigens Type of diagnosis Validation method Sample No Accuracy Action time Paper type Stability Sample dilution Buffer Ref A, B, Rh Forward volunteers records 5 - - Whatman No. 4 - 1/2 PBS* (Khan et al. 2010) A, B, Rh Forward gel assay test and conventional slide test 100 100% 1 min Whatman No. 4 and Kleeenex paper towel 7 Days in 4 °C 1/1 NSS (Al-Tamimi et al. 2012) A, B, Rh Forward gel card assay 99 100% 20 Sec + Washing Kleeenex paper towel - 1/1 NSS (Li et al. 2012) A, B, Rh Forward - - - - Kleeenex paper towel - 45/100 PSS (Li et al. 2013) A, B, Rh Forward gel card assay 98 100% 1.5 min Kleeenex paper towel - 85/100 PBS (Guan et al. 2014b) C, E, c, e, K, Jka, Jkb, M, N, S, P1, and Lea Forward gel card assay 266 100% - Kleeenex paper towel - 1/1 NSS (Li et al. 2014b) A, B, Rh Forward and Reverse conventional slide test 96 ≈ 91% 10 min Whatman No. 1 21 Days in 4 °C 1/2 NSS (Noiphung et al. 2015) C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S and s, P1, Lea and Leb Forward - 478 - - Kleeenex paper towel - 1/1 NSS, PBS (Then et al. 2015) A, B Forward and Reverse conventional slide test 76 100% 5-8 min Whatman No. 4 38 Days in 4 °C 1/4, 1/1 NSS (Songjaroen and Laiwattanapaisal 2016) D, K Forward volunteers records 210 - 7.5 min Kleenex paper towel - 1/1 NSS (Yeow et al. 2016) A, B, c, e, D, C, E, M, N, S, s, P1, Jka, Jkb, Lea, Leb, Fya, and Fyb Forward and Reverse gel card assay 3550 ≈100% 30 s Fiber glass and cotton linter 180 Days in 25 °C 45/100, 1/1 PBS (Zhang et al. 2017) A, B Forward conventional slide test 598 100% 3 min Whatman No. 113 14 Day in 4 °C 1/1 NSS (Songjaroen et al. 2018) A, B, Rh Forward conventional slide test - - 30 Sec + Washing Unrefined sisal paper - 1/2 NSS (Casals-Terré et al. 2019) A, B, Rh Forward - - - - Whatman No.1 - 1/1 NSS (Ansari et al. 2020) ABO & Rh Forward and Reverse conventional slide test - 100% Unrefined Eucalyptus papers - 1/2 NSS, PBS (Casals-Terré et al. 2020) A, B, Rh Forward - - - 30 Sec + Washing Whatman No. 4 modified with chitosan ≥ 100 days in 25 °C 1/1 NSS (Parween et al. 2020) *phosphate buffer saline, normal saline solution.

Evaluating the effect of a mixture of two main conjugated linoleic acid isomers on hepatic steatosis in HepG2 cellular model.

Hepatic steatosis is an early form of non-alcoholic fatty liver disease (NAFLD), caused by abnormal fat deposition in the hepatocytes. Conjugated linoleic acid (CLA) is a group of positional and geometric dienoic isomers of linoleic acid that attract significant attention because of its beneficial effects on chronic diseases such as cancer, obesity, and metabolic syndrome. This study examined the influence of a mixture of two main CLA isomers (CLA-mix) on lipid accumulation and lipid metabolism-related genes using HepG2 cells treated with palmitic acid (PA) as an in vitro model for hepatic steatosis. Methods and Results: HepG2 cells were treated for 24 h: control (BSA), model (BSA + PA), and treated groups (BSA-PA + non-toxic concentrations of CLA-mix). Intracellular lipid deposition, triglyceride (TG), total cholesterol (TC) and gene expression were measured by Oil-Red O staining, colorimetric assay kits and real-time PCR, respectively. CLA-mix at high concentrations had significantly decreased intracellular total lipid and TG deposition compared to the model group. However, none of the CLA-mix concentrations had a significant effect on the intracellular TC level. CLA-mix significantly increased the expression of some genes mainly regulated by PPARα but did not alter the expression of lipogenesis-related genes. Conclusions: These results demonstrate that high concentrations of CLA-mix protect against hepatic steatosis and play a role in regulating fatty acid oxidation and bile excretion through the PPARα pathway. It is suggested that the effect of different ratios of two main CLA isomers on the amount and ratio of bile compounds be investigated in future studies.

The possible effect of silver nanoparticles on glyceraldehyde-3-phosphate dehydrogenase activity and formation of amyloid-like aggregates in MCF-7 cell line.

Silver nanoparticles (AgNPs) are widely used in medicine, however, the underlying mechanisms of their action on cellular signaling have not been completely determined, and fundamental studies are required to clarify them. We aimed to investigate AgNPs effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as both the internal control gene and the redox-sensitive enzyme involved in apoptosis-related pathways and the formation of amyloid aggregates. To achieve this purpose, MCF-7 cells were treated with different concentrations (0, 3, 22, and 200 µg/ml) of AgNPs and then cell viability, generation of reactive oxygen species (ROS), induction of apoptosis, expression of GAPDH gene, the formation of amyloid aggregates, and GAPDH activity were assessed. The results indicated that treatment with AgNPs significantly reduced cell viability and increased apoptosis in a dose-dependent manner. The ROS levels increased at lower concentrations of AgNPs (up to 22 µg/ml) and during short-term exposure (30 min). The level of GAPDH gene expression was significantly upregulated by 1.22, 1.47, and 1.56 fold, in the concentrations of 3, 22, and 200 µg/ml, respectively. The amount of amyloid aggregates was significantly increased in a dose-dependent manner. The results of enzyme activity showed that AgNPs were affected on the activity of GAPDH protein, however, it has fluctuated that could not be interpreted by our limited data. In conclusion, our results suggested that AgNPs could affect the GAPDH gene expression and enzyme activity, therefore the selection of GAPDH as a gene and protein internal control in the (AgNPs)-related studies requires careful consideration. Additionally, AgNPs may cause apoptosis due to the increase in the production of amyloid aggregates.

Influence of plasma circulating exosomes obtained from obese women on tumorigenesis and tamoxifen resistance in MCF-7 cells.

Obesity is associated with breast cancer aggressiveness and drug resistance. Although the underlying mechanisms are unknown, recent studies indicated that exosomes have a principal contributory role in obesity-associated metabolic complications. Hence, we investigated whether obesity can mediate breast cancer progression and resistance to tamoxifen by plasma-derived-exosomes from obese women or not. Plasma exosomes isolated from five normal-weight (N-Exo) and five obese women (O-Exo) were characterized for size, zeta potential, and CD63 expression. After the treatment of MCF-7 cells with N-Exo and O-Exo, cell proliferation, migration, invasion as well as levels of MMP-9 and MMP-2 were evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound healing, transwell, and zymography methods, respectively. For evaluating resistance to tamoxifen, the cell viability, apoptosis, and the p53 protein were evaluated using the MTT assay, flow cytometry, and western blot methods, respectively. Cell proliferation, migration, and invasion were significantly increased in the cells treated with O-Exo than untreated cells (p = .001, p = .018, p = .034, respectively). Levels of MMP-2 and MMP-9 were remarkably increased in the cells treated with O-Exo in comparison with ones treated with N-Exo (p = .040, p = .043, respectively). As for resistance to tamoxifen, O-Exo had significantly the greater anti-apoptotic effects in comparison with the N-Exo group (p = .013). Besides, p53 levels were significantly decreased in the cells treated with O-Exo than ones treated with N-Exo (p = .045). The cell viability was significantly more in cells treated with O-Exo in comparison with the cells only treated with tamoxifen (p = .040). Our findings demonstrated that circulating exosomes derived from obese women could lead to tumorigenesis and tamoxifen resistance in breast cancer cells. However, more studies are needed to establish this notion.

mZD7349 peptide-conjugated PLGA nanoparticles directed against VCAM-1 for targeted delivery of simvastatin to restore dysfunctional HUVECs.

Endothelial dysfunction is initial and critical step of atherosclerosis. Impaired bioavailability of endothelial nitric oxide synthase (eNOS) is one of the main reasons of endothelial dysfunction. Improving bioavailability of eNOS by increasing its expression or activity using statins is an effective therapeutic strategy in restoring endothelial dysfunction. In this study, simvastatin (SIM) as a poorly water-soluble drug was loaded in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (SIM-PLGA-NPs). NPs were then conjugated with mZD7349 peptide (mZD7349-SIM-PLGA-NPs) and directed against vascular cell adhesion molecule 1 (VCAM-1). In vitro evaluation of the NPs for targeted delivery of SIM was performed on activated Human Umbilical Cord Vascular Endothelial Cells (HUVECs) by tumor necrosis factor alpha (TNF-α). Effect of mZD7349-SIM-PLGA-NPs and SIM-PLGA-NPs was compared on eNOS phosphorylation (ser-1177). Results of western blot showed SIM post-treatment increased significantly phosphor-eNOS (Ser1177) expression but no total eNOS expression. The study showed that mZD7349-SIM-PLGA-NPs have particle size, zeta potential value, polydispersity index (PDI) and encapsulation efficacy % of 233±18nm, -9.6±1.1mV, 0.59±0.066 and 69±17.3%, respectively. Also phosphor-eNOS (Ser1177) expression in activated HUVECs treated with mZD7349-SIM-PLGA-NPs was significantly (p<0.05) better than treated cells with SIM-PLGA-NPs. The results suggest that mZD7349-SIM-PLGA-NPs may be usable as an appropriate drug carrier for restoring endothelial dysfunction.

Potential of mZD7349-conjugated PLGA nanoparticles for selective targeting of vascular cell-adhesion molecule-1 in inflamed endothelium.

Early diagnosis and restoring normal function of dysfunctional endothelium is an attractive strategy for prevention of inflammatory diseases such as atherosclerosis. Inhibition of cell adhesion in the process of atherosclerosis plaque formation, mediated by peptide antagonists of very late antigen-4 (VLA-4) has already been developed and evaluated both in vitro and in vivo. In this study, for the first time, modified ZD7349 (mZD7349) peptide, as an antagonist for VLA-4, was used for targeting fluorescein isothiocyanate-loaded poly (DL-lactic-co-glycolic acid) nanoparticles (FITC-PLGA NPs). Rate of binding and internalization of mZD7349-NPs to activated human umbilical vein endothelial cells (HUVECs) were compared with that of untargeted. Effects of temperature reduction and clathrin-mediated endocytosis inhibitor (0.45M sucrose) were also studied on the binding and internalization of mZD7349-NPs and NPs. Results showed that binding of the conjugated NPs could be significantly blocked by pre-incubating cells with the free peptide, suggesting that the binding of NPs is mediated by attaching the surface peptide to VCAM-1 on HUVECs. Also, conjugated FITC-loaded NPs were shown to be rapidly endocytosized to a greater extent than the unconjugated ones. The binding and internalization of mZD7349-NPs and NPs were slowed down at low temperature and in the presence of sucrose with greater reductions for mZD7349-NPs. To conclude, the peptide-NPs targeting the VCAM-1 is suggested as a theranostic carrier for lesions upregulating VCAM-1.

Exosomes isolated from metabolically unhealthy normal weight and overweight phenotypes deteriorate the ER/PR positive breast cancer behavior.

Purpose: Obesity has been linked to a higher risk of postmenopausal breast cancer Yet, research indicates an opposite correlation between obesity and premenopausal breast cancer risk. Various obesity phenotypes based on metabolic health could play a significant part. This study aims to assess how plasma exosomes taken from women with varying obesity phenotypes impact MCF-7 cell migration, matrix metalloproteinase-2 activity, and apoptosis. Methods: The characterization of isolated exosomes and their internalization into MCF-7 cells was evaluated. The treatment of MCF-7 cells with exosomes isolated from different groups was done. Migration, the activity of MMP-2, mRNA expression of Bax and Bcl-2, protein expression of p-53 and Thr55 p-p53, and apoptosis were assessed. Results: Isolated exosomes from unhealthy obese individuals increase MCF-7 cell migration. Regarding MMP activities, unhealthy normal weight and overweight and healthy obese groups isolated exosomes increase the MMP-2 activity than the treated group with exosomes isolated from counterpart groups. Furthermore, unhealthy normal weight and overweight and healthy obese obtained exosomes decrease apoptosis compared to counterpart groups. Conclusion: Altogether, plasma exosomes derived from both unhealthy individuals with normal weight and overweight status, as well as those with unhealthy obesity, negatively impacted the behavior of estrogen/progesterone receptor-positive breast cancer cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-023-01295-1.

S-Allylcysteine, a garlic compound, increases ABCA1 expression in human THP-1 macrophages.

ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cholesterol efflux to apoA-I in lipid-loaded macrophages, which is the first step of reverse cholesterol transport in vivo and a critical step in preventing atherosclerosis. Enhanced ABCA1 expression may inhibit foam cell formation and consequently reduce atherogenic risk. The purpose of this study was to investigate the effect of S-allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract, on the expression of ATP-binding cassette transporter A1 in human THP-1 macrophages. The human monocyte THP-1 cells were differentiated to macrophage cells in the presence of phorbol 12-myristate13-acetate (PMA). Macrophage cells were then treated with different concentrations (10, 20 and 40 mM) of SAC for 24 h. Total RNA of treated macrophages was extracted and analyzed with real-time RT-PCR. ABCA1 protein expression was also analyzed with western blotting. Results showed that SAC increased the ABCA1 mRNA (1.82-, 2.07- and 2.23-fold) and protein (1.37-, 1.55- and 2.08-fold) expression in macrophage THP-1 cells compared with control (untreated cells). Results suggested that SAC can increase ABCA1 expression in macrophages and may be beneficial in promoting reverse cholesterol efflux.

The effect of silymarin (Silybum marianum) on human skin fibroblasts in an in vitro wound healing model.

CONTEXT: Silymarin, a flavonolignan from Silybum marianum (L.) Gaertn. (Asteraceae), has been reported to have antioxidant and anti-inflammatory properties. Therefore, it may be worthwhile to study the effect of silymarin on wound healing. OBJECTIVE: To evaluate the effect of silymarin on human fibroblast cells in an in vitro model of wound healing. MATERIALS AND METHODS: Human fibroblast cells were treated with different concentrations (4.5, 9, 18, 36 µg/mL) of silymarin. The effects of silymarin on cell viability, proliferation, collagen synthesis, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2'-deoxy-uridine, hydroxyproline analysis and real-time PCR, respectively. The effect of silymarin on cellular antioxidant status was determined by protection against hydrogen peroxide (H2O2)-induced cell injury and free radical scavenging activity (ABTS assay) of the cells. RESULTS: Results of the present study indicate that pretreatment of fibroblast cells with silymarin significantly protected cells against H2O2-induced injury (p < 0.05). After an 18 h treatment of cells with 36 µg/mL silymarin, total antioxidant capacity of cells significantly increased (p < 0.05). Furthermore, pretreatment of human fibroblast cells with silymarin significantly inhibited lipopolysaccharide (LPS)-induced COX-2 mRNA expression (p < 0.001). There was no significant difference in fibroblast proliferation and collagen synthesis between treatment and control groups (p > 0.05). DISCUSSION AND CONCLUSION: Silymarin may be useful as a therapeutic agent for the treatment of cutaneous wounds through its antioxidation and anti-inflammation effects.

Apoptotic effects of human amniotic fluid mesenchymal stem cells conditioned medium on human MCF-7 breast cancer cell line.

Introduction: Breast cancer, as the most common malignancy among women, is shown to have a high mortality rate and resistance to chemotherapy. Research has shown the possible inhibitory role of Mesenchymal stem cells in curing cancer. Thus, the present work used human amniotic fluid mesenchymal stem cell-conditioned medium (hAFMSCs-CM) as an apoptotic reagent on the human MCF-7 breast cancer cell line. Methods: Conditioned medium (CM) was prepared from hAFMSCs. After treating MCF-7 cells with CM, a number of analytical procedures (MTT, real-time PCR, western blot, and flow cytometry) were recruited to evaluate the cell viability, Bax and Bcl-2 gene expression, P53 protein expression, and apoptosis, respectively. Human fibroblast cells (Hu02) were used as the negative control. In addition, an integrated approach to meta-analysis was performed. Results: The MCF-7 cells' viability was decreased significantly after 24 hours (P < 0.0001) and 72 hours (P < 0.05) of treatment. Compared with the control cells, Bax gene's mRNA expression increased and Bcl-2's mRNA expression decreased considerably after treating for 24 hours with 80% hAFMSCs-CM (P = 0.0012, P < 0.0001, respectively); an increasing pattern in P53 protein expression could also be observed. The flow cytometry analysis indicated apoptosis. Results from literature mining and the integrated meta-analysis showed that hAFMSCs-CM is able to activate a molecular network where Bcl2 downregulation stands in harmony with the upregulation of P53, EIF5A, DDB2, and Bax, leading to the activation of apoptosis. Conclusion: Our finding demonstrated that hAFMSCs-CM presents apoptotic effect on MCF-7 cells; therefore, the application of hAFMSCs-CM, as a therapeutic reagent, can suppress breast cancer cells' viabilities and induce apoptosis.

The Ability of Antibody against Truncated Matrix Metalloproteinase 9 Peptide to Evaluate the Native Protein on Tear Drops of Dry Eye Disease Patients by a Point-of-Care Diagnostic System.

Purpose: To obtain a reactive and specific antibody against truncated matrix metalloproteinase 9 (MMP-9), that has reactivity toward the native protein. Precision, accuracy, specificity, and sensitivity were evaluated using a point-of-care test. Methods: An in silico study was used to confirm the anti peptide truncated MMP-9 is against native MMP-9. After an antibody titer assessment, purification, and characterization, the anti MMP-9 was assessed. The cut-off value was determined using the purified gelatinases of the supernatant HCT 116 cell line. The supernatant was purified by preparative native-polyacrylamide gel electrophoresis based on charge and size of the proteins. Furthermore, quality control (QC) of the results were performed following standard densitometry methods. Results: A truncated MMP-9 is the major epitope peptide that can trigger the immune system to scavenge for a specific and reactive antibody against the native MMP-9. The MMP-9 native protein is purified from the supernatant of the HCT 116 cell line and the commercially available, full-length MMP-9. The cut-off value was estimated at 30 µg/mL. QC results indicated that the specificity was 80%, sensitivity was 96.7%, accuracy was 91%, and precision was 91.66%. The area under curve was 0.827 (P < 0.001). The positive predictive value was 83%, and the negative predictive value was 96%. Conclusions: The antibody against the synthetic epitope peptide can detect the native MMP-9. Native MMP-9 is considered the main biomarker in an immunoassay POCT and is used to diagnose dry eye disease (DED). In accordance with QC results, MMP-9 point of care test can be utilized for screening patients suffering from DED.

Stability improvement of immobilized lactoperoxidase using polyaniline polymer.

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55 °C, which has been increased about 10 °C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60 days whereas the native enzyme lost 80 % of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K(m) and K(m.app) were calculated to be 0.6 and 0.4; also V(max) and V(max.app) were 1.3 and 0.9 respectively.

A theoretical strategy for acceleration of human immune response against SARS-CoV-2: a fusion protein harboring virus-binding and pre-exposed antigen domains.

Coronavirus disease 2019 (COVID-19) is a respiratory disease that outbreaks since December 2019 and spread globally. Various methods have been used to treat SARS-CoV-2 that is generally based on the information obtained from the therapeutic approaches used for SARS-COV and MERS patients. In this article, we introduce a theoretical strategy in which a two-domain fusion protein presents the virus to the immune system. This fusion protein contains a viral-binding domain such as the ACE2 domain and a domain such as the hepatitis B antigen that has previously been exposed to the immune system. This two-domain fusion protein, could be called "virus-presenting fusion protein", would attach to the virus spike protein via the ACE2 domain while the hepatitis B antigen would be bound by anti-hepatitis B antibodies facilitating the opsonization and presentation of the virus to the immune system. We believe that this virus-presenting fusion protein will accelerate the immune response to the SARS-CoV-2 virus.

Positive Correlation between Circulating Fetuin-A and Severity of Coronary Artery Disease in Men.

OBJECTIVE: Fetuin-A serves a dual function; its high levels are associated with metabolic syndrome, type 2 diabetes, obesity, insulin resistance, and nonalcoholic fatty liver disease, and on the other hand, it serves as a potent inhibitor of ectopic vascular calcification. Due to the opposing findings, the aim of the current study was to investigate serum fetuin-A levels in men with coronary artery disease (CAD). METHODS: In the case-control study, anthropometric and biochemical parameters were determined in 83 men (43 CAD patients and 40 control subjects). At last, the serum fetuin-A levels were measured using the fetuin-A human enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: A significant difference was detected among the two groups for triglyceride and cholesterol levels (P=0.003 and P=0.002, respectively). The mean fetuin-A levels were determined 230.57 ± 63.76 and 286.35 ± 64.07 µg/ml for the control group and the CAD patients, respectively (P<0.001). Fetuin- A was significantly correlated to the severity of CAD (r 0.393, P<0.001) and associated with the risk of CAD in subjects (OR [CI] = 1. 144 [1.060-1. 235]; p = 0.001). A cut-off value of 237.4 µg/ml had good sensitivity (76.7%) and specificity (65.0%) for differentiating between two groups [area under curve (AUC) = 0.732 (CI=0.621-0.842); p < 0.001]. CONCLUSION: Our results indicated that fetuin-A levels were positively correlated to the severity of CAD. The findings suggest that there is a possible link between pathogenic mechanisms of atherosclerosis and fetuin-A; however, more investigations are needed in this regard.

Anti-Inflammatory Effects of Plasma Circulating Exosomes Obtained from Normal-Weight and Obese Subjects on Hepatocytes.

INTRODUCTION: Obesity is a disorder with low-grade chronic inflammation that plays a key role in hepatic inflammation and steatosis. Moreover, there are studies to support the role of exosomes in cellular communications, the regulation of metabolic homeostasis and immunomodulatory activity. Accordingly, we aimed to evaluate the influence of plasma circulating exosomes derived from females with normal-weight and obesity on the secretion of inflammatory cytokines in human liver cells. METHODS: Plasma circulating exosomes were isolated from four normal (N-Exo) and four obese (OExo) women. The exosomes were characterized and approved for CD63 expression (common exosomal protein marker) and morphology/size using the western blot and TEM methods, respectively. The exosomes were used for the stimulation of HepG2 cells in vitro. After 24 h of incubation, the protein levels of TNF-α, IL-6, and IL-1ß were measured in the culture supernatant of HepG2 cells using the ELISA kit. RESULTS: The protein levels of IL-6 and TNF-α in the cells treated with O-Exo and N-Exo reduced significantly in comparison with the control group (P=0.039 and P<0.001 respectively), while significant differences were not found between normal and obese groups (P=0.808, and P=0.978 respectively). However, no significant differences were found among the three groups in terms of IL-1ß levels (P=0.069). Based on the correlation analysis, the protein levels of IL-6 were positively correlated with TNF-α (r 0.978, P<0.001). CONCLUSION: These findings suggest that plasma circulating exosomes have probably antiinflammatory properties independent of body mass index and may decrease the secretion of inflammatory cytokines in the liver. However, further in vitro and in vivo investigations are needed to address the anti-inflammatory function of N-Exo and O-Exo in human liver cells and/or other cells.

Exogenous coenzyme Q10 modulates MMP-2 activity in MCF-7 cell line as a breast cancer cellular model.

BACKGROUND/AIMS: Matrix Metalloproteinases 2 is a key molecule in cellular invasion and metastasis. Mitochondrial ROS has been established as a mediator of MMP activity. Coenzyme Q(10) contributes to intracellular ROS regulation. Coenzyme Q(10) beneficial effects on cancer are still in controversy but there are indications of Coenzyme Q(10) complementing effect on tamoxifen receiving breast cancer patients. METHODS: In this study we aimed to investigate the correlation of the effects of co-incubation of coenzyme Q10 and N-acetyl-L-cysteine (NAC) on intracellular H2O2 content and Matrix Metalloproteinase 2 (MMP-2) activity in MCF-7 cell line. RESULTS AND DISCUSSION: Our experiment was designed to assess the effect in a time and dose related manner. Gelatin zymography and Flowcytometric measurement of H2O2 by 2'7',-dichlorofluorescin-diacetate probe were employed. The results showed that both coenzyme Q10 and N-acetyl-L-cysteine reduce MMP-2 activity along with the pro-oxidant capacity of the MCF-7 cell in a dose proportionate manner. CONCLUSIONS: Collectively, the present study highlights the significance of Coenzyme Q(10) effect on the cell invasion/metastasis effecter molecules.

Specific cellular internalization and pH-responsive behavior of doxorubicin loaded PLGA-PEG nanoparticles targeted with anti EGFRvIII antibody.

AIM: Antibody-conjugated nanoparticles have attracted much attention in the field of cancer treatment due to the enhancement of the tumor cell response to anticancer drugs as well as reducing the side effects of chemotherapeutic agents on healthy tissues. However, most studies in this field generally mentioned the specific cellular uptake of conjugated nanoparticles. In this study, we loaded doxorubicin (DXR: as an effective antineoplastic agent) in PLGA-PEG (D,L-lactic-co-glycolic acid)-(polyethylene glycol) biocompatible polymeric nanoparticles (NPs) and then conjugated with anti-EGFRvIII antibody. The resulting nanoparticles had remarkable sensitivity to pH decrease and were capable of targeting specific cells. MATERIALS AND METHODS: To this aim, PLGA-PEG-COOH was used for the synthesis of nanoparticles and stabilized by polyvinyl alcohol (PVA) according to the nanoprecipitation method. The carboxylic groups on the surface of PLGA-PEG NPs were activated by EDC/NHS and covalently conjugated to amino groups of the monoclonal antibody. The prepared NPs were characterized by Zetasizer and transmission electron microscopy (TEM). The resulting NPs were evaluated in terms of entrapment efficiency (EE), drug loading efficiency (DLE), drug-release profile, and cell internalization. Intrinsic cytotoxicity was assessed by the MTT, apoptosis (Annexin V-PI) and cell cycle assays. KEY FINDINGS: The in vitro drug release assessment of conjugated particles (MAb-DXR-PLGA NPs) showed a slow sustained DXR release in physiological pH (7.4) values, while the initial drug release was markedly higher (the 1.9 fold) in acidic pH (6.5) ranges. The selectivity for cellular internalization of MAb-DXR-PLGA NPs into U87MG vIII cells (overexpressing EGFRvIII) in comparison with U87MG cells (lacking EGFRvIII expression) was also confirmed. The MTT assay demonstrated that the cytotoxicity of MAb-DXR-PLGA NPs against U87MG vIII cells was more pronounced when compared with BSA-DXR-PLGA NPs. The results of the MTT assay were also confirmed by apoptosis and cell cycle assays. SIGNIFICANCE: Our findings suggest that the designed anti-EGFRvIII MAb-DXR-PLGA NPs could be considered as a proper option for targeted drug delivery systems due to pH sensitivity and specific cellular internalization.

Effect of circulating exosomes derived from normal-weight and obese women on gluconeogenesis, glycogenesis, lipogenesis and secretion of FGF21 and fetuin A in HepG2 cells.

BACKGROUND: It is generally accepted that obesity can lead to metabolic disorders such as NAFLD and insulin resistance. However, the underlying mechanism has been poorly understood. Moreover, there is evidence to support the possible role of exosomes in the metabolic homeostasis regulation. Accordingly, we aimed to determine the effect of plasma circulating exosomes derived from obese and normal-weight women on insulin signaling and the secretion of hepatokines in human liver cells. METHODS: Plasma exosomes isolated from four obese (O-Exo) women and four normal-weight (N-Exo) female candidates were characterized for size, zeta potential, and CD63 protein expression and were used for stimulation of HepG2 cells. Then, cell viability, as well as levels of glycogen and triglyceride (TG), were evaluated. Levels of fetuin-A and FGF21 were measured using the ELISA kit. Expression of glucose 6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (PEPCK) genes were determined using qRT-PCR. Western blot analysis was carried out to evaluating the phosphorylation of GSK3ß. RESULTS: The TG levels increased significantly in the cells treated with O-Exo than the control (vehicle) group (P = 0.005) and normal-weight group (P = 0.018). Levels of p-GSK3ß and glycogen were significantly reduced by O-Exo in comparison with control (P = 0.002, P = 0.018, respectively). The mRNA expression of G6pase and PEPCK enzymes increased in the cells treated with O-Exo in comparison with the vehicle group (P = 0.017, P = 0.010, respectively). The levels of FGF21 in the supernatant of cells treated with O-Exo and N-Exo were significantly lower than the control group (P = 0.007). CONCLUSION: It appears that obesity-related circulating exosomes can impair insulin signaling pathways and associated components, increase intracellular TG content, and decrease FGF21 secretion in the hepatocytes.