Search results for: HEK293 cells

Authors: Sylwia K. Naliwajko, Justyna Moskwa, Patryk Nowakowski, Renata Markiewicz-Zukowska, Krystyna Gromkowska-Kepka, Anna Puscion-Jakubik, Maria H. Borawska

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Amygdalin is found in many fruit seeds, including apricot, peach, quince, apples, and almonds. Amygdalin (also named vitamin B17), as well as its sources, are commonly used as an alternative therapy or prevention of cancer. The potential activity of amygdalin is related to its enzymatic degradation to the hydrogen cyanide. Hydrogen cyanide is a toxic substance that causes liver and nerves damage, fever, coma or even death. Amygdalin is much better tolerated after intravenous than oral administration. The aim of this study was to examine the influence of amygdalin on glioblastoma multiforme cell lines. Three glioblastoma multiforme cell lines – U87MG, T98, LN18 were incubated (48 h) with amygdalin in concentrations 100, 250, 500, 1000 and 2000 µg/mL. The MTT (Thiazolyl Blue Tetrazolium Bromide) test and DNA binding test by [3H]-thymidine incorporation were used to determine the anti-proliferative activity of amygdalin. The secretion of metalloproteinases (MMP2 and MMP-9) from U87MG cells was estimated by gelatin zymography. The statistical analysis was performed using Statistica v. 13.0 software. The data was presented as a % of control. Amygdalin did not show significant inhibition of viability of all the glioblastoma cells in concentrations 100, 250, 500, 1000 µg/mL. In 2000 µg/mL there were significant differences compared to the control, but inhibition of viability was less than 20% (more than 80% of control). The average viability of U87MG cells was 92,0±4%, T98G: 85,8±3% and LN18: 94,7±2% of the control. There was no dose-response viability, and IC50 value was not recognized. DNA binding in U87MG cells was not inhibited (109,0±3 % of control). After treatment with amygdalin, we observed significantly increased secretion of MMP2 and MMP9 in U87MG cells (130,3±14% and 112,0±5% of control, respectively). Our results suggest that amygdalin has no anticancer activity in glioblastoma cell lines.

Keywords: amygdalin, anticancer, cell line, glioblastoma

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Authors: Mohammadjavad Sotoudeheian, Soroush Nematollahi

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Crohn’s disease (CD) is a chronic gastrointestinal segment inflammation encompassing immune dysregulation in a genetically susceptible individual in response to the environmental triggers and interaction between the microbiome and immune system. Uncontrolled inflammation leads to long-term complications, including fibrotic strictures and enteric fistulae. Increased production of Th1 and Th17-cell cytokines and defects in T-regulatory cells have been associated with CD. Th17-cells are essential for protection against extracellular pathogens, but their atypical activity can cause autoimmunity. Intrinsic defects in the control of programmed cell death in the mucosal T-cell compartment are strongly implicated in the pathogenesis of CD. The apoptosis defect in mucosal T-cells in CD has been endorsed as an imbalance of the Bcl-2 and the Bax. The immune system encounters foreign antigens through microbial colonization of mucosal surfaces or infections. In addition, FOSL downregulated IL-26 expression, a cytokine that marks inflammatory Th17-populations in patients suffering from CD. Furthermore, the expression of IL-23 is associated with the transcription factor primary leucine zipper transcription factor ATF-like (Batf). Batf-deficiency demonstrated the crucial role of Batf in colitis development. Batf and IL-23 mediate their effects by inducing IL-6 production. Strong association of IL-23R, Stat3, and Stat4 with IBD susceptibility point to a critical involvement of T-cells. IL-23R levels in transfer fistula were dependent on the AP-1 transcription factor JunB that additionally controlled levels of RORγt by facilitating DNA binding of Batf. T lymphocytes lacking JunB failed to induce IL-23- and Th17-mediated experimental colitis highlighting the relevance of JunB for the IL-23/ Th17 pathway. The absence of T-bet causes unrestrained Th17-cell differentiation. T-cells are central parts of immune-mediated colon fistula. Especially Th17-cells were highly prevalent in inflamed IBD tissues, as RORγt is effective in preventing colitis. Intraepithelial lymphocytes (IEL) contain unique T-cell subsets, including cells expressing RORγt. Increased activated Th17 and decreased T-regulatory cells in inflamed intestinal tissues had been seen. T-cells differentiate in response to many cytokines, including IL-1β, IL-6, IL-23, and TGF-β, into Th17-cells, a process which is critically dependent on the Batf. IL-23 promotes Th17-cell in the colon. Batf manages the generation of IL-23 induced IL-23R+ Th17-cells. Batf is necessary for TGF-β/IL-6-induced Th17-polarization. Batf-expressing T-cells are the core of T-cell-mediated colitis. The human-specific parts of three AP-1 transcription factors, FOSL1, FOSL2, and BATF, are essential during the early stages of Th17 differentiation. BATF supports the Th17 lineage. FOSL1, FOSL2, and BATF make possession of regulatory loci of genes in the Th17 lineage cascade. The AP1 transcription factor Batf is identified to control intestinal inflammation and seems to regulate pathways within lymphocytes, which could theoretically control the expression of several genes. It shows central regulatory properties over Th17-cell development and is intensely upregulated within IBD-affected tissues. Here, we demonstrated that targeting Batf in IBD appears as a therapeutic approach that reduces colitogenic T-cell activities during fistula formation while aiming to affect inflammation in the gut epithelial cells.

Keywords: immune system, Crohn’s Disease, BATF, T helper cells, Bcl, interleukin, FOSL

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Authors: S. A. Hashemvarzi, A. Heidarianpour, Z. Fallahmohammadi, M. Pourghasem, M. Kaviani

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Introduction: Parkinson’s disease (PD) is a progressive neurodegenerative in the central nervous system characterized by the loss of dopaminergic neurons in the substantia nigra resulting in loss of dopamine release in the striatum. Non-drug treatment options such as Stem cell transplantation and exercise have been considered for treatment of Parkinson's disease. Purpose: The purpose of this study was to evaluate the effect of aerobic exercise after bone marrow stem cells transplantation on recovery of dopaminergic neurons and promotion of angiogenesis markers in the striatum of parkinsonian rats. Materials and Methods: 42 male Wistar rats were divided randomly into six groups: Normal (N), Sham (S), Parkinson’s (P), Stem cells transplanted Parkinson’s (SP), Exercised Parkinson’s (EP) and Stem cells transplanted + Exercised Parkinson’s (SEP). To create a model of Parkinson's, the striatum was destroyed by injection of 6-hydroxy-dopamine into the striatum through stereotaxic apparatus. Stem cells were derived from the bone marrow of femur and tibia of male rats with 6-8 weeks old. After cultivation, approximately 5×105 cells in 5 microliter of medium were injected into the striatum of rats through the channel. Aerobic exercise was included 8 weeks of running on the treadmill with a speed of 15 meters per minute. At the end, all subjects were decapitated and striatum tissues were separately isolated for measurement of vascular endothelial growth factor (VEGF), dopamine (DA) and tyrosine hydroxylase (TH) levels. Results: VEGF, DA and TH levels in the striatum of parkinsonian rats significantly increased in treatment groups (SP, EP and SEP), especially in SEP group compared to P group after treatment (P<0.05). Conclusion: The findings implicate that the BMSCs transplantation in combination with exercise would have synergistic effects leading to functional recovery, dopaminergic neurons recovery and promotion of angiogenesis marker in the striatum of parkinsonian rats.

Keywords: stem cells, treadmill training, neurotrophic factors, Parkinson

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Authors: Shima Fasahat

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This research is an experimental research which was done about microbial fuel cells in order to study them for electricity generating and wastewater treatment. These days, it is very important to find new, clean and sustainable ways for energy supplying. Because of this reason there are many researchers around the world who are studying about new and sustainable energies. There are different ways to produce these kind of energies like: solar cells, wind turbines, geothermal energy, fuel cells and many other ways. Fuel cells have different types one of these types is microbial fuel cell. In this research, an MFC was built in order to study how it can be used for electricity generating and wastewater treatment. The microbial fuel cell which was used in this research is a reactor that has two tanks with a catalyst solution. The chemical reaction in microbial fuel cells is a redox reaction. The microbial fuel cell in this research is a two chamber MFC. Anode chamber is an anaerobic one (ABR reactor) and the other chamber is a cathode chamber. Anode chamber consists of stabilized sludge which is the source of microorganisms that do redox reaction. The main microorganisms here are: Propionibacterium and Clostridium. The electrodes of anode chamber are graphite pages. Cathode chamber consists of graphite page electrodes and catalysts like: O₂, KMnO₄ and C₆N₆FeK₄. The membrane which separates the chambers is Nafion117. The reason of choosing this membrane is explained in the complete paper. The main goal of this research is to generate electricity and treating wastewater. It was found that when you use electron receptor compounds like: O_2, MnO₄, C₆N₆FeK₄ the velocity of electron receiving speeds up and in a less time more current will be achieved. It was found that the best compounds for this purpose are compounds which have iron in their chemical formula. It is also important to pay attention to the amount of nutrients which enters to bacteria chamber. By adding extra nutrients in some cases the result will be reverse.  By using ABR the amount of chemical oxidation demand reduces per day till it arrives to a stable amount.

Keywords: anaerobic baffled reactor, bioenergy, electrode, energy efficient, microbial fuel cell, renewable chemicals, sustainable

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Authors: T. H. Huang, C. L. Fern, Y. K. Tseng

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The design and reliability of solar photovoltaic modules are crucial to the development of solar energy, and efforts are still being made to extend the life of photovoltaic modules to improve their efficiency because natural aging is time-consuming and does not provide manufacturers and investors with timely information, accelerated aging is currently the best way to estimate the life of photovoltaic modules. Bifacial solar cells not only absorb light from the front side but also absorb light reflected from the ground on the back side, surpassing the performance of single-sided solar cells. Due to the asymmetry of the two sides of the light, in addition to the difference in photovoltaic conversion efficiency, there will also be differences in heat distribution, which will affect the electrical properties and material structure of the bifacial solar cell itself. In this study, there are two types of experimental samples: packaged and unpackaged and then irradiated with UVC light sources and halogen lamps for accelerated aging, as well as a control group without aging. After two weeks of accelerated aging, the bifacial solar cells were visual observation, and infrared thermal images were taken; then, the samples were subjected to IV measurement, and samples were taken for SEM, Raman, and XRD analyses in order to identify the defects that lead to failure and chemical changes, as well as to analyze the reasons for the degradation of their characteristics. From the results of the analysis, it is found that aging will cause carbonization of the polymer material on the surface of bifacial solar cells, and the crystal structure will be affected.

Keywords: bifacial solar cell, accelerated aging, temperature, characterization, electrical measurement

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Authors: Hamed A. Keykha, Hadi Miri

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This paper describes a new method of soil stabilisation, electro-biogrouting (EBM), for improvement of soft soil with low hydraulic conductivity. This method uses an applied voltage gradient across the soil to induce the ions and bacteria cells through the soil matrix, resulting in CaCO3 precipitation and an increase of the soil shear strength in the process. The EBM were used effectively with two injection methods; bacteria injection and products of bacteria injection. The bacteria cells, calcium ions and urea were moved across the soil by electromigration and electro osmotic flow respectively. The products of bacteria (CO3-2) were moved by electromigration. The results showed that the undrained shear strength of the soil increased from 6 to 65 and 70 kPa for first and second injection method respectively. The injection of carbonate solution and calcium could be effectively flowed in the clay soil compare to injection of bacteria cells. The detection of CaCO3 percentage and its corresponding water content across the specimen showed that the increase of undrained shear strength relates to the deposit of calcite crystals between soil particles.

Keywords: Sporosarcina pasteurii, electrophoresis, electromigration, electroosmosis, biocement

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Authors: Moustafa Elhamouly, Masayuki Shimada

Abstract:

The production of competent oocytes is essential for reproductivity in mammals. Maintenance of mitochondrial efficiency is required to supply the ATP necessary for granulosa cell proliferation during the follicular development process. Treatment with Pyrroloquinoline quinone (PQQ) has been reported to increase the number of ovulated oocytes and pups per delivery in mice by maintaining healthy mitochondrial function. This study aimed to elucidate how PQQ maintains mitochondrial function during ovarian follicle growth. To do this, both in vitro and in vivo experiments were performed with granulosa cells from superovulated immature (3-week-old) mice that were pretreated with or without PQQ. The effects of PQQ on beta-oxidation, mitochondrial function, mitophagy, and mitochondrial biogenesis were examined. PQQ increased beta-oxidation-related genes and CPT1 protein content in granulosa cells and this was associated with a decreased phosphorylation of P38 signaling protein. Using the fatty acid oxidation assay on the flux analyzer, PQQ increased the reliance of beta-oxidation on the endogenous fatty acids and was associated with a mild UCP-dependant mitochondrial uncoupling, ATP production, mitophagy, and mitochondrial biogenesis. PQQ also increased the expression of endogenous antioxidant enzymes. Thus, PQQ induced beta-oxidation in growing granulosa cells relying on endogenous fatty acids. And reduced the Reactive oxygen species (ROS) production by inducing a mild mitochondrial uncoupling with keeping high mitochondrial function. Damaged mitochondria were recycled by the induced mitophagy and replaced by the increased mitochondrial biogenesis. Collectively, PQQ may enhance reproductivity by maintaining the efficiency of mitochondria to produce enough ATP required for normal folliculogenesis.

Keywords: granulosa cells, mitochondrial uncoupling, mitophagy, pyrroloquinoline quinone (PQQ), reactive oxygen species (ROS).

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Authors: Jae-Hyeon Kim, Michael Lee

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Intrinsic and acquired resistance limits the therapeutic benefits of oncogenic BRAF inhibitors in melanoma. MicroRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation. Thus, we investigated miRNA expression patterns in melanoma cell lines to identify candidate biomarkers for acquired resistance to BRAF inhibitor. Here, we used Affymetrix miRNA V3.0 microarray profiling platform to compare miRNA expression levels in three cell lines containing BRAF inhibitor-sensitive A375P BRAF V600E cells, their BRAF inhibitor-resistant counterparts (A375P/Mdr), and SK-MEL-2 BRAF-WT cells with intrinsic resistance to BRAF inhibitor. The miRNAs with at least a two-fold change in expression between BRAF inhibitor-sensitive and –resistant cell lines, were identified as differentially expressed. Averaged intensity measurements identified 138 and 217 miRNAs that were differentially expressed by 2 fold or more between: 1) A375P and A375P/Mdr; 2) A375P and SK-MEL-2, respectively. The hierarchical clustering revealed differences in miRNA expression profiles between BRAF inhibitor-sensitive and –resistant cell lines for miRNAs involved in intrinsic and acquired resistance to BRAF inhibitor. In particular, 43 miRNAs were identified whose expression was consistently altered in two BRAF inhibitor-resistant cell lines, regardless of intrinsic and acquired resistance. Twenty five miRNAs were consistently upregulated and 18 downregulated more than 2-fold. Although some discrepancies were detected when miRNA microarray data were compared with qPCR-measured expression levels, qRT-PCR for five miRNAs (miR-3617, miR-92a1, miR-1246, miR-1936-3p, and miR-17-3p) results showed excellent agreement with microarray experiments. To further investigate cellular functions of miRNAs, we examined effects on cell proliferation. Synthetic oligonucleotide miRNA mimics were transfected into three cell lines, and proliferation was quantified using a colorimetric assay. Of the 5 miRNAs tested, only miR-1246 altered cell proliferation of A375P/Mdr cells. The transfection of miR-1246 mimic strongly conferred PLX-4720 resistance to A375P/Mdr cells, implying that miR-1246 upregulation confers acquired resistance to BRAF inhibition. We also found that PLX-4720 caused much greater G2/M arrest in A375P/Mdr cells transfected with miR-1246mimic than that seen in scrambled RNA-transfected cells. Additionally, miR-1246 mimic partially caused a resistance to autophagy induction by PLX-4720. These results indicate that autophagy does play an essential death-promoting role inPLX-4720-induced cell death. Taken together, these results suggest that miRNA expression profiling in melanoma cells can provide valuable information for a network of BRAF inhibitor resistance-associated miRNAs.

Keywords: microRNA, BRAF inhibitor, drug resistance, autophagy

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Authors: Kumaran Narayanan, Andrew N. Osahor

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E. coli has been engineered by our group and by others as a vector to deliver DNA into cultured human and animal cells. However, so far conditions to improve gene delivery using this vector have not been investigated, resulting in a major gap in our understanding of the requirements for this vector to function optimally. Our group recently published novel data showing that simple addition of the DNA transfection reagent Lipofectamine increased the efficiency of the E. coli vector by almost 3-fold, providing the first strong evidence that further optimization of bactofection is possible. This presentation will discuss advances that demonstrate the effects of several intracellular strategies that improve the efficiency of this vector. Conditions that promote endosomal escape of internalized bacteria to evade lysosomal destruction after entry in the cell, a known obstacle limiting this vector, are elucidated. Further, treatments that increase bacterial lysis so that the vector can release its transgene into the mammalian environment for expression will be discussed. These experiments will provide valuable new insight to advance this E. coli system as an important class of vector technology for genetic correction of human disease models in cells and whole animals.

Keywords: DNA, E. coli, gene expression, vector

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Authors: Chang-Hui Shen, Paulina Konarzewska

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Cell homeostasis is regulated by vacuolar activity and it has been shown that lipid composition of the vacuole plays an important role in vacuolar function. The major phosphoinositide species present in the vacuolar membrane include phosphatidylinositol 3,5-biphosphate (PI(3,5)P₂) which is generated from PI(3)P controlled by Fab1p. Deletion of FAB1 gene reduce the synthesis of PI(3,5)P₂ and thus result in enlarged or fragmented vacuoles, with neutral vacuolar pH due to reduced vacuolar H⁺-ATPase activity. These mutants also exhibited poor growth at high extracellular pH and in the presence of CaCl₂. Conversely, VPS34 regulates the synthesis of PI(3)P from phosphatidylinositol (PI), and the lack of Vps34p results in the reduction of vacuolar activity. Although the cellular observations are clear, it is still unknown about the molecular mechanism between the phospholipid biosynthesis pathway and vacuolar activity. Since both VPS34 and FAB1 are important in vacuolar activity, we hypothesize that the molecular mechanism of vacuolar function might be regulated by the transcriptional regulators of phospholipid biosynthesis. In this study, we study the role of the major phospholipid biosynthesis transcription factor, INO2, in the regulation of vacuolar activity. We first performed qRT-PCR to examine the effect of Ino2p on the expression of VPS34 and FAB1. Our results showed that VPS34 was upregulated in the presence of inositol for both WT and ino2Δ cells. However, FAB1 was only upregulated significantly in ino2Δ cells. This indicated that Ino2p might be the negative regulator for FAB1 expression. Next, growth sensitivity experiment showed that WT, vma3Δ, and ino2Δ grew well in growth medium buffered to pH 5.5 containing 10 mM CaCl₂. As cells were switched to growth medium buffered to pH 7 containing CaCl₂ WT, ino2Δ and opi1Δ showed growth reduction, whereas vma3Δ was completely nonviable. As the concentration of CaCl₂ was increased to 60 mM, ino2Δ cells showed moderate growth reduction compared to WT. This result suggests that ino2Δ cells have better vacuolar activity. Microscopic analysis and vacuolar acidification were employed to further elucidate the importance of INO2 in vacuolar homeostasis. Analysis of vacuolar morphology indicated that WT and vma3Δ cells displayed vacuoles that occupied a small area of the cell when grown in media buffered to pH 5.5. Whereas, ino2Δ displayed fragmented vacuoles. On the other hand, all strains grown in media buffered to pH 7, exhibited enlarged vacuoles that occupied most of the cell’s surface. This indicated that the presence of INO2 may play negative effect in vacuolar morphology when cells are grown in media buffered to pH 5.5. Furthermore, vacuolar acidification assay showed that only vma3Δ cells displayed notably less acidic vacuoles as cells were grown in media buffered to pH 5.5 and pH 7. Whereas, ino2Δ cells displayed more acidic pH compared to WT at pH7. Taken together, our results demonstrated the molecular mechanism of the vacuolar activity regulated by the phospholipid biosynthesis transcription factors Ino2p. Ino2p negatively regulates vacuolar activity through the expression of FAB1.

Keywords: vacuole, phospholipid, homeostasis, Ino2p, FAB1

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Authors: Apurva Gupta, Surendra Singh

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Spirulina spp., as a promising source of many commercially valuable products, is grown photo autotrophically in open ponds and raceways on a large scale. However, the economic exploitation in an open system seems to have been limited because of lack of multiple stress-tolerant strains. The present study aims to isolate a stable stress tolerant mutant of Spirulina platensis with improved growth rate and enhanced potential to produce its commercially valuable bioactive compounds. N-methyl-n'-nitro-n-nitrosoguanidine (NTG) at 250 μg/mL (concentration permitted 1% survival) was employed for chemical mutagenesis to generate random mutants and screened against NaCl. In a preliminary experiment, wild type S. platensis was treated with NaCl concentrations from 0.5-1.5 M to calculate its LC₅₀. Mutagenized colonies were then screened for tolerance at 0.8 M NaCl (LC₅₀), and the surviving colonies were designated as NaCl tolerant mutants of S. platensis. The mutant cells exhibited 1.5 times improved growth against NaCl stress as compared to the wild type strain in control conditions. This might be due to the ability of the mutant cells to protect its metabolic machinery against inhibitory effects of salt stress. Salt stress is known to adversely affect the rate of photosynthesis in cyanobacteria by causing degradation of the pigments. Interestingly, the mutant cells were able to protect its photosynthetic machinery and exhibited 4.23 and 1.72 times enhanced accumulation of Chl a and phycobiliproteins, respectively, which resulted in enhanced rate of photosynthesis (2.43 times) and respiration (1.38 times) against salt stress. Phycocyanin production in mutant cells was observed to enhance by 1.63 fold. Nitrogen metabolism plays a vital role in conferring halotolerance to cyanobacterial cells by influx of nitrate and efflux of Na+ ions from the cell. The NaCl tolerant mutant cells took up 2.29 times more nitrate as compared to the wild type and efficiently reduce it. Nitrate reductase and nitrite reductase activity in the mutant cells also improved by 2.45 and 2.31 times, respectively against salt stress. From these preliminary results, it could be deduced that enhanced nitrogen uptake and its efficient reduction might be a reason for adaptive and halotolerant behavior of the S. platensis mutant cells. Also, the NaCl tolerant mutant of S. platensis with significant improved growth and phycocyanin accumulation compared to the wild type can be commercially promising.

Keywords: chemical mutagenesis, NaCl tolerant mutant, nitrogen metabolism, photosynthetic machinery, phycocyanin

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Authors: Yu Wen Wang, Shyh Ming Kuo, Hsia Ying Cheng, Yu Chiuan Wu

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Liver fibrosis is caused by the activation of hepatic stellate cells in the liver to secrete excessive and deposition of extracellular matrix. In recent years, many treatment strategies have been developed to reduce the activation of hepatic stellate cells and therefore to increase the decomposition of extracellular matrix. Bacopa monnieri, an herbaceous plant of the scrophulariaceae, containing saponins and glycosides, which with antioxidant, anti-inflammation, pain relief and free radical scavenging characteristics. This study was to evaluate the inhibition of hepatic stellate cell activity by Bacopa monnieri extract and its therapeutic potential in treating thioacetamide-induced liver fibrosis in rats. The results showed that the IC50 of Bacopa monnieri extract was 0.39 mg/mL. Bacopa monnieri extract could effectively reduce H2O2-induced hepatic stellate cells inflammation. In the TAA-induced liver fibrosis animal studies, albumin secretion recovered to normal level after treated with Bacopa monnieri extract for 2-w, and fibrosis related proteins, α-SMA and TGF-1levels decreased indicating the extract exerted therapeutic effect on the liver fibrosis. However, inflammatory factors TNF- obviously decreased after 4-w treatment. In summary, we could successfully extract the main component-Bacopaside I from the plant and acquired a potential therapy using this component in treating TAA-induced liver fibrosis in rat.

Keywords: anti-inflammatory, Bacopa monnieri, fibrosis, hepatic stellate cells, water extract

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Authors: Purwati, Sony Wibisono, Ari Sutjahjo, Askandar T. J., Fedik A. Rantam

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Diabetes mellitus type 2 (T2DM), also known as hyperglycemia, results from insulin resistance and relative insulin deficiency. Diabetes mellitus is the main cause of premature death, particularly among individuals under the age of 70 years old. Mesenchymal stem cells (MSCs) can release bioactive molecules that promote tissue repair and regeneration. Hence, in this research, we evaluated the potential of autologous adipose-derived mesenchymal stem cells (AD-MSCs) in 40 patients of phase I clinical trial in T2DM with various ages between 30-79 years. AD-MSCs are transferred through catheterization. MSCs were validated by measures of CD105+ and CD34- expression. The result showed that after AD-MSCs transplantation, blood glucose levels (fasting and 2-hour postprandial) and insulin levels were significantly decreasing. Besides that, the level of HbA1c significantly decreased after three months of AD-MSCs injection and increasing level of c-peptide after injection. Thus, we conclude that AD-MSCs injection has the potential for T2DM therapy.

Keywords: glucose, hyperglycemia, MSCs, T2DM

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Authors: D. Pradhan, G. Tripathy, S. Pradhan

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Objectives: Imperviousness gainst estrogen treatments is a noteworthy reason for infection backslide and mortality in estrogen receptor alpha (ERα)- positive breast diseases. Tamoxifen or estrogen withdrawal builds the reliance of breast malignancy cells on INT3 flagging. Here, we researched the commitment of Quercetin and INT3 motioning in endocrine-safe breast tumor cells. Methods: We utilized two models of endocrine treatments safe (ETR) breast tumor: Tamoxifen-safe (TamR) and long haul estrogen-denied (LTED) MCF7 cells. We assessed the transitory and intrusive limit of these cells by Transwell cells. Articulation of epithelial to mesenchymal move (EMT) controllers and in addition INT3 receptors and targets were assessed by constant PCR and western smudge investigation. Besides, we tried in-vitro hostile to Quercetin monoclonal Antibodies (mAbs) and Gamma Secretase Inhibitors (GSIs) as potential EMT inversion remedial specialists. At last, we created stable Quercetin overexpressing MCF7 cells and assessed their EMT components and reaction to Tamoxifen. Results: We found that ETR cells procured an Epithelial to Mesenchymal move (EMT) phenotype and showed expanded levels of Quercetin and INT3 targets. Interestingly, we distinguished more elevated amount of INT3 however lower levels of INT1 and INT3 proposing a change to motioning through distinctive INT3 receptors after obtaining of resistance. Against Quercetin monoclonal antibodies and the GSI PF03084014 were powerful in obstructing the Quercetin/INT3 pivot and in part repressing the EMT process. As a consequence of this, cell relocation and attack were weakened and the immature microorganism like populace was essentially decreased. Hereditary hushing of Quercetin and INT3 prompted proportionate impacts. At long last, stable overexpression of Quercetin was adequate to make MCF7 lethargic to Tamoxifen by INT3 initiation. Conclusions: ETR cells express abnormal amounts of Quercetin and INT3, whose actuation eventually drives intrusive conduct. Hostile to Quercetin mAbs and GSI PF03084014 lessen articulation of EMT particles decreasing cell obtrusiveness. Quercetin overexpression instigates Tamoxifen resistance connected to obtaining of EMT phenotype. Our discovering propose that focusing on Quercetin and INT3 warrants further clinical Correlation as substantial restorative methodologies in endocrine-safe breast.

Keywords: endocrine, epithelial, mesenchymal, INT3, quercetin, MCF7

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Authors: Haiyan Wang, Dongyun Wang, Yongyong Yan, Richard T. Jaspers, Gang Wu

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Mesenchymal stem cell and 3D printing-based bone tissue engineering present a promising technique to repair large-volume bone defects. Its success is highly dependent on cell attachment, spreading, osteogenic differentiation, and in vivo survival of stem cells on 3D-printed scaffolds. In this study, human salivary histatin-1 (Hst1) was utilized to enhance the interactions between human adipose-derived stem cells (hASCs) and 3D-printed β-tricalcium phosphate (β-TCP) bioceramic scaffolds. Fluorescent images showed that Hst1 significantly enhanced the adhesion of hASCs to both bioinert glass and 3D-printed β-TCP scaffold. In addition, Hst1 was associated with significantly higher proliferation and osteogenic differentiation of hASCs on 3D-printed β-TCP scaffolds. Moreover, coating 3D-printed β-TCP scaffolds with histatin significantly promotes the survival of hASCs in vivo. The ERK and p38 but not JNK signaling was found to be involved in the superior adhesion of hASCs to β-TCP scaffolds with the aid of Hst1. In conclusion, Hst1 could significantly promote the adhesion, spreading, osteogenic differentiation, and in vivo survival of hASCs on 3D-printed β-TCP scaffolds, bearing a promising application in stem cell/3D printing-based constructs for bone tissue engineering.

Keywords: 3d printing, adipose-derived stem cells, bone tissue engineering, histatin-1, osteogenesis

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Authors: Jagdish S. Patel, Piyush Chudasama

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Context: Over-expression of P-glycoprotein (P-gp) plays critical role in absorption of many drug candidates which results into lower bioavailability of the drug. P-glycoprotein also over expresses in many pathological conditions like diabetes, affecting the drug therapy. Modulation of P-gp expression using inhibitors can help in designing novel formulation enhancing the bioavailability of the drug in question. Objectives: The main focus of the study was to develop advanced glycation end products (AGEs) induced P-gp over expression in Caco-2 cells. Curcumin, piperine and epigallocatechin gallate were used to evaluate their P-gp inhibitory action using combinatorial approach. Materials and methods: Methylglyoxal (MG) induced P-gp over expression was checked in Caco-2 cells using real time PCR. P-gp inhibitory effects of the phytochemicals were measured after induction with MG alone and in combination of any two compounds. Cytotoxicity of each of the phytochemical was evaluated using MTT assay. Results: Induction with MG (100mM) significantly induced the over expression of P-glycoprotein in Caco-2 cells after 24 hr. Curcumin, piperine and epigallocatechin gallate alone significantly reduced the level of P-gp within 6 hr of treatment period monitored by real time PCR. The combination of any two phytochemical also down regulated the expression of P-gp in cells. Combinations of Curcumin and epigallocatechin gallate have shown significant down regulation when compared with other two combinations. Conclusions: Combinatorial approach for down regulating the expression of P-gp, in pathological conditions like diabetes, has demonstrated promising approach for therapeutic purpose.

Keywords: p-glycoprotein, curcumin, piperine, epigallocatechin gallate, p-gp inhibition

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Authors: Li Chen, Feng Zhang, Shizhong Zheng

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SaikosaponinD (SSD) is a major component of saikosaponins isolated from Bupleurumfalactum. Our current study was to examine the toxic effect of SSD on liver cells and explore the possible mechanism. The results demonstrated that SSD induced mouse liver injury and led to apoptosis in LO2 cells. HE staining and TUNEL analyses showed that SSD stimulated liver injury and hepatocyte apoptosis in vivo. Subsequent experiments showed that SSD down-regulated Bcl-2 but up-regulated Bax. In vitro, SSD-treated LO2 cells exhibited apparent down-regulated expression of p-p38. Moreover, PDGF-βR agonist PDGF-BB alone significantly upregulated p38 phosphorylation, while combined with SSD, p38 phosphorylation expression was reduced. Furthermore, shRNA-mediated PDGF-βR knockdown augmented the inactivation of p-p38 and Bcl2 but abrogated the activation of Bax, these results were more obvious when shRNA combined with SSD. These data indicated that SSD stimulated liver injury and apoptosis in hepatocytes and PDGF-βR /p38 pathway may be the potential mechanistic.

Keywords: saikosaponin D, hepatotoxicity, liver injury, apoptosis, platelet-derived growth factor-β receptor, p38

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Authors: Maryam Zahedi Fard, Nazia Abdul Majid, Hapipah Mohd Ali, Mahmood Ameen Abdulla

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New quinazoline schiff base 3-(5-bromo-2-hydroxy-3-methoxybenzylideneamino)-2-(5-bromo-2-hydroxy-3-methoxyphenyl)-2,3-dihydroquinazolin-4(1H)-one was investigated for anticancer activity against MCF-7 human breast cancer cell line with involved mechanism of apoptosis. The compound demonstrated a remarkable antiproliferative effect, with an IC50 value of 3.41 ± 0.34, after 72 hours of treatment. Morphological apoptotic features in treated MCF-7 cells were observed by AO/PI staining. Furthermore, treated MCF-7 cells subjected to apoptosis death, as exhibited by perturbation of mitochondrial membrane potential and cytochrome c release as well as increase in ROS generation. We also found activation of caspases 3/7 and -9. Moreover, acute toxicity test demonstrated the nontoxic nature of the compound in mice. Our results showed the selected compound significantly induce apoptosis in MCF-7 cells via intrinsic pathway, which might be considered as a potent candidate for further in vivo and clinical breast cancer studies.

Keywords: antiproliferative effect, MCF-7 human breast cancer cell line, apoptosis, caspases

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Authors: Lise P. Labéjof, Ivna Mororó, Raquel G. Bastos, Maria Isabel G. Severo, Arno H. de Oliveira

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This work presents an alternative mode of intestine mucosa renewal that may allow to better understand the total loss of villi after irradiation. It was tested a morphological method of 3d reconstruction using micrographs of serial sections of rat duodenum. We used hundreds of sections of each specimen of duodenum placed on glass slides and examined under a light microscope. Those containing the detachment, approximately a dozen, were chosen for observation under a transmission electron microscope (TEM). Each of these sections was glued on a block of epon resin and recut into a hundred of 60 nm-thick sections. Ribbons of these ultrathin sections were distributed on a series of copper grids in the same order of appearance than during the process of microstomia. They were then stained by solutions of uranyl and lead salts and observed under a TEM. The sections were pictured and the electron micrographs showing signs of cells detachment were transferred into two softwares, ImageJ to align the cellular structures and Reconstruct to realize the 3d reconstruction. It has been detected epithelial cells that exhibited all signs of programmed cell death and localized at the villus-crypt junction. Their nucleus was irregular in shape with a condensed chromatin in clumps. Their cytoplasm was darker than that of neighboring cells, containing many swollen mitochondria. In some places of the sections, we could see intercellular spaces enlarged by the presence of shrunk cells which displayed a plasma membrane with an irregular shape in thermowell as if the cell interdigitations would distant from each other. The three-dimensional reconstruction of the crypts has allowed observe gradual loss of intercellular contacts of crypt cells in the longitudinal plan of the duodenal mucosa. In the transverse direction, there was a gradual increase of the intercellular space as if these cells moved away from one another. This observation allows assume that the gradual remoteness of the cells at the villus-crypt junction is the beginning of the mucosa detachment. Thus, the shrinking of cells due to apoptosis is the way that they detach from the mucosa and progressively the villi also. These results are in agreement with our initial hypothesis and thus have demonstrated that the villi become detached from the mucosa at the villus-crypt junction by the programmed cell death process. This type of loss of entire villus helps explain the rapid denudation of the intestinal mucosa in case of irradiation.

Keywords: 3dr, transmission electron microscopy, ionizing radiations, rat small intestine, apoptosis

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Authors: Vasudha Devi, Arul Amuthan, K. Narayanan, Praveen KS, Venkata Rao J

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Background: Siddha Medicine is one of the Indian traditional medical systems, in which the cancer disease is mentioned as 'putrunoi' which literally means the disease of growth like termite mound. There are number of formulations available for the treatment of cancer disease. Neeradi muthu vallathymezugu (NMV) and thamira kattu chendooram (TKC) are two drugs commonly prescribed by Siddha physicians. These drugs have been clinically reported to be safe and effective when given orally. Though these formulations are in practice for centuries, no efforts have been made to standardize them and explore their anti-cancer potential systematically. Objective: To compare the cytotoxic activity of NMV and TKC with doxorubicin using cancer cell lines. Materials and methods: For this study, ethanol extract of NMV was taken, whereas TKC was used as such. In vitro cytotoxic activity was evaluated by sulphorhodamine (SRB) assay against human hepatic cancer cells (HepG2), human breast cancer cells (MCF-7) and human cervical cancer cells [KeLa]. Doxorubicin was used as the standard. The SRB assay is based on the ability of cellular proteins to bind with sulphorhodamine-B. The number of live cells in drug treated cell lines directly affects the color formation in the assay, which is estimated calorimetrically by measuring the absorbance at 540 nm to calculate the cytotoxicity (inhibitory concentration - IC50 value) of the drug. Results: The IC50values of NMV, TKC and doxorubicin against HepG2 were 3.08 µg/ml, 20.21 µg/ml and 1.21µg/ml respectively. In MCF-7, it was 11.75 µg/ml, 17.67 µg/ml and 2.8µg/ml. In HeLa, the values were 24.76 µg/ml, 73.35 µg/ml and 1.12µg/ml. Conclusions: The study proves the possible anti-cancer potential of these two formulations. Compared to TKC, NMV showed good cytotoxic effect even at low dose. Human hepatic cancer cells responded well even at very low dose, when compared to other cancer cells. Though, cytotoxic potential of these compounds was found to be less compared to doxorubicin, the isolated lead compound may have the potential to be used as an anticancer drug clinically.

Keywords: Neeradi muthu vallathymezugu (Hydnocarpus laurifolia), thamira kattu chendooram, cytotoxicity, in-vitro, Siddha Medicine

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Authors: Yihenew Simegniew Birhan, Hsieh Chih Tsai

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The recent advancements in synthetic chemistry and nanotechnology fostered the development of different nanocarriers for enhanced intracellular delivery of pharmaceutical agents to tumor cells. Polymeric micelles (PMs), characterized by small size, appreciable drug loading capacity (DLC), better accumulation in tumor tissue via enhanced permeability and retention (EPR) effect, and the ability to avoid detection and subsequent clearance by the mononuclear phagocyte (MNP) system, are convenient to improve the poor solubility, slow absorption and non-selective biodistribution of payloads embedded in their hydrophobic cores and hence, enhance the therapeutic efficacy of chemotherapeutic agents. Recently, redox-responsive polymeric micelles have gained significant attention for the delivery and controlled release of anticancer drugs in tumor cells. In this study, we synthesized redox-responsive diselenide bond containing amphiphilic polymer, Bi(mPEG-PLGA)-Se₂ from mPEG-PLGA, and 3,3'-diselanediyldipropanoic acid (DSeDPA) using DCC/DMAP as coupling agents. The successful synthesis of the copolymers was verified by different spectroscopic techniques. Above the critical micelle concentration, the amphiphilic copolymer, Bi(mPEG-PLGA)-Se₂, self-assembled into stable micelles. The DLS data indicated that the hydrodynamic diameter of the micelles (123.9 ± 0.85 nm) was suitable for extravasation into the tumor cells through the EPR effect. The drug loading content (DLC) and encapsulation efficiency (EE) of DOX-loaded micelles were found to be 6.61 wt% and 54.9%, respectively. The DOX-loaded micelles showed initial burst release accompanied by sustained release trend where 73.94% and 69.54% of encapsulated DOX was released upon treatment with 6mM GSH and 0.1% H₂O₂, respectively. The biocompatible nature of Bi(mPEG-PLGA)-Se₂ copolymer was confirmed by the cell viability study. In addition, the DOX-loaded micelles exhibited significant inhibition against HeLa cells (44.46%), at a maximum dose of 7.5 µg/mL. The fluorescent microscope images of HeLa cells treated with 3 µg/mL (equivalent DOX concentration) revealed efficient internalization and accumulation of DOX-loaded Bi(mPEG-PLGA)-Se₂ micelles in the cytosol of cancer cells. In conclusion, the intelligent, biocompatible, and the redox stimuli-responsive behavior of Bi(mPEG-PLGA)-Se₂ copolymer marked the potential applications of diselenide-linked mPEG-PLGA micelles for the delivery and on-demand release of chemotherapeutic agents in cancer cells.

Keywords: anticancer drug delivery, diselenide bond, polymeric micelles, redox-responsive

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Authors: Ayaz Ahmad

Abstract:

TOPIC: A study of the formation ,existness and stability of localised pulses in pde Ayaz Ahmad ,NITP, Abstract:In this paper we try to govern the evolution deterministic variable over space and time .We analysis the behaviour of the model which allows us to predict and understand the possible behaviour of the physical system .Bifurcation theory provides a basis to systematically investigate the models for invariant sets .Exploring the behaviour of PDE using bifurcation theory which provides many challenges both numerically and analytically. We use the derivation of a non linear partial differential equation which may be written in this form ∂u/∂t+c ∂u/∂x+∈(∂^3 u)/(∂x^3 )+¥u ∂u/∂x=0 We show that the temperature increased convection cells forms. Through our work we look for localised solution which are characterised by sudden burst of aeroidic spatio-temporal evolution. Key word: Gaussian pulses, Aeriodic ,spatio-temporal evolution ,convection cells, nonlinearoptics, Dr Ayaz ahmad Assistant Professor Department of Mathematics National institute of technology Patna ,Bihar,,India 800005 [email protected] +91994907553

Keywords: Gaussian pulses, aeriodic, spatio-temporal evolution, convection cells, nonlinear optics

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Authors: Kuan-Hao Tsui, Li-Te Lin, Chia-Jung Li

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The combination of Y-27632 and Vitamin C significantly enhances the quality of aging germ cells by reducing reactive oxygen species (ROS) production, restoring mitochondrial membrane potential balance, and promoting mitochondrial fusion. The age-related decline in oocyte quality contributes to reduced fertility, increased aneuploidy, and diminished embryo quality, with mitochondrial dysfunction in both oocytes and granulosa cells being a key factor in this decline. Experiments on aging germ cells investigated the effects of the Y-27632 and Vitamin C combination. In vivo studies involved aged mice to assess oocyte maturation and ROS accumulation during culture. The assessment included mitochondrial activity, ROS levels, mitochondrial membrane potential, and mitochondrial dynamics. Cellular energy metabolism and ATP production were also measured. The combination treatment effectively addressed mitochondrial dysfunction and regulated cellular energy metabolism, promoting oxygen respiration and increasing ATP production. In aged mice, this supplement treatment enhanced in vitro oocyte maturation and prevented ROS accumulation in aging oocytes during culture. While these findings are promising, further research is needed to explore the long-term effects and potential side effects of the Y-27632 and Vitamin C combination. Additionally, translating these findings to human subjects requires careful consideration. Overall, the study suggests that the Y-27632 and Vitamin C combination could be a promising intervention to mitigate aging-related dysfunction in germ cells, potentially enhancing oocyte quality, particularly in the context of in vitro fertilization.

Keywords: ovarian aging, supplements, ROS, mitochondria

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Authors: Daniel Aberdam

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Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by reduced visual acuity. Patients have also progressive disorders including cataract, glaucoma and corneal abnormalities making their condition very challenging to manage. Aniridia-related keratopathy (ARK), caused by a combination of factors including limbal stem-cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface, affects up to 95% of patients. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub-epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. Although animal models partially recapitulate this disease, there is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9 technology) to introduce a nonsense mutation found in patients into one allele of the PAX6 gene into limbal stem cells. Resulting mutated clones, expressing half of the amount of PAX6 protein and thus representative of haploinsufficiency were further characterized. Sequencing analysis showed that no off-target mutations were induced. The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also reduced. Remarkably, addition of soluble recombinant PAX6 protein into the culture medium was sufficient to activate endogenous PAX6 gene and, as a consequence, rescue the phenotype. It strongly suggests that our in vitro model recapitulates well the epithelial defect and becomes a powerful tool to identify drugs that could rescue the corneal defect in patients. Furthermore, we demonstrate that the homeotic transcription factor Pax6 is able to be uptake naturally by recipient cells to function into the nucleus.

Keywords: Pax6, crispr/cas9, limbal stem cells, aniridia, gene therapy

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Authors: Thomais Tsoulia, Arvind Y. M. Sundaram, Stine Braaen, Øyvind Haugland, Espen Rimstad, Øystein Wessel, Maria K. Dahle

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Fish red blood cells (RBC) are nucleated, and in addition to their function in gas exchange, they have been characterized as mediators of immune responses. Salmonid RBC are the major target cells of Piscineorthoreovirus (PRV), a virus associated with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon. The activation of antiviral response genesin RBChas previously been described in ex vivo and in vivo PRV-infection models, but not explored in the initial virus encounter phase. In the present study, mRNA transcriptome responses were explored in erythrocytes from individual fish, kept ex vivo, and exposed to purified PRV for 24 hours. The responses were compared to responses in macrophage-like salmon head kidney (SHK-1) and endothelial-like Atlantic salmon kidney (ASK) cells, none of which support PRV replication. The comparative analysis showed that the antiviral response to PRV was strongest in the SHK-1 cells, with a set of 80 significantly induced genes (≥ 2-fold upregulation). In RBC, 46 genes were significantly upregulated, while ASK cells were not significantly responsive. In particular, the transcriptome analysis of RBC revealed that PRV significantly induced interferon regulatory factor 1 (IRF1) and interferon-induced protein with tetratricopeptide repeats 5-like (IFIT9). However, several interferon-regulated antiviral genes which have previously been reported upregulated in PRV infected RBC in vivo (myxovirus resistance (Mx), interferon-stimulated gene 15 (ISG15), toll-like receptor 3 (TLR3)), were not significantly induced after 24h of virus stimulation. In contrast to RBC, these antiviral response genes were significantly upregulated in SHK-1. These results confirm that RBC are involved in the innate immune response to viruses, but with a delayed antiviral response compared to SHK-1. A notable difference is that interferon regulatory factor 1 (IRF-1) is the most strongly induced gene in RBC, but not among the significantly induced genes in SHK-1. Putative differences in the binding, recognition, and response to PRV, and any link to effects on the ability of PRV to replicate remains to be explored.

Keywords: antiviral responses, atlantic salmon, piscine orthoreovirus-1, red blood cells, RNA-seq

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Authors: Sahar Rakhshan, Simonetta Geninatti Crich, Diego Alberti, Rachele Stefania

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The combination of imaging and therapeutic agents in the same smart nanoparticle is a promising option to perform a minimally invasive imaging guided therapy. In this study, Low density lipoproteins (LDL), one of the most attractive biodegradable and biocompatible nanoparticles, were used for the simultaneous delivery of Paclitaxel (PTX), a hydrophobic antitumour drug and an amphiphilic contrast agent, Gd-AAZTA-C17, in B16-F10 melanoma cell line. These cells overexpress LDL receptors, as assessed by Flow cytometry analysis. PTX and Gd-AAZTA-C17 loaded LDLs (LDL-PTX-Gd) have been prepared, characterized and their stability was assessed under 72 h incubation at 37 ◦C and compared to LDL loaded with Gd-AAZTA-C17 (LDL-Gd) and LDL-PTX. The cytotoxic effect of LDL-PTX-Gd was evaluated by MTT assay. The anti-tumour drug loaded into LDLs showed a significantly higher toxicity on B16-F10 cells with respect to the commercially available formulation Paclitaxel Kabi (PTX Kabi) used in clinical applications. It was possible to demonstrate a high uptake of LDL-Gd in B16-F10 cells. As a consequence of the high cell uptake, melanoma cells showed significantly high cytotoxic effect when incubated in the presence of PTX (LDL-PTX-Gd). Furthermore, B16-F10 have been used to perform Magnetic Resonance Imaging. By the analysis of the image signal intensity, it was possible to extrapolate the amount of internalized PTX indirectly by the decrease of relaxation times caused by Gd, proportional to its concentration. Finally, the treatment with PTX loaded LDL on B16-F10 tumour bearing mice resulted in a marked reduction of tumour growth compared to the administration of PTX Kabi alone. In conclusion, LDLs are selectively taken-up by tumour cells and can be successfully exploited for the selective delivery of Paclitaxel and imaging agents.

Keywords: low density lipoprotein, melanoma cell lines, MRI, paclitaxel, personalized medicine application, theragnostic System

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Authors: Damião Sousa, Hasan Turkez, Ozlem Tozlu, Tamires Lima

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Glioblastoma (GBM) is an aggressive cancer from the brain and with high prevalence and significant morbimortality. Therefore, it is necessary to investigate new therapeutic options against this pathology. Thus, the purpose of this study was to evaluate the antitumor activity from Mentha crispa essential oil (MCEO), its major constituent rotundifolone (ROT) and a series of six analogues on human U87MG glioblastoma cell line. The antitumor effects of the compounds on human U87MG-GBM cell line were assessed using in vitro cell viability assays. In addition, biosafety tests were performed on cultured human blood cells. The data show that MCEO, 1,2-perillaldehyde epoxide (EPER1) and perillaldehyde (PALD) were the most cytotoxic compounds against the U87MG cells, with IC50 values of 16.263, 15.087 and 14.888 μg/mL, respectively. The treatment with MCEO, EPER1 and PALD did not lead to damage in blood cells. These chemical analogues may be useful as prototypes for development of novel antitumor drugs due to their promising activities and toxicological safety.

Keywords: antitumor activity, cancer, natural products, terpenes

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Authors: Ainur Mukhambetova, Miras Karzhauov, Vyacheslav Ogay

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Introduction: Mesenchymal stem cells (MSCs) have the capacity to be differentiated into several cell lineages and are a promising source for cell therapy and tissue engineering. However, currently most MSCs culturing protocols use media supplemented with fetal bovine serum (FBS), which limits their application in clinic due to the possibility of zoonotic infections, contamination and immunological reactions. Consequently, formulating effective serum free culture medium becomes one of the important problems in contemporary cell biotechnology. Objectives: The aim of this study was to define an optimal serum-free medium for culturing of periosteum derived MSCs. Materials and methods: The MSCs were extracted from human periosteum and transferred to the culture flasks pretreated with CELLstart™. Immunophenotypic characterization, proliferation and in vitro differentiation of cells grown on STEM PRO® MSC SFM were compared to the cells cultured in the standard FBS containing media. Chromosome analysis and flow cytometry were also performed. Results: We have shown that cells were grown on STEM PRO® MSC SFM retained all the morphological, immunophenotypic (CD73, CD90, CD105, vimentin and Stro-1) and cell differentiation characteristics specific to MSCs. Chromosome analysis indicated no anomalies in the chromosome structure. Flow cytometry showed a high expression of cell adhesion molecules CD44 (98,8%), CD90 (97,4%), CD105 (99,1%). In addition, we have shown that cell is grown on STEM PRO® MSC SFM have higher proliferation capacity compared to cell expanded on standard FBS containing the medium. Conclusion: We have shown that STEM PRO® MSC SFM is optimal for culturing periosteum derived human MSCs which subsequently can be safely used in cell therapy.

Keywords: cell technologies, periosteum-derived MSCs, regenerative medicine, serum-free medium

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Authors: Eduardo Kanagushiku Pereira, Frank Gregory Cavalcante da Silva, Barbara Soares Gonçalves, Ana Lúcia Bergamasco Galastri, Ronei Luciano Mamoni

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The inflammatory response initiates after the recognition of pathogens by receptors expressed by innate immune cells. Among these receptors, the NLRP3 was associated with the recognition of pathogenic fungi in experimental models. NLRP3 operates forming a multiproteic complex called inflammasome, which actives caspase-1, responsible for the production of the inflammatory cytokines IL-1beta and IL-18. In this study, we aimed to investigate the involvement of NLRP3 in the inflammatory response elicited in macrophages against Paracoccidioides brasiliensis (Pb), the etiologic agent of PCM. Macrophages were differentiated from THP-1 cells by treatment with phorbol-myristate-acetate. Following differentiation, macrophages were stimulated by Pb yeast cells for 24 hours, after previous treatment with specific NLRP3 (3,4-methylenedioxy-beta-nitrostyrene) and/or caspase-1 (VX-765) inhibitors, or specific inhibitors of pathways involved in NLRP3 activation such as: Reactive Oxigen Species (ROS) production (N-Acetyl-L-cysteine), K+ efflux (Glibenclamide) or phagossome acidification (Bafilomycin). Quantification of IL-1beta and IL-18 in supernatants was performed by ELISA. Our results showed that the production of IL-1beta and IL-18 by THP-1-derived-macrophages stimulated with Pb yeast cells was dependent on NLRP3 and caspase-1 activation, once the presence of their specific inhibitors diminished the production of these cytokines. Furthermore, we found that the major pathways involved in NLRP3 activation, after Pb recognition, were dependent on ROS production and K+ efflux. In conclusion, our results showed that NLRP3 participates in the recognition of Pb yeast cells by macrophages, leading to the activation of the NLRP3-inflammasome and production of IL-1beta and IL-18. Together, these cytokines can induce an inflammatory response against P. brasiliensis, essential for the establishment of the initial inflammatory response and for the development of the subsequent acquired immune response.

Keywords: inflammation, IL-1beta, IL-18, NLRP3, Paracoccidioidomycosis

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Authors: May Fadheel Estephan, Richard Perks

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Context: Cancer is a serious health concern that affects millions of people worldwide. Early detection and treatment are essential for improving patient outcomes. However, current methods for cancer detection have limitations, such as low sensitivity and specificity. Research Aim: The aim of this study was to develop an optical sensor for cancer detection using elastic light scattering spectroscopy (ELSS). ELSS is a noninvasive optical technique that can be used to characterize the size and concentration of particles in a solution. Methodology: An optical probe was fabricated with a 100-μm-diameter core and a 132-μm centre-to-centre separation. The probe was used to measure the ELSS spectra of polystyrene spheres with diameters of 2, 0.8, and 0.413 μm. The spectra were then analysed to determine the size and concentration of the spheres. Findings: The results showed that the optical probe was able to differentiate between the three different sizes of polystyrene spheres. The probe was also able to detect the presence of polystyrene spheres in suspension concentrations as low as 0.01%. Theoretical Importance: The results of this study demonstrate the potential of ELSS for cancer detection. ELSS is a noninvasive technique that can be used to characterize the size and concentration of cells in a tissue sample. This information can be used to identify cancer cells and assess the stage of the disease. Data Collection: The data for this study were collected by measuring the ELSS spectra of polystyrene spheres with different diameters. The spectra were collected using a spectrometer and a computer. Analysis Procedures: The ELSS spectra were analysed using a software program to determine the size and concentration of the spheres. The software program used a mathematical algorithm to fit the spectra to a theoretical model. Question Addressed: The question addressed by this study was whether ELSS could be used to detect cancer cells. The results of the study showed that ELSS could be used to differentiate between different sizes of cells, suggesting that it could be used to detect cancer cells. Conclusion: The findings of this research show the utility of ELSS in the early identification of cancer. ELSS is a noninvasive method for characterizing the number and size of cells in a tissue sample. To determine cancer cells and determine the disease's stage, this information can be employed. Further research is needed to evaluate the clinical performance of ELSS for cancer detection.

Keywords: elastic light scattering spectroscopy, polystyrene spheres in suspension, optical probe, fibre optics

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