Search results for: toll-like receptor signaling

Authors: S. Ben-Shabat, T. Turgeman, O. Leubinski, N. Roth-Bejerano, V. Kagan-Zur, Y. Sitrit

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The ectomycorrhizal (ECM) desert truffle Terfezia boudieri produces edible fruit bodies and forms symbiosis with its host plant Helianthemum sessiliflorum (Cistaceae) in the Negev desert of Israel. The symbiosis is vital for both partners' survival under desert conditions. Under desert habitat conditions, ECMs must form symbiosis before entering the dry season. To secure a successful encounter, in the course of evolution, both partners have responded by evolving special signals exchange that facilitates recognition. Members of the Cistaceae family serve as host plants for many important truffles. Conceivably, during evolution a common molecule present in Cistaceae plants was recruited to facilitate successful encounter with ectomycorrhizas. Arbuscular vesicular fungi (AM) are promiscuous in host preferences, in contrast, ECM fungi show specificity to host plants. Accordingly, we hypothesize that H. sessiliflorum secretes a chemotrophic-signaling, which is common to plants hosting ECM fungi belonging to the Pezizales. However, thus far no signaling molecules have been identified in ECM fungi. We developed a bioassay for chemotrophic activity. Fractionation of root exudates revealed a substance with chemotrophic activity and molecular mass of 534. Following the above concept, screening the transcriptome of Terfezia, grown under chemoattraction, discovered genes showing high homology to G proteins-coupled receptors of plant pathogens involved in positive chemotaxis and chemotaxis suppression. This study aimed to identify the active molecule using analytical methods (LC-MS, NMR etc.). This should contribute to our understanding of how ECM fungi communicate with their hosts in the rhizosphere. In line with the ability of Terfezia to form also endomycorrhizal symbiosis like AM fungi, analysis of the mechanisms may likewise be applicable to AM fungi. Developing methods to manipulate fungal growth by the chemoattractant can open new ways to improve inoculation of plants.

Keywords: chemotrophic signal, Helianthemum sessiliflorum, Terfezia boudieri, ECM

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Authors: Shamim Mushtaq, Moazzam Shahid

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Breast cancer (BC) claims the lives of half a million women every year and is the most common cause of death in the developing world. In 2019, it was estimated that BC alone accounts for 15% of all cancer deaths in younger women (aged < 45 years old) with advanced-stage lung metastasis. According to the World Health Organization & International Union against Cancer, in Asia, a high number of cancer-related deaths will be observed in 2020, whereas the burden will be reduced in Western countries due to awareness about the disease, better health facilities and advanced treatments. In the last 15 years, it has been reported that the incidence of BC has increased by 1.1% among Asian compared to the US population from 2003 to 2012. To date, several BC biological subtypes have been reported so far, which are associated with different treatment responses. The heterogeneity and diversity of BC reflected these different subtypes, including Luminal A (23.7% prevalence) and B (38.8% prevalence) that have pathological estrogen receptor (ER+)-positive tumors, the human epidermal growth factor receptor 2 (HER2) (11.2% prevalence) and triple-negative breast cancer (TNBC) (25% prevalence). According to Shaukat Khanum Memorial Cancer Hospital and Research Centre – Pakistan, ten years of data showed that among 636 BC patients, 30.5% had TNBC who were <40 years of age, which is an extremely alarming situation. Therefore, there is a dire need to explore and develop therapeutic targets for the treatment of early TNBC. Since the last decade, unfortunately, there has been little success in understanding the complexity of TNBC and in discovering new biological therapeutic targets. However, conventional chemotherapy is the only choice of treatment for TNBC patients. Many investigators revealed advances in multi-omics (multiple "omes", e.g., genome, proteome, transcriptome, epigenome, and microbiome) which were later identified as actionable targets and increased prevalence in TNBC patients. However, various drugs have been identified so far which are related to a particular diagnostic and prognostic biomarker. For example, Epidermal growth factor receptor ( EGFR or ErbB-1), HER-2/neu (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4). Protein Transglin-2 (TAGLN 2 ) and Profilins-1 (Pfn-1 ) are the ubiquitously expressed large family of proteins present in all eukaryotes, enabling actin cytoskeletal reorganization. It is known that the oncogenic transformation of cells is accompanied by alteration in the actin cytoskeleton. There are causal connections between altered expression of actin cytoskeletal regulators and cancer progression. Our case-control study identified TAGLN-2 and Pfn-1 proteins in TNBC blood by mass spectrometry. Both TAGLN-2 and Pfn-1 proteins are differentially expressed in early and advanced stages of TNBS patients, which could be potential predictors or therapeutic targets for TNBC.

Keywords: TNBC, blood biomarkers, mass spectrometry, qPCR, ELISA

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Authors: Katarzyna Bialik-Wąs, Klaudia Pluta, Dagmara Malina, Małgorzata Miastkowska

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In recent years, biocompatible hydrogels have been used more and more in medical applications, especially as modern dressings and drug delivery systems. The main goal of this research was the characteristics of bio-hybrid hydrogel materials incorporated with the nanocarrier-drug system, which enable the release in a gradual and prolonged manner, up to 7 days. Therefore, the use of such a combination will provide protection against mechanical damage and adequate hydration. The proposed bio-hybrid hydrogels are characterized by: transparency, biocompatibility, good mechanical strength, and the dual release system, which allows for gradual delivery of the active substance, even up to 7 days. Bio-hybrid hydrogels based on sodium alginate (SA), poly(vinyl alcohol) (PVA), glycerine, and Aloe vera solution (AV) were obtained through the chemical crosslinking method using poly(ethylene glycol) diacrylate as a crosslinking agent. Additionally, a nanocarrier-drug system was incorporated into SA/PVA/AV hydrogel matrix. Here, studies were focused on the release profiles of active substances from bio-hybrid hydrogels using the USP4 method (DZF II Flow-Through System, Erweka GmbH, Langen, Germany). The equipment incorporated seven in-line flow-through diffusion cells. The membrane was placed over support with an orifice of 1,5 cm in diameter (diffusional area, 1.766 cm²). All the cells were placed in a cell warmer connected with the Erweka heater DH 2000i and the Erweka piston pump HKP 720. The piston pump transports the receptor fluid via seven channels to the flow-through cells and automatically adapts the setting of the flow rate. All volumes were measured by gravimetric methods by filling the chambers with Milli-Q water and assuming a density of 1 g/ml. All the determinations were made in triplicate for each cell. The release study of the model active substance was carried out using a regenerated cellulose membrane Spectra/Por®Dialysis Membrane MWCO 6-8,000 Carl Roth® Company. These tests were conducted in buffer solutions – PBS at pH 7.4. A flow rate of receptor fluid of about 4 ml /1 min was selected. The experiments were carried out for 7 days at a temperature of 37°C. The released concentration of the model drug in the receptor solution was analyzed using UV-Vis spectroscopy (Perkin Elmer Company). Additionally, the following properties of the modified materials were studied: physicochemical, structural (FT-IR analysis), morphological (SEM analysis). Finally, the cytotoxicity tests using in vitro method were conducted. The obtained results exhibited that the dual release system allows for the gradual and prolonged delivery of the active substances, even up to 7 days.

Keywords: wound dressings, SA/PVA hydrogels, nanocarrier-drug system, USP4 method

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Authors: Jade Edey, Andrew Bennett, Gareth Hathway

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Nuclear receptor-related 1 protein (also known as Nurr1 or NR4A2) is an orphan nuclear receptor which plays a vital role in the development, survival and maintenance of dopaminergic (DA) neurons particularly in the substantia nigra (SN). Increasing research has investigated Nurr1’s additional role within microglia and astrocytes where it has been suggested to act as a negative regulator of inflammation; potentially offering neuroprotection. Considering both DA neurodegeneration and neuroinflammation are commonly accepted constituents of Parkinson’s Disease (PD), understanding the mechanisms by which Nurr1 regulates inflammatory processes could provide an attractive therapeutic target. Nurr1 regulates inflammation via a transrepressive mechanism possibly dependent upon SUMOylation. In addition, Nurr1 can transactivate numerous genes involved in DA synthesis, such as Tyrosine Hydroxylase (TH). A C-terminal splice variant of Nurr1, Nurr-1a, has been reported in both neuronal and glial cells. However, research into its transcriptional activity is minimal. We employed in vitro methods such as SUMO-Pulldown experiments alongside Luciferase reporter assays to investigate the SUMOylation status and transactivation capabilities of Nurr1 and Nurr-1a respectively. The SUMO-Pulldown assay demonstrated Nurr-1a undergoes significantly more SUMO modification than its full-length variant. Consequently, despite having less transcriptional activation than Nurr1, Nurr1a may play a more prominent role in repression of microglial inflammation. Contrary to published literature we also identified that SUMOylation enhances transcriptional activation by Nurr1 and Nurr1a. SUMOylation-dependent increases in Nurr1 and Nurr1a transcriptional activation were only evident in neuronal SHSY5Y cells but not in HEK293 cells. This research provides novel insight into the regulation of Nurr-1a and indicates differential effects of SUMOylation dependent regulation in neuronal and inflammatory cells.

Keywords: nuclear receptors, Parkinson’s disease, inflammation, transcriptional regulation

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Authors: Yusuf E. Mustapha, Inioluwa A Akindoyeni, Oluwatoyin G. Ezekiel, Ifeoluwa O. Awogbindin, Ebenezer O. Farombi

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Background: Parkinson's disease (PD) is the most prevalent motor disorder. Available therapies are palliative with no effect on disease progression. Kolaviron (KV), a natural anti-inflammatory and antioxidant agent, has been reported to possess neuroprotective effects in Parkinsonian flies and rats. Objective: The present study investigates the neuroprotective effect of KV, focusing on the DJ1/Nrf2 signaling pathway. Methodology: All-trans retinoic acid (ATRA, 10 mg/kg, i.p.) was used to inhibit Nrf2. Murine model of PD was established with four doses of MPTP (20 mg/kg i.p.) at 2 hours interval. MPTP mice were pre-treated with either KV (200 mg/kg/day p.o), ATRA, or both conditions for seven days before PD induction. Motor behaviour was evaluated, and markers of oxidative stress/damage and its regulators were assessed with immunofluorescence and ELISA techniques. Results: MPTP-treated mice covered less distance with reduced numbers of anticlockwise rotations, heightened freezing, and prolonged immobility when compared to control. However, KV significantly attenuated these deficits. Pretreatment of MPTP mice with KV upregulated Nrf2 expression beyond MPTP level with a remarkable reduction in Keap1 expression and marked elevation of DJ-1 level, whereas co-administration with ATRA abrogated these effects. KV treatment restored MPTP-mediated depletion of endogenous antioxidant, striatal oxidative stress, oxidative damage, and inhibition of acetylcholinesterase activity. However, ATRA treatment potentiated acetylcholinesterase inhibition and attenuated the protective effect of KV on the level of nitric oxide and activities of catalase and superoxide dismutase. Conclusion: Kolaviron protects Parkinsonian mice by stabilizing and activating the Nrf2 signaling pathway. Thus, kolaviron can be explored as a pharmacological lead in PD management.

Keywords: Garcinia kola, Kolaviron, Parkinson Disease, Nrf2, behavioral incompetence, neurodegeneration

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Authors: Sima Parvizi Omran, Rezvan Tavakoli, Mahnaz Safari, Mohammadreza Aghasadeghi, Abolfazl Fateh, Pooneh Rahimi

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Background: SARS-CoV-2, a single-stranded RNA betacoronavirus causes the global outbreak of coronavirus disease 2019 (COVID-19). Several clinical and scientific concerns are raised by this pandemic. Genetic factors can contribute to pathogenesis and disease susceptibility. There are single nucleotide polymorphisms (SNPs) in many of the genes in the immune system that affect the expression of specific genes or functions of some proteins related to immune responses against viral infections. In this study, we analyzed the impact of polymorphism in the interferon alpha and beta receptor subunit 2 (IFNAR2) and dipeptidyl peptidase 9 (Dpp9) genes and clinical parameters on the susceptibility and resistance to Coronavirus disease (COVID-19). Methods: A total of 330- SARS-CoV-2 positive patients (188 survivors and 142 nonsurvivors) were included in this study. All single-nucleotide polymorphisms (SNPs) on IFNAR2 (rs2236757) and Dpp9 (rs2109069) were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results: In survivor patients, the frequency of the favourable genotypes of IFNAR2 SNP (rs2236757 GC) was significantly higher than in nonsurvivor patients, and also Dpp9 (rs2109069 AT) genotypes were associated with the severity of COVID-19 infection. Conclusions: This study demonstrated that the severity of COVID- 19 patients was strongly associated with clinical parameters and unfavourable IFNAR2, Dpp9 SNP genotypes. In order to establish the relationship between host genetic factors and the severity of COVID-19 infection, further studies are needed in multiple parts of the world.

Keywords: SARS-CoV-2, COVID-19, interferon alpha and beta receptor subunit 2, dipeptidyl peptidase 9, single-nucleotide polymorphisms

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Authors: Tarana Siddika, Ilka U. Heinemann, Patrick O’Donoghue

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Protein kinase B (AKT1) is a serine/threonine kinase and central transducer of cell survival pathways. Typical approaches to study AKT1 biology in cells rely on growth factor or insulin stimulation that activates AKT1 via phosphorylation at two key regulatory sites (Threonine308, Serine473), yet cell stimulation also activates many other kinases and fails to differentiate the effect of the two main activating sites of AKT1 on downstream substrate phosphorylation and cell growth. While both AKT1 activating sites are associated with disease and used as clinical markers, in some cancers, high levels of Threonine308 phosphorylation are associated with poor prognosis while in others poor survival correlates with high Serine473 levels. To produce cells with specific AKT1 activity, a system was developed to deliver active AKT1 to human cells. AKT1 phospho-variants were produced from Escherichia coli with programmed phosphorylation by genetic code expansion. Tagging of AKT1 with an N-terminal cell penetrating peptide tag derived from the human immunodeficiency virus trans-activator of transcription (TAT) helped to enter AKT1 proteins in mammalian cells. The TAT-tag did not alter AKT1 kinase activity and was necessary and sufficient to rapidly deliver AKT1 protein variants that persisted in human cells for 24 h without the need to use transfection reagents. TAT-pAKT1T308, TAT-pAKT1S473 and TAT-pAKT1T308S473 proteins induced selective phosphorylation of the known AKT1 substrate GSK-3αβ, and downstream stimulation of the AKT1 pathway as evidenced by phosphorylation of ribosomal protein S6 at Serine240/244 in transfected cells. Increase in cell growth and proliferation was observed due to the transfection of different phosphorylated AKT1 protein variants compared to cells with TAT-AKT1 protein. The data demonstrate efficient delivery of AKT1 with programmed phosphorylation to human cells, thus establishing a cell-based model system to investigate signaling that is dependent on specific AKT1 activity and phosphorylation.

Keywords: cell penetrating peptide, cell signaling, protein kinase b (AKT1), phosphorylation

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Authors: Mohammad M. Aria, Fatma Öz, Yashar Esmaeilian, Marco Carofiglio, Valentina Cauda, Özlem Yalçın

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Photoelectrical stimulation of cells with semiconductor organic polymers have been shown promising applications in neuroprosthetics such as retinal prosthesis. Photoelectrical stimulation of the cell membranes can be induced through a photo-electric charge separation mechanism in the semiconductor materials, and it can alter intracellular calcium level through both stimulation of voltage-gated ion channels and increase of intracellular reactive oxygen species (ROS) level. On the other hand, targeting voltage-gated ion channels in cancer cells to induce cell apoptosis through calcium signaling alternation is an effective mechanism which has been explained before. In this regard, remote control of the voltage-gated ion channels aimed to alter intracellular calcium by using photo-active organic polymers can be novel technology in cancer therapy. In this study, we used P (ITO/Indium thin oxide)/P3HT(poly(3-hexylthiophene-2,5-diyl)) and PN (ITO/ZnO/P3HT) photovoltaic junctions to stimulate MDA-MB-231 breast cancer cells. We showed that the photo-stimulation of breast cancer cells through photo capacitive current generated by the photovoltaic junctions are able to excite the cells and alternate intracellular calcium based on the calcium imaging (at 8mW/cm² green light intensity and 10-50 ms light durations), which has been reported already to safety stimulate neurons. The control group did not undergo light treatment and was cultured in T-75 flasks. We detected 20-30% cell death for ITO/P3HT and 51-60% cell death for ITO/ZnO/P3HT samples in the light treated MDA-MB-231 cell group. Western blot analysis demonstrated poly(ADP-ribose) polymerase (PARP) activated cell death in the light treated group. Furthermore, Annexin V and PI fluorescent staining indicated both apoptosis and necrosis in treated cells. In conclusion, our findings revealed that the photoelectrical stimulation of cells (through long time overstimulation) can induce cell death in cancer cells.

Keywords: Ca²⁺ signaling, cancer therapy, electrically excitable cells, photoelectrical stimulation, voltage-gated ion channels

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Authors: Ann Ying-An Chen, Yi-Lun Tsai, Hso-Chi Chaung

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An understanding of pathogens and the immune system has played an utmost important role in agricultural research for the development of vaccinations. The immunological synapse, cell to cell interaction play a crucial role in triggering the body's immune system, such as activation between antigen-presenting cells (APCs) and different subsets of T-cell. If these interactions are regulated appropriately, the host has the ability to defend itself against a wide spectrum of infectious pathogens. The aim of this study is to establish and to characterize a porcine immune synapse system by co-culturing T cell/APC. In this study, blood samples were collected from specific-pathogen-free piglets, and peripheral blood mononuclear cells (PBMC) were separated by using Ficoll-Pague. The PBMC were then stained with CD4 (FITC) and CD25 (PE) antibodies. Different subsets of T cells sorted by fluorescence-activated cell sorting flow cytometer were co-cultured for 24 hrs with alveolar macrophages, and the profiles of cytokine secretion and mRNA transcription levels of Toll-like receptors were examined after. Results showed that the three stages of immune synapse were clearly visible and identified under both transmission and scanning electron microscope (TEM and SEM). The significant interaction differences in toll-like receptor expressions within the co-cultured cell system were observed. The TLR7 mRNA expressions in CD4+CD25- cells were lower than those in CD4+CD25+ and CD4 -CD25+. Interestingly, the IL-10 production levels in CD4+CD25- cells (7.732 pg/mL) were significantly higher than those of CD4+CD25+ (2.636 pg/mL) and CD4 -CD25+ (2.48 pg/mL). These findings demonstrated that a clear understanding of the porcine immune synapse system can contribute greatly for further investigations on the mechanism of T-cell activation, which can benefit in the discovery of potential adjuvant candidate or effective antigen epitopes in the development of vaccinations with high efficacy.

Keywords: antigen-presenting cells, immune synapse, pig, T subsets, toll-like receptor

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Authors: Akanksha Agrawal, Richa Rathor, Geetha Suryakumar

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Muscle represents about ¾ of the body mass, and a healthy muscular system is required for human performance. A healthy muscular system is dynamically balanced via the catabolic and anabolic process. High altitude associated hypoxia altered this redox balance via producing reactive oxygen and nitrogen species that ultimately modulates protein structure and function, hence, disrupts proteostasis or protein homeostasis. The mechanism by which proteostasis is clinched includes regulated protein translation, protein folding, and protein degradation machinery. Perturbation in any of these mechanisms could increase proteome imbalance in the cellular processes. Altered proteostasis in skeletal muscle is likely to be responsible for contributing muscular atrophy in response to hypoxia. Therefore, we planned to elucidate the mechanism involving altered proteostasis leading to skeletal muscle atrophy under chronic hypobaric hypoxia. Material and Methods-Male Sprague Dawley rats weighing about 200-220 were divided into five groups - Control (Normoxic animals), 1d, 3d, 7d and 14d hypobaric hypoxia exposed animals. The animals were exposed to simulated hypoxia equivalent to 282 torr pressure (equivalent to an altitude of 7620m, 8% oxygen) at 25°C. On completion of chronic hypobaric hypoxia (CHH) exposure, rats were sacrificed, muscle was excised and biochemical, histopathological and protein synthesis signaling were studied. Results-A number of changes were observed with the CHH exposure time period. ROS was increased significantly on 07 and 14 days which were attributed to protein oxidation via damaging muscle protein structure by oxidation of amino acids moiety. The oxidative damage to the protein further enhanced the various protein degradation pathways. Calcium activated cysteine proteases and other intracellular proteases participate in protein turnover in muscles. Therefore, we analysed calpain and 20S proteosome activity which were noticeably increased at CHH exposure as compared to control group representing enhanced muscle protein catabolism. Since inflammatory markers (myokines) affect protein synthesis and triggers degradation machinery. So, we determined inflammatory pathway regulated under hypoxic environment. Other striking finding of the study was upregulation of Akt/PKB translational machinery that was increased on CHH exposure. Akt, p-Akt, p70 S6kinase, and GSK- 3β expression were upregulated till 7d of CHH exposure. Apoptosis related markers, caspase-3, caspase-9 and annexin V was also increased on CHH exposure. Conclusion: The present study provides evidence of disrupted proteostasis under chronic hypobaric hypoxia. A profound loss of muscle mass is accompanied by the muscle damage leading to apoptosis and cell death under CHH. These cellular stress response pathways may play a pivotal role in hypobaric hypoxia induced skeletal muscle atrophy. Further research in these signaling pathways will lead to development of therapeutic interventions for amelioration of hypoxia induced muscle atrophy.

Keywords: Akt/PKB translational machinery, chronic hypobaric hypoxia, muscle atrophy, protein degradation

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Authors: Natalia V. Beloglazova, Pieterjan Lenain, Martin Hedstrom, Dietmar Knopp, Sarah De Saeger

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In recent years polycyclic aromatic hydrocarbons (PAHs), which represent a hazard to humans and entire ecosystem, have been receiving an increased interest due to their mutagenic, carcinogenic and endocrine disrupting properties. They are formed in all incomplete combustion processes of organic matter and, as a consequence, ubiquitous in the environment. Benzo(a)pyrene (BaP) is on the priority list published by the Environmental Agency (US EPA) as the first PAH to be identified as a carcinogen and has often been used as a marker for PAHs contamination in general. It can be found in different types of water samples, therefore, the European Commission set up a limit value of 10 ng L–1 (10 ppt) for BAP in water intended for human consumption. Generally, different chromatographic techniques are used for PAHs determination, but these assays require pre-concentration of analyte, create large amounts of solvent waste, and are relatively time consuming and difficult to perform on-site. An alternative robust, stand-alone, and preferably cheap solution is needed. For example, a sensing unit which can be submerged in a river to monitor and continuously sample BaP. An affinity sensor based on capacitive transduction was developed. Natural antibodies or their synthetic analogues can be used as ligands. Ideally the sensor should operate independently over a longer period of time, e.g. several weeks or months, therefore the use of molecularly imprinted polymers (MIPs) was discussed. MIPs are synthetic antibodies which are selective for a chosen target molecule. Their robustness allows application in environments for which biological recognition elements are unsuitable or denature. They can be reused multiple times, which is essential to meet the stand-alone requirement. BaP is a highly lipophilic compound and does not contain any functional groups in its structure, thus excluding non-covalent imprinting methods based on ionic interactions. Instead, the MIPs syntheses were based on non-covalent hydrophobic and π-π interactions. Different polymerization strategies were compared and the best results were demonstrated by the MIPs produced using electropolymerization. 4-vinylpyridin (VP) and divinylbenzene (DVB) were used as monomer and cross-linker in the polymerization reaction. The selectivity and recovery of the MIP were compared to a non-imprinted polymer (NIP). Electrodes were functionalized with natural receptor (monoclonal anti-BaP antibody) and with MIPs selective towards BaP. Different sets of electrodes were evaluated and their properties such as sensitivity, selectivity and linear range were determined and compared. It was found that both receptor can reach the cut-off level comparable to the established ML, and despite the fact that the antibody showed the better cross-reactivity and affinity, MIPs were more convenient receptor due to their ability to regenerate and stability in river till 7 days.

Keywords: antibody, benzo(a)pyrene, capacitive sensor, MIPs, river water

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Authors: Nadia H. Mohammed, Anmar Al-Taie, Fadia H. Al-Sultany

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Although breast cancer prevalence continues to increase, mortality has been decreasing as a result of early detection and improvement in adjuvant systemic therapy. Nevertheless, this disease required further efforts to understand and identify the associated potential risk factors that could play a role in the prevalence of this malignancy among Iraqi women. The objective of this study was to assess the perception of certain predictive risk factors on the prevalence of breast cancer types among a sample of Iraqi women diagnosed with breast cancer. This was a retrospective observational study carried out at National Cancer Research Center in College of Medicine, Baghdad University from November 2017 to January 2018. Data of 100 patients with breast cancer whose biopsies examined in the National Cancer Research Center were included in this study. Data were collected to structure a detailed assessment regarding the patients’ demographic, medical and cancer records. The majority of study participants (94%) suffered from ductal breast cancer with mean age 49.57 years. Among those women, 48.9% were obese with body mass index (BMI) 35 kg/m². 68.1% of them had positive family history of breast cancer and 66% had low parity. 40.4% had stage II ductal breast cancer followed by 25.5% with stage III. It was found that 59.6% and 68.1% had positive oestrogen receptor sensitivity and positive human epidermal growth factor (HER2/neu) receptor sensitivity respectively. In regard to the impact of prediction of certain variables on the incidence of ductal breast cancer, positive family history of breast cancer (P < 0.0001), low parity (P< 0.0001), stage I and II breast cancer (P = 0.02) and positive HER2/neu status (P < 0.0001) were significant predictive factors among the study participants. The results from this study provide relevant evidence for a significant positive and potential association between certain risk factors and the prevalence of breast cancer among Iraqi women.

Keywords: Ductal Breast Cancer, Hormone Sensitivity, Iraq, Risk Factors

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Authors: Eashwar V. Somasundaram, Raoul R. Wadhwa, Jacob G. Scott

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The use of radiomics in measuring geometric properties of tumor images such as size, surface area, and volume has been invaluable in assessing cancer diagnosis, treatment, and prognosis. In addition to analyzing geometric properties, radiomics would benefit from measuring topological properties using persistent homology. Intuitively, features uncovered by persistent homology may correlate to tumor structural features. One example is necrotic cavities (corresponding to 2D topological features), which are markers of very aggressive tumors. We develop a data pipeline in R that clusters tumors images based on persistent homology is used to identify meaningful clinical distinctions between tumors and possibly new relationships not captured by established clinical categorizations. A preliminary analysis was performed on 16 Magnetic Resonance Imaging (MRI) breast tissue segments downloaded from the 'Investigation of Serial Studies to Predict Your Therapeutic Response with Imaging and Molecular Analysis' (I-SPY TRIAL or ISPY1) collection in The Cancer Imaging Archive. Each segment represents a patient’s breast tumor prior to treatment. The ISPY1 dataset also provided the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status data. A persistent homology matrix up to 2-dimensional features was calculated for each of the MRI segmentation. Wasserstein distances were then calculated between all pairwise tumor image persistent homology matrices to create a distance matrix for each feature dimension. Since Wasserstein distances were calculated for 0, 1, and 2-dimensional features, three hierarchal clusters were constructed. The adjusted Rand Index was used to see how well the clusters corresponded to the ER/PR/HER2 status of the tumors. Triple-negative cancers (negative status for all three receptors) significantly clustered together in the 2-dimensional features dendrogram (Adjusted Rand Index of .35, p = .031). It is known that having a triple-negative breast tumor is associated with aggressive tumor growth and poor prognosis when compared to non-triple negative breast tumors. The aggressive tumor growth associated with triple-negative tumors may have a unique structure in an MRI segmentation, which persistent homology is able to identify. This preliminary analysis shows promising results in the use of persistent homology on tumor imaging to assess the severity of breast tumors. The next step is to apply this pipeline to other tumor segment images from The Cancer Imaging Archive at different sites such as the lung, kidney, and brain. In addition, whether other clinical parameters, such as overall survival, tumor stage, and tumor genotype data are captured well in persistent homology clusters will be assessed. If analyzing tumor MRI segments using persistent homology consistently identifies clinical relationships, this could enable clinicians to use persistent homology data as a noninvasive way to inform clinical decision making in oncology.

Keywords: cancer biology, oncology, persistent homology, radiomics, topological data analysis, tumor imaging

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Authors: Abdolamir Allameh, Seyyed Mortaza Haghgoo, Adnan Khosravi, Esmaeil Mortaz, Mihan Pourabdollah-Toutkaboni, Sharareh Seifi

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Non-Small Cell Lung Carcinoma (NSCLC) is associated with a number of gene mutations in epidermal growth factor receptor (EGFR). The prognostic significance of mutations in exons 19 and 21, together with serum levels of EGFR, amphiregulin (AR), and Transforming Growth Factor-alpha (TGF-α) are implicated in diagnosis and treatment. The aim of this study was to examine the relationship of EGFR mutations in selected exons with the expression of relevant ligands in sera samples of NSCLC patients. For this, a group of NSCLC patients (n=98) referred to the hospital for lung surgery with a mean age of 59±10.5 were enrolled (M/F: 75/23). Blood specimen was collected from each patient. Besides, formalin fixed paraffin embedded tissues were processed for DNA extraction. Gene mutations in exons 19 and 21 were detected by direct sequencing, following DNA amplification which was done by PCR (Polymerase Chain Reaction). Also, serum levels of EGFR, AR, and TGF-α were measured by ELISA. The results of our study show that EGFR mutations were present in 37% of Iranian NSCLC patients. The most frequently identified mutations were deletions in exon 19 (72.2%) and substitutions in exon 21 (27.8%). The most frequently identified alteration, which is considered as a rare mutation, was the E872K mutation in exon 21, which was found in 90% (9 out of 10) cases. EGFR mutation detected in exon 21 was significantly (P<0.05) correlated with the levels of its ligands, EGFR and TGF-α in serum samples. Furthermore, it was found that increased serum AR (>3pg/ml) and TGF-α (>10.5 pg/ml) were associated with shorter overall survival (P<0.05). The results clearly showed a close relationship between EGFR mutations and serum EGFR and serum TGF-α. Increased serum EGFR was associated with TGF-α and AR and linked to poor prognosis of NSCLC. These findings are implicated in clinical decision-making related to EGFR-Tyrosine kinase inhibitors (TKIs).

Keywords: lung cancer, Iranian patients, epidermal growth factor, mutation, prognosis

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Authors: Ankush M. Dewle, Suditi Bhattacharya, Prachi R. Abhang, Savita Datar, Ajay J. Jog, Rupesh K. Srivastava, Geetanjali Tomar

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Objectives: The aim of this study was to investigate the quality of mesenchymal stem cells (MSCs) obtained from ageing gingival tissues, in order to suggest their potential role in autologous stem cell therapy for old individuals. Methods: MSCs were isolated from gingival tissues of young (18-45 years) and old (above 45 years) donors by enzymatic digestion. MSCs were analysed for cfu-f, surface marker expression by flow-cytometry and multilineage differentiation potential. The angiogenic potential was compared in a chick embryo yolk sac membrane model. The aging and differentiation markers including SA-β-galactosidase and p21 respectively were analysed by staining and flow-cytometry analysis. Additionally, osteogenic markers such as glucocorticoid receptor (GR), vitamin D receptor (VDR) were measured by flow-cytometry and RT-qPCR was performed for quantification of osteogenic gene expression. Alizarin Red S and alkaline phosphatase (ALP) activity were also quantitated. Results: Gingival MSCs (GMSCs) from both the age groups were similar in their morphology and displayed cfu-f. They had similar expression of MSC surface markers and p21, comparable rate of proliferation and differentiated to all the four lineages. GMSCs from young donors had a higher adipogenic differentiation potential as compared to the old GMSCs. Moreover, these cells did not display a significant difference in ALP activity probably due to comparable expression of GR, VDR, and osteogenic genes. Conclusions: Ageing of GMSCs occurs at a much slower rate than stem cells from other sources. Thus we suggest GMSCs as an excellent candidate for autologous stem cell therapy in degenerative diseases of elderly individuals. Clinical Significance: GMSCs could help overcome the setbacks in clinical implementation of autologous stem cell therapy for regenerative medicine in all age group of patient.

Keywords: bone regeneration, cell therapy, senescence, stem cell

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Authors: Peter David Reiss

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The concept of the psychosis prodrome has allowed for the identification of adolescent and young adult patients who have a significantly elevated risk of developing schizophrenia spectrum disorders. A number of different interventions have been tested in order to prevent or delay progression of symptoms. To date, there has been no consistent meta-analytical evidence to support efficacy of antipsychotic treatment for patients in the prodromal state, and their use remains therefore inconclusive. Although antipsychotics may manage symptoms transiently, they have not been found to prevent or delay onset of psychotic disorders. Furthermore, pharmacological intervention in high-risk individuals remains controversial, because of the antipsychotic side effect profile in a population in which only about 20 to 35 percent will eventually convert to psychosis over a two-year period, with even after two years conversion rates not exceeding 30 to 40 percent. This general estimate is additionally problematic, in that it ignores the fact that there is significant variation in individual risk among clinical high-risk cases. The current lack of reliable tests for at-risk patients makes it difficult to justify individual treatment decisions. Preventive treatment should ideally be dictated by an individual’s risk while minimizing potentially harmful medication exposure. This requires more accurate predictive assessments by using valid and accessible prognostic markers. The following will compare prediction and risk modification potential of behavioral biomarkers such as disturbances of basic sense of self and emotion awareness, neurocognitive biomarkers such as attention, working and declarative memory, and neurophysiological biomarkers such as glutamatergic abnormalities and NMDA receptor dysfunction. Identification of robust biomarkers could therefore not only provide more reliable means of psychosis prediction, but also help test and develop new clinical interventions targeted at the prodromal state.

Keywords: at-risk mental state, biomarkers, glutamatergic system, NMDA receptor, psychosis prodrome, schizophrenia

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Authors: Saad Mahmud Khan, Sarah El Hajj Chehadeh, Mehera Abdulrahman, Wael Osman, Habiba Al Safar

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Obesity is a non-communicable disease that is widely prevalent with approximately 600 million people classified as obese worldwide. Its etiology is multifactorial and involves a complex interplay between genes and the environment. Over the past few decades, obesity rates among the Emirati population have been increasing. The aim of this study was to investigate the association of candidate gene single nucleotide polymorphisms (SNPs), namely the fat mass and obesity associated (FTO) gene SNP rs9939609 and Vitamin D Receptor (VDR) gene SNP rs1544410, with obesity in the UAE population. Methods: This is a case-control study in which 414 individuals were enrolled during their routine visit to endocrinology clinics in Abu Dhabi, United Arab Emirates between the period of June 2012 and December 2013. Several biochemical tests and clinical assessments along with a lifestyle questionnaire for each participant were completed at the clinic. Genomic DNA was extracted from saliva samples of 201 obese, 114 overweight and 99 normal subjects. Genotyping for the variants was performed using TaqMan assay. Results: The mean Body Mass Index (BMI) ± SD for the obese, overweight, and normal subjects was 35.76 ± 4.54, 27.53 ± 1.45 and 22.69 ± 1.84 kg/m2, respectively. Increasing BMI values were associated with an increase in values for systolic blood pressure, diastolic blood pressure, HbA1c, and triglycerides. The SNP rs9939609 in the FTO gene was found to be significantly associated with the BMI (p=0.028), with the minor allele A having a clear additive effect on BMI values. No significant association was detected between BMI and rs1544410 of the VDR gene. Conclusions: Our study findings indicate that the minor allele A of the rs9939609 has a significant association with increasing BMI values. In addition, our findings support the fact that increasing BMI is associated with increasing risks of other comorbidities such as higher blood pressure, poorer glycemic control and higher triglycerides.

Keywords: body mass index, FTO gene, obesity, rs9939609, United Arab Emirates

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Authors: Valeria Arcucci, Musarat Ishaq, Steven A. Stacker, Greg J. Goodall, Marc G. Achen

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Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGF-C and VEGF-D, are key drivers of lymphatic remodelling, the means by which many signaling pathways in endothelial cells are coordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. We seek to understand the broader molecular mechanisms that control cancer metastasis, and are focusing on microRNAs, which coordinately regulate signaling pathways involved in complex biological responses in health and disease. Here, using small RNA sequencing, we found that a specific microRNA, miR-132, is upregulated in expression in lymphatic endothelial cells (LECs) in response to the lymphangiogenic growth factors. Interestingly, ectopic expression of miR-132 in LECs in vitro stimulated proliferation and tube formation of these cells. Moreover, miR-132 is expressed in lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGF-D by immunohistochemical analysis on tumour tissue microarrays. In order to dissect the complexity of regulation by miR-132 in lymphatic biology, we performed Argonaute HITS-CLIP, which led us to identify the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different pathways mainly involved in cell proliferation and regulation of the extracellular matrix and cell-cell junctions. We are now exploring the functional significance of miR-132 targets in the biology of LECs using biochemical techniques, functional in vitro cell assays and in vivo lymphangiogenesis assays. This project will ultimately define the molecular regulation of lymphatic remodelling by miR-132, and thereby identify potential therapeutic targets for drugs designed to restrict the growth and remodelling of tumour lymphatics resulting in metastatic spread.

Keywords: argonaute HITS-CLIP, cancer, lymphatic remodelling, miR-132, VEGF

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Authors: Marek Kuźniewski, Magdalena B. Kaziuk , Danuta Fedak, Paulina Dumnicka, Ewa Stępień, Beata Kuśnierz-Cabala, Władysław Sułowicz

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Background: The high prevalence of cardiovascular morbidity and mortality among patients with chronic kidney disease (CKD) is observed especially in those undergoing dialysis. Osteoprotegerin (OPG) and its ligands, receptor activator of nuclear factor kappa-B ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have been associated with cardiovascular complications. Our aim was to study their role as cardiovascular risk factors in stage 5 CKD patients. Methods: OPG, RANKL and TRAIL concentrations were measured in 69 hemodialyzed CKD patients and 35 healthy volunteers. In CKD patients, cardiovascular dysfunction was assessed with aortic pulse wave velocity (AoPWV), carotid artery intima-media thickness (CCA-IMT), coronary artery calcium score (CaSc) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) serum concentration. Cardiovascular and overall mortality data were collected during a 7-years follow-up. Results: OPG plasma concentrations were higher in CKD patients comparing to controls. Total soluble RANKL was lower and OPG/RANKL ratio higher in patients. Soluble TRAIL concentrations did not differ between the groups and OPG/TRAIL ratio was higher in CKD patients. OPG and OPG/TRAIL positively predicted long-term mortality (all-cause and cardiovascular) in CKD patients. OPG positively correlated with AoPWV, CCA-IMT and NT-proBNP whereas OPG/TRAIL with AoPWV and NT-proBNP. Described relationships were independent of classical and non-classical cardiovascular risk factors, with exception of age. Conclusions: Our study confirmed the role of OPG as a biomarker of cardiovascular dysfunction and a predictor of mortality in stage 5 CKD. OPG/TRAIL ratio can be proposed as a predictor of cardiovascular dysfunction and mortality.

Keywords: osteoprotegerin, tumor necrosis factor-related apoptosis-inducing ligand, receptor activator of nuclear factor kappa-B ligand, hemodialysis, chronic kidney disease, cardiovascular disease

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Authors: Axel Mancebo, Ana M. Bada, Angel Casacó, Bárbara González, Avelina León, María E. Arteaga, Consuelo González, Belinda Sánchez, Adriana Carr, Nuris Ledón, Arianna Iglesias

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Despite the many preventive and therapeutic modalities aimed at curing cancer, it remains as a serious world health problem. Promising recent developments suggest that cancer immunotherapy may be the next great hope for cancer treatment. EGFRs are receptor tyrosine kinases and it is considered an important therapeutic target related with tumor progression, and several types of molecular therapies, including monoclonal antibodies, small molecules, and vaccines, have been developed to target the HER family of receptors. On the other hand, gangliosides are membrane glycosphingolipids that contain two variants of sialic acid, the N-acetylated (NeuAc) and the N-glycolylated (NeuGc) variant. The high expression of this antigen-specific molecule has been associated with malignant tumor progression and immunosuppressive mechanisms, so ganglioside could be considered as the target for cancer immunotherapy. We have been working for several years in the safety evaluation of cancer vaccines targeting these two systems, the EGF receptor and ganglioside. We presented in this work results of repeated dose toxicity studies performed in Sprague Dawley rats and Cynomolgus monkeys, including clinical observations, body weight and rectal temperature measuring, clinical pathology analysis, gross necropsy and histological examination in rodent studies, and immunological evaluation. Immunizations were capable of inducing mainly inflammatory effects at the injection site, with findings largely attributable to the adjuvants used and probably enhanced by the immunological properties of the antigens. In general, these vaccines were shown to be well tolerated, and these studies in relevant species allow treating cancer patients with tumors during long periods with relative weight safety margin.

Keywords: cancer vaccines, safety, toxicology, rats, non human primates

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Authors: Norihiro Kato, Yuriko Takayama

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Some gram-negative bacteria enable the simultaneous activation of gene expression involved in N-acylhomoserine lactone (AHL) dependent cell-to-cell communication system. Such regulatory system for the bacterial group behavior is termed as quorum sensing (QS) because a diffusible AHL signal can accumulate around the cell during the increase of the cell density and trigger activation of the sequential QS process. By blocking the QS, the expression of diverse genes related to infection, antibiotic production, and biofilm formation is inhibited. Conditioning of QS by regulation of the DNA-receptor-AHL interaction is a potential target for enhancing host defenses against pathogenicity. We focused on engineered application of transcriptional regulator SpnR produced in opportunistic human pathogen Serratia marcescens. The SpnR can interact with AHL signals at an N-terminal domain and also with a promoter region of a QS target gene at a C-terminal domain. As the initial process of the QS activation, the SpnR forms a complex with the AHL to enhance the expression of pig cluster; the SpnR normally acts as an activator for the expression of the QS-dependent gene. In this research, we attempt to artificially control QS by changing the role of SpnR. The QS-dependent prodigiosin production is expected to inhibit by externally added SpnR in the culture broth of AS-1 strain because the AHL concentration was kept below the threshold by AHL-SpnR complex formation. Maltose-binding protein (MBP)-tagged SpnR (MBP-SpnR) was overexpressed in Escherichia coli and purified using an affinity chromatography equipped with an amylose resin column. The specific interaction between AHL and MBP-SpnR was demonstrated by quartz crystal microbalance (QCM) sensor. AHL with amino end-group was coupled with COOH-terminated self-assembled monolayer prepared on a gold electrode of 27-MHz quartz crystal sensor using water-soluble carbodiimide. After the injection of MBP-SpnR into a cup-type sensor cell filled with the buffer solution, time course of resonant frequency change (ΔFs) was determined. A decrease of ΔFs clearly showed the uptake of MBP-SpnR onto the AHL-immobilized electrode. Furthermore, no binding affinity was observed after the heat-inactivation of MBP-SpnR at 80ºC. These results suggest that MBP-SpnR possesses a specific affinity for AHL. MBP-SpnR was added to the culture medium as an AHL trap to study inhibitory effects on intracellularly accumulated prodigiosin. With approximately 2 µM MBP-SpnR, the amount of prodigiosin induced was half that of the control without any additives. In conclusion, the function of SpnR could be switched by adding it to the cell culture. Exogenously added MBP-SpnR possesses high affinity for AHL derived from cells and acts as an inhibitor of AHL-mediated QS.

Keywords: intracellular signaling, microbial biotechnology, quorum sensing, transcriptional regulator

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Authors: Afsaneh Mohammadnejad, Marianne Nygaard, Jan Baumbach, Shuxia Li, Weilong Li, Jesper Lund, Jacob v. B. Hjelmborg, Lene Christensen, Qihua Tan

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Cognitive impairment in the elderly is a key issue affecting the quality of life. Despite a strong genetic background in cognition, only a limited number of single nucleotide polymorphisms (SNPs) have been found. These explain a small proportion of the genetic component of cognitive function, thus leaving a large proportion unaccounted for. We hypothesize that one reason for this missing heritability is the misspecified modeling in data analysis concerning phenotype distribution as well as the relationship between SNP dosage and the phenotype of interest. In an attempt to overcome these issues, we introduced a model-free method based on the generalized correlation coefficient (GCC) in a genome-wide association study (GWAS) of cognitive function in twin samples and compared its performance with two popular linear regression models. The GCC-based GWAS identified two genome-wide significant (P-value < 5e-8) SNPs; rs2904650 near ZDHHC2 on chromosome 8 and rs111256489 near CD6 on chromosome 11. The kinship model also detected two genome-wide significant SNPs, rs112169253 on chromosome 4 and rs17417920 on chromosome 7, whereas no genome-wide significant SNPs were found by the linear mixed model (LME). Compared to the linear models, more meaningful biological pathways like GABA receptor activation, ion channel transport, neuroactive ligand-receptor interaction, and the renin-angiotensin system were found to be enriched by SNPs from GCC. The GCC model outperformed the linear regression models by identifying more genome-wide significant genetic variants and more meaningful biological pathways related to cognitive function. Moreover, GCC-based GWAS was robust in handling genetically related twin samples, which is an important feature in handling genetic confounding in association studies.

Keywords: cognition, generalized correlation coefficient, GWAS, twins

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Authors: Natalia Moreno-Castellanos, Amaia Rodriguez, Gema Frühbeck

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Introduction: In adipocytes, the cytoskeleton undergoes important expression and distribution in adipocytes rearrangements during adipogenesis and in obesity. Indeed, a role for these proteins in the regulation of adipocyte differentiation and response to insulin has been demonstrated. Recently, septins have been considered as new components of the cytoskeletal network that interact with other cytoskeletal elements (actin and tubulin) profoundly modifying their dynamics. However, these proteins have not been characterized as yet in adipose tissue. In this work, were examined the cellular, molecular and functional features of a member of this family, septin 11 (SEPT11), in adipocytes and evaluated the impact of obesity on the expression of this protein in human adipose tissue. Methods: Adipose gene and protein expression levels of SEPT11 were analysed in human samples. SEPT11 distribution was evaluated by immunocytochemistry, electronic microscopy, and subcellular fractionation techniques. GST-pull down, immunoprecipitation and a Yeast-Two Hybrid (Y2H) screening were used to identify the SEPT11 interactome. Gene silencing was employed to assess the role of SEPT11 in the regulation of insulin signaling and lipid metabolism in adipocytes. Results: SEPT11 is expressed in human adipocytes, and its levels increased in both omental and subcutaneous adipose tissue in obesity, with SEPT11 mRNA content positively correlating with parameters of insulin resistance in subcutaneous fat. In non-stimulated adipocytes, SEPT11 immunoreactivity showed a ring-like distribution at the cell surface and associated to caveolae. Biochemical analyses showed that SEPT11 interacted with the main component of caveolae, caveolin-1 (CAV1) as well as with the fatty acid-binding protein, FABP5. Notably, the three proteins redistributed and co-localized at the surface of lipid droplets upon exposure of adipocytes to oleate. In this line, SEPT11 silencing in 3T3-L1 adipocytes impaired insulin signaling and decreased insulin-induced lipogenesis. Conclusions: Those findings demonstrate that SEPT11 is a novel component of the adipocyte cytoskeleton that plays an important role in the regulation of lipid traffic, metabolism and can thus represent a potential biomarker of insulin resistance in obesity in adipocytes through its interaction with both CAV1 and FABP5.

Keywords: caveolae, lipid metabolism, obesity, septins

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Authors: Chengcao Sun, Cuili Yang, Shujun Li, Ruilin Xue, Liang Wang, Yongyong Xi, Dejia Li

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Backgrounds: Sulforaphane, one of the most important isothiocyanates in the human diet, is known to have chemopreventive and antioxidant activities in different tissues via activation of NF-E2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). However, its effects on muscular dystrophy remain unknown. This work was undertaken to evaluate the effects of Sulforaphane on Duchenne muscular dystrophy (DMD). Methods: 4-week-old mdx mice were treated with SFN by gavage (2 mg/kg body weight per day) for 8 weeks. Blood was collected from eye socket every week, and tibial anterior, extensor digitorum longus, gastrocnemius, soleus, triceps brachii muscles and heart samples were collected after 8-week gavage. Force measurements and mice exercise capacity assays were detected. GSH/GSSG ratio, TBARS, CK and LDH levels were analyzed by spectrophotometric methods. H&E staining was used to analyze histological and morphometric of skeletal muscles of mdx mice, and Evas blue dye staining was made to detect sarcolemmal integrity of mdx mice. Further, the role of Sulforaphane on Nrf2/ARE signaling pathway was analyzed by ELISA, western blot and qRT-PCR. Results: Our results demonstrated that SFN treatment increased the expression and activity of muscle phase II enzymes NQO1 and HO-1 with Nrf2 dependent manner. SFN significantly increased skeletal muscle mass, muscle force (~30%), running distance (~20%) and GSH/GSSG ratio (~3.2 folds) of mdx mice, and decreased the activities of plasma creatine phosphokinase (CK) (~45%) and lactate dehydrogenase (LDH) (~40%), gastrocnemius hypertrophy (~25%), myocardial hypertrophy (~20%) and MDA levels (~60%). Further, SFN treatment also reduced the central nucleation (~40%), fiber size variability, inflammation and improved the sarcolemmal integrity of mdx mice. Conclusions: Collectively, these results show that SFN can improve muscle function, pathology and protect dystrophic muscle from oxidative damage in mdx mice through Nrf2 signaling pathway, which indicate Nrf2 may have clinical implications for the treatment of patients with muscular dystrophy.

Keywords: sulforaphane, duchenne muscular dystrophy, Nrf2, oxidative stress

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Authors: Anupalli Roja Rani, Pavithra Dasari

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Background: Among several, breast cancer has emerged as the most common female cancer in developing countries. It is the most common cause of cancer-related deaths worldwide among women. It is a molecularly and clinically heterogeneous disease. Moreover, it is a hormone–dependent tumor in which estrogens can regulate the growth of breast cells by binding with estrogen receptors (ERs). Moreover, the use of natural products in cancer therapeutics is due to their properties of biocompatibility and less toxicity. Plants are the vast reservoirs for various bioactive compounds. Coleus barbatus (Lamiaceae) contains anticancer properties against several cancer cell lines. Method: In the present study, an attempt is being made to enrich the knowledge of the anticancer activity of pure compounds extracted from Coleus barbatus (Andrew). On human breast cancer cell lines MCF-7. Here in, we are assessing the antiproliferative activity of Coleus barbatus (Andrew) plant extracts against MCF 7 and also evaluating their toxicity in normal human mammary cell lines such as Human Mammary Epithelial Cells (HMEC). The active fraction of plant extract was further purified with the help of Flash chromatography, Medium Pressure Liquid Chromatography (MPLC) and preparative High-Performance Liquid Chromatography (HPLC). The structure of pure compounds will be elucidated by using modern spectroscopic methods like Nuclear magnetic resonance (NMR), Electrospray Ionisation Mass Spectrometry (ESI-MS) methods. Later, the growth inhibition morphological assessment of cancer cells and cell cycle analysis of purified compounds were assessed using FACS. The growth and progression of signaling molecules HER2, GRP78 was studied by secretion assay using ELISA and expression analysis by flow cytometry. Result: Cytotoxic effect against MCF-7 with IC50 values were derived from dose response curves, using six concentrations of twofold serially diluted samples, by SOFTMax Pro software (Molecular device) and respectively Ellipticine and 0.5% DMSO were used as a positive and negative control. Conclusion: The present study shows the significance of various bioactive compounds extracted from Coleus barbatus (Andrew) root material. It acts as an anti-proliferative and shows cytotoxic effects on human breast cancer cell lines MCF7. The plant extracts play an important role pharmacologically. The whole plant has been used in traditional medicine for decades and the studies done have authenticated the practice. Earlier, as described, the plant has been used in the ayurveda and homeopathy medicine. However, more clinical and pathological studies must be conducted to investigate the unexploited potential of the plant. These studies will be very useful for drug designing in the future.

Keywords: coleus barbatus, HPLC, MPLC, NMR, MCF7, flash chromatograph, ESI-MS, FACS, ELISA.

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Authors: Nadia Naseem, Farwa Batool, Nasir Mehmood, AbdulHannan Nagi

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Background: The incidence and mortality of breast cancer vary significantly in geographically distinct populations. In Pakistan, breast cancer has shown an increase in incidence in young females and is characterized by more aggressive behavior. The tumor suppressor TP53 gene is a crucial genetic factor that plays a significant role in breast carcinogenesis. This study investigated the TP53 mutations in molecular subtypes of both nodes negative and positive breast cancer in young Pakistani patients. Material and Methods: p53, Estrogen Receptor (ER), Progesterone Receptor (PR), Her-2 neu and Ki 67 expressions were analyzed immunohistochemically in a series of 75 node negative (A) and 75 node positive (B) young (aged: 19-40 years) breast cancer patients diagnosed between 2014 to 2017 at two leading hospitals of Punjab, Pakistan. Tumor tissue specimens and peripheral blood samples were examined for TP53 mutations by direct sequencing of the gene (exons 4-9). The relation of TP53 mutations to these markers and clinicopathological data was investigated. Results: Mean age of the patients was 32.4 + 9.1 SD. Invasive breast carcinoma was the most frequent histological variant (A=92%, B=94.6%). Grade 3 carcinoma was the commonest grade (A=72%, B=81.3%). Triple negative cases (ER-, PR-, Her-2) formed most of the molecular subtypes (A=44%, B=50.6%). A total of 17.2% (A: 6.6%, B: 10.6%) patients showed TP53 mutations. Mutations were significantly more frequent in triple negative cases (A: 74.8%, B: 62.2%) compared to HER2-positive patients (P < 0.0001). In the multivariate analysis of the whole patient group, the independent prognosticator were triple negative cases (P=0.021), TP53 overexpression by IHC (P=0.001) and advanced-stage disease (P=0.007). No statistically significant correlation between TP53 mutations and clinicopathological parameters was found (P < 0.05). Conclusions: It is concluded that TP53 mutations are infrequently present in breast carcinoma of young Pakistani population and there was no significant correlation between p53 mutation and early onset disease. Immunohistochemically detected TP53 expression in our resource-constrained to set up can be beneficial in predicting mutations at the younger age in our population.

Keywords: immunohistochemistry (IHC), invasive breast carcinoma (IBC), Pakistan, TP53

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Authors: Frida Carrillo Morales, Maria Maricela Carrasco Yépez, Saúl Rojas Hernández

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Naegleria fowleri is the causative agent of primary amebic meningoencephalitis, this disease is acute and fulminant that affects humans. It has been reported that despite the existence of therapeutic options against this disease, its mortality rate is 97%. Therefore, the need arises to have vaccines that confer protection against this disease and, in addition to developing adjuvants to enhance the immune response. In this regard, in our work group, we obtained a peptide designed from the membrane protein MP2CL5 of Naegleria fowleri called Smp145 that was shown to be immunogenic; however, it would be of great importance to enhance its immunological response, being able to co-administer it with a non-toxic adjuvant. Therefore, the objective of this work was to carry out the bioinformatic design of a peptide of the Naegleria fowleri membrane protein MP2CL5 conjugated with a non-toxic modified adjuvant from the native A1 structure of Cholera Toxin. For which different bioinformatics tools were used to obtain a model with a modification in amino acid 61 of the A1 subunit of the CT (CTA1), to which the Smp145 peptide was added and both molecules were joined with a 13-glycine linker. As for the results obtained, the modification in CTA1 bound to the peptide produces a reduction in the toxicity of the molecule in in silico experiments, likewise, the prediction in the binding of Smp145 to the receptor of B cells suggests that the molecule is directed in specifically to the BCR receptor, decreasing its native enzymatic activity. The stereochemical evaluation showed that the generated model has a high number of adequately predicted residues. In the ERRAT test, the confidence with which it is possible to reject regions that exceed the error values was evaluated, in the generated model, a high score was obtained, which determines that the model has a good structural resolution. Therefore, the design of the conjugated peptide in this work will allow us to proceed with its chemical synthesis and subsequently be able to use it in the mouse meningitis protection model caused by N. fowleri.

Keywords: immunology, vaccines, pathogens, infectious disease

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Authors: Bruna Lima Gomes, Vanessa Caroline De Barros Bonato, Luciano Freschi, Eduardo Purgatto

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Plant hormones are long known to be tightly associated with fruit development and are involved in controlling various aspects of fruit ripening. For fleshy fruits, ripening is characterized for changes in texture, color, aroma and other parameters that markedly contribute to its quality. Ethylene is one of the major players regulating the ripening-related processes, but emerging evidences suggest that auxin is also part of this dynamic control. Thus, the aim of this study was providing new insights into the auxin role during ripening and the hormonal interplay between auxin and ethylene. For that, tomato fruits (Micro-Tom) were collected at mature green stage and separated in four groups: one for indole-3-acetic acid (IAA) treatment, one for ethylene, one for a combination of IAA and ethylene, and one for control. Hormone solution was injected through the stylar apex, while mock samples were injected with buffer only. For ethylene treatments, fruits were exposed to gaseous hormone. Then, fruits were left to ripen under standard conditions and to assess ripening development, hue angle was reported as color indicator and ethylene production was measured by gas chromatography. The transcript levels of three ripening-related ethylene receptors (LeETR3, LeETR4 and LeETR6) were evaluated by RT-qPCR. Results showed that ethylene treatment induced ripening, stimulated ethylene production, accelerated color changes and induced receptor expression, as expected. Nonetheless, auxin treatment showed the opposite effect once fruits remained green for longer time than control group and ethylene perception has changed, taking account the reduced levels of receptor transcripts. Further, treatment with both hormones revealed that auxin effect in delaying ripening was predominant, even with higher levels of ethylene. Altogether, the data suggest that auxin modulates several aspects of the tomato fruit ripening modifying the ethylene perception. The knowledge about hormonal control of fruit development will help design new strategies for effective manipulation of ripening regarding fruit quality and brings a new level of complexity on fruit ripening regulation.

Keywords: ethylene, auxin, fruit ripening, hormonal crosstalk

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Authors: Chengcao Sun, Shujun Li, Dejia Li

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Sulforaphane (SFN) possesses powerful chemo-preventive effects and plays a crucial role on oxidative stress and inflammatory. In our recent study, SFN treatment could relieve muscular dystrophy in mdx mice by activating Nrf2 (NF-E2 related factor 2). Moreover, our findings indicated that SFN-activated Nrf2 alleviated muscle inflammation in dystrophin-deficient mdx mice through suppressing NF-κB signaling pathway. Collectively, SFN-induced Nrf2 molecular pathway might be a promising approach for treatment of the patients with Duchenne muscular dystrophy.

Keywords: sulforaphane, Duchenne muscular dystrophy, Nrf2, inflammation, fibrosis, oxidative stress

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Authors: Ginger Egberts, Fred Vermolen, Paul van Zuijlen

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One of the common complications in post-burn scars is contractions. Depending on the extent of contraction and the wound dimensions, the contracture can cause a limited range-of-motion of joints. A one-dimensional morphoelastic continuum hypothesis-based model describing post-burn scar contractions is considered. The beauty of the one-dimensional model is the speed; hence it quickly yields new results and, therefore, insight. This model describes the movement of the skin and the development of the strain present. Besides these mechanical components, the model also contains chemical components that play a major role in the wound healing process. These components are fibroblasts, myofibroblasts, the so-called signaling molecules, and collagen. The dermal layer is modeled as an isotropic morphoelastic solid, and pulling forces are generated by myofibroblasts. The solution to the model equations is approximated by the finite-element method using linear basis functions. One of the major challenges in biomechanical modeling is the estimation of parameter values. Therefore, this study provides a comprehensive description of skin mechanical parameter values and a sensitivity analysis. Further, since skin mechanical properties change with aging, it is important that the model is feasible for predicting the development of contraction in burn patients of different ages, and hence this study provides a feasibility study. The variability in the solutions is caused by varying the values for some parameters simultaneously over the domain of computation, for which the results of the sensitivity analysis are used. The sensitivity analysis shows that the most sensitive parameters are the equilibrium concentration of collagen, the apoptosis rate of fibroblasts and myofibroblasts, and the secretion rate of signaling molecules. This suggests that most of the variability in the evolution of contraction in burns in patients of different ages might be caused mostly by the decreasing equilibrium of collagen concentration. As expected, the feasibility study shows this model can be used to show distinct extents of contractions in burns in patients of different ages. Nevertheless, contraction formation in children differs from contraction formation in adults because of the growth. This factor has not been incorporated in the model yet, and therefore the feasibility results for children differ from what is seen in the clinic.

Keywords: biomechanics, burns, feasibility, fibroblasts, morphoelasticity, sensitivity analysis, skin mechanics, wound contraction

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