Search results for: ctDNA methylation

Authors: Neda Gegar Goshe, M. Moradi, Hossein Rassi

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The antibacterial properties of macrolide antibiotics (such as erythromycin and tylosin) depend ultimately on the glycosylation of otherwise inactive polyketide lactones. Among the sugars commonly found in such macrolides are various 6-deoxyhexoses including the 3-dimethylamino sugars mycaminose and desosamine (4-deoxymycaminose). Some macrolides (such as tylosin) possess multiple sugar moieties, whereas others (such as erythromycin) have two sugar substituents. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors-malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA-used by modular PKS, and is a host that is amenable to genetic manipulation. As patterns of glycosylation markedly influence a macrolide's drug activity, there is considerable interest in the possibility of using combinatorial biosynthesis to generate new pairings of polyketide lactones with sugars, especially 6-deoxyhexoses. Here, we report a successful attempt to alter the aminodeoxyhexose-biosynthetic capacity of Streptomyces fradiae (a producer of tylosin) by importing genes from the erythromycin producer Saccharopolyspora erythraea. The bio transformation of erythromycin-D into the desired major component erythromycin-A involves two final enzymatic reactions, EryK-catalyzed hydroxylation at the C-12 position of the aglycone and EryG-catalyzed O methylation at the C-3 position of macrose. This engineered S. fradiae produced substantial amounts of two potentially useful macrolides that had not previously been obtained by fermentation.

Keywords: tylosin, eryK and eryG genes, streptomyces fradiae

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Authors: Aniket Kumar, Jacob Peedicayil

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Major depressive disorder (MDD) is a common and important psychiatric disorder. Several clinical features of MDD suggest an epigenetic basis for its pathogenesis. Since epigenetics (heritable changes in gene expression not involving changes in DNA sequence) may underlie the pathogenesis of MDD, epigenetic drugs such as DNA methyltransferase inhibitors (DNMTi) and histone deactylase inhibitors (HDACi) may be useful for treating MDD. The available literature indexed in Pubmed on preclinical drug trials of epigenetic drugs for the treatment of MDD was investigated. The search terms we used were ‘depression’ or ‘depressive’ and ‘HDACi’ or ‘DNMTi’. Among epigenetic drugs, it was found that there were 3 preclinical trials using HDACi and 3 using DNMTi for the treatment of MDD. All the trials were conducted on rodents (mice or rats). The animal models of depression that were used were: learned helplessness-induced animal model, forced swim test, open field test, and the tail suspension test. One study used a genetic rat model of depression (the Flinders Sensitive Line). The HDACi that were tested were: sodium butyrate, compound 60 (Cpd-60), and valproic acid. The DNMTi that were tested were: 5-azacytidine and decitabine. Among the three preclinical trials using HDACi, all showed an antidepressant effect in animal models of depression. Among the 3 preclinical trials using DNMTi also, all showed an antidepressant effect in animal models of depression. Thus, epigenetic drugs, namely, HDACi and DNMTi, may prove to be useful in the treatment of MDD and merit further investigation for the treatment of this disorder.

Keywords: DNA methylation, drug discovery, epigenetics, major depressive disorder

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Authors: Anjani Kumar Tiwari, Neelam Kumari, Anil Mishra

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The family of phosphodiesterases (PDEs) plays a critical role in control of the level, localization, and duration of intracellular 3’-5’-cyclic adenosine monophosphate (cAMP) and 3’-5’-cyclic guanosine monophosphate (cGMP) signals by specifically hydrolyzing these cyclic nucleotides. As the involvement of cyclic nucleotide second messengers in cell signaling and homeostasis is established, the regulation of these pathways in the brain by various PDE isoforms is an area of considerable interest, as they are involved in nearly all brain functions and in the etiology of neuropsychiatric diseases. The PDE10A isoform, isolated from different species and characterized regarding structure and function, has received much attention in recent years, particularly in the context of schizophrenia and Huntington’s disease, which are both related to a role of PDE10A in the regulation of striatal dopaminergic neurotransmission. Quinazoline analogue 1-(4-methoxyphenyl)-6,7-dimethoxyquinazoline, was evaluated as specific PET marker for phosphodiesterase (PDE) 10A. Here, we report the radiosynthesis of [11C]2 and the in vitro and in vivo evaluation of [11C]2 as a potential positron emission tomography (PET) radiotracer for imaging PDE10A in the central nervous system (CNS). The radiosynthesis of [11C]2 was achieved by O-methylation of the corresponding des-methyl precursor with [11C]methyl iodide. [11C]2 was obtained with ∼50% radiochemical yield. PET imaging studies in rat brain displayed initial specific uptake with very rapid clearance of [11C]2 from brain. Though [11C]2 is not an ideal radioligand for clinical imaging of PDE10A in the CNS. Modified analogue of quinazoline having a higher potency for inhibiting PDE10A and improved pharmacokinetic properties will be necessary for imaging this enzyme with PET.

Keywords: PDE10A, PET, radiotracer, quinazoline

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Authors: Nathalie Baeza-Kallee, Raphaël Bergès, Victoria Hein, Stéphanie Cabaret, Jeremy Garcia, Abigaëlle Gros, Emeline Tabouret, Aurélie Tchoghandjian, Carole Colin, Dominique Figarella-Branger

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Glioblastoma (GBM) contains cancer stem cells that are resistant to treatment. GBM cancer stem cell expresses glycolipids recognized by the A2B5 antibody. A2B5, induced by the enzyme ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyl transferase 3 (ST8Sia3), plays a crucial role in the proliferation, migration, clonogenicity, and tumorigenesis of GBM cancer stem cells. Our aim was to characterize the resulting effects of neuraminidase that remove A2B5 in order to target GBM cancer stem cells. To this end, we set up a GBM organotypic slice model; quantified A2B5 expression by flow cytometry in U87-MG, U87-ST8Sia3, and GBM cancer stem cell lines, treated or not by neuraminidase; performed RNAseq and DNA methylation profiling; and analyzed the ganglioside expression by liquid chromatography-mass spectrometry in these cell lines, treated or not with neuraminidase. Results demonstrated that neuraminidase decreased A2B5 expression, tumor size, and regrowth after surgical removal in the organotypic slice model but did not induce a distinct transcriptomic or epigenetic signature in GBM CSC lines. RNAseq analysis revealed that OLIG2, CHI3L1, TIMP3, TNFAIP2, and TNFAIP6 transcripts were significantly overexpressed in U87-ST8Sia3 compared to U87-MG. RT-qPCR confirmed these results and demonstrated that neuraminidase decreased gene expression in GBM cancer stem cell lines. Moreover, neuraminidase drastically reduced ganglioside expression in GBM cancer stem cell lines. Neuraminidase, by its pleiotropic action, is an attractive local treatment against GBM.

Keywords: cancer stem cell, ganglioside, glioblastoma, targeted treatment

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Authors: Parvin Goli, Amirhosein Kefayat, Rezvan Goli

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Background: It is well established that parents can influence their offspring's neurodevelopment. It is shown that paternal environment and lifestyle is beneficial for the progeny's fitness and might affect their metabolic mechanisms; however, the effects of paternal exercise on the brain in the offspring have not been explored in detail. Objective: This study aims to review the impact of paternal physical exercise on memory and learning, neuroplasticity, as well as DNA methylation levels in the off-spring's hippocampus. Study design: In this systematic review and meta-analysis, an electronic literature search was conducted in databases including PubMed, Scopus, and Web of Science. Eligible studies were those with an experimental design, including an exercise intervention arm, with the assessment of any type of memory function, learning ability, or any type of brain plasticity as the outcome measures. Standardized mean difference (SMD) and 95% confidence intervals (CI) were computed as effect size. Results: The systematic review revealed the important role of environmental enrichment in the behavioral development of the next generation. Also, offspring of exercised fathers displayed higher levels of memory ability and lower level of brain-derived neurotrophic factor. A significant effect of paternal exercise on the hippocampal volume was also reported in the few available studies. Conclusion: These results suggest an intergenerational effect of paternal physical activity on cognitive benefit, which may be associated with hippocampal epigenetic programming in offspring. However, the biological mechanisms of this modulation remain to be determined.

Keywords: hippocampal plasticity, learning ability, memory, parental exercise

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Authors: Abbas Pourreza

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MicroRNAs (miRNAs) are small single-stranded non-coding RNAs. They are almost 18-25 nucleotides long and very conservative through evolution. They are involved in adjusting the expression of numerous genes due to the existence of a complementary region, generally in the 3' untranslated regions (UTR) of target genes, against particular mRNAs in the cell. Also, miRNAs have been proven to be involved in cell development, differentiation, proliferation, and apoptosis. More than 2000 miRNAs have been recognized in human cells, and these miRNAs adjust approximately one-third of all genes in human cells. Dysregulation of miRNA originated from abnormal DNA methylation patterns of the locus, cause to down-regulated or overexpression of miRNAs, and it may affect tumor formation or development of it. Breast cancer (BC) is the most commonly identified cancer, the most prevalent cancer (23%), and the second-leading (14%) mortality in all types of cancer in females. BC can be classified based on the status (+/−) of the hormone receptors, including estrogen receptor (ER), progesterone receptor (PR), and the Receptor tyrosine-protein kinase erbB-2 (ERBB2 or HER2). Currently, there are four main molecular subtypes of BC: luminal A, approximately 50–60 % of BCs; luminal B, 10–20 %; HER2 positive, 15–20 %, and 10–20 % considered Basal (triple-negative breast cancer (TNBC)) subtype. Aberrant expression of miR-145, miR-21, miR-10b, miR-125a, and miR-206 was detected by Stem-loop real-time RT-PCR in BC cases. Breast tumor formation and development may result from down-regulation of a tumor suppressor miRNA such as miR-145, miR-125a, and miR-206 and/or overexpression of an oncogenic miRNA such as miR-21 and miR-10b. MiR-125a, miR-206, miR-145, miR-21, and miR-10b are hugely predicted to be new tumor markers for the diagnosis and prognosis of BC. MiR-21 and miR-125a could play a part in the treatment of HER-2-positive breast cancer cells, while miR-145 and miR-206 could speed up the evolution of cure techniques for TNBC. To conclude, miRNAs will be presented as hopeful molecules to be used in the primary diagnosis, prognosis, and treatment of BC and battle as opposed to its developed drug resistance.

Keywords: breast cancer, HER2 positive, miRNA, TNBC

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Authors: Atena Sadat Hosseini, Mohammadhossein Habibi

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Acute myeloid leukemia (AML) is the most typically diagnosed leukemia. In older adults, AML imposes a dismal outcome. AML originates with a dominant mutation, then adds collaborative, transformative mutations leading to myeloid transformation and clinical/biological heterogeneity. Several chemotherapeutic drugs are used for this cancer. These drugs are naturally associated with several side effects, and finding a more accurate molecular mechanism of these drugs can have a significant impact on the selection and better candidate of drugs for treatment. In this study, we evaluated bortezomibin myeloma cells using bioinformatics analysis and evaluation of RNA-Seq data. Then investigated the molecular pathways proteins- proteins interactions associated with this chemotherapy drug. A total of 658upregulated genes and 548 downregulated genes were sorted.AUF1 (hnRNP D0) binds and destabilizes mRNA, degradation of GLI2 by the proteasome, the role of GTSE1 in G2/M progression after G2 checkpoint, TCF dependent signaling in response to WNT demonstrated in upregulated genes. Besides insulin resistance, AKT phosphorylates targets in the nucleus, cytosine methylation, Longevity regulating pathway, and Signal Transduction of S1P Receptor were related to low expression genes. With respect to this results, HIST2H2AA3, RP11-96O20.4, ZED9, PRDX1, and DOK2, according to node degrees and betweenness elements candidates from upregulated genes. in the opposite side, PYURF, NRSN1, FGF23, UPK3BL, and STAG3 were a prominent role in downregulated genes. Sum up, Using in silico analysis in the present study, we conducted a precise study ofbortezomib molecular mechanisms in myeloma cells. so that we could take further evaluation to discovermolecular cancer therapy. Naturally, more additional experimental and clinical procedures are needed in this survey.

Keywords: myeloma cells, acute myeloid leukemia, bioinformatics analysis, bortezomib

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Authors: Ramin Ghasemi Shayan, Morteza Janebifam

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Tumors are regularly inhomogeneous. Provincial varieties in death, metabolic action, multiplication and body part are watched. There’s expanding proof that strong tumors may contain subpopulations of cells with various genotypes and phenotypes. These unmistakable populaces of malignancy cells can connect during a serious way and may contrast in affectability to medications. Most tumors show organic heterogeneity1–3 remembering heterogeneity for genomic subtypes, varieties inside the statement of development variables and genius, and hostile to angiogenic factors4–9 and varieties inside the tumoural microenvironment. These can present as contrasts between tumors in a few people. for instance, O6-methylguanine-DNA methyltransferase, a DNA fix compound, is hushed by methylation of the quality advertiser in half of glioblastoma (GBM), adding to chemosensitivity, and improved endurance. From the outset, there includes been specific enthusiasm inside the usage of dissemination weighted imaging (DWI) and dynamic complexity upgraded MRI (DCE-MRI). DWI sharpens MRI to water dispersion inside the extravascular extracellular space (EES) and is wiped out with the size and setup of the cell populace. Additionally, DCE-MRI utilizes dynamic obtaining of pictures during and after the infusion of intravenous complexity operator. Signal changes are additionally changed to outright grouping of differentiation permitting examination utilizing pharmacokinetic models. PET scan modality gives one of a kind natural particularity, permitting dynamic or static imaging of organic atoms marked with positron emanating isotopes (for example, 15O, 18F, 11C). The strategy is explained to a colossal radiation portion, which points of confinement rehashed estimations, particularly when utilized together with PC tomography (CT). At long last, it's of incredible enthusiasm to quantify territorial hemoglobin state, which could be joined with DCE-CT vascular physiology estimation to create significant experiences for understanding tumor hypoxia.

Keywords: heterogeneity, computerized tomography scan, magnetic resonance imaging, PET

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Authors: Fatemeh Zeinali Sehrig

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Background: miRNAs play an important role in the post-transcriptional regulation of genes, including genes involved in DNA methylation (DNMTs), and are also important regulators of oncogenic pathways. The study of microRNAs and DNMTs in breast cancer allows the development of targeted treatments and early detection of this cancer. Methods and Materials: Clinical Patients and Samples: Institutional guidelines, including ethical approval and informed consent, were followed by the Ethics Committee (Ethics code: IR.IAU.TABRIZ.REC.1401.063) of Tabriz Azad University, Tabriz, Iran. In this study, tissues of 100 patients with breast cancer and tissues of 100 healthy women were collected from Noor Nejat Hospital in Tabriz. The basic characteristics of the patients with breast cancer included: 1)Tumor grade(Grade 3 = 5%, Grade 2 = 87.5%, Grade 1 = 7.5%), 2)Lymph node(Yes = 87.5%, No = 12.5%), 3)Family cancer history(Yes = 47.5%, No = 41.3%, Unknown = 11.2%), 4) Abortion history(Yes = 36.2%).In silico methods (data gathering, process, and build networks): Gene Expression Omnibus (GEO), a high-throughput genomic database, was queried for miRNAs expression profiles in breast cancer. For Experimental protocol Tissue Processing, Total RNA isolation, complementary DNA(cDNA) synthesis, and quantitative real time PCR (QRT-PCR) analysis were performed. Results: In the present study, we found significant (p.value<0.05) changes in the expression level of miRNAs and DNMTs in patients with breast cancer. In bioinformatics studies, the GEO microarray data set, similar to qPCR results, showed a decreased expression of miRNAs and increased expression of DNMTs in breast cancer. Conclusion: According to the results of the present study, which showed a decrease in the expression of miRNAs and DNMTs in breast cancer, it can be said that these genes can be used as important diagnostic and therapeutic biomarkers in breast cancer.

Keywords: gene expression omnibus, microarray dataset, breast cancer, miRNA, DNMT (DNA methyltransferases)

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Authors: Luciana P. Mazur, Tatiana A. Pozdniakova, Rui A. R. Boaventura, Vitor J. P. Vilar

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The principal mechanism of metal ions sequestration by brown algae involves the formation of complexes between the metal ion and functional groups present on the cell wall of the biological material. To understand the role of functional groups on copper(II) uptake by Ascophyllum nodosum, some functional groups were chemically modified. The esterification of carboxylic groups was carried out by suspending the biomass in a methanol/HCl solution under stirring for 48 h and the blocking of the sulfonic groups was performed by repeating the same procedure for 4 cycles of 48 h. The methylation of amines was conducted by suspending the biomass in a formaldehyde/formic acid solution under shaking for 6 h and the chemical modification of sulfhydryl groups on the biomass surface was achieved using dithiodipyridine for 1 h. Equilibrium sorption studies for Cu2+ using the raw and esterified algae were performed at pH 2.0 and 4.0. The experiments were performed using an initial copper concentration of 300 mg/L and algae dose of 1.0 g/L. After reaching the equilibrium, the metal in solution was quantified by atomic absorption spectrometry. The biological material was analyzed by Fourier Transform Infrared Spectroscopy and Potentiometric Titration techniques for functional groups identification and quantification, respectively. The results using unmodified algae showed that the maximum copper uptake capacity at pH 4.0 and 2.0 was 1.17 and 0.52 mmol/g, respectively. At acidic pH values most carboxyl groups are protonated and copper sorption suffered a significant reduction of 56%. Blocking the carboxylic, sulfonic, amines and sulfhydryl functional groups, copper uptake decreased by 24/26%, 69/81%, 1/23% and 40/27% at pH 2.0/4.0, respectively, when compared to the unmodified biomass. It was possible to conclude that the carboxylic and sulfonic groups are the main functional groups responsible for copper binding (>80%). This result is supported by the fact that the adsorption capacity is directly related to the presence of carboxylic groups of the alginate polymer, and the second most abundant acidic functional group in brown algae is the sulfonic acid of fucoidan that contributes, to a lower extent, to heavy metal binding, particularly at low pH.

Keywords: biosorption, brown marine macroalgae, copper, ion-exchange

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Authors: Jeena Gupta

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In the today’s world of pharmaceutical products, one should not forget the healing properties of inexpensive food materials especially spices. They are known to possess hidden pharmaceutical ingredients, imparting them the qualities of being anti-microbial, anti-oxidant, anti-inflammatory and anti-carcinogenic. Further aberrant epigenetic regulatory mechanisms like DNA methylation, histone modifications or altered microRNA expression patterns, which regulates gene expression without changing DNA sequence, contribute significantly in the development of various diseases. Changing lifestyles and diets exert their effect by influencing these epigenetic mechanisms which are thus the target of dietary phytochemicals. Bioactive components of plants have been in use since ages but their potential to reverse epigenetic alterations and prevention against diseases is yet to be explored. Spices being rich repositories of many bioactive constituents are responsible for providing them unique aroma and taste. Some spices like curcuma and garlic have been well evaluated for their epigenetic regulatory potential, but for others, it is largely unknown. We have evaluated the biological activity of phyto-active components of Fennel, Cardamom and Fenugreek by in silico molecular modeling, in vitro and in vivo studies. Ligand-based similarity studies were conducted to identify structurally similar compounds to understand their biological phenomenon. The database searching has been done by using Fenchone from fennel, Sabinene from cardamom and protodioscin from fenugreek as a query molecule in the different small molecule databases. Moreover, the results of the database searching exhibited that these compounds are having potential binding with the different targets found in the Protein Data Bank. Further in addition to being epigenetic modifiers, in vitro study had demonstrated the antimicrobial, antifungal, antioxidant and cytotoxicity protective effects of Fenchone, Sabinene and Protodioscin. To best of our knowledge, such type of studies facilitate the target fishing as well as making the roadmap in drug design and discovery process for identification of novel therapeutics.

Keywords: epigenetics, spices, phytochemicals, fenchone

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Authors: Keethadath Arshad, Kappalli Sudha

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Mud crab, Scylla Serrata, a commercially important shellfish with high global demand appears to be the rich source of dietary fatty acids. Its increased production through aquaculture and highly perishable nature would necessitate improved techniques for their proper preservation. Optimized irradiation has been identified as an effective method to facilitate safety and extended shelf life for a broad range of the perishable food items including finfishes and shellfishes. The present study analyzed the effects of dose-dependent gamma irradiation on the fatty acid profile of the muscle derived from the candidate species (S. serrata) at both qualitative and quantitative levels. Wild grown, average sized, intermolt male S. Serrata were gamma irradiated (^60C, 3.8kGy/ hour) at the dosage of 0.5kGy, 1.0kGy and 2.0kGy using gamma chamber. Total lipid extracted by Folch method, after methylation, were analyzed for the presence fatty acids adopting Gas Chromatograph equipped with flame ionization detector by comparing with the authentic FAME reference standards. The tissue from non-irradiated S. serrata showed the presence of 12 SFA, 6 MUFA, 8PUFA and 2 TF; PUFA includes medicinally important ω-3 FA such as C18:3, C20:5 and C22:6 and ω-6 FA such as γ- C18:3 and C20:2. Dose-dependent gamma irradiation reduced the number of detectable fatty acids (10, 8 and 8 SFA, 6, 6 and 5MUFA, 7, 7, and 6 PUFA and 1, 1, and 0 TF in 0.5kGy, 1.0kGy and 2kGy irradiated samples respectively). Major fatty acids detected in both irradiated and non-irradiated samples were as follows: SFA- C16:0, C18:0, C22:0 and C14:0; MUFA - C18:1 and C16:1and PUFA- C18:2, C20:5, C20:2 and C22:6. Irradiation doses ranging from 1-2kGy substantially reduced the ω-6 C18:3 and ω-3 C18:3. However, the omega fatty acids such as C20:5, C22:6 and C20:2 could survive even after 2kGy irradiation. Significantly, trans fat like C18:2T and C18:1T were completely disappeared upon 2kGy irradiation. From the overall observations made from the present study, it is suggested that irradiation dose up to 1kGy is optimum to maintain the fatty acid profile and eradicate the trans fat of the muscle derived from S. serrata.

Keywords: fatty acid profile, food preservation, gamma irradiation, scylla serrata

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Authors: Menna Ewida, Dalal Abou El-Ella, Dina Lasheen, Huessin El-Subbagh

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Introduction: Vascular Endothelial Growth Factor receptor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis to create new blood vessels. VEGF family binds to three trans-membrane tyrosine kinase receptors,Dihydrofolate reductase (DHFR) is an enzyme of crucial importance in medicinal chemistry. DHFR catalyzes the reduction 7,8 dihydro-folate to tetrahydrofolate and intimately couples with thymidylate synthase which is a pivotal enzyme that catalysis the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) utilizing N5,N10-methylene tetrahydrofolate as a cofactor which functions as the source of the methyl group. Purpose: Novel substituted Thiazole agents were designed as DHFR and VEGF-TK inhibitors with increased synergistic activity and decreased side effects. Methods: Five series of compounds were designed with a rational that mimic the pharmacophoric features present in the reported active compounds that target DHFR & VEGFR. These molecules were docked against Methotrexate & Sorafenib as controls. An in silico ADMET study was also performed to validate the bioavailability of the newly designed compounds. The in silico molecular docking & ADMET study were also applied to the non-classical antifolates for comparison. The interaction energy comparable to that of MTX for DHFRI and Sorafenib for VEGF-TKI activity were recorded. Results: Compound 5 exhibited the highest interaction energy when docked against Sorafenib, While Compound 9 showed the highest interaction energy when docked against MTX with the perfect binding mode. Comparable results were also obtained for the ADMET study. Most of the compounds showed absorption within (95-99) zone which varies according to the type of substituents. Conclusions: The Substituted Thiazole Analogues could be a suitable template for antitumor drugs that possess enhanced bioavailability and act as DHFR and VEGF-TK inhibitors.

Keywords: anti-tumor agents, DHFR, drug design, molecular modeling, VEGFR-TKIs

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Authors: Y. Landkocz, C. Lepers, P.J. Martin, B. Fougère, F. Roy Saint-Georges. A. Verdin, F. Cazier, F. Ledoux, D. Courcot, F. Sichel, P. Gosset, P. Shirali, S. Billet

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In 2013, the International Agency for Research on Cancer (IARC) classified air pollution and fine particles as carcinogenic to humans. Causal relationships exist between elevated ambient levels of airborne particles and increase of mortality and morbidity including pulmonary diseases, like lung cancer. However, due to a double complexity of both physicochemical Particulate Matter (PM) properties and tumor mechanistic processes, mechanisms of action remain not fully elucidated. Furthermore, because of several common properties between air pollution PM and tobacco smoke, like the same route of exposure and chemical composition, potential mechanisms of synergy could exist. Therefore, smoking could be an aggravating factor of the particles toxicity. In order to identify some mechanisms of action of particles according to their size, two samples of PM were collected: PM0.03 2.5 and PM0.33 2.5 in the urban-industrial area of Dunkerque. The overall cytotoxicity of the fine particles was determined on human bronchial cells (BEAS-2B). Toxicological study focused then on the metabolic activation of the organic compounds coated onto PM and some genetic and epigenetic changes induced on a co-culture model of BEAS-2B and alveolar macrophages isolated from bronchoalveolar lavages performed in smokers and non-smokers. The results showed (i) the contribution of the ultrafine fraction of atmospheric particles to genotoxic (eg. DNA double-strand breaks) and epigenetic mechanisms (eg. promoter methylation) involved in tumor processes, and (ii) the influence of smoking on the cellular response. Three main conclusions can be discussed. First, our results showed the ability of the particles to induce deleterious effects potentially involved in the stages of initiation and promotion of carcinogenesis. The second conclusion is that smoking affects the nature of the induced genotoxic effects. Finally, the in vitro developed cell model, using bronchial epithelial cells and alveolar macrophages can take into account quite realistically, some of the existing cell interactions existing in the lung.

Keywords: air pollution, fine and ultrafine particles, genotoxic and epigenetic alterations, smoking

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Authors: Milda Nainyte, Viktoras Masevicius

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Biological methylation is a methyl group transfer from S-adenosyl-L-methionine (AdoMet) onto N-, C-, O- or S-nucleophiles in DNA, RNA, proteins or small biomolecules. The reaction is catalyzed by enzymes called AdoMet-dependent methyltransferases (MTases), which represent more than 3 % of the proteins in the cell. As a general mechanism, the methyl group from AdoMet replaces a hydrogen atom of nucleophilic center producing methylated DNA and S-adenosyl-L-homocysteine (AdoHcy). Recently, DNA methyltransferases have been used for the sequence-specific, covalent labeling of biopolymers. Two types of MTase catalyzed labeling of biopolymers are known, referred as two-step and one-step. During two-step labeling, an alkylating fragment is transferred onto DNA in a sequence-specific manner and then the reporter group, such as biotin, is attached for selective visualization using suitable chemistries of coupling. This approach of labeling is quite difficult and the chemical hitching does not always proceed at 100 %, but in the second step the variety of reporter groups can be selected and that gives the flexibility for this labeling method. In the one-step labeling, AdoMet analog is designed with the reporter group already attached to the functional group. Thus, the one-step labeling method would be more comfortable tool for labeling of biopolymers in order to prevent additional chemical reactions and selection of reaction conditions. Also, time costs would be reduced. However, effective AdoMet analog appropriate for one-step labeling of biopolymers and containing cleavable bond, required for reduction of PCR interferation, is still not known. To expand the practical utility of this important enzymatic reaction, cofactors with activated sulfonium-bound side-chains have been produced and can serve as surrogate cofactors for a variety of wild-type and mutant DNA and RNA MTases enabling covalent attachment of these chains to their target sites in DNA, RNA or proteins (the approach named methyltransferase-directed Transfer of Activated Groups, mTAG). Compounds containing hex-2-yn-1-yl moiety has proved to be efficient alkylating agents for labeling of DNA. Herein we describe synthetic procedures for the preparation of N-biotinoyl-N’-(pent-4-ynoyl)cystamine starting from the coupling of cystamine with pentynoic acid and finally attaching the biotin as a reporter group. The synthesis of the first AdoMet based cofactor containing a cleavable reporter group and appropriate for one-step labeling was developed.

Keywords: adoMet analogs, DNA alkylation, cofactor, methyltransferases

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Authors: Sridhar A. Malkaram, Tamer E. Fandy

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Purpose: 5-Azacytidine (5AC) and decitabine (DC) are DNA hypomethylating agents. We recently demonstrated that both drugs increase the enzymatic activity of the histone deacetylase enzyme SIRT6. Accordingly, we are comparing the changes H3K9 acetylation changes in the whole genome induced by both drugs using leukemia cells. Description of Methods & Materials: Mononuclear cells from the bone marrow of six de-identified naive acute myeloid leukemia (AML) patients were cultured with either 500 nM of DC or 5AC for 72 h followed by ChIP-Seq analysis using a ChIP-validated acetylated-H3K9 (H3K9ac) antibody. Chip-Seq libraries were prepared from treated and untreated cells using SMARTer ThruPLEX DNA- seq kit (Takara Bio, USA) according to the manufacturer’s instructions. Libraries were purified and size-selected with AMPure XP beads at 1:1 (v/v) ratio. All libraries were pooled prior to sequencing on an Illumina HiSeq 1500. The dual-indexed single-read Rapid Run was performed with 1x120 cycles at 5 pM final concentration of the library pool. Sequence reads with average Phred quality < 20, with length < 35bp, PCR duplicates, and those aligning to blacklisted regions of the genome were filtered out using Trim Galore v0.4.4 and cutadapt v1.18. Reads were aligned to the reference human genome (hg38) using Bowtie v2.3.4.1 in end-to-end alignment mode. H3K9ac enriched (peak) regions were identified using diffReps v1.55.4 software using input samples for background correction. The statistical significance of differential peak counts was assessed using a negative binomial test using all individuals as replicates. Data & Results: The data from the six patients showed significant (Padj<0.05) acetylation changes at 925 loci after 5AC treatment versus 182 loci after DC treatment. Both drugs induced H3K9 acetylation changes at different chromosomal regions, including promoters, coding exons, introns, and distal intergenic regions. Ten common genes showed H3K9 acetylation changes by both drugs. Approximately 84% of the genes showed an H3K9 acetylation decrease by 5AC versus 54% only by DC. Figures 1 and 2 show the heatmaps for the top 100 genes and the 99 genes showing H3K9 acetylation decrease after 5AC treatment and DC treatment, respectively. Conclusion: Despite the similarity in hypomethylating activity and chemical structure, the effect of both drugs on H3K9 acetylation change was significantly different. More changes in H3K9 acetylation were observed after 5 AC treatments compared to DC. The impact of these changes on gene expression and the clinical efficacy of these drugs requires further investigation.

Keywords: DNA methylation, leukemia, decitabine, 5-Azacytidine, epigenetics

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Authors: Huixia Shi, Can Hu, Jun Zhu, Hongling Guo, Haiyan Li, Hongyan Du

Abstract:

The identification of human body fluids during forensic investigations is a critical step to determine key details, and present strong evidence to testify criminal in a case. With the popularity of DNA and improved detection technology, the potential question must be revolved that whether the suspect’s DNA derived from saliva or semen, menstrual or peripheral blood, how to identify the red substance or aged blood traces on the spot is blood; How to determine who contribute the right one in mixed stains. In recent years, molecular approaches have been developing increasingly on mRNA, miRNA, DNA methylation and microbial markers, but appear expensive, time-consuming, and destructive disadvantages. Physicochemical methods are utilized frequently such us scanning electron microscopy/energy spectroscopy and X-ray fluorescence and so on, but results only showing one or two characteristics of body fluid itself and that out of working in unknown or mixed body fluid stains. This paper focuses on using chemistry methods Raman spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to discriminate species of peripheral blood, menstrual blood, semen, saliva, vaginal secretions, urine or sweat. Firstly, non-destructive, confirmatory, convenient and fast Raman spectroscopy method combined with more accurate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method can totally distinguish one from other body fluids. Secondly, 11 spectral signatures and specific metabolic molecules have been obtained by analysis results after 70 samples detected. Thirdly, Raman results showed peripheral and menstrual blood, saliva and vaginal have highly similar spectroscopic features. Advanced statistical analysis of the multiple Raman spectra must be requested to classify one to another. On the other hand, it seems that the lactic acid can differentiate peripheral and menstrual blood detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, but that is not a specific metabolic molecule, more sensitivity ones will be analyzed in a forward study. These results demonstrate the great potential of the developed chemistry methods for forensic applications, although more work is needed for method validation.

Keywords: body fluids, identification, Raman spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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Authors: K. K. Balavenkatraman, B. Bertschi, K. Bigot, A. Grevot, A. Doelemeyer, S. D. Chibout, A. Wolf, F. Pognan, N. Manevski, O. Kretz, P. Swart, K. Litherland, J. Ashton-Chess, B. Ling, R. Wettstein, D. J. Schaefer

Abstract:

Skin toxicity is poorly detected during preclinical studies, and drug-induced side effects in humans such as rashes, hyperplasia or more serious events like bullous pemphigus or toxic epidermal necrolysis represent an important hurdle for clinical development. In vitro keratinocyte-based epidermal skin models are suitable for the detection of chemical-induced irritancy, but do not recapitulate the biological complexity of full skin and fail to detect potential serious side-effects. Normal healthy skin explants may represent a valuable complementary tool, having the advantage of retaining the full skin architecture and the resident immune cell diversity. This study investigated several conditions for the maintenance of good morphological structure after several days of culture and the retention of phase II metabolism for 24 hours in skin explants in vitro. Human skin samples were collected with informed consent from patients undergoing plastic surgery and immediately transferred and processed in our laboratory by removing the underlying dermal fat. Punch biopsies of 4 mm diameter were cultured in an air-liquid interface using transwell filters. Different cultural conditions such as the effect of calcium, temperature and cultivation media were tested for a period of 14 days and explants were histologically examined after Hematoxylin and Eosin staining. Our results demonstrated that the use of Williams E Medium at 32°C maintained the physiological integrity of the skin for approximately one week. Upon prolonged incubation, the upper layers of the epidermis become thickened and some dead cells are present. Interestingly, these effects were prevented by addition of EGFR inhibitors such as Afatinib or Erlotinib. Phase II metabolism of the skin such as glucuronidation (4-methyl umbeliferone), sulfation (minoxidil), N-acetyltransferase (p-toluidene), catechol methylation (2,3-dehydroxy naphthalene), and glutathione conjugation (chlorodinitro benzene) were analyzed by using LCMS. Our results demonstrated that the human skin explants possess metabolic activity for a period of at least 24 hours for all the substrates tested. A time course for glucuronidation with 4-methyl umbeliferone was performed and a linear correlation was obtained over a period of 24 hours. Longer-term culture studies will indicate the possible evolution of such metabolic activities. In summary, these results demonstrate that human skin explants maintain a normal structure for several days in vitro and are metabolically active for at least the first 24 hours. Hence, with further characterisation, this model may be suitable for the study of drug-induced toxicity.

Keywords: human skin explant, phase II metabolism, epidermal growth factor receptor, toxicity

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Authors: Yujie Yuan, Yiyang Fan, Hong Fan

Abstract:

Objective: The RNA methylation recognition protein Aly/REF export factor (ALYREF) is considered one type of “reader” protein acting as a recognition protein of m5C, has been reported involved in several biological progresses including cancer initiation and progression. 5-methylcytosine (m5C) is a conserved and prevalent RNA modification in all species, as accumulating evidence suggests its role in the promotion of tumorigenesis. It has been claimed that ALYREF mediates nuclear export of mRNA with m5C modification and regulates biological effects of cancer cells. However, the systematical regulatory pathways of ALYREF in cancer tissues have not been clarified, yet. Methods: The expression level of ALYREF in pan-cancer and their normal tissues was compared through the data acquired from The Cancer Genome Atlas (TCGA). The University of Alabama at Birmingham Cancer data analysis Portal UALCAN was used to analyze the relationship between ALYREF and clinical pathological features. The relationship between the expression level of ALYREF and prognosis of pan-cancer, and the correlation genes of ALYREF were figured out by using Gene Expression Correlation Analysis database GEPIA. Immune related genes were obtained from TISIDB (an integrated repository portal for tumor-immune system interactions). Immune-related research was conducted by using Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) and TIMER. Results: Based on the data acquired from TCGA, ALYREF has an obviously higher-level expression in various types of cancers compared with relevant normal tissues excluding thyroid carcinoma and kidney chromophobe. The immunohistochemical images on The Human Protein Atlas showed that ALYREF can be detected in cytoplasm, membrane, but mainly located in nuclear. In addition, a higher expression level of ALYREF in tumor tissue generates a poor prognosis in majority of cancers. According to the above results, cancers with a higher expression level of ALYREF compared with normal tissues and a significant correlation between ALYREF and prognosis were selected for further analysis. By using TISIDB, we found that portion of ALYREF co-expression genes (such as BIRC5, H2AFZ, CCDC137, TK1, and PPM1G) with high Pearson correlation coefficient (PCC) were involved in anti-tumor immunity or affect resistance or sensitivity to T cell-mediated killing. Furthermore, based on the results acquired from GEPIA, there was significant correlation between ALYREF and PD-L1. It was exposed that there is a negative correlation between the expression level of ALYREF and ESTIMATE score. Conclusion: The present study indicated that ALYREF plays a vital and universal role in cancer initiation and progression of pan-cancer through regulating mitotic progression, DNA synthesis and metabolic process, and RNA processing. The correlation between ALYREF and PD-L1 implied ALYREF may affect the therapeutic effect of immunotherapy of tumor. More evidence revealed that ALYREF may play an important role in tumor immunomodulation. The correlation between ALYREF and immune cell infiltration level indicated that ALYREF can be a potential therapeutic target. Exploring the regulatory mechanism of ALYREF in tumor tissues may expose the reason for poor efficacy of immunotherapy and offer more directions of tumor treatment.

Keywords: ALYREF, pan-cancer, immunotherapy, PD-L1

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Authors: Ricardo Alberto Chiong Zevallos, Eduardo Moraes Rego Reis

Abstract:

Pancreatic adenocarcinoma (PDAC) patients have a less than 10% 5-year survival rate. PDAC has no defined diagnostic and prognostic biomarkers. Gemcitabine is the first-line drug in PDAC and several other cancers. Long non-coding RNAs (lncRNAs) contribute to the tumorigenesis and are potential biomarkers for PDAC. Although lncRNAs aren’t translated into proteins, they have important functions. LncRNAs can decoy or recruit proteins from the epigenetic machinery, act as microRNA sponges, participate in protein translocation through different cellular compartments, and even promote chemoresistance. The chromatin remodeling enzyme EZH2 is a histone methyltransferase that catalyzes the methylation of histone 3 at lysine 27, silencing local expression. EZH2 is ambivalent, it can also activate gene expression independently of its histone methyltransferase activity. EZH2 is overexpressed in several cancers and interacts with lncRNAs, being recruited to a specific locus. EZH2 can be recruited to activate an oncogene or silence a tumor suppressor. The lncRNAs misregulation in cancer can result in the differential recruitment of EZH2 and in a distinct epigenetic landscape, promoting chemoresistance. The relevance of the EZH2-lncRNAs interaction to chemoresistant PDAC was assessed by Real Time quantitative PCR (RT-qPCR) and RNA Immunoprecipitation (RIP) experiments with naïve and gemcitabine-resistant PDAC cells. The expression of several lncRNAs and EZH2 gene targets was evaluated contrasting naïve and resistant cells. Selection of candidate genes was made by bioinformatic analysis and literature curation. Indeed, the resistant cell line showed higher expression of chemoresistant-associated lncRNAs and protein coding genes. RIP detected lncRNAs interacting with EZH2 with varying intensity levels in the cell lines. During RIP, the nuclear fraction of the cells was incubated with an antibody for EZH2 and with magnetic beads. The RNA precipitated with the beads-antibody-EZH2 complex was isolated and reverse transcribed. The presence of candidate lncRNAs was detected by RT-qPCR, and the enrichment was calculated relative to INPUT (total lysate control sample collected before RIP). The enrichment levels varied across the several lncRNAs and cell lines. The EZH2-lncRNA interaction might be responsible for the regulation of chemoresistance-associated genes in multiple cancers. The relevance of the lncRNA-EZH2 interaction to PDAC was assessed by siRNA knockdown of a lncRNA, followed by the analysis of the EZH2 target expression by RT-qPCR. The chromatin immunoprecipitation (ChIP) of EZH2 and H3K27me3 followed by RT-qPCR with primers for EZH2 targets also assess the specificity of the EZH2 recruitment by the lncRNA. This is the first report of the interaction of EZH2 and lncRNAs HOTTIP and PVT1 in chemoresistant PDAC. HOTTIP and PVT1 were described as promoting chemoresistance in several cancers, but the role of EZH2 is not clarified. For the first time, the lncRNA LINC01133 was detected in a chemoresistant cancer. The interaction of EZH2 with LINC02577, LINC00920, LINC00941, and LINC01559 have never been reported in any context. The novel lncRNAs-EZH2 interactions regulate chemoresistant-associated genes in PDAC and might be relevant to other cancers. Therapies targeting EZH2 alone weren’t successful, and a combinatorial approach also targeting the lncRNAs interacting with it might be key to overcome chemoresistance in several cancers.

Keywords: epigenetics, chemoresistance, long non-coding RNAs, pancreatic cancer, histone modification

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Authors: Zakia Hbellaq

Abstract:

The Mediterranean region is considered a hotbed for climate change. Morocco is a semi-arid Mediterranean country facing water shortages and poor water quality. Its limited water resources limit the activities of various economic sectors. Most of Morocco's territory is in arid and desert areas. The potential water resources are estimated at 22 billion m3, which is equivalent to about 700 m3/inhabitant/year, and Morocco is in a state of structural water stress. Strictly speaking, the Kingdom of Morocco is one of the “very riskiest” countries, according to the World Resources Institute (WRI), which oversees the calculation of water stress risk in 167 countries. The surprising results of the Institute (WRI) rank Morocco as one of the riskiest countries in terms of water scarcity, ranking 3.89 out of 5, thus occupying the 23rd place out of a total of 167 countries, which indicates that the demand for water exceeds the available resources. Agriculture with a score of 3.89 is most affected by water stress from irrigation and places a heavy burden on the water table. Irrigation is an unavoidable technical need and has undeniable economic and social benefits given the available resources and climatic conditions. Irrigation, and therefore the agricultural sector, currently uses 86% of its water resources, while industry uses 5.5%. Although its development has undeniable economic and social benefits, it also contributes to the overfishing of most groundwater resources and the surprising decline in levels and deterioration of water quality in some aquifers. In this context, REUSE is one of the proposed solutions to reduce the water footprint of the agricultural sector and alleviate the shortage of water resources. Indeed, wastewater reuse, also known as REUSE (reuse of treated wastewater), is a step forward not only for the circular economy but also for the future, especially in the context of climate change. In particular, water reuse provides an alternative to existing water supplies and can be used to improve water security, sustainability, and resilience. However, given the introduction of organic trace pollutants or, organic micro-pollutants, the absorption of emerging contaminants, and decreasing salinity, it is possible to tackle innovative capabilities to overcome these problems and ensure food and health safety. To this end, attention will be paid to the adoption of an integrated and attractive approach, based on the reinforcement and optimization of the treatments proposed for the elimination of the organic load with particular attention to the elimination of emerging pollutants, to achieve this goal. , membrane bioreactors (MBR) as stand-alone technologies are not able to meet the requirements of WHO guidelines. They will be combined with heterogeneous Fenton processes using persulfate or hydrogen peroxide oxidants. Similarly, adsorption and filtration are applied as tertiary treatment In addition, the evaluation of crop performance in terms of yield, productivity, quality, and safety, through the optimization of Trichoderma sp strains that will be used to increase crop resistance to abiotic stresses, as well as the use of modern omics tools such as transcriptomic analysis using RNA sequencing and methylation to identify adaptive traits and associated genetic diversity that is tolerant/resistant/resilient to biotic and abiotic stresses. Hence, ensuring this approach will undoubtedly alleviate water scarcity and, likewise, increase the negative and harmful impact of wastewater irrigation on the condition of crops and the health of their consumers.

Keywords: water scarcity, food security, irrigation, agricultural water footprint, reuse, emerging contaminants

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