Search results for: cytotoxic

Authors: S. Kudlacik-Kramarczyk, M. Kedzierska, B. Tyliszczak

Abstract:

Recently, the continuous development of medicine and related sciences has been observed. Particular emphasis is directed on the development of biomaterials, i.e., non-toxic, biocompatible and biodegradable materials that may improve the effectiveness of treatment as well as the comfort of patients. This is particularly important in the case of cancer treatment. Currently, there are many methods of cancer treatment based primarily on chemotherapy and the surgical removal of the tumor, but it is worth noting that these therapies also cause many side effects. Among women, the most common cancer is breast cancer. It may be completely cured, but the consequence of treatment is partial or complete breast mastectomy and radiation therapy, which results in severe skin burns. The skin of the patient after radiation therapy is very burned, and therefore requires intensive care and high frequency of dressing changes. The traditional dressing adheres to the burn wounds and does not absorb adequate amount of exudate from injuries and the patient is forced to change the dressing every 2 hours. Therefore, the main purpose was to develop an innovative combination of dressing material with drug carriers that may be used in anti-cancer therapy. The innovation of this solution is the combination of these two products into one system, i.e., a transdermal system with the possibility of a controlled release of the drug- cytostatic. Besides, the possibility of modifying the hydrogel matrix with aloe vera juice provides this material with new features favorable from the point of view of healing processes of burn wounds resulting from the radiation therapy. In this study, hydrogel materials containing protein spheres with the active substance have been obtained as a result of photopolymerization process. The reaction mixture consisting of the protein (albumin) spheres incorporated with cytostatic, chitosan, adequate crosslinking agent and photoinitiator has been subjected to the UV radiation for 2 minutes. Prepared materials have been subjected to the numerous studies including the analysis of cytotoxicity using murine fibroblasts L929. Analysis was conducted based on the mitochondrial activity test (MTT reduction assay) which involves the determining the number of cells characterized by proper metabolism. Hydrogel materials obtained using different amount of crosslinking agents have been subjected to the cytotoxicity analysis. According to the standards, tested material is defined as cytotoxic when the viability of cells after 24 h incubation with this material is lower than 70%. In the research, hydrogel polymer materials containing protein spheres incorporated with the active substance, i.e. a cytostatic, have been developed. Such a dressing may support the treatment of cancer due to the content of the anti-cancer drug - cytostatic, and may also provide a soothing effect on the healing of the burn wounds resulted from the radiation therapy due to the content of aloe vera juice in the hydrogel matrix. Based on the conducted cytotoxicity studies, it may be concluded that the obtained materials do not adversely affect the tested cell lines, therefore they can be subjected to more advanced analyzes.

Keywords: hydrogel polymers, cytostatics, drug carriers, cytotoxicity

Procedia PDF Downloads 107

Authors: Lijuan Li

Abstract:

Nitric oxide (NO) has become a fascinating entity in biological chemistry over the past few years. It is a gaseous lipophilic radical molecule that plays important roles in several physiological and pathophysiological processes in mammals, including activating the immune response, serving as a neurotransmitter, regulating the cardiovascular system, and acting as an endothelium-derived relaxing factor. NO functions in eukaryotes both as a signal molecule at nanomolar concentrations and as a cytotoxic agent at micromolar concentrations. The latter arises from the ability of NO to react readily with a variety of cellular targets leading to thiol S-nitrosation, amino acid N-nitrosation, and nitrosative DNA damage. Nitric oxide can readily bind to metals to give metal-nitrosyl (M-NO) complexes. Some of these species are known to play roles in biological NO storage and transport. These complexes have different biological, photochemical, or spectroscopic properties due to distinctive structural features. These recent discoveries have spawned a great interest in the development of transition metal complexes containing NO, particularly its iron complexes that are central to the role of nitric oxide in the body. Spectroscopic evidence would appear to implicate species of “Fe(NO)2+” type in a variety of processes ranging from polymerization, carcinogenesis, to nitric oxide stores. Our research focuses on isolation and structural studies of non-heme iron nitrosyls that mimic biologically active compounds and can potentially be used for anticancer drug therapy. We have shown that reactions between Fe(NO)2(CO)2 and a series of imidazoles generated new non-heme iron nitrosyls of the form Fe(NO)2(L)2 [L = imidazole, 1-methylimidazole, 4-methylimidazole, benzimidazole, 5,6-dimethylbenzimidazole, and L-histidine] and a tetrameric cluster of [Fe(NO)2(L)]4 (L=Im, 4-MeIm, BzIm, and Me2BzIm), resulted from the interactions of Fe(NO)2 with a series of substituted imidazoles was prepared. Recently, a series of sulfur bridged iron di nitrosyl complexes with the general formula of [Fe(µ-RS)(NO)2]2 (R = n-Pr, t-Bu, 6-methyl-2-pyridyl, and 4,6-dimethyl-2-pyrimidyl), were synthesized by the reaction of Fe(NO)2(CO)2 with thiols or thiolates. Their structures and properties were studied by IR, UV-vis, 1H-NMR, EPR, electrochemistry, X-ray diffraction analysis and DFT calculations. IR spectra of these complexes display one weak and two strong NO stretching frequencies (νNO) in solution, but only two strong νNO in solid. DFT calculations suggest that two spatial isomers of these complexes bear 3 Kcal energy difference in solution. The paramagnetic complexes [Fe2(µ-RS)2(NO)4]-, have also been investigated by EPR spectroscopy. Interestingly, the EPR spectra of complexes exhibit an isotropic signal of g = 1.998 - 2.004 without hyperfine splitting. The observations are consistent with the results of calculations, which reveal that the unpaired electron dominantly delocalize over the two sulfur and two iron atoms. The difference of the g values between the reduced form of iron-sulfur clusters and the typical monomeric di nitrosyl iron complexes is explained, for the first time, by of the difference in unpaired electron distributions between the two types of complexes, which provides the theoretical basis for the use of g value as a spectroscopic tool to differentiate these biologically active complexes.

Keywords: di nitrosyl iron complex, metal nitrosyl, non-heme iron, nitric oxide

Procedia PDF Downloads 276

Authors: Sajmina Khatun, Monika Pebam, Aravind Kumar Rengan

Abstract:

Photothermal therapy, a subset of nanomedicine, takes advantage of light-absorbing agents to generate localized heat, selectively eradicating cancer cells. This innovative approach minimizes damage to healthy tissues and offers a promising avenue for targeted cancer treatment. Unlike conventional therapies, photothermal therapy harnesses the power of light to combat malignancies precisely and effectively, showcasing its potential to revolutionize cancer treatment paradigms. The combined strengths of nanomedicine and photothermal therapy signify a transformative shift toward more effective, targeted, and tolerable cancer treatments in the medical landscape. Utilizing natural products becomes instrumental in formulating diverse bioactive medications owing to their various pharmacological properties attributed to the existence of phenolic structures, triterpenoids, and similar compounds. Hyptis suaveolens, commonly known as pignut, stands as an aromatic herb within the Lamiaceae family and represents a valuable therapeutic plant. Flourishing in swamps and alongside tropical and subtropical roadsides, these noxious weeds impede the development of adjacent plants. Hyptis suaveolens ranks among the most globally distributed alien invasive species. The present investigation revealed that a versatile, biodegradable liposome nanosystem (HIL NPs), incorporating bioactive molecules from Hyptis suaveolens, exhibits effective bioavailability to cancer cells, enabling tumor ablation upon near-infrared (NIR) laser exposure. The components within the nanosystem, specifically the bioactive molecules from Hyptis, function as anticancer agents, aiding in the photothermal ablation of highly metastatic lung cancer cells. Despite being a prolific weed impeding neighboring plant growth, Hyptis suaveolens showcases therapeutic benefits through its bioactive compounds. The obtained HIL NPs, characterized as a photothermally active liposome nanosystem, demonstrate a pronounced fluorescence absorption peak in the NIR range and achieve a high photothermal conversion efficiency under NIR laser irradiation. Transmission electron microscopy (TEM) and particle size analysis reveal that HIL NPs possess a spherical shape with a size of 141 ± 30 nm. Moreover, in vitro assessments of HIL NPs against lung cancer cell lines (A549) indicate effective anticancer activity through a combined cytotoxic effect and hyperthermia. Tumor ablation is facilitated by apoptosis induced by the overexpression of ɣ-H2AX, arresting cancer cell proliferation. Consequently, the multifunctional and biodegradable nanosystem (HIL NPs), incorporating bioactive compounds from Hyptis, provides valuable perspectives for developing an innovative therapeutic strategy originating from a challenging weed. This approach holds promise for potential applications in both bioimaging and the combined use of phyto-photothermal therapy for cancer treatment.

Keywords: bioactive liposome, hyptis suaveolens, photothermal therapy, lung cancer

Procedia PDF Downloads 54

Authors: Sahar Nasr, Lin Li, Edwin Wang

Abstract:

Bladder cancer (BLCA) is known as the 13th cause of death among cancer patients worldwide, and ~575,000 new BLCA cases are diagnosed each year. Urothelial carcinoma (UC) is the most prevalent subtype among BLCA patients, which can be categorized into muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC). Currently, various therapeutic options are available for UC patients, including (1) transurethral resection followed by intravesical instillation of chemotherapeutics or Bacillus Calmette-Guérin for NMIBC patients, (2) neoadjuvant platinum-based chemotherapy (NAC) plus radical cystectomy is the standard of care for localized MIBC patients, and (3) systematic chemotherapy for metastatic UC. However, conventional treatments may lead to several challenges for treating patients. As an illustration, some patients may suffer from recurrence of the disease after the first line of treatment. Recently, immune checkpoint therapy (ICT) has been introduced as an alternative treatment strategy for the first or second line of treatment in advanced or metastatic BLCA patients. Although ICT showed lucrative results for a fraction of BLCA patients, ~80% of patients were not responsive to it. Therefore, novel treatment methods are required to augment the ICI response rate within BLCA patients. It has been shown that the infiltration of T-cells into the tumor microenvironment (TME) is positively correlated with the response to ICT within cancerous patients. Therefore, the goal of this study is to enhance the infiltration of cytotoxic T-cells into TME through the identification of target genes within the tumor that are responsible for the non-T-cell inflamed TME and their inhibition. BLCA bulk RNA-sequencing data from The Cancer Genome Atlas (TCGA) and immune score for TCGA samples were used to determine the Pearson correlation score between the expression of different genes and immune score for each sample. The genes with strong negative correlations were selected (r < -0.2). Thereafter, the correlation between the expression of each gene and survival in BLCA patients was calculated using the TCGA data and Cox regression method. The genes that are common in both selected gene lists were chosen for further analysis. Afterward, BLCA bulk and single-cell RNA-sequencing data were ranked based on the expression of each selected gene and the top and bottom 25% samples were used for pathway enrichment analysis. If the pathways related to the T-cell infiltration (e.g., antigen presentation, interferon, or chemokine pathways) were enriched within the low-expression group, the gene was included for downstream analysis. Finally, the selected genes will be used to calculate the correlation between their expression and the infiltration rate of the activated CD+8 T-cells, natural killer cells and the activated dendric cells. A list of potential target genes has been identified and ranked based on the above-mentioned analysis and criteria. SUN-1 got the highest score within the gene list and other identified genes in the literature as benchmarks. In conclusion, inhibition of SUN1 may increase the tumor-infiltrating lymphocytes and the efficacy of ICI in BLCA patients. BLCA tumor cells with and without SUN-1 CRISPR/Cas9 knockout will be injected into the syngeneic mouse model to validate the predicted SUN-1 effect on increasing tumor-infiltrating lymphocytes.

Keywords: data analysis, gene expression analysis, gene identification, immunoinformatic, functional genomics, transcriptomics

Procedia PDF Downloads 131

Authors: Rafia S. Al-lamki, Jun Wang, Simon Pacey, Jordan Pober, John R. Bradley

Abstract:

Tumor necrosis factor alpha (TNF), signaling through TNFR2, may act an autocrine growth factor for renal tubular epithelial cells. Clear cell renal carcinomas (ccRCC) contain cancer stem cells (CSCs) that give rise to progeny which form the bulk of the tumor. CSCs are rarely in cell cycle and, as non-proliferating cells, resist most chemotherapeutic agents. Thus, recurrence after chemotherapy may result from the survival of CSCs. Therapeutic targeting of both CSCs and the more differentiated bulk tumor populations may provide a more effective strategy for treatment of RCC. In this study, we hypothesized that TNFR2 signaling will induce CSCs in ccRCC to enter cell cycle so that treatment with ligands that engage TNFR2 will render CSCs susceptible to chemotherapy. To test this hypothesis, we have utilized wild-type TNF (wtTNF) or specific muteins selective for TNFR1 (R1TNF) or TNFR2 (R2TNF) to treat either short-term organ cultures of ccRCC and adjacent normal kidney (NK) tissue or cultures of CD133+ cells isolated from ccRCC and adjacent NK, hereafter referred to as stem cell-like cells (SCLCs). The effect of cyclophosphamide (CP), currently an effective anticancer agent, was tested on CD133+SCLCs from ccRCC and NK before and after R2TNF treatment. Responses to TNF were assessed by flow cytometry (FACS), immunofluorescence, and quantitative real-time PCR, TUNEL, and cell viability assays. Cytotoxic effect of CP was analyzed by Annexin V and propidium iodide staining with FACS. In addition, we assessed the effect of TNF on isolated SCLCs differentiation using a three-dimensional (3D) culture system. Clinical samples of ccRCC contain a greater number SCLCs compared to NK and the number of SCSC increases with higher tumor grade. Isolated SCLCs show expression of stemness markers (oct4, Nanog, Sox2, Lin28) but not differentiation markers (cytokeratin, CD31, CD45, and EpCAM). In ccRCC organ cultures, wtTNF and R2TNF increase CD133 and TNFR2 expression and promote cell cycle entry whereas wtTNF and R1TNF increase TNFR1 expression and promote cell death of SCLCs. Similar findings are observed in SCLCs isolated from NK but the effect was greater in SCLCs isolated from ccRCC. Application of CP distinctly triggered apoptotic and necrotic cell death in SLCSs pre-treatment with R2TNF as compared to CP treatment alone, with SCLCs from ccRCC more sensitive to CP compared to SLCS from NK. Furthermore, TNF promotes differentiation of SCLCs to an epithelial phenotype in 3D cultures, confirmed by cytokeratin expression and loss of stemness markers Nanog and Sox2. The differentiated cells show positive expression of TNF and TNFR2. These findings provide evidence that selective engagement of TNFR2 drive CSCs to cell proliferation/differentiation, and targeting of cycling cells with TNFR2 agonist in combination with anti-cancer agents may be a potential therapy for RCC.

Keywords: cancer stem cells, ccRCC, cell cycle, cell death, TNF, TNFR1, TNFR2, CD133

Procedia PDF Downloads 241

Authors: A. Matiushkina, A. Bazhenova, I. Litvinov, E. Kornilova, A. Dubavik, A. Orlova

Abstract:

Magnetic-luminescent complexes based on superparamagnetic iron oxide nanoparticles (SPIONs) and semiconductor quantum dots (QDs) have been recognized as a new class of materials that have high potential in modern medicine. These materials can serve for theranostics of oncological diseases, and also as a target agent for drug delivery. They combine the qualities characteristic of magnetic nanoparticles, that is, magneto-controllability and the ability to local heating under the influence of an external magnetic field, as well as phosphors, due to luminescence of which, for example, early tumor imaging is possible. The complexity of creating complexes is the energy transfer between particles, which quenches the luminescence of QDs in complexes with SPIONs. In this regard, a relatively new type of alloyed (CdₓZn₁₋ₓSeᵧS₁₋ᵧ)-ZnS QDs is used in our work. The presence of a sufficiently thick gradient semiconductor shell in alloyed QDs makes it possible to reduce the probability of energy transfer from QDs to SPIONs in complexes. At the same time, Forster Resonance Energy Transfer (FRET) is a perfect instrument to confirm the formation of complexes based on QDs and different-type energy acceptors. The formation of complexes in the aprotic bipolar solvent dimethyl sulfoxide is ensured by the coordination of the carboxyl group of the stabilizing QD molecule (L-cysteine) on the surface iron atoms of the SPIONs. An analysis of the photoluminescence (PL) spectra has shown that a sequential increase in the SPIONs concentration in the samples is accompanied by effective quenching of the luminescence of QDs. However, it has not confirmed the formation of complexes yet, because of a decrease in the PL intensity of QDs due to reabsorption of light by SPIONs. Therefore, a study of the PL kinetics of QDs at different SPIONs concentrations was made, which demonstrates that an increase in the SPIONs concentration is accompanied by a symbatic reduction in all characteristic PL decay times. It confirms the FRET from QDs to SPIONs, which indicates the QDs/SPIONs complex formation, rather than a spontaneous aggregation of QDs, which is usually accompanied by a sharp increase in the percentage of the QD fraction with the shortest characteristic PL decay time. The complexes have been studied by the magnetic circular dichroism (MCD) spectroscopy that allows one to estimate the response of magnetic material to the applied magnetic field and also can be useful to check SPIONs aggregation. An analysis of the MCD spectra has shown that the complexes have zero residual magnetization, which is an important factor for using in biomedical applications, and don't contain SPIONs aggregates. Cell penetration, biocompatibility, and stability of QDs/SPIONs complexes in cancer cells have been studied using HeLa cell line. We have found that the complexes penetrate in HeLa cell and don't demonstrate cytotoxic effect up to 25 nM concentration. Our results clearly demonstrate that alloyed (CdₓZn₁₋ₓSeᵧS₁₋ᵧ)-ZnS QDs can be successfully used in complexes with SPIONs reached new hybrid nanostructures, which combine bright luminescence for tumor imaging and magnetic properties for targeted drug delivery and magnetic hyperthermia of tumors. Acknowledgements: This work was supported by the Ministry of Science and Higher Education of Russian Federation, goszadanie no. 2019-1080 and was financially supported by Government of Russian Federation, Grant 08-08.

Keywords: alloyed quantum dots, magnetic circular dichroism, magneto-luminescent complexes, superparamagnetic iron oxide nanoparticles

Procedia PDF Downloads 86

Authors: Yuliya Y. Gavrichenko

Abstract:

Background and Aims: The increasing bacterial resistance to almost all the available antibiotics has encouraged scientists to search for alternative sources of antibacterial agents. Nowadays, it is known that many plant secondary metabolites have diverse biological activity. These compounds can be potentially active against human bacterial and viral infections. Extended research has been carried out to explore the use of the luminescent bacterial test as a rapid, accurate and inexpensive method to assess the antibacterial properties and to predict the biological activity spectra for plant origin substances. Method: Botanical material of fifteen species was collected from their natural and cultural habitats on the Crimean peninsula. The aqueous extracts of following plants were tested: Robinia pseudoacacia L., Sideritis comosa, Cotinus coggygria Scop., Thymus serpyllum L., Juglans regia L., Securigera varia L., Achillea millefolium L., Phlomis taurica, Corylus avellana L., Sambucus nigra L., Helichrysum arenarium L., Glycyrrhiza glabra L., Elytrigia repens L., Echium vulgare L., Conium maculatum L. The test was carried out using luminous strains of marine bacteria Photobacterium leiognathi, which was isolated from the Sea of Azov as well as four Escherichia coli MG1655 recombinant strains harbouring Vibrio fischeri luxCDABE genes. Results: The bactericidal capacity of plant extracts showed significant differences in the study. Cotinus coggygria, Phlomis taurica, Juglans regia L. proved to be the most toxic to P. leiognathi. (EC50 = 0.33 g dried plant/l). Glycyrrhiza glabra L., Robinia pseudoacacia L., Sideritis comosa and Helichrysum arenarium L. had moderate inhibitory effects (EC50 = 3.3 g dried plant/l). The rest of the aqueous extracts have decreased the luminescence of no more than 50% at the lowest concentration (16.5 g dried plant/l). Antibacterial activity of herbal extracts against constitutively luminescent E. coli MG1655 (pXen7-lux) strain was observed at approximately the same level as for P. leiognathi. Cotinus coggygria and Conium maculatum L. extracts have increased light emission in the mutant E. coli MG1655 (pFabA-lux) strain which is associated with cell membranes damage. Sideritis comosa, Phlomis taurica, Juglans regia induced SOS response in E. coli (pColD-lux) strain. Glycyrrhiza glabra L. induced protein damage response in E. coli MG1655 (pIbpA-lux) strain. Conclusion: The received results have shown that the plants’ extracts had nonspecific antimicrobial effects against both E. coli (pXen7-lux) and P. leiognathi biosensors. Mutagenic, cytotoxic and protein damage effects have been observed. In general, the bioluminescent inhibition test result correlated with the traditional use of screened plants. It leads to the following conclusion that whole-cell luminescent biosensors could be the indicator of overall plants antibacterial capacity. The results of the investigation have shown a possibility of bioluminescent method in medicine and pharmacy as an approach to research the antibacterial properties of medicinal plants.

Keywords: antibacterial property, bioluminescence, medicinal plants, whole-cell biosensors

Procedia PDF Downloads 95

Authors: Maria Zulema Morquecho Vega, Fabiola CarolinaMiranda Castro, Rafael Verdugo Miranda, Ignacio Yocupicio Villegas, Ana lidia Barron Raygoza, Martin enrique MArquez Cordova, Jose Alberto Duarte Moller

Abstract:

Background: Anemopsis californica, also called (tame grass) belongs to the Saururaceae family small, green plant. The blade is long and wide. Gives a white flower. The plant population is only found in humid, swampy habitats, it grows where there is water, along the banks of streams and water holes. In the winter, it dries up. The leaves, rhizomes, or roots of this plant have been used to treat a range of diseases. Some of its healing properties are used to treat wounds, cold and flu symptoms, spasmodic cough, infection, pain and inflammation, burns, swollen feet, as well as lung ailments, asthma, circulatory problems (varicose veins), rheumatoid arthritis, purifies blood, helps in urinary and digestive tract diseases, sores and healing, for headache, sore throat, diarrhea, kidney pain. The tea made from the leaves and roots is used to treat uterine cancer, womb cancer, relieves menstrual pain and stops excessive bleeding after childbirth. It is also used as a gynecological treatment for infections, hemorrhoids, candidiasis and vaginitis. Objective: To study the cytotoxicity of gels prepared with silver nanoparticles in AC extract combined with chitosan, collagen and hyaluronic acid as an alternative therapy for skin conditions. Methods: The Ag NPs were synthesized according to the following method. A 0.3 mg/mL solution is prepared in 10 ml of deionized water, adjust to pH 12 with NaOH, stirring is maintained constant magnetic and a temperature of 80 °C. Subsequently, 100 ul of a 0.1 M AgNO3 solution and kept stirring constantly for 15 min. Once the reaction is complete, measurements are performed by UV-Vis. A gel was prepared in a 5% solution of acetic acid with the respective nanoparticles and AC extract of silver in the extract of AC. Chitosan is added until the process begins to occur gel. At that time, collagen will be added in a ratio of 3 to 5 drops, and later, hyaluronic acid in 2% of the total compound formed. Finally, after resting for 24 hours, the cytotoxic effect of the gels was studied. in the presence of highly positive bacteria Staphylococcus aureus and highly negative for Escherichia coli. Cultures will be incubated for 24 hours in the presence of the compound and compared with the reference. Results: Silver nanoparticles obtained had a spherical shape and sizes among 20 and 30 nm. UV-Vis spectra confirm the presence of silver nanoparticles showing a surface plasmon around 420 nm. Finally, the test in presence of bacteria yield a good antibacterial property of this nanocompound and tests in people were successful. Conclusion: Gel prepared by biogenic synthesis shown beneficious effects in severe acne, acne vulgaris and wound healing with diabetic patients.

Keywords: anemopsis californica, nanomedicina, biotechnology, biomedicine

Procedia PDF Downloads 64

Authors: Suikinai Nobre Santos, Ranko Gacesa, Paul F. Long, Itamar Soares de Melo

Abstract:

Extremophilic microorganism are adapted to biotopes combining several stress factors (temperature, pressure, radiation, salinity and pH), which indicate the richness valuable resource for the exploitation of novel biotechnological processes and constitute unique models for investigations their biomolecules (1, 2). The above information encourages us investigate bioprospecting synthesized compounds by a noval actinomycete, designated thermotolerant Streptomyces caatingaensis CMAA 1322, isolated from sample soil tropical dry forest (Caatinga) in the Brazilian semiarid region (3-17°S and 35-45°W). This set of constrating physical and climatic factores provide the unique conditions and a diversity of well adapted species, interesting site for biotechnological purposes. Preliminary studies have shown the great potential in the production of cytotoxic, pesticidal and antimicrobial molecules (3). Thus, to extend knowledge of the genes clusters responsible for producing biosynthetic pathways of natural products in strain CMAA1322, whole-genome shotgun (WGS) DNA sequencing was performed using paired-end long sequencing with PacBio RS (Pacific Biosciences). Genomic DNA was extracted from a pure culture grown overnight on LB medium using the PureLink genomic DNA kit (Life Technologies). An approximately 3- to 20-kb-insert PacBio library was constructed and sequenced on an 8 single-molecule real-time (SMRT) cell, yielding 116,269 reads (average length, 7,446 bp), which were allocated into 18 contigs, with 142.11x coverage and N50 value of 20.548 bp (BioProject number PRJNA288757). The assembled data were analyzed by Rapid Annotations using Subsystems Technology (RAST) (4) the genome size was found to be 7.055.077 bp, comprising 6167 open reading frames (ORFs) and 413 subsystems. The G+C content was estimated to be 72 mol%. The closest-neighbors tool, available in RAST through functional comparison of the genome, revealed that strain CMAA1322 is more closely related to Streptomyces hygroscopicus ATCC 53653 (similarity score value, 537), S. violaceusniger Tu 4113 (score value, 483), S. avermitilis MA-4680 (score value, 475), S. albus J1074 (score value, 447). The Streptomyces sp. CMAA1322 genome contains 98 tRNA genes and 135 genes copies related to stress response, mainly osmotic stress (14), heat shock (16), oxidative stress (49). Functional annotation by antiSMASH version 3.0 (5) identified 41 clusters for secondary metabolites (including two clusters for lanthipeptides, ten clusters for nonribosomal peptide synthetases [NRPS], three clusters for siderophores, fourteen for polyketide synthetase [PKS], six clusters encoding a terpene, two clusters encoding a bacteriocin, and one cluster encoding a phenazine). Our work provide in comparative analyse of genome and extract produced (data no published) by lineage CMAA1322, revealing the potential of microorganisms accessed from extreme environments as Caatinga” to produce a wide range of biotechnological relevant compounds.

Keywords: caatinga, streptomyces, environmental stresses, biosynthetic pathways

Procedia PDF Downloads 213

Authors: V. Cesaroni, C. Girometta, A. Bernicchia, M. Brusoni, F. Corana, R. M. Baiguera, C. M. Cusaro, M. L. Guglielminetti, B. Mannucci, H. Kawagishi, C. Perini, A. M. Picco, P. Rossi, E. Salerni, E. Savino

Abstract:

Fungi of the genus Hericium contain various compounds with antibacterial activity, cytotoxic effect on cancer cells and bioactive molecules. Some of the active metabolites stimulate the synthesis of the Nerve Growth Factor (NGF). Recently, the effect of dietary supplement based on Hericium erinaceus on recognition memory and on hippocampal mossy fiber-CA3 neurotransmission was published. The aim of this study was to investigate the presence of Hericium species on Italian territory in order to isolate the strains for further studies and applications. The first step was to collect Hericium sporophores in Tuscany: H. alpestre Pers., H. coralloides (Scop.) Pers. and H. erinaceus (Bull.) Pers. were the species present. The strains of H. alpestre (H.a.1), H. coralloides (H.c.1) and H. erinaceus (H.e.1 & H.e.2) have been isolated in pure culture and preserved in the collection of the University of Pavia (MicUNIPV). The DNA sequences obtained from the strains were compared to other sequences found in international databases. Therefore, it was possible to construct a phylogenetic tree that highlights the clear separation in clades of the sequences and the molecular identification of our strains with the species of Hericium considered. The second step was to cultivate indoor and outdoor H. erinaceus in order to obtain as many sporophores as possible for further chemical analysis. All the procedures for H. erinaceus cultivation have been followed. Among the available recipes for indoor H. erinaceus cultivation, it was used a substrate formulation contained 70% oak sawdust, 20% rice bran, 10% wheat straw, 1% CaCO3 and 1% sucrose. The bioactive compounds present in the mycelia and in the sporophores of H. erinaceus were chemically analyzed in collaboration with the Centro Grandi Strumenti of the University of Pavia using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). The materials to be analyzed were previously freeze-dried and then extracted with an alcoholic procedure. Preliminary chromatographic analysis revealed the presence of potentially bioactive and structurally different secondary metabolites such as polysaccharides, erinacins, ericenones, steroids and other terpenoids. Ericenones C and D (in sporophores) and erinacin A (in mycelium) have been identified by comparison with the respective standards. These molecules are known to have effects on the Central Nervous System (CNS) cells, which is the main objective of our studies. Thanks to the high sensitivity in the detection of bioactive compounds of H. erinaceus, it will be possible to use the To obtain lyophilized mycelium and the respective culture broth, 4 small pieces (about 5 mm2) of the respective H.e.1 or H.c.1 strains, taken from the margin of growing cultures (MEA), were inoculated into 1 liter of 2% ME (malt extract, Biokar Diagnostics). The static liquid cultures were kept at 24 °C in the dark chamber and fungi grew for one month. 10 replicates for each strain have been done. The method proposed as an analytical screening protocol to determine the optimal growth conditions of the fungus and to improve the production chain of H. erinaceus. These results encourage to carry out chemical analyzes also on H. alpestre and H. coralloides in order to evaluate the presence of bioactive compounds in these two species.

Keywords: Hericium species, Hercium erinaceus bioactive compounds, medicinal mushrooms, mushroom cultivation

Procedia PDF Downloads 114

Authors: Sami El Khatib, Agnès Leroux, Jean-Louis Merlin, François Guillemin, Marie-Ange D’Hallewin

Abstract:

Photodynamic therapy (PDT) is a treatment modality based on the cytotoxic effect occurring on the target tissues by interaction of a photosensitizer with light in the presence of oxygen. One of the major advances in PDT can be attributed to the use of topical aminolevulinic (ALA) to induce Protoporphyrin IX (PpIX) for the treatment of early stage cancers as well as diagnosis. ALA is a precursor of the heme synthesis pathway. Locally delivered to the target tissue ALA overcomes the negative feedback exerted by heme and promotes the transient formation of PpIX in situ to reach critical effective levels in cells and tissue. Whereas early steps of the heme pathway occur in the cytosol, PpIX synthesis is shown to be held in the mitochondrial membranes and PpIX fluorescence is expected to accumulate in close vicinity of the initial building site and to progressively diffuse to the neighboring cytoplasmic compartment or other lipophylic organelles. PpIX is known to be highly reactive and will be degraded when irradiated with light. PpIX photobleaching is believed to be governed by a singlet oxygen mediated mechanism in the presence of oxidized amino acids and proteins. PpIX photobleaching and subsequent spectral phototransformation were described widely in tumor cells incubated in vitro with ALA solution, or ex vivo in human and porcine mucosa superfused with hexylaminolevulinate (hALA). PpIX photobleaching was also studied in vivo, using animal models such as normal or tumor mice skin and orthotopic rat bladder model. Hexyl aminolevulinate a more potent lipophilic derivative of ALA was proposed as an adjunct to standard cystoscopy in the fluorescence diagnosis of bladder cancer and other malignancies. We have previously reported the effectiveness of hALA mediated PDT of rat bladder cancer. Although normal and tumor bladder epithelium exhibit similar fluorescence intensities after intravesical instillation of two hALA concentrations (8 and 16 mM), the therapeutic response at 8mM and 20J/cm2 was completely different from the one observed at 16mM irradiated with the same light dose. Where the tumor is destroyed, leaving the underlying submucosa and muscle intact after an 8 mM instillation, 16mM sensitization and subsequent illumination results in the complete destruction of the underlying bladder wall but leaves the tumor undamaged. The object of the current study is to try to unravel the underlying mechanism for this apparent contradiction. PpIX extraction showed identical amounts of photosensitizer in tumor bearing bladders at both concentrations. Photobleaching experiments revealed mono-exponential decay curves in both situations but with a two times faster decay constant in case of 16mM bladders. Fluorescence microscopy shows an identical fluorescence pattern for normal bladders at both concentrations and tumor bladders at 8mM with bright spots. Tumor bladders at 16 mM exhibit a more diffuse cytoplasmic fluorescence distribution. The different response to PDT with regard to the initial pro-drug concentration can thus be attributed to the different cellular localization.

Keywords: bladder cancer, hexyl-aminolevulinate, photobleaching, confocal fluorescence microscopy

Procedia PDF Downloads 376

Authors: Deepanwita Deka, Dhruva Kumar Jha

Abstract:

Endophytes are the microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects. Endophytes are rich source of therapeutic substances like antimicrobial, anticancerous, herbicidal, insecticidal, immunomodulatory compounds. Brucea mollis, commonly known as Quinine in Assam, belonging to the family Simaroubaceae, is a shrub or small tree, recorded as endangered species in North East India by CAMP survey in 2003. It is traditionally being used as antimalarial and antimicrobial agent and has antiplasmodial, cytotoxic, anticancer, diuretic, cardiovascular effect etc. Being endangered and medicinal; this plant may host certain noble endophytes which need to be studied in depth. The aim of the present study was isolation and identification of potent endophytic fungi from Brucea mollis, an endangered medicinal plant, to protect it from extinction due to over use for medicinal purposes. Aseptically collected leaves, barks and roots samples of healthy plants were washed and cut into a total of 648 segments of about 2 cm long and 0.5 cm broad with sterile knife, comprising 216 segments each from leaves, barks and roots. These segments were surface sterilized using ethanol, mercuric chloride (HgCl2) and aqueous solution of sodium hypochlorite (NaClO). Different media viz., Czapeck-Dox-Agar (CDA, Himedia), Potato-Dextrose-Agar (PDA, Himedia), Malt Extract Agar (MEA, Himedia), Sabourad Dextrose Agar (SDA, Himedia), V8 juice agar, nutrient agar and water agar media and media amended with plant extracts were used separately for the isolation of the endophytic fungi. A total of 11 fungal species were recovered from leaf, bark and root tissues of B. mollis. The isolates were screened for antimicrobial, antioxidant and enzymatic activities using certain protocols. Cochliobolus geniculatus was identified as the most dominant species. The mycelia sterilia (creamy white) showing highest inhibitory activity against Candida albicans (MTCC 183) was induced to sporulate using modified PDA media. The isolate was identified as Geosmithia pallida. The internal transcribed spacer of rDNA was sequenced for confirmation of the taxonomic identity of the sterile mycelia (creamy white). The internal transcribed spacer r-DNA sequence was submitted to the NCBI (KU693285) for the first time from India. G. pallida and Penicillium showed highest antioxidant activity among all the isolates. The antioxidant activity of G. pallida and Penicillium didn’t show statistically significant difference (P˃0.05). G. pallida, Cochliobolus geniculatus and P. purpurogenum respectively showed highest cellulase, amylase and protease activities. Thus, endopytic fungal isolates may be used as potential natural resource of pharmaceutical importance. The endophytic fungi, Geosmithia pallida, may be used for synthesis of pharmaceutically important natural products and consequently can replace plants hitherto used for the same purpose. This study suggests that endophytes should be investigated more aggressively to better understand the endophyte biology of B. mollis.

Keywords: Antimicrobial activity, antioxidant activity, Brucea mollis, endophytic fungi, enzyme activity, Geosmithia pallida

Procedia PDF Downloads 151

Authors: Mohammadjavad Sotoudeheian, Navid Farahmandian

Abstract:

Kawasaki disease (KD) is the primary cause of acquired pediatric heart disease as an acute vasculitis. In children with prolonged fever, rash, and inflammation of the mucosa KD must be considered as a clinical diagnosis. There is a persuasive suggestion of immune-mediated damage as the pathophysiologic cascade of KD. For example, the invasion of cytotoxic T-cells supports a viral etiology and the inflammasome of the innate immune system is a critical component in the vasculitis formation in KD. Animal models of KD propose the cytokine profiles, such as increased IL-1 and GM-CSF, which cause vascular damage. CRP and IFN-γ elevated expression and the upregulation of IL-6, and IL-10 production are also described in previous studies. Untreated KD is a critical risk factor for coronary artery diseases and myocardial infarction. Vascular damage may encompass amplified T-cell activity. SMAD3 is an essential molecule in down-regulating T-cells and increasing expression of FoxP3. It has a critical effect in the differentiation of regulatory T-cells. The discrepancy of regulatory T-cells and pro-inflammatory Th17 has been studied in acute coronary syndrome during KD. However in the coronary artery damaged lymphocytes and IgA plasma cells are seen at the lesion locations, the major immune cells in the coronary lesions are monocytes/macrophages and neutrophils. These cells secrete TNF-α, and activates matrix metalloprotease (MMP)-9, reducing the integrity of vessels and prompting patients to arise aneurysm. MMPs can break down the components of the extracellular matrix and assist immune cell movement. IVIG as an effective form of treatment clarified the role of the immune system, which may target pathogenic antigens and regulate cytokine production. Several reports have revealed that in the coronary arteries, high expression of MMP-9 in monocyte/macrophage results in pathologic cascades. Curcumin is a potent antioxidant and anti-inflammatory molecule. Curcumin decreases the production of reactive oxygen and nitrogen species and inhibits transcription factors like AP-1 and NF-κB. Curcumin also contains the characteristics of inhibitory effects on MMPs, especially MMP-9. The upregulation of MMP-9 is an important cellular response. Curcumin treatment caused a reverse effect and down-regulates MMP-9 gene expression which may fund the anti-inflammatory effect. Curcumin inhibits MMP-9 expression via PKC and AMPK-dependent pathways in Human monocytes cells. Elevated expression and activity of MMP-9 are correlated with advanced vascular lesions. AMPK controls lipid metabolism and oxidation, and protein synthesis. AMPK is also necessary for the MMP-9 activity and THP-1 cell adhesion to endothelial cells. Curcumin was shown to inhibit the activation of AMPKα. Compound C (AMPK inhibitor) inhibits MMP-9 expression level. Therefore, through inactivating AMPKs and PKC, curcumin decreases the MMP-9 level, which results in inhibiting monocyte/macrophage differentiation. Compound C also suppress the phosphorylation of three major classes of MAP kinase signaling, suggesting that curcumin may suppress MMP-9 level by inactivation of MAPK pathways. MAPK cascades are activated to induce the expression of MMP-9. Curcumin inhibits MAPKs phosphorylation, which contributes to the down-regulation of MMP-9. This study demonstrated that the potential inhibitory properties of curcumin over MMP-9 lead to a therapeutic strategy to reduce the risk of coronary artery involvement during KD.

Keywords: MMP-9, coronary artery aneurysm, Kawasaki’s disease, curcumin, AMPK, immune system, NF-κB, MAPK

Procedia PDF Downloads 273

Authors: Manuel I. Capel, Antonio Tomeu, Alberto Salguero

Abstract:

Tumor growth from a transformed cancer-cell up to a clinically apparent mass spans through a range of spatial and temporal magnitudes. Through computer simulations, Cellular Automata (CA) can accurately describe the complexity of the development of tumors. Tumor development prognosis can now be made -without making patients undergo through annoying medical examinations or painful invasive procedures- if we develop appropriate CA-based software tools. In silico testing mainly refers to Computational Biology research studies of application to clinical actions in Medicine. To establish sound computer-based models of cellular behavior, certainly reduces costs and saves precious time with respect to carrying out experiments in vitro at labs or in vivo with living cells and organisms. These aim to produce scientifically relevant results compared to traditional in vitro testing, which is slow, expensive, and does not generally have acceptable reproducibility under the same conditions. For speeding up computer simulations of cellular models, specific literature shows recent proposals based on the CA approach that include advanced techniques, such the clever use of supporting efficient data structures when modeling with deterministic stochastic cellular automata. Multiparadigm and multiscale simulation of tumor dynamics is just beginning to be developed by the concerned research community. The use of stochastic cellular automata (SCA), whose parallel programming implementations are open to yield a high computational performance, are of much interest to be explored up to their computational limits. There have been some approaches based on optimizations to advance in multiparadigm models of tumor growth, which mainly pursuit to improve performance of these models through efficient memory accesses guarantee, or considering the dynamic evolution of the memory space (grids, trees,…) that holds crucial data in simulations. In our opinion, the different optimizations mentioned above are not decisive enough to achieve the high performance computing power that cell-behavior simulation programs actually need. The possibility of using multicore and GPU parallelism as a promising multiplatform and framework to develop new programming techniques to speed-up the computation time of simulations is just starting to be explored in the few last years. This paper presents a model that incorporates parallel processing, identifying the synchronization necessary for speeding up tumor growth simulations implemented in Java and C++ programming environments. The speed up improvement that specific parallel syntactic constructs, such as executors (thread pools) in Java, are studied. The new tumor growth parallel model is proved using implementations with Java and C++ languages on two different platforms: chipset Intel core i-X and a HPC cluster of processors at our university. The parallelization of Polesczuk and Enderling model (normally used by researchers in mathematical oncology) proposed here is analyzed with respect to performance gain. We intend to apply the model and overall parallelization technique presented here to solid tumors of specific affiliation such as prostate, breast, or colon. Our final objective is to set up a multiparadigm model capable of modelling angiogenesis, or the growth inhibition induced by chemotaxis, as well as the effect of therapies based on the presence of cytotoxic/cytostatic drugs.

Keywords: cellular automaton, tumor growth model, simulation, multicore and manycore programming, parallel programming, high performance computing, speed up

Procedia PDF Downloads 212

Authors: Demetrio L. Valle Jr., Juliana Janet M. Puzon, Windell L. Rivera

Abstract:

From the various medicinal plants available in the Philippines, crude ethanol extracts of twelve (12) Philippine medicinal plants, namely: Senna alata L. Roxb. (akapulko), Psidium guajava L. (bayabas), Piper betle L. (ikmo), Vitex negundo L. (lagundi), Mitrephora lanotan (Blanco) Merr. (Lanotan), Zingiber officinale Roscoe (luya), Curcuma longa L. (Luyang dilaw), Tinospora rumphii Boerl (Makabuhay), Moringga oleifera Lam. (malunggay), Phyllanthus niruri L. (sampa-sampalukan), Centella asiatica (L.) Urban (takip kuhol), and Carmona retusa (Vahl) Masam (tsaang gubat) were studied. In vitro methods of evaluation against selected Gram-positive and Gram-negative multidrug-resistant (MDR), bacteria were performed on the plant extracts. Although five of the plants showed varying antagonistic activities against the test organisms, only Piper betle L. exhibited significant activities against both Gram-negative and Gram-positive multidrug-resistant bacteria, exhibiting wide zones of growth inhibition in the disk diffusion assay, and with the lowest concentrations of the extract required to inhibit the growth of the bacteria, as supported by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Further antibacterial studies of the Piper betle L. leaf, obtained by three extraction methods (ethanol, methanol, supercritical CO2), revealed similar inhibitory activities against a multitude of Gram-positive and Gram-negative MDR bacteria. Thin layer chromatography (TLC) assay of the leaf extract revealed a maximum of eight compounds with Rf values of 0.92, 0.86, 0.76, 0.53, 0.40, 0.25, 0.13, and 0.013, best visualized when inspected under UV-366 nm. TLC- agar overlay bioautography of the isolated compounds showed the compounds with Rf values of 0.86 and 0.13 having inhibitory activities against Gram-positive MDR bacteria (MRSA and VRE). The compound with an Rf value of 0.86 also possesses inhibitory activity against Gram-negative MDR bacteria (CRE Klebsiella pneumoniae and MBL Acinetobacter baumannii). Gas Chromatography-Mass Spectrometry (GC-MS) was able to identify six volatile compounds, four of which are new compounds that have not been mentioned in the medical literature. The chemical compounds isolated include 4-(2-propenyl)phenol and eugenol; and the new four compounds were ethyl diazoacetate, tris(trifluoromethyl)phosphine, heptafluorobutyrate, and 3-fluoro-2-propynenitrite. Phytochemical screening and investigation of its antioxidant, cytotoxic, possible hemolytic activities, and mechanisms of antibacterial activity were also done. The results showed that the local variant of Piper betle leaf extract possesses significant antioxidant, anti-cancer and antimicrobial properties, attributed to the presence of bioactive compounds, particularly of flavonoids (condensed tannin, leucoanthocyanin, gamma benzopyrone), anthraquinones, steroids/triterpenes and 2-deoxysugars. Piper betle L. is also traditionally known to enhance wound healing, which could be primarily due to its antioxidant, anti-inflammatory and antimicrobial activities. In vivo studies on mice using 2.5% and 5% of the ethanol leaf extract cream formulations in the excised wound models significantly increased the process of wound healing in the mice subjects, the results and values of which are at par with the current antibacterial cream (Mupirocin). From the results of the series of studies, we have definitely proven the value of Piper betle L. as a source of bioactive compounds that could be developed into therapeutic agents against MDR bacteria.

Keywords: Philippine herbal medicine, multidrug-resistant bacteria, Piper betle, TLC-bioautography

Procedia PDF Downloads 716

Authors: Kyriaki Hatziagapiou, Eleni Kakouri, Konstantinos Bethanis, Alexandra Nikola, Eleni Koniari, Charalabos Kanakis, Elias Christoforides, George Lambrou, Petros Tarantilis

Abstract:

Medulloblastoma is a highly invasive tumour, as it tends to disseminate throughout the central nervous system early in its course. Despite the high 5-year-survival rate, a significant number of patients demonstrate serious long- or short-term sequelae (e.g., myelosuppression, endocrine dysfunction, cardiotoxicity, neurological deficits and cognitive impairment) and higher mortality rates, unrelated to the initial malignancy itself but rather to the aggressive treatment. A strong rationale exists for the use of Crocus sativus L (saffron) and its bioactive constituents (crocin, crocetin, safranal) as pharmaceutical agents, as they exert significant health-promoting properties. Crocins are water soluble carotenoids. Unlike other carotenoids, crocins are highly water-soluble compounds, with relatively low toxicity as they are not stored in adipose and liver tissues. Crocins have attracted wide attention as promising anti-cancer agents, due to their antioxidant, anti-inflammatory, and immunomodulatory effects, interference with transduction pathways implicated in tumorigenesis, angiogenesis, and metastasis (disruption of mitotic spindle assembly, inhibition of DNA topoisomerases, cell-cycle arrest, apoptosis or cell differentiation) and sensitization of cancer cells to radiotherapy and chemotherapy. The current research aimed to study the potential cytotoxic effect of crocins on TE671 medulloblastoma cell line, which may be useful in the optimization of existing and development of new therapeutic strategies. Crocins were extracted from stigmas of saffron in ultrasonic bath, using petroleum-ether, diethylether and methanol 70%v/v as solvents and the final extract was lyophilized. Identification of crocins according to high-performance liquid chromatography (HPLC) analysis was determined comparing the UV-vis spectra and the retention time (tR) of the peaks with literature data. For the biological assays crocin was diluted to nuclease and protease free water. TE671 cells were incubated with a range of concentrations of crocins (16, 8, 4, 2, 1, 0.5 and 0.25 mg/ml) for 24, 48, 72 and 96 hours. Analysis of cell viability after incubation with crocins was performed with Alamar Blue viability assay. The active ingredient of Alamar Blue, resazurin, is a blue, nontoxic, cell permeable compound virtually nonfluorescent. Upon entering cells, resazurin is reduced to a pink and fluorescent molecule, resorufin. Viable cells continuously convert resazurin to resorufin, generating a quantitative measure of viability. The colour of resorufin was quantified by measuring the absorbance of the solution at 600 nm with a spectrophotometer. HPLC analysis indicated that the most abundant crocins in our extract were trans-crocin-4 and trans-crocin-3. Crocins exerted significant cytotoxicity in a dose and time-dependent manner (p < 0.005 for exposed cells to any concentration at 48, 72 and 96 hours versus cells not exposed); as their concentration and time of exposure increased, the reduction of resazurin to resofurin decreased, indicating reduction in cell viability. IC50 values for each time point were calculated ~3.738, 1.725, 0.878 and 0.7566 mg/ml at 24, 48, 72 and 96 hours, respectively. The results of our study could afford the basis of research regarding the use of natural carotenoids as anticancer agents and the shift to targeted therapy with higher efficacy and limited toxicity. Acknowledgements: The research was funded by Fellowships of Excellence for Postgraduate Studies IKY-Siemens Programme.

Keywords: crocetin, crocin, medulloblastoma, saffron

Procedia PDF Downloads 184