scholarly journals Isolation and identification of enteroviruses from sewage and sewage-contaminated water samples from Ibadan, Nigeria, 2012-2013

2017 ◽  
Author(s):  
Johnson Adekunle Adeniji ◽  
Adetunji Oladapo Adewale ◽  
Temitope Oluwasegun Cephas Faleye ◽  
Moses Olubusuyi Adewumi
Keyword(s):  
Cell Lines ◽  
Rna Extraction ◽  
Contaminated Water ◽  
Rt Pcr ◽  
Isolation And Identification ◽  
Wild Poliovirus ◽  
Southwest Nigeria ◽  
Species Specific ◽  
Mcf 7 ◽  
Ibadan Nigeria

AbstractIn 2010, we described sewage contaminated water (SCW) bodies that consistently yielded enteroviruses (EVs) in enterovirus surveillance (ES) sites in Lagos, Nigeria. By 2012, we demonstrated the presence and circulation of Wild Poliovirus 3 (WPV3) in these ES sites. Here we describe ES sites that consistently yield EVs in Ibadan metropolis southwest Nigeria.Twenty-five ES samples were collected by grab method from nine sites between October, 2012 and March, 2013. Samples were concentrated and four (RD, HEp2C, MCF-7 and L20B) different cell lines used for virus isolation from the concentrates. Isolates were subjected to RNA extraction, cDNA synthesis, PanEnterovirus 5l-UTR and VP1 assays. Unidentifiable isolates were further subjected to species-specific RT-PCR assays. Amplicons were sequenced, isolates identified and subjected to phylogenetic analysis.Twenty-five isolates were recovered from 8 (32%) of the 25 ES samples collected. Twenty-three of the isolates were identified as EVs by the PanEntero5l-UTR assay. Thirteen (57%) of the 23 EVs were positive for the VP1 assay, and identified as Coxsackievirus B3 (CVB3) (1 isolate), CVB6 (1 isolate), E6 (2 isolates), E7 (5 isolates), E11 (1 isolate), E12 (1 isolate) and E13 (2 isolates). None and 2 (25%) of the remaining isolates were positive for the EV-B and EV-C assays, respectively. The 2 EV-C positive enteroviruses were isolated on MCF-7.This study describes three very productive ES sites, and documents the presence of CVB3, CVB6, E6, E7, E11, E12 and, E13 in Ibadan, Nigeria. It shows that including other cell lines in EV isolation protocols can broaden the diversity of EV types recoverable.

scholarly journals Anticancer Potential of Biogenic Silver Nanoparticles: A Mechanistic Study

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 707
Author(s):  
Mohd Shahnawaz Khan ◽  
Alya Alomari ◽  
Shams Tabrez ◽  
Iftekhar Hassan ◽  
Rizwan Wahab ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Cancer Cells ◽  
Cell Lines ◽  
Human Life ◽  
Mechanistic Study ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Cell Viability Assay ◽  
Hct 116 ◽  
Mcf 7

The continuous loss of human life due to the paucity of effective drugs against different forms of cancer demands a better/noble therapeutic approach. One possible way could be the use of nanostructures-based treatment methods. In the current piece of work, we have synthesized silver nanoparticles (AgNPs) using plant (Heliotropiumbacciferum) extract using AgNO3 as starting materials. The size, shape, and structure of synthesized AgNPs were confirmed by various spectroscopy and microscopic techniques. The average size of biosynthesized AgNPs was found to be in the range of 15 nm. The anticancer potential of these AgNPs was evaluated by a battery of tests such as MTT, scratch, and comet assays in breast (MCF-7) and colorectal (HCT-116) cancer models. The toxicity of AgNPs towards cancer cells was confirmed by the expression pattern of apoptotic (p53, Bax, caspase-3) and antiapoptotic (BCl-2) genes by RT-PCR. The cell viability assay showed an IC50 value of 5.44 and 9.54 µg/mL for AgNPs in MCF-7 and HCT-116 cell lines respectively. We also observed cell migration inhibiting potential of AgNPs in a concentration-dependent manner in MCF-7 cell lines. A tremendous rise (150–250%) in the production of ROS was observed as a result of AgNPs treatment compared with control. Moreover, the RT-PCR results indicated the difference in expression levels of pro/antiapoptotic proteins in both cancer cells. All these results indicate that cell death observed by us is mediated by ROS production, which might have altered the cellular redox status. Collectively, we report the antimetastasis potential of biogenic synthesized AgNPs against breast and colorectal cancers. The biogenic synthesis of AgNPs seems to be a promising anticancer therapy with greater efficacy against the studied cell lines.

Antioxidant, Anti-Inflammatory and Cytotoxic Activities of Jasminum multiflorum (Burm. F.) Andrews Leaves towards MCF-7 Breast Cancer and HCT 116 Colorectal Cell Lines and Identification of Bioactive Metabolites

2021 ◽  
Vol 21 ◽  
Author(s):  
Seham Salah El-Hawary ◽  
Hala M. EL-Hefnawy ◽  
Samir Mohamed Osman ◽  
Mohamed A. El-Raey ◽  
Fatma Alzahraa Mokhtar ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Mass Spectroscopy ◽  
Cytotoxic Activities ◽  
Isolation And Identification ◽  
Antioxidant Power ◽  
Hct 116 ◽  
Anti Inflammatory ◽  
Β Carotene ◽  
Mcf 7

Background: The plants of high phenolic contents are perfect antioxidant and anti-inflammatory candidates and participate in biological studies as effective agents towards different cancer cell lines. Objective: To investigate the antioxidant, anti-inflammatory, and cytotoxic activities of the hydromethanolic leaf extract of Jasminum multiflorum (Burm. f.) Andrews. (J. multiflorum), and phenolic profiling of the extract. Methods: The antioxidant activity for the extract was estimated using β-Carotene-linoleic and ferric reducing antioxidant power (FRAP) assays. The anti-inflammatory activity was evaluated by histamine release assay. Cytotoxicity of J. multiflorum was performed using a neutral red uptake assay towards breast cancer (MCF-7) and colorectal cancer (HCT 116) cell lines. Phenolic profiling of the leaves was characterized using high performance liquid chromatography coupled to photodiode array detector-mass spectroscopy-mass spectroscopy (HPLC-PDA-MS/MS), and chromatographic isolation and identification of the isolated compounds were performed using spectroscopic and NMR data, and virtual docking was performed to the isolated compounds against HSP90 (HEAT SHOCK PROTEIN 90). Results : At a concentration of 75 µg mL-1, J. multiflorum extract showed high antioxidant power; 68.23±0.35 % inhibition and 60.30±0.60 a TEAC (µmol Trolox g-1) for β-Carotene-linoleic assay and FRAP assay; respectively, and possessed anti-inflammatory activity with IC50 67.2 µg/ml. J. multiflorum showed high cytotoxic activity with IC50 of 24.81 µg/ml and 11.38 µg/ml for MCF-7 and HCT 116 cell lines, respectively. HPLC-PDA-MS/MS analysis tentatively identified 39 compounds; major compounds are secoiridoid glycosides, kaempferol, and quercetin glycosides, in addition to simple phenylethanoid compounds. Isolation of active metabolites was performed and led to the isolation and identification of four compounds. On the basis of docking study using HSP90 legend, kaempferol neohesperidoside showed a high cytotoxic potential supported by a high affinity score towards HSP90 legend protein. Conclusion: Jasminum multiflorum is a good candidate to isolate cytotoxic agents.

Investigation on the mechanism of dichloroacetate (DCA) induced apoptosis in breast cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14637-e14637
Author(s):  
L. Ko ◽  
J. Allalunis-Turner
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Glucose Metabolism ◽  
Cell Lines ◽  
Pyruvate Dehydrogenase ◽  
Breast Cancer Cell Lines ◽  
Mitochondrial Activity ◽  
Rt Pcr ◽  
Induced Apoptosis ◽  
Mcf 7

e14637 Background: DCA is a generic, orally available, small molecule metabolic modulator that has an established history in the treatment of mitochondrial diseases and lactic acidosis. DCA inhibits pyruvate dehydrogenase kinase (PDK), an inhibitor of pyruvate dehydrogenase, a key enzyme in glucose metabolism. DCA preferentially shifts glucose metabolism in cancer cells from glycolysis to glucose oxidation, reversing the unique aerobic glycolysis found in solid tumors. DCA induced apoptosis in cancer cells as evidenced by the efflux of cytochrome c and apoptosis-inducing factor from the mitochondria. Recent studies suggested apoptosis induction by DCA in glioblastoma, endometrial, prostate, and non-small cell lung cancers. In this study we attempt to establish a link between DCA and apoptosis in breast cancer cell lines. Methods: Six breast cancer cell lines were investigated (BT474, MCF-7, MDA-MB231, MDA- MB468, SKBR3, T47D). Quantitative real-time polymerase chain reaction (RT-PCR) was performed using Taqman probes to identify increased DNA templates of PDK isoforms 1–4, using DCA concentrations from 0 to 20mM. Western blots were performed to identify increased expression of PDK isoforms and correlate findings with Taqman. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium) assays were performed to measure decreased mitochondrial activity and cytotoxicity. Flow cytometry using annexin V and propidium iodide was performed to measure apoptosis and cell death. Results: RT-PCR showed increased DNA expression of all PDK isoforms in MDA-MB231 cells after DCA treatment. The effect was most pronounced at 10mM concentration. 10mM of DCA also increased DNA expression of all PDK isoforms in MCF-7 cells, and PDK1 in T47D cells. MTS assays showed increased cell kill and decreased mitochondrial activity in all six cell lines, with IC50 ranging between 20mM and 30 mM. Flow cytometry showed increased apoptosis induced by DCA at IC50 for BT474 and MCF-7 cell lines. Conclusions: Apoptosis appears to play a role in the mechanism of DCA in breast cancer, with increased PDK isoform expressions, cytotoxicity and decreased mitochondrial activity. Data from flow cytometry suggested DCA-induced apoptosis in two cell lines. No significant financial relationships to disclose.

scholarly journals Isolation and Identification of Enteroviruses from Sewage and Sewage-Contaminated Water Samples from Ibadan, Nigeria, 2012-2013

2017 ◽  
Vol 06 (03) ◽  
Author(s):  
Johnson Adekunle Adeniji ◽  
Adetunji Oladapo Adewale ◽  
Temitope Oluwasegun Cephas Faleye ◽  
Moses Olubusuyi Adewumi
Keyword(s):  
Water Samples ◽  
Contaminated Water ◽  
Isolation And Identification ◽  
Ibadan Nigeria

scholarly journals Isolation and Identification of Enterovirus D111 in a Child with Acute Flaccid Paralysis in Nigeria

2018 ◽  
Author(s):  
T.O.C. Faleye ◽  
M.O. Adewumi ◽  
O.T. Olayinka ◽  
J.A Adeniji
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Illumina Sequencing ◽  
Acute Flaccid Paralysis ◽  
Rna Extraction ◽  
Flaccid Paralysis ◽  
Isolation And Identification ◽  
Test Kit ◽  
Group A

ABSTRACTThe WHO recommended cell-culture-based algorithm requires enterovirus (EV) isolates to produce reproducible cytopathic effect (R-CPE) in RD and/or L20b cell lines. Samples with non-reproducible CPE (NR-CPE) are considered negative for EVs. We investigated whether there could be EVs lurking in samples with NR-CPE.Fifty-nine (59) cell culture supernatants (CCS) (collected between 2016 and 2017) recovered from RD and L20b cell culture tubes with NR-CPE, were analyzed in this study. The tubes had been previously inoculated with stool suspension from children (<15 years) in Nigeria with acute flaccid paralysis (AFP). All CCS were screened for Enteric Adenoviruses and group A Rotavirus using a rapid immunochromatographic test kit. Subsequently, they were passaged in HEp-2 cell line. All isolates were subjected to RNA extraction, cDNA synthesis, three (5l-UTR, VP1 and EV Species C [EV-C]) different PCR assays and sequencing of amplicons. EVs were further subjected to Illumina sequencing.All CCS were negative for Adenoviruses and group A Rotaviruses. Four CCS produced R-CPE in HEp-2 cell line, three of which were positive for the 5l-UTR assay. Of the 3 isolates two and none were positive for the VP1 and EV-C assays, respectively. One of the two VP1 amplicons was successfully sequenced and identified as Echovirus 1(E1). Illumina sequencing of the three 5l- UTR positive isolates confirmed the E1 isolate and typed the remaining two as EV-Ds (94 and 111).We describe the first EV-D94 and 111 isolates of Nigerian origin. We also show that NR-CPE could sometimes be caused by EVs that do not produce R-CPE in RD and L20b cell lines but do so in other cell lines like HEp-2.

Validation of real-time RT-PCR for analysis of human breast cancer cell lines resistant or sensitive to treatment with antiestrogens.

2003 ◽  
pp. 409-418 ◽  
Author(s):  
P de Cremoux ◽  
C Tran-Perennou ◽  
B L Brockdorff ◽  
E Boudou ◽  
N Br√ºnner ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Real Time ◽  
Cell Lines ◽  
Model System ◽  
Model Systems ◽  
Breast Cancer Cell Lines ◽  
Antiestrogen Resistance ◽  
Human Breast ◽  
Rt Pcr ◽  
Mcf 7

Using a quantitative real-time RT-PCR technique we have compared the expression of a number of genes in two different human breast cancer model systems for development of acquired resistance to antiestrogens. The model system developed at the Danish Cancer Society comprises the cell lines MCF-7, MCF-7/TAMR-1, MCF-7/182R-6 and MCF-7/182R-7, and the model system developed at the Lombardi Cancer Research Center consists of the cell lines MCF-7/LCC1, MCF-7/LCC2 and MCF-7/LCC9. The findings on the well-known parameters estrogen receptor (ER)alpha, progesterone receptor (PR) and epidermal growth factor receptor (EGFR) are in good agreement with previous reports, thus documenting the usefulness of the real-time RT-PCR technique for multiparametric RNA analysis. The gene expression levels in the two model systems were found to be quite similar in relation to ERalpha, AIB1 (amplified in breast cancer-1), breast cancer antiestrogen resistance gene 1 (BCAR1) and ErbB-2 mRNA expression, whereas significant differences were observed on the expression of ERbeta, multidrug resistance gene 1 (MDR1), PR and EGFR. Furthermore, the presented data suggest that ERbeta, AIB1, BCAR1, CYP19 and MDR1 are unlikely to be causally involved in development of antiestrogen resistance in these breast cancer cell lines.

scholarly journals Phytochemical Study and Evaluation of the Cytotoxic Properties of Methanolic Extract from Baccharis obtusifolia

2018 ◽  
Vol 2018 ◽  
pp. 1-5 ◽  
Author(s):  
Juan Carlos Romero-Benavides ◽  
Gina C. Ortega-Torres ◽  
Javier Villacis ◽  
Sara L. Vivanco-Jaramillo ◽  
Karla I. Galarza-Urgilés ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Methanol Extract ◽  
Methanolic Extract ◽  
Chromatographic Purification ◽  
Isolation And Identification ◽  
Phytochemical Study ◽  
Mcf 7

Some species of the Baccharis genus have been shown to possess important biomedical properties, including cytotoxic activity. In this study, we examined the cytotoxic effect of methanol extract from Baccharis obtusifolia (Asteraceae) in cancer cell lines of prostate (PC-3), colon (RKO), astrocytoma (D-384), and breast (MCF-7). The methanolic extract displayed the largest substantial cytotoxic effect in lines of colon cancer (RKO) and cerebral astrocytoma (D-384). Chromatographic purification of the B. obtusifolia methanolic extract led to the isolation and identification of 5,4′-dihydroxy-7-methoxyflavone (1) and 5-hydroxy-7,4′-dimethoxyflavone (2) compounds of the flavonoid type.

scholarly journals First Report of Tomato Brown Rugose Fruit Virus on Tomato in Syria

Plant Disease ◽  
2021 ◽  
Author(s):  
Ziad M Hasan ◽  
Nidà Mohammed Salem ◽  
Imad D. Ismail ◽  
Insaf Akel ◽  
Ahmad Y Ahmad
Keyword(s):  
Mosaic Virus ◽  
Rna Extraction ◽  
Coastal Region ◽  
Rt Pcr ◽  
Specific Primers ◽  
Total Rna ◽  
First Report ◽  
Species Specific ◽  
Das Elisa ◽  
Total Rna Extraction

Tomato (Solanum lycopersicum L.) is an important vegetable crop worldwide. In spring and autumn 2017, virus-like symptoms were observed on greenhouse grown tomato plants in the east of Akkar plain (south of coastal region, Tartous governorate, Syria). These symptoms were: mild to severe mosaic on the apical leaves, brown necrosis on sepals, receptacle and flower’s cluster carrier, and severe symptoms of brown rugose and discoloration on fruit. During next growing seasons, disease spread was observed in most of Syrian coastal region with disease incidence ranged from 40% to 70% by 2020. Tomato brown rugose fruit virus (ToBRFV) was suspected as a main causal agent of the disease, especially since its first report in Jordan, a neighboring country (Salem et al. 2016), Palestine (Alkowni et al. 2019), Turkey (Fidan et al. 2019), Germany (Menzel et al. 2019), Italy (Panno et al. 2019), America (Camacho-Beltrán et al. 2019), Egypt (Amer and Mahmoud, 2020), and recently in Spain (Alfaro-Fernandez et al. 2021). In November and December 2020, seventy-one leaf samples from symptomatic plants (59 from Tartous and 12 from Lattakia governorates) and seven from asymptomatic ones (5 from Tartous and 2 from Lattakia) were collected and tested for the presence of ToBRFV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using ToBRFV-commercial kit (LOEWE® Biochemia, Germany) following the manufacturer’s instructions. Results showed, forty-three of symptomatic samples reacted positively (38 in Tartous and 5 in Lattakia) and none of asymptomatic ones. On the other hand, sap mechanical inoculation of 10 tomato cv. Mandaloun F1 (Enza Zaden, the Netherlands) plants using a positive tomato isolate gave systemic mosaic symptoms in all plants identical to those observed in the original plants in the field, after 13 days of inoculation, and necrotic local lesions on 10 plants of Nicotiana tabacum after 5 days, indicating the presence of a tobamovirus in general. ToBRFV infection was confirmed in all mechanically-inoculated plants by DAS-ELISA. Further tests were necessary to investigate ToBRFV presence, because of its serological relationships with another tobamoviruses. Six representative symptomatic samples (ELISA-positive) and two asymptomatic (ELISA-negative) samples were subjected to total RNA extraction using the SV-Total RNA Extraction kit (Promega, U.S.A.) following the manufacturer’s instructions. The samples were tested by two-step reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers and protocols for most common tomato-infecting viruses, including: tomato chlorosis virus and tomato infectious chlorosis virus (Dovas et al. 2002), pepino mosaic virus (PepMV) and tomato torrado virus (Wieczorek et al. 2013), alfalfa mosaic virus (Parrella et al. 2000), tomato spotted wilt virus (Salem et al. 2012) and a pair of primers: ToBRFV-F2 (5’-CATATCTCTCGACACCAGTAAAAGGACCCG-3’) and ToBRFV-R2 (5’-TCCGAGTATAGGAAGACTCTGGTTGGTC-3’) targeting a region of the RNA dependent RNA polymerase (RdRp), of the ToBRFV genome (KT383474; Salem et al. 2016). First-strand cDNA synthesis was carried out using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Promega) and random primer according to the manufacturer's protocol, then followed by PCR with the seven species-specific primers. Only ToBRFV was detected among all tested viruses in symptomatic samples (ELISA-positive), and none of the tested viruses was detected in the asymptomatic plants. To confirm the presence of ToBRFV, two selected RdRp-specific PCR amplicons (872 bp) were purified and ligated into pGEM T-Easy Vector (Promega), and three clones were sequenced (GenBank accession nos. MZ447794 to 96). BLASTn analysis showed that the nucleotide sequences are 99.77-100% identical and shared around 99% identity to RdRp of ToBRFV isolate (MT118666) from Turkey available in the GenBank. Accordingly, the presence of ToBRFV was confirmed by bioassays on indicator plants, DAS-ELISA, RT-PCR, and further sequencing. To our knowledge, this is the first report of ToBRFV infecting tomato in Syria, and this requires special emphasis for further investigations because of the virus severity, easy transmission ability and absent of commercial resistance varieties till now.

scholarly journals Evaluation of the effect of amygdalin on survivin gene expression in MCF-7 breast cancer cell line

Genetika ◽  
2018 ◽  
Vol 50 (2) ◽  
pp. 647-657
Author(s):  
Iman Shahrokhiyan ◽  
Abbas Doosti ◽  
Hossein Sazegar
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cancer Cells ◽  
Cell Lines ◽  
Mtt Assay ◽  
Breast Cancer Cells ◽  
Rt Pcr ◽  
Survivin Gene ◽  
Cell Groups ◽  
Mcf 7

The effects of amygdalin as an herbal substance on prevention of cancer cells proliferation it has been shown. Also, survivin gene is one of the important genes on inhibition of apoptosis and controlling of the cell cycle in cancer cell lines. For this purpose, the present study was undertaken for the first time to evaluate the effects of amygdalin on survivin gene expression in MCF-7 and HDF. The MCF-7 (Breast cancer cells) and normal HDF were treated by different doses of amygdalin (0.5 to 10 mg/ml). Then, the MTT assay was done on each cell groups on 24, 48, and 72 h after treatment. In each period time the cells were detached and used for RNA extraction and cDNA synthesis. Finally, the survivin gene expression in each cell groups was evaluated by quantitative real-time PCR (q-RT-PCR) analysis. The MTT assay was showed that 5 mg/ml of amygdalin concentration at 48 hours after treatment it was suitable for evaluation of this compound on MCF-7 and HDF cell lines. The survey results of q-RT-PCR were showed in both amygdalin-treated MCF-7 (MCF-7+AMG) compare to the treated HDF (HDF-AMG as an control group) the expression level of survivin gene were decreased but this reduction only in MCF-7+AMG group was statistically significant (p <0.05). Our findings indicated that amygdalin in particular; decrease the survivin mRNA in MCF-7 breast cancer cells compare to normal healthy cells and leads to apoptosis and the death of cancer cells. It is suggested that in future study it must be better the effects of amygdalin supplemented with surfactant investigate against other cancer cells and evaluate the more molecular markers to specify the anti-cancer activity potential of this herbal compound.

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