Unaccounted uncertainty from qPCR efficiency estimates entails uncontrolled false positive rates

BACKGROUND: Accurate adjustment for the amplification efficiency (AE) is an important part of real-time quantitative polymerase chain reaction (qPCR) experiments. The most commonly used correction strategy is to estimate the AE by dilution experiments and use this as a plug-in when efficiency correcting the ΔΔC(q). Currently, it is recommended to determine the AE with high precision as this plug-in approach does not account for the AE uncertainty, implicitly assuming an infinitely precise AE estimate. Determining the AE with such precision, however, requires tedious laboratory work and vast amounts of biological material. Violation of the assumption leads to overly optimistic standard errors of the ΔΔC(q), confidence intervals, and p-values which ultimately increase the type I error rate beyond the expected significance level. As qPCR is often used for validation it should be a high priority to account for the uncertainty of the AE estimate and thereby properly bounding the type I error rate and achieve the desired significance level. RESULTS: We suggest and benchmark different methods to obtain the standard error of the efficiency adjusted ΔΔC(q) using the statistical delta method, Monte Carlo integration, or bootstrapping. Our suggested methods are founded in a linear mixed effects model (LMM) framework, but the problem and ideas apply in all qPCR experiments. The methods and impact of the AE uncertainty are illustrated in three qPCR applications and a simulation study. In addition, we validate findings suggesting that MGST1 is differentially expressed between high and low abundance culture initiating cells in multiple myeloma and that microRNA-127 is differentially expressed between testicular and nodal lymphomas. CONCLUSIONS: We conclude, that the commonly used efficiency corrected quantities disregard the uncertainty of the AE, which can drastically impact the standard error and lead to increased false positive rates. Our suggestions show that it is possible to easily perform statistical inference of ΔΔC(q), whilst properly accounting for the AE uncertainty and better controlling the false positive rate

Fusaric acid Fumonisin B1 CO -treatment regulates AMPK signalling and induces Apoptosis in HEPG2 cells.

Masters Degree. University of KwaZulu-Natal, Durban.Background/Aim: Fusaric acid (FA) and Fumonisin B1 (FB1) are mycotoxins produced by Fusarium fungal species. These mycotoxins are major contaminants of maize and contribute to toxicity in animals and humans. The main mechanisms of FA and FB1 toxicity involve the induction of oxidative stress and apoptosis; however, FA was additionally found to chelate divalent cations, whereas FB1 inhibits sphingolipid synthesis. AMPK is an energy sensor involved in regulating cell proliferation. AMPK targets the transcription factors, p53 and FOXO3a that play a major role in apoptosis. To date numerous studies have investigated the individual effects of FA and FB1, however, their combined synergistic effects are unclear. This study investigated the effect of FA and FB1 co-treatment on AMPK-induced apoptosis in liver HepG2 cells. Methods: HepG2 cells were cultured and co-treated with various concentrations (5, 27, 100μM and combined 104μM FA and 200μM FB1 IC50s) of FA and FB1 for 24 hrs. Cytotoxic effects of FA and FB1 on HepG2 cells were determined using the MTT assay. The TBARS assay was used to determine oxidative stress. Western blot was used to determine protein expression of AMPK, p-AMPK and p53, whereas q-PCR was used to measure FOXO3a mRNA expression. LDH assay was used to measure membrane integrity. ATP levels and activity of caspases -3/7, -8 and -9 were measured using luminometry. Results: A combination of FA and FB1 decreased cell viability in a dose dependant manner. An IC50 of 27μM for FA and FB1 was obtained. ATP levels were significantly increased at 5μM and 27μM, whereas at 100μM and combined IC50s were significantly decreased (p<0.0001). Oxidative stress was significantly increased in FA and FB1 treated cells in a dose dependent manner (p<0.0001). The protein expression of total AMPK was decreased at 5μM, but increased at 27μM, 100μM and combined IC50s in relation to control (p<0.0001).p- AMPK showed a significant decrease (p<0.0001) in all FA and FB1 treated samples despite the increase in the expression of total AMPK. FOXO3a mRNA expression was decreased at 5μM and at combined IC50s, with the decrease being significant at 5μM. The results also indicated an increase at 27μM and 100μM (p<0.0001). p53 protein expressions were significantly decreased in all samples (p<0.0001). Caspase -3/7, -8 and -9 were significantly increased at 5-100μM and decreased at combined IC50s in HepG2 cells. In FA and FB1 samples, LDH levels were significantly decreased at 5μM and 27μM, and significantly increased at 100μM and combined IC50s (p<0.0001). Conclusion: FA and FB1 co-treatments suppressed AMPK signalling by downregulating p- AMPK and induced apoptosis and/necrosis in HepG2 cells

The Effects of Quorum Sensing Molecules on Streptococcus mutans Biofilm

The use of quorum quenching (QQ) molecules to prevent biofilm formation has recently gained momentum. Interrupting the quorum sensing (QS) pathways of cariogenic bacteria like Streptococcus mutans could reduce the formation of dental plaque on teeth, lowering the incidences of caries and periodontitis. This research studied the effects of three QQ molecules (p-coumaric acid, tyrosol, and farnesol) on S. mutans biofilm formation on biotic and abiotic surfaces. This research specifically studied the changes in eDNA, protein, carbohydrate, and viable cells in the extracellular matrix (ECM) of the biofilm. The gene expression of gtfb was studied to determine if the QQ molecules played a role in gene expression. All three QQ molecules showed reduction on different components of the S. mutans biofilm. Tyrosol reduced the greatest amount of overall biomass when the biofilm was formed on examples of both biotic and abiotic surfaces. Tyrosol also had the highest reduction of proteins in biofilms formed on plastic and the total number of viable cells in biofilms formed on 20% porosity hydroxyapatite (HA) disks. p-Coumaric acid reduced the most eDNA and the number of viable cells in the biofilm when it was formed on plastic. p-Coumaric acid also decreased the eDNA and carbohydrate concentrations the most in biofilms formed on 20% porosity HA disks. Additionally, p-coumaric acid reduced the number of viable cells in the biofilm formed on full density HA disks. Farnesol decreased the most carbohydrates concentration on both plastic and full density HA disks. Farnesol decreased the most eDNA on full density HA disks. Farnesol was the only QQ molecule to lower the gene expression of gtfb in the biofilm. All three QQ molecules decreased some individual components of the S. mutans biofilm, but tyrosol was the overall best QQ molecule for total lessening of biomass and components of the ECM

Tilapia lake virus (TiLV) : utvikling av PCRbaserte diagnostiske metoder og studier av infeksjonsmekanismer

Tilapia lake virus (TiLV) is an emerging virus of wild and farmed tilapiines responsible for causing mortalities and significant economic losses to the aquaculture industry. Since its first report in Israel, the virus has been reported in four continents (Asia, Africa, South and North America) to cause mortalities ranging from the lower extreme of 5-20% and higher extremes of up to 90%. The infection status in many countries is not yet known and implementation of surveillance programs has been recommended. Lake Victoria was selected for TiLV surveillance because it contains a large number of tilapiine species of economic importance to the surrounding East African countries. Well-optimized tools for rapid detection and quantification of the virus are also needed. Moreover, the mode of virus uptake in cells and importance for replication is not known. Therefore, this thesis aimed at understanding the possible presence of TiLV in Lake Victoria in East Africa, to develop tools for detection and quantification of the virus and to shed light of virus uptake mechanisms in permissive cells in vitro. In this thesis, a standard RT-PCR and a quantitative real-time RT-PCR (qRT-PCR) were developed based on all the ten segments of TiLV. The standard PCR was used to screen Nile tilapia (Oreochromis niloticus) from Lake Victoria in Eastern Africa. The quantitative real-time RT-PCR was developed based on virus supernatant of known titre and against samples of unknown virus titre originating from infected TFC #10 cells and fish organs. The virus’ ability to hemagglutinate avian and piscine erythrocytes was assessed, and the modulation of ammonium chloride on uptake and replication of TiLV in E-11 cells was studied. The findings reported in study I showed that primers designed from segment two of TiLV for a standard RT-PCR were the best at detecting TiLV in the infected cells and Nile tilapia organs. TiLV genome was detected in 28 Nile tilapias (14. 66%, N = 191) in which 17.78% (N=45) were from wild fish and 13.70% (N=146) from farmed fish (cage farming). The genomes of circulating TiLV in Nile tilapia from Lake Victoria were identical to those detected from Israel (98%), Ecuador (98%), Thailand (96%), Peru (96%) and USA (97%). TiLV was not grown from infected fish and thus its ability to cause disease in Nile tilapia was not studied. Therefore, I am recommending further studies to fulfil Koch’s postulates. The data reported in study II showed that the developed and optimized quantitative real-time RT-PCR detected TiLV in virus supernatants of known titre and in organs of unknown titre from infected Nile tilapia. The developed assay is sensitive and specific to TiLV with all the primers efficiency being within the range of 95-105%, except primers targeting segment ten that gave an efficiency of 93%. The intra- and inter-assay coefficient of variation ranged between 0.00% ~ 2.63% and 0.00% ~ 5.92%, respectively, which is within the recommended range (below 5%) for an assay to be repeatable and reproducible. The detection limit of 2 TCID50/ml was found for primers targeting segments 1, 2, 3, 4 and 9 while lower detection limit of 20 TCID50/ml was found for primers targeting segment 5, 6, 7, 8 and 10. Overall primers targeting segment 3 had the highest detection limit and primers targeting segment 7 had the lowest detection limit. Interestingly, despite the two primer sets (for segment 3 and 7) having different TiLV detection limits they had an equal amplification efficiency of 98%. Therefore, primer optimization for qRT-PCR is important to optimize assay sensitivity. The study reported in paper III was directed at understanding the hemagglutination property of TiLV using avian and piscine erythrocytes, and the infection mechanisms in E-11 cells. TiLV did not hemagglutinate erythrocytes from any of the species tested indicating that the virus lack hemagglutinin. Further, the study has shown that ammonium chloride does not affect the replication of TiLV in E-11 cells indicating that the virus is not using the endocytic pathway during internalization. Taken together, the two observations suggest that, TiLV is not taken up by receptor-mediated endocytosis during internalization into E-11 cells. Thus, further studies are needed to unravel the uptake mechanism(s), which is the important information for controlling the virus by antiviral agents or immunoprophylaxis.Tilapia lake virus (TiLV) er et fremvoksende virus som infiserer ville arter og oppdrettsarter av tilapia og gir dødelighet og betydelige økonomiske tap i oppdrett. Siden den første beskrivelsen av sykdommen fra Israel, har viruset blitt påvist på fire kontinenter (Asia, Afrika, Sør- og Nord-Amerika) og gir dødelighet fra 5-20% opp mot 90%. Forekomst av viruset er ikke kjent i mange av de landene som driver tilapiaoppdrett, og det er nødvendig å etablere bedre overvåknings- og kontrollprogrammer i disse landene. I denne studien ble Lake Victoria valgt for TiLV-screening fordi den inneholder et stort antall tilapia-arter som er av økonomisk betydning for de omkringliggende østafrikanske landene. Det er også behov for optimaliserte metoder for rask deteksjon og kvantifisering av viruset. Hvordan viruset tas opp i cellene og hvordan det replikerer er ikke kjent. I denne avhandlingen ble det gjennomført studier for å forstå forekomst av TiLV i Lake Victoria i Øst-Afrika, det ble etablert verktøy for påvisning og kvantifisering av viruset med molekylærbiologiske metoder, og det ble gjennomført studier for å bedre forstå opptaksmekanismer i celler under infeksjonen. Det ble utviklet en standard RT-PCR og en kvantitativ RT-PCR (qRT-PCR) basert på alle de ti segmentene til viruset. Standard PCR ble brukt til å undersøke Nile tilapia (Oreochromis niloticus) fra Victoriasjøen. Den kvantitative RT-PCR metoden ble testet mot kjent og ukjent virustiter fra henholdsvis infiserte TFC# 10 celler og organer fra infiserte fisk. Hemagglutinering av røde blodlegemer fra hønsefugl og fisk ble også undersøkt, samt effekten av ammoniumklorid på replikasjonen av TiLV i E-11-celler. Resultatene i studie I viste at primere designet fra segment 2 benyttet for påvisning med standard RT-PCR var best egnet til å påvise TiLV i infiserte cellene og organer fra infisert fisk. TiLV fra Victoriasjøen ble påvist i 28 fisk (14. 66%, N = 191) hvor 17,78% (N = 45) var fra villfisk og 13,70% (N = 146) fra oppdrettsfisk. De sekvensene som ble påvist i Nil-tilapia fra Victoriasjøen var tilnærmet identiske med de som ble påvist i Israel (98%), Ecuador (98%), Thailand (96%), Peru (96%) og USA (97%). TiLV ble ikke dyrket eller testet med tanke på virulens/evne til å forårsake sykdom i Nil-tilapia, og derfor anbefaler jeg videre studier for å oppfylle Kochs postulater. I studie II ble det etablert en ny og optimalisert kvantitativ RT-PCR metode for påvisning av TiLV genom i prøver fra infiserte celler med kjent titer (mengde virus) og fra organer fra infisert Nil-tilapia uten kjent titer. Metoden som ble utviklet er sensitiv og spesifikk for TiLV, og primer-effektiviteten var innenfor et akseptabelt område, 95-105%, bortsett fra primere rettet mot segment 10 (93%). Variasjonskoeffisienten for intra- og inter-analyse varierte mellom henholdsvis 0,00% ~ 2,63% og 0,00% ~ 5,92%, som er innenfor det anbefalte området (under 5%) for at en analyse skal anses som repeterbar og reproduserbar. Sensitiviteten til metoden var 2 TCID50/ml, og primere spesifikke for segmentene 1, 2, 3, 4 og 9 gav samme resultat. En nedre deteksjonsgrense på 20 TCID50/ml ble påvist for primere rettet mot segment 5, 6, 7, 8 og 10. Primere spesifikke for mot segment 3 gav høyest sensitivitet og primere segment 7 den laveste. Begge primersettene hadde en effektivitet på 98%. I artikkel III var målsettingen å forstå hemagglutinasjonsegenskapen til TiLV ved bruk av erythrocytter fra hønsefugl, tilapia og laks, samt betydningen av endocytose i tidlig fase av infeksjonen i E-11-celler. TiLV gir ikke hemagglutinering av erytrocytter fra noen av de testede artene, noe som indikerer at viruset mangler hemagglutinin. Studien har også vist at ammoniumklorid ikke påvirker, dvs. hemmer eller forsinker replikasjonen av TiLV i E-11-celler, noe som indikerer at viruset ikke tas opp ved endocytose. Samlet antyder de to observasjonene at TiLV ikke blir tatt opp av reseptormediert endocytose i E-11-celler. De gjennomførte studiene viser at det er behov for å forstå opptaksmekanismen(e) til virus, som er en viktig informasjonen for å kontrollere virusinfeksjonen med anti-virale midler eller vaksiner

Investigation of anticancer activity and mechanisms of Meso- 2,3-dimercaptosuccinic acid (DMSA) in glioblastoma cell lines

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Master of Philosophy.Glioblastoma (GBM) is an aggressive and the most prevalent form of brain tumour. It is characterized by high intertumoral and intratumoral heterogeneity and a low survival rate. Since 2005, the current gold standard of care for treating newly diagnosed GBM has remained the same: maximal safe resection, concomitant chemoradiation (CRT) with temozolomide (TMZ), and adjuvant TMZ. Thus, there is an urgent need to develop more therapeutic interventions for GBM to improve the disease prognosis. Our research group at the University of Wolverhampton has been working on repurposing known FDA approved drugs for cancer treatment. Previous studies from our group have shown that Disulfiram (DS), an FDA approved anti alcoholism drug has excellent anticancer activity against a wide range of cancers with its metal binding capability through its unique dithiol structure. Hence our group is interested in identifying clinically used dithiol compounds which are structurally similar to DS and has metal binding ability to repurpose them as potential anticancer drugs. This study focussed on determining the anticancer activity of Dimercaptosuccinic acid (DMSA), an FDA approved, orally administrated heavy metal chelator, clinically used to treat heavy metal poisoning. DMSA in combination with copper (Cu) exhibits cytotoxicity activity in GBM cell lines. DMSA+Cu, in combination with the conventional GBM drugs such as TMZ, Lomustine (CCNU) and Carmustine (BCNU), showed synergistic cytotoxicity and sensitized the GBM cell lines to first line drugs tested in this study. This study further explored the possible mechanisms of action of DMSA+Cu such as generation of reactive oxygen species (ROS), induction of DNA damage and cell death, inhibition of hypoxia induced stemness and epithelial to mesenchymal transition characteristics, which are the core anticancer mechanisms of DS+Cu. Collectively, our results show that DMSA+Cu induces DNA damage and cytotoxicity in GBM cell lines, in addition to synergistic enhancement of anti-GBM drugs. However, the mechanism of action of DMSA+Cu is very different to that of DS+Cu, especially we observed that DMSA+Cu does not target hypoxia induced cancer stem cell population or generate ROS to induce cytotoxicity. Thus, further studies on the mechanisms of DMSA+Cu induced cytotoxicity will translate DMSA as a potentially novel repurposed therapeutic agent for GBM treatment, due to its easy availability and established safety data

Molecular-genetics studies of organophosphate induced neurodegeneration in differentiating mammalian cell lines and neural progenitor stem cells

Organophosphorus (OP) pesticides are widely used despite evidence they cause neurotoxicity after exposure. The primary target for OPs is acetylcholinesterase, but there is evidence they can inhibit other cellular proteins including cytoskeletal and axon growth associated proteins, which are implicated in nervous system development. Furthermore, little is known about the ability of OPs to cause genotoxicity. The objectives of this study were to evaluate the effects of selected OPs on neurite outgrowth, expression of cytoskeletal proteins and associated gene expression levels, and to investigate histone deacetylation (HDAC) activity in three types of differentiating cell models. The initial findings indicated that cell viability was unaffected by exposure to 1, 3 and 10 µM CPF, CPO and PSP in N2a and C6 cells. A high content assay was sensitive enough to rapidly detect and quantify morphological changes, including inhibition of neurite number and length. Western blot and ELISA analysis in N2a and C6 cells revealed reduced levels of the selected cytoskeletal and associated regulatory proteins (MAP-2, Tau, βIII-tubulin, GAP43, NFH and GFAP) following the treatment with at least one concentration of CPF, CPO and PSP, which could be linked to the inhibition of neurite outgrowth. Using quantitative RT-PCR analysis on the total RNA of the genes MAP-2, TUBB3, MAPT, NEFH, GAP-43, and GFAP showed a good correlation between the altered protein expression and regulation of gene levels for most markers, which suggests these OPs can cause genotoxic effects. Increased levels of HDAC activity were observed for all OPs in rodent cell lines, suggesting that epigenetic effects may be at least partly involved in some gene expression changes. RT-PCR analysis of TUBB3, NEFH, and GFAP was also carried out in ReNcell CX cells, a co-culture of neuronal and glial cells, and showed down-regulation of gene levels for at least one concentration of all the OPs, as well as increasing the level of HDAC activity in a similar pattern to the results for N2a and C6 cells. Taken together, the data in this thesis suggest a novel action of OPs altering HDAC activity, which can be correlated to some of the observed changes in gene and protein levels of selected cytoskeletal and associated regulatory proteins that can be linked to the observed disruption of neurite outgrowth and neural development. Further work is needed to identify other molecular targets invoved in these phenomena, particularly when there is no correlation with HDAC activity changes