The outbreak of 2019 novel coronavirus disease (Covid-19) caused by SARS-CoV-2 has spread rapidly, inducing a progressive growth in infected patients number. Social isolation (lockdown) has been assessed to prevent and control virus diffusion, leading to a worldwide financial and political crisis. Currently, SARS-CoV-2 RNA detection in nasopharyngeal swab takes place by real-time PCR (RT-qPCR). However, molecular tests can give some false-negative results. In this context, serological assays can be useful to detect IgG/IgM antibodies, to assess the degree of immunization, to trace the contacts, and to support the decision to re-admit people at work. A lot of serological diagnostic kits have been proposed on the market but validation studies have not been published for many of them. The aim of our work was to compare and to evaluate different assays analytical performances (two different immunochromatographic cards, an immunofluorescence chromatographic card, and a chemiluminescence-automated immunoassay) on 43 positive samples with RT-qPCR-confirmed SARS-CoV-2 infection and 40 negative control subjects. Our data display excellent IgG/IgM specificities for all the immunocromatographic card tests (100% IgG and 100% IgM) and for the chemiluminescence-automated assay (100% IgG and 94% IgM); IgG/IgM sensitivities are moderately lower for all methods, probably due to the assay viral antigen's nature and/or to the detection time of nasopharyngeal swab RT-qPCR, with respect to symptoms onset. Given that sensitivities (around 94% and 84% for IgG and IgM, respectively) implicate false-negative cases and given the lack of effective vaccines or treatments, the only currently available procedure to reduce SARS-CoV-2 transmission is to identify and isolate persons who are contagious. For this reason, we would like to submit a flowchart in which serological tests, integrated with nasopharyngeal swab RT-qPCR, are included to help social and work activities implementation after the pandemic acute phase and to overcome lockdown.
This article belongs to the Special Issue Advances in the Toxicity Mechanisms of Microcystins Revealed by Omics Approaches ; Microcystins (MCs) are hepatotoxins produced by some cyanobacteria. They are cyclic peptides that inhibit the serine/threonine protein phosphatases (PPs) PP1 and PP2A, especially PP2A. The inhibition of PP2A triggers a series of molecular events, which are responsible for most MC cytotoxic and genotoxic effects on animal cells. It is also known that MCs induce oxidative stress in cells due to the production of reactive oxygen species (ROS). However, a complete characterization of the toxic effects of MCs is still not accomplished. This study aimed to clarify additional molecular mechanisms involved in MC-LR toxicity, using Saccharomyces cerevisiae as eukaryotic model organism. First, a shotgun proteomic analysis of S. cerevisiae VL3 cells response to 1 nM, 10 nM, 100 nM, and 1 µM MC-LR was undertaken and compared to the control (cells not exposed to MC-LR). This analysis revealed a high number of proteins differentially expressed related with gene translation and DNA replication stress; oxidative stress; cell cycle regulation and carbohydrate metabolism. Inference of genotoxic effects of S. cerevisiae VL3 cells exposed to different concentrations of MC-LR were evaluated by analyzing the expression of genes Apn1, Apn2, Rad27, Ntg1, and Ntg2 (from the Base Excision Repair (BER) DNA repair system) using the Real-Time RT-qPCR technique. These genes displayed alterations after exposure to MC-LR, particularly the Apn1/Apn2/Rad27, pointing out effects of MC-LR in the Base Excision Repair system (BER). Overall, this study supports the role of oxidative stress and DNA replication stress as important molecular mechanisms of MC-LR toxicity. Moreover, this study showed that even at low-concentration, MC-LR can induce significant changes in the yeast proteome and in gene expression. ; Key Contribution: The results of this study reveal molecular mechanisms of MC-LR toxicity in yeast related with the production of ROS, oxidative stress, and genotoxicity. ; This research was funded by the European Regional Development Fund (ERDF) through the COMPETE e Operational Competitiveness Program and national funds through FCT (Foundation for Science and Technology) grant to E. Valerio (SFRH/BPD/75922/2011). It was also funded by the Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by FCT—Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the program PT2020. This work has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 823860 (TOXICROP). ; info:eu-repo/semantics/publishedVersion
In last five years, the Africa has faced two outbreaks of Zaire ebolavirus. These outbreaks have been the largest so far, and latest outbreak is still ongoing and affecting the Democratic Republic of the Congo. We tested in parallel three different Zaire ebolavirus (EBOV) realtime RT-PCRs targeting the nucleoprotein gene (EBOV NP-RT-qPCRs) described by Trombley et al. (2010); Huang et al. (2012) and Weidmann et al. (2004). These assays are used regularly in diagnostic laboratories. The limit of detection (LOD), intra-assay repeatability using different matrixes, sensitivity and specificity were determined. In addition, the primers and probes were aligned with the sequences available in ongoing and past outbreaks in order to check the mismatches. The specificity of all three EBOV NP-RT-qPCRs were excellent (100 %), and LODs were under or 10 copies per PCR reaction. Intra-assay repeatability was good in all assays, however the Ct-values were bit higher using the EDTA-blood based matrix. All of the primers and probes in EBOV NP-RT-qPCR assays have one or more mismatches in the probes and primers when the 2267 Zaire EBOV NP sequences, including strains Ituri from DRC outbreak (year 2018), was aligned. The EBOV strain of Bikoro (year 2018) circulating in DRC was 100 % match in Trombley and Weidmann assay, but had one mismatch in Huang assay. ; Peer reviewed
The erupt of Covid-19 sickness realized by Covid-19has blowout quickly, encouraging the dynamic improvement in corrupted positive case number. Social separation (lockdown) was reviewed to hinder and control disease scattering, provoking a general cash related and political crisis. At present, Covid-19RNA area in nasopharyngeal swab occurs by nonstop PCR (RT-qPCR). In any case, sub-nuclear tests can give few fake negative outcomes. In our current one of a kind circumstances, serological measures may remain useful to recognize IgG/IgM antibodies, to assess the degree of immunization, to follow the contacts, and to push the decision to re-yield people at work. A lot of serological suggestive units were projected accessible yet endorsement looks at have not been appropriated for countless them. The current research was conducted at Mayo Hospital, Lahore from February 2020 to June 2020. Our data show astounding IgG/IgM specificities for all the immunosera motographic card tests and for the chemiluminescence-automated look at; IgG/IgM sensitivities are unobtrusively lesser for whole strategies, apparently on account of the test viral antigen's tendency or conceivably to the acknowledgment time of nasopharyngeal swab RT-qPCR, concerning signs starting. Given that sensitivities (around 96% and 86% for IgG additionally, IgM, independently) catch sham negative respondents also given the nonattendance of incredible antibodies or drugs, primary by and by open strategy to diminish Covid-19transmission is to perceive and withdraw individuals who are irresistible. Along these lines, we should introduce a flowchart where serological tests, composed with nasopharyngeal swab RT-qPCR, are joined to aid social in addition work practices utilization afterwards epidemic exceptional stage in addition to beat lockdown. Keywords: Instrument, Lock down, Covid-19, Corona Virus.
Mycotoxins contaminants, one of the most serious global challenges, have been attracted more and more attention from International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) and scientists in food safety sciences. Many countries and organizations have established the maximum contamination level of the main mycotoxins at very low values. Traditional analytic strategies are mainly based on instrumental quantitative method and immunoassays approaches. Aptamer, a novel molecules recognition element like or even superior to antibodies, has attracted more and more attentions for scientists. Therefore, specific aptamers could be employed to construct biosensors for the detection of mycotoxins. The objective of this thesis is to develop novel aptamer-based biosensors for sensitive determination of trace levels of mycotoxins and to provide a promising application of these aptasensors for more hazard factors in food safety sciences. The main contents and results are as follows: (1) Aptamer-based biosensor for detection of mycotoxins Mycotoxins are a large types of secondary metabolites appeared by fungi, they pose a great hazard and toxic reactions to human and animals. A majority of countries and regulators, such as European Union, have established series of requirements and set the maximum tolerated levels. The development of high sensitive and specific analytical platform for mycotoxins is much in demand to address new challenges for food safety in worldwide. Due to the superiority of simple, rapid, and low-cost characteristics, aptamer-based biosensors are successfully developed for the detection of various mycotoxins with high sensitivity and selectivity compared with traditional instrumental methods and immunological approaches. In this article, we discuss and analyze the development of aptasensors for mycotoxins determination in food and agricultural products during the last eleven years and cover the literatures from the first report in 2008 until today. In addition, challenges and future trends for the selection of aptamers towards various mycotoxins and aptasensors for multi-mycotoxins analysis are summarized. Given the promising development and potential application of aptasensors, the future researches will witness the great practicability of aptamer-based biosensor for food safety field. (2) A qPCR aptasensor for sensitive detection of aflatoxin M1 Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, imposes serious health hazards. AFM1 had previously been classified as a group 2B carcinogen (IARC, 1993) and has been classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) (IARC, 2002). Determination of AFM1 thus plays an important role for quality control of food safety. In this work, a sensitive and reliable aptasensor was developed for the detection of AFM1. The immobilization of aptamer through a strong interaction with biotin–streptavidin was used as a molecular recognition element, and its complementary ssDNA was employed as the template for a real-time quantitative polymerase chain reaction (RT-qPCR) amplification. Under optimized assay conditions, a linear relationship (ranging from 1.0×10-4 to 1.0 µg L-1) was achieved with a limit of detection (LOD) down to 0.03 ng L-1. In addition, the aptasensor developed here exhibits high selectivity for AFM1 over other mycotoxins and small effects from cross-reaction with structural analogs. The method proposed here has been successfully applied to quantitative determination of AFM1 in infant rice cereal and infant milk powder samples. Results demonstrated that the current approach is potentially useful for food safety analysis, and it could be extended to a large number of targets. (3) A novel graphene oxide-based aptasensor for amplified fluorescent detection of aflatoxin M1 in milk powder In this paper, a rapid and sensitive fluorescent aptasensor for the detection of aflatoxin M1 (AFM1) in milk powder has been developed. Graphene oxide (GO) was employed to quench the fluorescence of carboxyfluorescein-labelled aptamer and protect the aptamer from nuclease cleavage. Upon the addition of AFM1, a formation of AFM1/aptamer complex resulted in the aptamer detached from the surface of GO, then the aptamer was cleaved by DNase I and the target AFM1 was released for a new cycle, which led to a great signal amplification and high sensitivity. Under optimized conditions, the GO-based detection of the aptasensor exhibited a linear response to AFM1 in a dynamic range from 0.2 to 10 μg/kg, with a limit of detection (LOD) of 0.05 μg/kg. Moreover, the developed aptasensor showed a high specificity towards AFM1 without interference from other mycotoxins. In addition, the technique has been successfully applied for detection of AFM1 in infant milk powder samples. This aptasensor proposed here offers a promising technology for food safety and can be extended to various targets. (4) Graphene oxide driven fluorescent aptasensor for the detection of fumonisin B1 Fumonisin B1 (FB1), one of the most toxic mycotoxins, has been designated as possible 2B group carcinogen by the International Agency for Research on Cancer (IARC) in 2002. Therefore, simple, sensitive and specific approaches for the detection of FB1 are much in demand. In this study, a novel aptasensor was introduced for FB1 analysis based on graphene oxide (GO) and DNase I signal amplification. GO was adopted as a fluorescence quencher against ROX-modified aptamer and a protectant for the aptamer from cleavaging by DNase I for subsequent target cycling and signal amplification detection. This proposed sensing strategy exhibited a good linearity for FB1 determination in the dynamic range from 0.5 to 20 ng mL-1 with a good correlation of R2 = 0.995. Its detection of limit was established at 0.15 ng mL-1 (S/N = 3). The specific analysis indicated that this aptasensor was selective for FB1 other than other mycotoxins. In addition, the practical application in real samples of this aptasensor for the detection of FB1 was investigated. The sensing platform proposed here was useful for a potential application in the field of food safety for mycotoxins analysis. (5) Articles highlights and future perspective By using the novel aptamer-based biosensors, we developed several approaches for the detection of the most toxic mycotoxins for food safety. In addition, these sensing strategies could be applied for more hazard factors determination by simple replacement of the specific aptamers. More importantly, though the practical applicability, feasibility, and accuracy of these proposed aptasensors were investigated and evaluated through the analysis of the spiked samples experiments, the future's researches were needed for a validation of these aptasensors with real contaminated samples to determine the performances such as limit of detection and quantification, precision, trueness, accuracy, etc. Through the method validation, these aptasensors will be widely used for the detection of mycotoxins. In addition, future direction will focus on the simplification of analytic principle and devices and the combination of novel aptamers with new materials and techniques to improve the analytical performance and market practicality of aptasensors.
Calls for greater data access and research transparency have emerged on many fronts within professional social science. For example, the American Political Science Association (APSA) recently adopted new guidelines for data access and research transparency. APSA has also appointed the Data Access and Research Transparency (DA-RT) ad hoc committee to continue exploring these issues. DA-RT sponsored this symposium. In addition, funding agencies like the National Institutes for Health (NIH) and the National Science Foundation (NSF) have expanded requirements for data management and data distribution. These pressures present challenges to researchers, but they also present opportunities.
Calls for greater data access and research transparency have emerged on many fronts within professional social science. For example, the American Political Science Association (APSA) recently adopted new guidelines for data access and research transparency. APSA has also appointed the Data Access and Research Transparency (DA-RT) ad hoc committee to continue exploring these issues. DA-RT sponsored this symposium. In addition, funding agencies like the National Institutes for Health (NIH) and the National Science Foundation (NSF) have expanded requirements for data management and data distribution. These pressures present challenges to researchers, but they also present opportunities. Adapted from the source document.
Critical deliberations concerning the Data Archiving and Research Transparency effort (DA-RT) which had been set in motion within the context of the American Political Science Association's (APSA) Qualitative and Multi-Methods Research (QMMR) Section had, by the Fall of 2015, resulted in multiple conference workshops and panels, email exchanges, webpage and listserv posts, and various Section newsletter publications. Most of these seemed to come from Comparative Government and International Relations (IR) scholars, who are the mainstays of the QMMR Section. Researchers in other subfields of political science—notably, public policy, public administration, public law, and political theory—were less often heard from among those deliberations. And so Peri Schwartz-Shea and I, both of us working in the first two of those subfields, convened a roundtable at the 2016 Western Political Science Association (WPSA) meeting, "Engaging DA-RT: Critical Assessments from Public Policy and Political Theory," to address this gap. The essays in this symposium—by Renee Cramer (Drake University), Samantha Majic (John Jay College, CUNY), Amy Cabrera Rasmussen (California State UniversityLong Beach), Peregrine Schwartz-Shea (University of Utah), and Nancy J. Hirschmann (University of Pennsylvania), ordered by appearance here—were developed from those roundtable presentations. (Amy T. Linch [Pennsylvania State University] was also a member of the roundtable, but she has not joined in this written compendium.) As panel chair, I set the stage for the discussion; and it is those comments that I present here, expanded to situate DA-RT in its contemporary context.
Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r2 = 0.99). The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs (miR-93, miR-25, miR-106a, miR-17-5p and miR-26a) in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies (let-7a, miR-17-5p and miR-25 in skeletal muscle; miR-17-5p, miR-93 and miR-26a in kidney, miR-26a, miR-103 and let-7a in liver, let-7a, miR-25 and miR-106a in ovary and miR-103, let-7a and miR-93 in uterus). Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken. ; This work was supported by the projects AGL2007-66371-C02-01 and AGL2010-22358-C02-01 and by the Consolider-Ingenio 2010 program (CSD2007-00036) from Ministerio de Ciencia e Innovación. OT is recipient of PhD fellowship (Programa de Formación al Personal Investigador - FPI) from the Spanish government. ; Peer reviewed
Peste des petits ruminants (PPR) disease was first confirmed in Tanzania in 2008 in sheep and goats in Ngorongoro District, northern Tanzania, and is now endemic in this area. This study aimed to characterise PPR disease in pastoralist small ruminant flocks in Ngorongoro District. During June 2015, 33 PPR-like disease reports were investigated in different parts of the district, using semi-structured interviews, clinical examinations, PPR virus rapid detection test (PPRV-RDT), and laboratory analysis. Ten flocks were confirmed as PPRV infected by PPRV-RDT and/or real-time reverse transcription-polymerase chain reaction (RT-qPCR), and two flocks were co-infected with bluetongue virus (BTV), confirmed by RT-qPCR. Phylogenetic analysis of six partial N gene sequences showed that the PPR viruses clustered with recent lineage III Tanzanian viruses, and grouped with Ugandan, Kenyan and Democratic Republic of Congo isolates. No PPR-like disease was reported in wildlife. There was considerable variation in clinical syndromes between flocks: some showed a full range of PPR signs, while others were predominantly respiratory, diarrhoea, or oro-nasal syndromes, which were associated with different local disease names (olodua&mdash ; a term for rinderpest, olkipiei&mdash ; lung disease, oloirobi&mdash ; fever, enkorotik&mdash ; diarrhoea). BTV co-infection was associated with severe oro-nasal lesions. This clinical variability makes the field diagnosis of PPR challenging, highlighting the importance of access to pen-side antigen tests and multiplex assays to support improved surveillance and targeting of control activities for PPR eradication.
