Enhanced analysis of real-time PCR data by using a variable efficiency model : FPK-PCR

Current methodology in real-time Polymerase chain reaction (PCR) analysis performs well provided PCR efficiency remains constant over reactions. Yet, small changes in efficiency can lead to large quantification errors. Particularly in biological samples, the possible presence of inhibitors forms a challenge. We present a new approach to single reaction efficiency calculation, called Full Process Kinetics-PCR (FPK-PCR). It combines a kinetically more realistic model with flexible adaptation to the full range of data. By reconstructing the entire chain of cycle efficiencies, rather than restricting the focus on a 'window of application', one extracts additional information and loses a level of arbitrariness. The maximal efficiency estimates returned by the model are comparable in accuracy and precision to both the golden standard of serial dilution and other single reaction efficiency methods. The cycle-to-cycle changes in efficiency, as described by the FPK-PCR procedure, stay considerably closer to the data than those from other S-shaped models. The assessment of individual cycle efficiencies returns more information than other single efficiency methods. It allows in-depth interpretation of real-time PCR data and reconstruction of the fluorescence data, providing quality control. Finally, by implementing a global efficiency model, reproducibility is improved as the selection of a window of application is avoided

Optimization of a viability PCR method for the detection of Listeria monocytogenes in food samples

Postprint (author's final draft

Simulation of between repeat variability in real time PCR reactions

While many decisions rely on real time quantitative PCR (qPCR) analysis few attempts have hitherto been made to quantify bounds of precision accounting for the various sources of variation involved in the measurement process. Besides influences of more obvious factors such as camera noise and pipetting variation, changing efficiencies within and between reactions affect PCR results to a degree which is not fully recognized. Here, we develop a statistical framework that models measurement error and other sources of variation as they contribute to fluorescence observations during the amplification process and to derived parameter estimates. Evaluation of reproducibility is then based on simulations capable of generating realistic variation patterns. To this end, we start from a relatively simple statistical model for the evolution of efficiency in a single PCR reaction and introduce additional error components, one at a time, to arrive at stochastic data generation capable of simulating the variation patterns witnessed in repeated reactions (technical repeats). Most of the variation in C-q values was adequately captured by the statistical model in terms of foreseen components. To recreate the dispersion of the repeats' plateau levels while keeping the other aspects of the PCR curves within realistic bounds, additional sources of reagent consumption (side reactions) enter into the model. Once an adequate data generating model is available, simulations can serve to evaluate various aspects of PCR under the assumptions of the model and beyond

Validation of an enzyme-linked immunosorbent assay for the quantification of human IgG directed against the repeat region of the circumsporozoite protein of the parasite Plasmodium falciparum.

BACKGROUND: Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines. METHODS: The validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum. RESULTS: The anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time. CONCLUSIONS: This ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine

The role of aggregates in the thermal stability of Mg-PSZ refractories for vacuum induction melting

Mg-PSZ refractories used as vacuum induction melting crucibles are particle-reinforced composites with aggregate and matrix phases comprising fused zirconia. Three commercial varieties were cycled eight times to service temperatures and their microstructural and thermomechanical evolution investigated, with focus placed on the aggregate populations. Two refractories, with large aggregates of similar size, were found to retain stiffness after cycling but in the refractory containing aggregates with high stabiliser levels, reaction between the stabiliser and Al and Si impurities produced secondary phases. Volume changes accompanying formation of these phases, and subsequent thermal expansion mismatches, led to aggregate break-up with consequent reductions in refractory toughness and strength. Secondary phases developed only rarely in the aggregates (with lower levels of stabiliser) of the second refractory. These aggregates remained intact and the refractory retained its toughness and strength. A third refractory contained small, unstabilised aggregates in a stabilised matrix and the strain mismatches that ensued during polymorphic transformation damaged microstructural interfaces. Refractory stiffness halved within eight cycles and toughness and strength were lost. All three refractories displayed R-curve behaviour and quasi-stable fracture curves were observed during bend tests. The study shows that when using fused zirconia aggregates to design refractories, engineers need to i) limit stabiliser concentrations - a difference of just ±1 wt% Mg (in the presence of impurity elements) may determine whether secondary phase formation occurs and ii) eliminate alumina and silica impurities when possible through substitution of zircon sand with baddeleyite as the source for fused zirconia.Open Acces

How to perform RT-qPCR accurately in plant species?: a case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

Background: Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. Results: An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. Conclusions: Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway

The State of the Science of Natural Family Planning Fifty Years after Humane Vitae: A Report from NFP Scientists’ Meeting Held at the US Conference of Catholic Bishops, April 4, 2018

A one-day meeting of physicians, professional nurses, and scientists actively involved in Natural Family Planning (NFP) research was held to review the state of the science of NFP and consider future priorities. The meeting had four objectives: (i) determine the gaps in research evidence for secure methods of NFP among women of all reproductive categories, (ii) determine the gaps in the research and development of new technology for providing NFP services, (iii) determine the gaps in the research that determine the benefits and challenges with use of NFP among married couples, and (iv) provide prioritized ideas for future research needs from the analysis of evidence gaps from objectives above. This article summarizes the discussion and conclusions drawn from topics reviewed. While much has been accomplished in the fifty years since Humane vitae, there are still many gaps to address. Five areas for future research in NFP were identified as high priority: (1) well-designed method effectiveness studies among various reproductive categories including important subpopulations (postpartum, perimenopause, posthormonal contraceptive), normally cycling women (especially US women), and comparative studies between NFP methods; (2) validation studies to establish the benefit of charting fertility signs (both currently known and potential new indicators) as a screening tool for women’s health issues; (3) ongoing independent evaluation of fertility monitoring apps to provide users perspective on the relative merits of each and to identify those most worthy of further effectiveness testing; (4) studies evaluating the impact of new technologies on NFP adoption, use, and persistence; and (5) creation of a shared database across various NFP methods to collaborate on shared research interests, longitudinal studies, and so on. This summarizes a meeting to review the scientific and medical progress related to natural family planning made in the 50 years since Humane Vitae and to define priorities for future work. Areas reviewed included the evidence for avoiding pregnancy in normally cycling, postpartum, and perimenopausal women, the impact of new technology, including fertility charting apps, on NFP, and the impact on relationships and personal well-being from use of NFP. Five priority focus areas for future research were also identified

Extraction of anthocyanins and total phenolic compounds from açai (euterpe oleracea mart.) using an experimental design methodology. part 1: Pressurized liquid extraction

Currently, açai is one of the most important fruits present in the world. Several studies have demonstrated its high content in phenolic compounds and anthocyanins. Both of them are responsible of interesting properties of the fruit such as anti-inflammatory, antioxidant or anticancer. In the present study, two optimized pressurized liquid extraction (PLE) methods have been developed for the extraction of anthocyanins and total phenolic compounds from açai. A full factorial design (Box-Behnken design) with six variables (solvent composition (25%-75% methanol-in-water), temperature (50-100°C), pressure (100-200 atm), purge time (30-90 s), pH (2-7) and flushing (50%-150%)) were employed. The percentage of methanol in the extraction solvent was proven to be the most significant variable for the extraction of anthocyanins. In the case of total phenolic compounds, the extraction temperature was the most influential variable. The developed methods showed high precision, with relative standard deviations (RSD) of less than 5%. The applicability of the methods was successfully evaluated in real samples. In conclusion, two rapid and reliable PLE extraction methods to be used for laboratories and industries to determine anthocyanins and total phenolic compounds in açai and its derived products were developed in this work

Capturing the ‘ome’ : the expanding molecular toolbox for RNA and DNA library construction

All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application

Efficacy of spa-therapy, mud-pack therapy, balneotherapy and mud-bath therapy in the management of knee osteoarthritis. A systematic review

Background: Osteoarthritis (OA) is the most common musculoskeletal disease in the world. OA is the result of an inflammatory and degenerative process affecting the entire joint. Osteoarthritis, especially involving the knee, has a relevant socio-economic impact in terms of drugs, hospital admissions, work absences and temporary or permanent invalidity. Therapy of knee osteoarthritis is based on pharmacological and non-pharmacological measures. Methods: We conducted a systematic review of the studies published between 2002 and 2017 on spa-therapy, mud-pack therapy, balneotherapy and mud-bath therapy in the treatment of knee osteoarthritis in order to investigate the evidence of the efficacy of such treatment on pain, functional limitation, drug use and quality of life. Overall, 35 studies were examined among which 12 were selected and included in the review if trial comparative. Each report was reviewed to identify the criteria used for study enrolment and for assignment to experimental vs control groups, sample size, type and characteristics of treatment, features of mineral water, control intervention, assessment point, endpoints, outcome measures, tests used for statistical analysis of the results. We have been able to illustrate the main results obtained in the individual studies and to elaborate these results in order to allow as much a unitary presentation as possible, and hence an overall judgment. Results: Because the studies we reviewed differed markedly from one another in terms of the methods used, we were unable to conduct a quantitative analysis (meta-analysis) of pooled data from the 12 studies. For the purposes of the present review, we re-evaluated the results of the different studies using the same statistical method, the Student’s t test, which is used to compare the means of two frequency distributions. Among all the studies, the most relevant indexes used to measure effectiveness of spa therapy were improved including VAS, Lequesne’s and WOMAC Score. Conclusions: The mud-pack therapy, balneotherapy, mud-bath therapy and spa therapy has proved to be effective in the treatment and in the secondary prevention of knee osteoarthritis, by reducing pain, non-steroidal antiinflammatory drug consumption, functional limitation and improving quality of life of affected patients. Is a noninvasive, complication-free, and cost-effective alternative modality for the conservative treatment of knee osteoarthritis