Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

<p>Abstract</p> <p>Background</p> <p>Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (<it>tCD2-luc2</it>). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (<it>luc2-IRES-tCD2</it>). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors.</p> <p>Results</p> <p>The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. <it>In vitro</it> and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the <it>luc2-IRES-tCD2</it> construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth.</p> <p>Conclusion</p> <p>Lentiviral transduction of the chimeric <it>tCD2-luc2 </it>fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for <it>in vivo </it>imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi-cassette vectors in combination with various inserts of interest.</p

Apolipoprotein B is regulated by gonadotropins and constitutes a predictive biomarker of IVF outcomes

International audienceAbstractBackgroundFollicular fluid (FF) is an important micro-environment influencing oocyte growth, its development competence, and embryo viability. The FF content analysis allows to identify new relevant biomarkers, which could be predictive of in vitro fertilization (IVF) outcomes. Inside ovarian follicle, the amount of FF components from granulosa cells (GC) secretion, could be regulated by gonadotropins, which play a major role in follicle development.MethodsThis prospective study included 61 female undergoing IVF or Intra-cytoplasmic sperm injection (ICSI) procedure. Apolipoprotein B (APOB) concentrations in follicular fluid and APOB gene and protein expression in granulosa cells from reproductively aged women undergoing an in vitro fertilization program were measured. The statistical analyses were performed according to a quartile model based on the amount of APOB level found in FF.ResultsAmounts of APOB were detected in human FF samples (mean ± SD: 244.6 ± 185.9 ng/ml). The odds of obtaining an oocyte in the follicle and a fertilized oocyte increased significantly when APOB level in FF was higher than 112 ng/ml [i.e., including in Quartile Q 2, Q3 and Q4] (p = 0.001; p < 0.001, respectively). The probabilities of obtaining an embryo and a top quality embryo on day 2, were significantly higher if APOB levels were within the ranges of 112 and 330 ng/ml (i.e. in Q2 and Q3) or 112 and 230 ng/ml (i.e. in Q2), respectively (p < 0.001; p = 0.047, respectively). In addition, our experiments in vitro indicated that APOB gene and protein expression, along with APOB content into culture were significantly under-expressed in GC upon stimulation with gonadotropins (follicular stimulating hormone: FSH and/or human chorionic gonadotropin: hCG).ConclusionWe are reporting a positive and statistically significant associations between APOB and oocyte retrieval, oocyte fertilization, and embryo quality. Using an experimental study component, the authors report significant reduced APOB expression and content for luteinized granulosa cells cultured in the presence of gonadotropins

Characterization of single chain antibody targets through yeast two hybrid

<p>Abstract</p> <p>Background</p> <p>Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful <it>in vitro </it>selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.</p> <p>Results</p> <p>We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.</p> <p>Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed.</p> <p>Conclusions</p> <p>Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.</p

Methodology to improve water and energy use by proper irrigationscheduling in pressurised networks

With the aim of reducing energy consumption and improving water use in pressurised irrigation systems, the methodology to minimise energy consumption by grouping intakes of pressurised irrigation networks into sectors, as developed by Jimenez Bello et al. (2010a), was modified to enable irrigation intakes to operate during the scheduled period for each intake instead of operating during restricted irrigation periods of the same length. Moreover, a method was developed to detect the maximum number of intakes that can operate without extra energy if the source has sufficient head to feed at least some of the intakes. These methods were applied to a Mediterranean irrigation system, where the total cropped area was mainly citrus orchards. In this case study, water was allocated to two groups of intakes, one fed by gravity and the other by pumps. A saving of 36.3 % was achieved by increasing the total volume supplied by gravity, decreasing the injection pump head, and improving the pump performance. Therefore, all the intakes only operated during the irrigation periods at the minimum required pressure.This research was supported by funds from Climate-KIC AGADAPT and from EU 7th Framework Programme FIGARO projects. The authors wish to acknowledge the support provided by Picassent Sector XI staff. The revision of this paper was funded by "The Universitat Politecnica de Valencia, Spain".Jiménez Bello, MA.; Royuela Tomás, Á.; Manzano Juarez, J.; García Prats, A.; Martínez Alzamora, F. (2015). Methodology to improve water and energy use by proper irrigationscheduling in pressurised networks. Agricultural Water Management. 149:91-101. doi:10.1016/j.agwat.2014.10.026S9110114

Random scenarios generation with minimun energy consumption model for sectoring optimization in pressurized irrigation networks using a simulated annealing approach

A pressurized irrigation network may operate in two ways, namely, on demand and organized under operating sectors. In the first case, the user decides when to irrigate, and the pumping station has to meet the discharge and pressure head requirements of the group of users that is demanding water at any time. In the second case, the operating hydrants at a given moment are previously established, which permits identification of scenarios related to lesser energy consumption. In this work, a new model was developed that identifies such scenarios. The optimization process is carried out by means of simulated annealing (SA). The model was applied to an example and the result obtained was compared with the same network operating on demand and sectorized using the criterion of hydrant elevation with respect to the pumping station. The scenario adopted for SA saved 11.8% and 15.5% in energy consumption compared with the two other scenarios, and decreased the installed power requirement by 38.3% and 21.6%, respectively.García Prats, A.; Guillem Picó, S.; Martínez Alzamora, F.; Jiménez Bello, MA. (2012). Random scenarios generation with minimun energy consumption model for sectoring optimization in pressurized irrigation networks using a simulated annealing approach. Journal of Irrigation and Drainage Engineering. 138(7):613-624. doi:10.1061/(ASCE)IR.1943-4774.0000452S613624138

Soil microbial CNP and respiration responses to organic matter and nutrient additions: evidence from a tropical soil incubation

Soil nutrient availability has a strong influence on the fate of soil carbon (C) during microbial decomposition, contributing to Earth's C balance. While nutrient availability itself can impact microbial physiology and C partitioning between biomass and respiration during soil organic matter decomposition, the availability of labile C inputs may mediate the response of microorganisms to nutrient additions. As soil organic matter is decomposed, microorganisms retain or release C, nitrogen (N) or phosphorus (P) to maintain a stoichiometric balance. Although the concept of a microbial stoichiometric homeostasis has previously been proposed, microbial biomass CNP ratios are not static, and this may have very relevant implications for microbial physiological activities. Here, we tested the hypothesis that N, P and potassium (K) nutrient additions impact C cycling in a tropical soil due to microbial stoichiometric constraints to growth and respiration, and that the availability of energy-rich labile organic matter in the soil (i.e. leaf litter) mediates the response to nutrient addition. We incubated tropical soil from French Guiana with a ¹³C labeled leaf litter addition and with mineral nutrient additions of +K, +N, +NK, +PK and +NPK for 30 days. We found that litter additions led to a ten-fold increase in microbial respiration and a doubling of microbial biomass C, along with greater microbial N and P content. We found some evidence that P additions increased soil CO² fluxes. Additionally, we found microbial biomass CP and NP ratios varied more widely than CN in response to nutrient and organic matter additions, with important implications for the role of microorganisms in C cycling. The addition of litter did not prime soil organic matter decomposition, except in combination with +NK fertilization, indicating possible P-mining of soil organic matter in this P-poor tropical soil. Together, these results point toward an ultimate labile organic substrate limitation of soil microorganisms in this tropical soil, but also indicate a complex interaction between C, N, P and K availability. This highlights the difference between microbial C cycling responses to N, P, or K additions in the tropics and explains why coupled C, N and P cycling modeling efforts cannot rely on strict microbial stoichiometric homeostasis as an underlying assumption

Pentalogy of Cantrell: two patients and a review to determine prognostic factors for optimal approach

Two patients with incomplete pentalogy of Cantrell are described. The first was a girl with a large omphalocele with evisceration of the heart, liver and intestines with an intact sternum. Echocardiography showed profound intracardiac defects. The girl died 33 h after birth. The second patient was a female fetus with ectopia cordis (EC) without intracardiac anomalies; a large omphalocele with evisceration of the heart, stomach, spleen and liver; a hypoplastic sternum and rib cage; and a scoliosis. The pregnancy was terminated. A review of patients described in the literature is presented with the intention of finding prognostic factors for an optimal approach to patients with the pentalogy of Cantrell. In conclusion the prognosis seems to be poorer in patients with the complete form of pentalogy of Cantrell, EC, and patients with associated anomalies. Intracardial defects do not seem to be a prognostic factor

The case for strategic international alliances to harness nutritional genomics for public and personal health

Nutrigenomics is the study of how constituents of the diet interact with genes, and their products, to alter phenotype and, conversely, how genes and their products metabolise these constituents into nutrients, antinutrients, and bioactive compounds. Results from molecular and genetic epidemiological studies indicate that dietary unbalance can alter gene-nutrient interactions in ways that increase the risk of developing chronic disease. The interplay of human genetic variation and environmental factors will make identifying causative genes and nutrients a formidable, but not intractable, challenge. We provide specific recommendations for how to best meet this challenge and discuss the need for new methodologies and the use of comprehensive analyses of nutrient-genotype interactions involving large and diverse populations. The objective of the present paper is to stimulate discourse and collaboration among nutrigenomic researchers and stakeholders, a process that will lead to an increase in global health and wellness by reducing health disparities in developed and developing countrie

Global Boundary Stratotype Section and Point (GSSP) for the Anthropocene Series: Where and how to look for potential candidates

International audienc