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Reuse of nanofibers in a Biological Warfare Agent detection nanophotonic sensing device
(CBRNE Research and Inovation, 2019-05-29) Peraile, Inés; Lorenzo Lozano, P.; González López, L.; Murillo, Nieves; Dabbagh Escalante, Nushin Alba; Rozas Sanz, Gabriel; Cabria Ramos, J. C.; Gil García, M.
"The use of biological injurious agent ravages not only at human population but also at livestock and crops [1], causing effects both on human health and on the economy [2]. Therefore, the fast and specific detection of these biological injurious agents is necessary in many fields such as environmental control, clinic diagnostic, food industry, Security and Defence. Therefore, much effort has been devoted to the development of new technologies for their detection and identification. The priority objective is to carry out a low cost, easy-of-use and specific device to be used on site and able to be miniaturized [3]. In order to meet this need, the specificity of the antigen-antibody binding is applied in a multitude of biosensors. Among these immuno-biosensors, biological warfare agent detection equipment stands out. Provided both the high costs for this equipment and the need for the first responders to move into the CBRN accident area, it could be desirable to have reusable detection devices.
In the OPTONANOSENS project, an immuno-biosensor with nanophotonic sensing has been developing. In this device, nylon nanofibers with 5% pyridine, manufactured by Tecnalia laid on a metallic support is used to immobilized antibodies [4,5]. The aims of this work are both to make a comparison between several procedures which let these nanofibers to be reused in different detection events and the optimisation of the more suitable procedure which allows a repetitive use and reliable of the biofunctionalized nanofibers in the final sensing device."
Rapid identification of Bacillus anthracis by real-time PCR with dual hybridization probes in environmental swabs
(Elsevier, 2017-11-04) Bassy Alvarez, Olga; Jiménez, Oscar; Ortega García, María Victoria; Granja, Carmen; Cabria Ramos, J. C.
In the present study, we report the development of a real-time PCR assay for the identification of Bacillus anthracis, based on the amplification of a unique chromosomal marker, the E4 sequence, with dual hybridization probes. The assay was evaluated using a panel of ten B. anthracis strains, two B. anthracis isolates from human clinical samples, 12 B. anthracis environmental swabs and 40 non- B. anthracis strains. All 12 B. anthracis strains and clinical isolates were correctly detected, and the method did not show cross-reactions with other micro-organisms. Likewise, the E4 sequence was not found in those strains of B. thuringiensis and B. cereus closely related (homology > 90%) to B. anthracis by computer analysis. On the other hand, this molecular assay showed a high analytical sensitivity, 3.5 genome equivalents per reaction at 95% probability. Furthermore, the real-time PCR assay allowed sequence-specific detection of the amplicon (melting peak with a Tm of 63.5 °C ± 0.5 °C) without post-amplification procedures, which offers an additional advantage over other qPCR assays for B. anthracis detection. Finally, the performance of the method was successfully evaluated in 12 environmental samples. In summary, we have developed a rapid and specific method for the molecular identification of Bacillus anthracis in environmental samples.
Recovery of Francisella tularensis from soil samples by filtration and detection by real-time PCR and cELISA
(Royal Society of Chemistry, 2008-01-17) Sellek Cano, Ricela; Jiménez, Oscar; Aizpurua, Carmen; Fernández Frutos, Begoña; De León, Patricia; Camacho, Maite; Fernández Moreira, Daniel; Ybarra, Carmen; Cabria Ramos, J. C.
The aim of this study was to develop a specific and highly sensitive method able to detect very low concentrations of Francisella tularensis in soil samples by real-time PCR (qPCR) with SYBR Green I. tul4gene, which encodes the 17-kDa protein (TUL4) in F. tularensis strains, was amplified using a LightCycler (LC) device. We achieved a detection limit of 0.69 fg of genomic DNA from F. tularensis subp. holarctica live vaccine strain (LVS), corresponding to a value less than 3.4 genome equivalents per reaction. The qPCR was shown to be specific, highly sensitive and reproducible. In addition, we evaluated 2 new methods for recovering bacteria from soil based on 1-step filtration using glass fiber filters and PVDF filters. These filtration methods enabled us to recover F. tularensis efficiently from soil samples. As few as 50 CFU per 0.5 g of soil were detected by qPCR. Capture enzyme-linked immunosorbent assay (cELISA) allowed us to detect and quantify the amount of bacteria recovered from soil by an immunological method. Although qPCR was more sensitive than cELISA, we did not observe substantial differences in the amount of bacteria quantified by both methods.
Preliminary validation of real-time PCR assays for the identification of Yersinia pestis
(De Gruyter Brill, 2008-07-21) Tomaso, Herbert; Jacob, Daniela; Eickhoff, Meike; Scholz, Holger; Al Dahouk, Sascha; Kattar, Mireille; Reischl, Udo; Plicka, Helga; Strand Olsen, Jaran; Nikkari, Simo; Matero, Pirjo; Beuret, Christian; Ciammaruconi, Andrea; Lista, Florigio; Gala, Jean Luc; Broll, Hermann; Appel, Bernd; Sellek Cano, Ricela; Ybarra, María del Carmen; Broekhuijsen, Martien; Indra, Alexander; Petersen, Roger; Neubauer, Heinrich
Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis.
Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values.
Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results.
Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.
Phenotypic and genetic analyses of 111 clinical and environmental O1, O139, and non-O1/O139 Vibrio cholerae strains from different geographical areas
(Cambridge University Press, 2012-08-05) Sellek Cano, Ricela; Niemcewicz, Marcin; Olsen, Jaran; Bassy Alvarez, Olga; Lorenzo Lozano, P.; Martí, L.; Roszowiak, A.; Kocik, J.; Cabria Ramos, J. C.
A total of 111 clinical and environmental O1, O139 and non-O1/O139 Vibrio cholerae strains isolated between 1978 and 2008 from different geographical areas were typed using a combination of methods: antibiotic susceptibility, biochemical test, serogroup, serotype, biotype, sequences containing variable numbers of tandem repeats (VNTRs) and virulence genes ctxA and tcpA amplification. As a result of the performed typing work, the strains were organized into four clusters: cluster A1 included clinical O1 Ogawa and O139 serogroup strains (ctxA + and tcpA +); cluster A2 included clinical non-O1/O139 strains (ctxA − and tcpA −), as well as environmental O1 Inaba and non-O1/O139 strains (ctxA − and tcpA −/tcpA +); cluster B1 contained two clinical O1 strains and environmental non-O1/O139 strains (ctxA − and tcpA +/tcpA −); cluster B2 contained clinical O1 Inaba and Ogawa strains (ctxA + and tcpA +). The results of this work illustrate the advantage of combining several typing methods to discriminate between clinical and environmental V. cholerae strains.
OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay
(Springer Nature Link, 2014-06-18) Paauw, Armand; Trip, Hein; Niemcewicz, Marcin; Sellek Cano, Ricela; Heng, Jonathan; Mars Groenendijk, Roos; De Jong, Ad; Majchrzykiewicz Koehorst, Joanna; Olsen, Jaran; Tsivtsivadze, Evgeni
Background
Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks.
Results
The use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay.
Conclusions
The extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved.
Two neighbours of the ultra-short-period Earth-sized planet K2-157 b in the warm Neptunian savanna
(EDP Sciences, 2025-07-22) Castro-González, Amadeo; Bouchy, F.; Correia, A. C. M.; Sozzetti, A.; Lillo Box, J.; Figueira, P.; Lavie, B.; Lovis, C.; Hobson, Melissa J.; Sousa, S. G.; Adibekyan, Vardan; Standing, Matthew R.; Hara, Nathan C.; Barrado, David; Silva, André; Bourrier, V.; Korth, J.; Santos, Nuno C.; Damasso, M.; Zapatero Osorio, María Rosa; Rodrigues, José; Alibert, Yann; Barros, S. C. C.; Cristiani, S.; Marcantonio, Paolo Di; González Hernández, J. I.; Lo Curto, G.; Martins, C. J. A. P.; Nunes, Nelson J.; Pallé, E.; Pepe, Francesco; Suárez Mascareño, A.; Tabernero, H. M.; Fundacao para a Ciencia e a Tecnologia (FCT); European Commission (EC); Centros de Excelencia Severo Ochoa, INSTITUTO DE ASTROFÍSICA DE CANARIAS (IAC), SEV-2015-0548
[Context] The formation and evolution of ultra-short-period (USP) rocky planets is poorly understood. However, it is widely thought that these planets could not have formed at their present-day close-in orbits, but instead migrated inwards through interactions with outer neighbours.
[Aims] We aim to confirm and characterise the USP Earth-sized validated planet K2-157 b (Porb = 8.8 h) and constrain the presence of additional companions in the system through radial velocity (RV) measurements.
[Methods] We measured 49 RVs with the ESPRESSO spectrograph and tested different planetary and non-planetary configurations to infer the model that best represents our data set. We derived the orbital and physical properties of the system through a global RV and transit model.
[Results] We detected two additional super-Neptune-mass planets located within the warm Neptunian savanna, K2-157 c (Porb,c = 25.942−0.044+0.045d, Mp,c sin i = 30.8 ± 1.9 M⊕) and K2-157 d (Porb,d = 66.50−0.59+0.71d, Mp,d sin i = 23.3 ± 2.5 M⊕). The joint analysis constrains the mass of K2-157 b at the 2.7σ level, Mp,b = 1.14−0.42+0.41 M⊕ (< 2.4 M⊕ at 3σ), which, together with the inferred radius, Rp,b = 0.935 ± 0.090 R⊕, make the planet compatible with a rocky composition with a likely (68% confidence) higher iron-to-silicate mass fraction than Earth. K2 data discard non-grazing transit configurations for K2-157 c (ic < 88.4° at 3σ), and ESPRESSO data constrain the eccentricities of K2-157 c and K2-157 d to ec < 0.2 and ed < 0.5 at 3σ. Our dynamical analysis indicates that the system is stable for eccentricities up to ec, ed ~ 0.3 and mutual inclinations up to ~60°. At a population level, we find that the trend that the closest USP planets tend to orbit late-type stars does not hold when scaling the orbital separation to the Roche limit, which suggests that the orbital distribution of the closest planets across spectral types is primarily determined by tidal disruption.
[Conclusions] The orbital architecture of K2-157 is unusual in the known exoplanet plethora, with only one similar case reported to date: 55 Cnc. The USP planets of these systems, being accompanied by massive, long-period, relatively spaced, and possibly misaligned neighbours, could have migrated inwards through eccentricity-based mechanisms triggered by secular interactions.
