A dual paper-based nucleic acid extraction method from blood in under ten minutes for point-of-care diagnostics.

Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the kelch 13 gene from P. falciparum. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution (eluted disk) as well as direct placement of the disk in the LAMP reaction (in situ disk). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually any POC setting. When combined with LAMP, our approach provides sample-to-result detection in under 35 minutes

Preliminary optical design of PANIC, a wide-field infrared camera for CAHA

In this paper, we present the preliminary optical design of PANIC (PAnoramic Near Infrared camera for Calar Alto), a wide-field infrared imager for the Calar Alto 2.2 m telescope. The camera optical design is a folded single optical train that images the sky onto the focal plane with a plate scale of 0.45 arcsec per 18 micron pixel. A mosaic of four Hawaii 2RG of 2k x 2k made by Teledyne is used as detector and will give a field of view of 31.9 arcmin x 31.9 arcmin. This cryogenic instrument has been optimized for the Y, J, H and K bands. Special care has been taken in the selection of the standard IR materials used for the optics in order to maximize the instrument throughput and to include the z band. The main challenges of this design are: to produce a well defined internal pupil which allows reducing the thermal background by a cryogenic pupil stop; the correction of off-axis aberrations due to the large field available; the correction of chromatic aberration because of the wide spectral coverage; and the capability of introduction of narrow band filters (~1%) in the system minimizing the degradation in the filter passband without a collimated stage in the camera. We show the optomechanical error budget and compensation strategy that allows our as built design to met the performances from an optical point of view. Finally, we demonstrate the flexibility of the design showing the performances of PANIC at the CAHA 3.5m telescope.Comment: This paper has been presented in the SPIE of Astronomical Telescopes and Instrumentation 2008 in Marseille (France

A dual-sensing thermo-chemical ISFET array for DNA-based diagnostics.

This paper presents a 32x32 ISFET array with in-pixel dual-sensing and programmability targeted for on-chip DNA amplification detection. The pixel architecture provides thermal and chemical sensing by encoding temperature and ion activity in a single output PWM, modulating its frequency and its duty cycle respectively. Each pixel is composed of an ISFET-based differential linear OTA and a 2-stage sawtooth oscillator. The operating point and characteristic response of the pixel can be programmed, enabling trapped charge compensation and enhancing the versatility and adaptability of the architecture. Fabricated in 0.18 μm standard CMOS process, the system demonstrates a quadratic thermal response and a highly linear pH sensitivity, with a trapped charge compensation scheme able to calibrate 99.5% of the pixels in the target range, achieving a homogeneous response across the array. Furthermore, the sensing scheme is robust against process variations and can operate under various supply conditions. Finally, the architecture suitability for on-chip DNA amplification detection is proven by performing Loop-mediated Isothermal Amplification (LAMP) of phage lambda DNA, obtaining a time-to-positive of 7.71 minutes with results comparable to commercial qPCR instruments. This architecture represents the first in-pixel dual thermo-chemical sensing in ISFET arrays for Lab-on-a-Chip diagnostics

¿Van las investigaciones en ciencias sociales y en humanidades en la dirección correcta?

Product of a post-rational vision and, therefore, apparently postmodern. Is the latter the central point, which prompts the question: how has this postmodern vision generated the increasing development of obscurantism, radical constructivism and relativism to the point of being filtered in academic spaces, reflecting today so much acceptance? That this work should not be understood as intolerance to freedom of expression, or a tendency towards scientific dogma, but rather, to problematize the correct path towards which work and academic life should be followed. Given what has been stated, it is proposed, -responding to the question of the topic that has been proposed- that indicated development it responds to a distorted and appropriate academic freedom to the needs of who expresses it, where the slogan; It is no longer how to approach understanding the world; rather, what is it that makes the academic decision and scientific and cultural production more viable for particularities.Producto de una visión post-racional y, por consiguiente, al parecer postmoderna. Es esto último el punto central, el cual propicia la pregunta ¿cómo esta visión postmoderna ha generado el acreciente desarrollo del obscurantismo, el constructivismo radical y el relativismo hasta el punto de estar filtrado en los espacios académicos reflejando hoy, tanta acogida? Que no se entienda el presente trabajo como intolerancia a la libertad de expresión, o una tendencia al dogma cientificista sino, problematizar el camino correcto hacia donde se debe seguir el trabajo y la vida académica. Dado lo planteado se propone, -respondiendo a la pregunta del tema que se ha propuesto- que indicado desarrollo responde a una libertad académica distorsionada y adecuada a las necesidades de quien la manifiesta, donde la consigna; ya no es, cómo aproximarnos a entender el mundo; sino, qué es lo que hace más viable para particularidades, la decisión académica y la producción científica y cultural

PANIC: the new panoramic NIR camera for Calar Alto

PANIC is a wide-field NIR camera, which is currently under development for the Calar Alto observatory (CAHA) in Spain. It uses a mosaic of four Hawaii-2RG detectors and covers the spectral range from 0.8-2.5 micron(z to K-band). The field-of-view is 30x30 arcmin. This instrument can be used at the 2.2m telescope (0.45arcsec/pixel, 0.5x0.5 degree FOV) and at the 3.5m telescope (0.23arcsec/pixel, 0.25x0.25 degree FOV). The operating temperature is about 77K, achieved by liquid Nitrogen cooling. The cryogenic optics has three flat folding mirrors with diameters up to 282 mm and nine lenses with diameters between 130 mm and 255 mm. A compact filter unit can carry up to 19 filters distributed over four filter wheels. Narrow band (1%) filters can be used. The instrument has a diameter of 1.1 m and it is about 1 m long. The weight limit of 400 kg at the 2.2m telescope requires a light-weight cryostat design. The aluminium vacuum vessel and radiation shield have wall thicknesses of only 6 mm and 3 mm respectively.Comment: This paper has been presented in the SPIE of Astronomical Telescopes and Instrumentation 2008 in Marseille (France

Effect of Co-Inoculation with Mycorrhiza and Rhizobia on the Nodule Trehalose Content of Different Bean Genotypes

Studies on Rhizobium-legume symbiosis show that trehalose content in nodules under drought stress correlates positively with an increase in plant tolerance to this stress. Fewer reports describe trehalose accumulation in mycorrhiza where, in contrast with rhizobia, there is no flux of carbohydrates from the microsymbiont to the plant. However, the trehalose dynamics in the Mycorrhiza-Rhizobium-Legume tripartite symbiosis is unknown. The present study explores the role of this tripartite symbiosis in the trehalose content of nodules grown under contrasting moisture conditions. Three wild genotypes (P. filiformis, P. acutifolis and P. vulgaris) and two commercial genotypes of Phaseolus vulgaris (Pinto villa and Flor de Mayo) were used. Co-inoculation treatments were conducted with Glomus intraradices and a mixture of seven native rhizobial strains, and trehalose content was determined by GC/MS. The results showed a negative effect of mycorrhizal inoculation on nodule development, as mycorrhized plants showed fewer nodules and lower nodule dry weight compared to plants inoculated only with Rhizobium. Mycorrhizal colonization was also higher in plants inoculated only with Glomus as compared to plants co-inoculated with both microsymbionts. In regard to trehalose, co-inoculation negatively affects its accumulation in the nodules of each genotype tested. However, the correlation analysis showed a significantly positive correlation between mycorrhizal colonization and nodule trehalose content

Allele-Specific Isothermal Amplification Method Using Unmodified Self-Stabilizing Competitive Primers.

Rapid and specific detection of single nucleotide polymorphisms (SNPs) related to drug resistance in infectious diseases is crucial for accurate prognostics, therapeutics and disease management at point-of-care. Here, we present a novel amplification method and provide universal guidelines for the detection of SNPs at isothermal conditions. This method, called USS-sbLAMP, consists of SNP-based loop-mediated isothermal amplification (sbLAMP) primers and unmodified self-stabilizing (USS) competitive primers that robustly delay or prevent unspecific amplification. Both sets of primers are incorporated into the same reaction mixture, but always targeting different alleles; one set specific to the wild type allele and the other to the mutant allele. The mechanism of action relies on thermodynamically favored hybridization of totally complementary primers, enabling allele-specific amplification. We successfully validate our method by detecting SNPs, C580Y and Y493H, in the Plasmodium falciparum kelch 13 gene that are responsible for resistance to artemisinin-based combination therapies currently used globally in the treatment of malaria. USS-sbLAMP primers can efficiently discriminate between SNPs with high sensitivity (limit of detection of 5 × 101 copies per reaction), efficiency, specificity and rapidness (<35 min) with the capability of quantitative measurements for point-of-care diagnosis, treatment guidance, and epidemiological reporting of drug-resistance

Amplification curve analysis: Data-driven multiplexing using real-time digital PCR

Information about the kinetics of PCR reactions are encoded in the amplification curve. However, in digital PCR (dPCR), this information is typically neglected by collapsing each amplification curve into a binary output (positive/negative). Here, we demonstrate that the large volume of raw data obtained from realtime dPCR instruments can be exploited to perform data-driven multiplexing in a single fluorescent channel using machine learning methods, by virtue of the information in the amplification curve. This new approach, referred to as amplification curve analysis (ACA), was shown using an intercalating dye (EvaGreen), reducing the cost and complexity of the assay and enabling the use of melting curve analysis for validation. As a case study, we multiplexed 3 carbapenem-resistant genes to show the impact of this approach on global challenges such as antimicrobial resistance. In the presence of single targets, we report a classification accuracy of 99.1% (N = 16188) which represents a 19.7% increase compared to multiplexing based on the final fluorescent intensity. Considering all combinations of amplification events (including coamplifications), the accuracy was shown to be 92.9% (N = 10383). To support the analysis, we derived a formula to estimate the occurrence of co-amplification in dPCR based on multivariate Poisson statistics, and suggest reducing the digital occupancy in the case of multiple targets in the same digital panel. The ACA approach takes a step towards maximizing the capabilities of existing real-time dPCR instruments and chemistries, by extracting more information from data to enable data-driven multiplexing with high accuracy. Furthermore, we expect that combining this method with existing probe-based assays will increase multiplexing capabilities significantly. We envision that once emerging point-of-care technologies can reliably capture real-time data from isothermal chemistries, the ACA method will facilitate the implementation of dPCR outside of the lab

4-1BBL as a Mediator of Cross-Talk between Innate, Adaptive, and Regulatory Immunity against Cancer

The ability of tumor cells to evade the immune system is one of the main challenges we confront in the fight against cancer. Multiple strategies have been developed to counteract this situation, including the use of immunostimulant molecules that play a key role in the anti-tumor immune response. Such a response needs to be tumor-specific to cause as little damage as possible to healthy cells and also to track and eliminate disseminated tumor cells. Therefore, the combination of immunostimulant molecules and tumor-associated antigens has been implemented as an antitumor therapy strategy to eliminate the main obstacles confronted in conventional therapies. The immunostimulant 4-1BBL belongs to the tumor necrosis factor (TNF) family and it has been widely reported as the most effective member for activating lymphocytes. Hence, we will review the molecular, pre-clinical, and clinical applications in conjunction with tumor-associated antigens in antitumor immunotherapy, as well as the main molecular pathways involved in this association