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Adenosine-5'-triphosphate (ATP) is the primary energy carrier in all living organisms, and its detection in living cells represents a well-established approach. ATP-driven bioluminescence (BL) relying on the D-luciferin-luciferase reaction is a bioanalytical tool widely employed for monitoring hygiene and microbial contamination of foods.Here, we report a straightforward method for ATP BL detection using an ATP sensing paper fabricated with an alternative freeze-dry procedure. The assay can be easily implemented in laboratories equipped with (i) freeze-drying, wax printing, and 3D printing technologies and (ii) instrumentation for BL detection such as benchtop luminometers and portable light detectors including a smartphone camera without the need for additional equipment., Elisa Michelini acknowledges support by MIUR and Prima (Partnership for Research and Innovation in the Mediterranean Area) with FEDKITO project. ICN2 is funded by CERCA programme, Generalitat de Catalunya. Grant SEV-2017-0706 funded by MCIN/AEI/10.13039/501100011033.

This method originated due to the need to quickly and sensitively detect Avocado sunblotch viroid (ASBVd) in nursery and field trees in California. Optimum sampling protocols were developed for leaf collection from different sized trees based on size and branching as well as for fruit. An ethanol containing buffered extract from 1 g of ground leaf tissue was used as the source of RNA. The extract was absorbed onto small pieces (disks) of Whatman No. 1 filter paper which were then washed and dried. RNA was eluted from the filter paper using sterile water and used as a template in a standard single-tube RT-PCR reaction. The RNA adsorbed on the filter paper disks was quite stable, and the disks could be stored for over 1 year and shipped worldwide at ambient temperature with no noticeable decline in the quality or quantity of the resulting RT-PCR products. The filter paper capture method was expanded to the detection of other viroids including Potato spindle tuber viroid, Peach latent mosaic viroid, and Chrysanthemum stunt viroid and was tested with some viruses as well with minor modifications of the standard protocol.

Spectral flow cytometry is a new technology that enables measurements of fluorescent spectra and light scattering properties in diverse cellular populations with high precision. Modern instruments allow simultaneous determination of up to 40+ fluorescent dyes with heavily overlapping emission spectra, discrimination of autofluorescent signals in the stained specimens, and detailed analysis of diverse autofluorescence of different cells—from mammalian to chlorophyll-containing cells like cyanobacteria. In this paper, we review the history, compare modern conventional and spectral flow cytometers, and discuss several applications of spectral flow cytometry.

The efficiency of genomic selection strongly depends on the prediction accuracy of the genetic merit of candidates. Numerous papers have shown that the composition of the calibration set is a key contributor to prediction accuracy. A poorly defined calibration set can result in low accuracies, whereas an optimized one can considerably increase accuracy compared to random sampling, for a same size. Alternatively, optimizing the calibration set can be a way of decreasing the costs of phenotyping by enabling similar levels of accuracy compared to random sampling but with fewer phenotypic units. We present here the different factors that have to be considered when designing a calibration set, and review the different criteria proposed in the literature. We classified these criteria into two groups: model-free criteria based on relatedness, and criteria derived from the linear mixed model. We introduce criteria targeting specific prediction objectives including the prediction of highly diverse panels, biparental families, or hybrids. We also review different ways of updating the calibration set, and different procedures for optimizing phenotyping experimental designs.

During budding yeast mitosis, duplicated chromosomes are aligned at the center of the metaphase mitotic spindle, and the centromeres are stretched by forces generated within the mitotic spindle. In response to these stretching forces, mechanical tension builds up in the centromeric chromatin. The magnitude of this tension is detected by the cell to signal the attachment configuration of the sister chromosomes: a high tension signal would indicate that sister chromosomes are properly attached to opposite spindle poles, while a low tension signal could indicate the lack of a bipolar attachment. A low tension signal drives the cell to correct improper attachments in metaphase, thus preventing potential errors in anaphase chromosome segregation. In this paper, we describe a microscopy-based method to directly measure the magnitude of centromere tension in budding yeast metaphase spindles. The advantage of this method is that quantitative tension estimates are obtained without perturbing spindle and/or chromosome structure, and as cells progress normally through mitosis.

International audience; Colloidal gels are formed through the aggregation of attractive particles, whose size ranges from 10 nm to a few micrometers, suspended in a liquid. Such gels are ubiquitous in everyday life applications, from food products to paints or construction materials, in particular thanks to their ability to easily “yield”, i.e., to turn from a solid to a liquid under the application of a weak external load. Understanding and controlling the mechanical response of colloidal gels is therefore of prime importance. Depending on the details of the system, however, the resulting gel networks present different microstructural organizations that may lead to widely different mechanical responses. This raises important challenges in fully characterizing yielding and in uncovering the mechanisms of nonlinear response in colloidal gels. In this paper, we distinguish between two classes of colloidal gels showing respectively reversible yielding, where the gel network reforms upon load release, and irreversible yielding, where the network is fully destroyed through fractures and phase separation. This broad, empirical distinction is achieved through rheology and local experiments at a mesoscopic scale, intermediate between the network characteristic size and the sample size. We further discuss how the observables derived from creep and fatigue experiments may be modeled to predict yielding and highlight open questions and future research directions in the domain.

Microarrays are broadly used in the omic investigation and have several areas of applications in biology and medicine, providing a significant amount of data for a single experiment. Different kinds of microarrays are available, identifiable by characteristics such as the type of probes, the surface used as support, and the method used for the target detection. To better deal with microarray datasets, the development of microarray data analysis protocols simple to use as well as able to produce accurate reports, and comprehensible results arise. The object of this paper is to provide a general protocol showing how to choose the best software tool to analyze microarray data, allowing to efficiently figure out genomic/pharmacogenomic biomarkers.

In this paper, we describe the application of electrochemical impedance spectroscopy (EIS) to characterize process of formation and properties of solid-supported tethered bilayer membranes on solid conducting substrates. Along with the description of experimental procedures to prepare substrates and self-assembly of phospholipid bilayers onto gold-coated glass slides, we describe the detailed protocols of EIS measurements. We demonstrate the utility of EIS in the evaluation of the properties of both molecular anchor layers used to immobilize tBLMs as well as characterization of tBLMs. We show that the EIS methodology extends the applicability of this technique well beyond the mere evaluation of electric parameters. Specifically, we demonstrate how by using EIS one may evaluate both density and size of water-filled defects (ion-channels) in tBLMs, to determine the structural mode (homogeneous, heterogeneous, or clustered) of distribution of defects in tBLMs. Our methodology can be applied in both basic protein membrane interaction studies, as well as in the development of precision biosensoric systems with tBLMs as a sensing element.

A wireless wearable sensor on a paper substrate was used to continuously monitor respiratory behavior that can extract and deliver clinically relevant respiratory parameters to a smartphone. Intended to be placed horizontally at the midpoint of the costal margin and the xiphoid process as determined through anatomical analysis and experimental test, the wearable sensor is compact at only 40 × 35 × 6 mm3 in size and 6.5 g weight including a 2.7 g lithium battery. The wearable sensor, consisting of an ultrasound emitter, an ultrasound receiver, wireless transmission system, and associated data acquisition, measures the linear change in circumference at the attachment location by recording and analyzing the changes in ultrasound pressure as the distance between the emitter and the receiver changes. Changes in ultrasound pressure corresponding to linear strain are converted to temporal lung volume data and are wirelessly transmitted to an associated custom-designed smartphone app. Processing the received data, the mobile app is able to display the temporal volume trace and the flow rate vs. volume loop graphs, which are standard plots used to analyze respiration. From the plots, the app is able to extract and display clinically relevant respiration parameters, including forced expiratory volume delivered in the first second of expiration (FEV1) and forced vital capacity (FVC). The sensor was evaluated with eight volunteers, showing a mean difference of the FEV1/FVC ratio as bounded by 0.00-4.25% when compared to the industry-standard spirometer results. By enabling continuous tracking of respiratory behavioral parameters, the wireless wearable sensor helps monitor the progression of chronic respiratory illnesses, including providing warnings to asthma patients and caregivers to pursue necessary medical assistance.

A powerful method for examining genetic fitness and function on a large scale is to couple saturating transposon mutagenesis with high-throughput sequencing (TnSeq). By mapping where transposon insertions can be tolerated in a genome, it is possible to analyze the fitness of every gene in a genome simultaneously under a given growth condition. While this technique can describe genes as essential or nonessential under those growth conditions, sufficient mutagenesis and sequencing depth can provide more subtle differences in fitness. In this paper, TnSeq was used to analyze gene fitness of two Alphaproteobacteria from different environments: the freshwater oligotroph Brevundimonas subvibrioides (Caulobacterales) and the soil plant pathogen Agrobacterium tumefaciens (Rhizobiales) for the purpose of comparing conservation of gene function.