The excessive use of antibiotics in food-producing animals causes a steady rise of multiple antibiotic resistance in foodborne bacteria. Next to sulfonamides, the most common antibiotics groups are fluoroquinolones, aminoglycosides, and ß-lactams. Therefore, there is a need for a quick, efficient, and low-cost detection procedure for antibiotics. In this study, we propose an inkjet-printed aptamer-based biosensor developed for the detection of the fluoroquinolone ciprofloxacin. Due to their extraordinary high affinity and specificity, aptamers are already widely used in various applications. Here we present a ciprofloxacin-binding RNA aptamer developed by systematic evolution of ligands by exponential enrichment (SELEX). We characterized the secondary structure of the aptamer and determined the KD to 36 nM that allow detection of antibiotic contamination in a relevant range. We demonstrate that RNA aptamers can be inkjet-printed, dried, and resolved while keeping their functionality consistently intact. With this proof of concept, we are paving the way for a potential range of additional aptamer-based, printable biosensors. The increasing antibiotic resistance of bacteria represents one of the main public health concerns. Excessive use of antibiotics both in therapy and food production is often assumed to be the main cause . An inevWith the discovery of SELEX , a technique that allows aptamer development for specific recognition of any target molecule, the possibility for a new type of biosensor has arisen ,4. AptamBiosensors that make use of an aptamer for target recognition are called aptasensors. With respect to their detection system, optical aptasensors can be subdivided into different subclasses: fluorescence (labeled and label-free), colorimetric , chemiluminescence and other optical aptasensors  . In recePrinting of biosensors is a way to easily and rapidly produce a device potentially capable of transferring a biosensing reaction from the test tube in the lab to a sensor for use in the field . If evenWe recently reported the selection of CFX-binding RNA aptamers aimed at the development of new synthetic riboswitches . In addiHere we exploit the CFX aptamer R10K6_V11 as CFX aptasensor. We performed a detailed structure–function analysis and characterized the fluorescence-based detector system. We then inkjet-printed the aptasensor onto paper-based support material. We analyzed functionality after printing and repeated drying–resolving cycles. The experiments show that the aptasensor can be inkjet-printed without damage and that drying and resolving of aptamers is possible while maintaining their functionality.2 and 100 mM KCl. For a size marker, radioactive-labeled RNA was subjected to alkaline hydroxylation by incubating it for 3 min at 96 °C in 50 mM Na2CO3 (pH 9.0). In addition, RNA was incubated with 20 U RNase T1 for 3 min at 55 °C to identify guanines [The RNA aptamer was dephosphorylated and subsequently 5’ labeled with 32[P]-γ-ATP as previously described . After pguanines . All reaBy measuring the fluorescence quenching as a function of RNA concentration in the presence of CFX or CFX-derivatives, the dissociation constants (KD) for RNA-ligand complexes were determined. The fluorescence intensities were measured on a Fluorolog FL3-22 (Horiba Jobin Yvon). Excitation and emission wavelengths for CFX and each CFX-derivative are given in 2 (pH 7.4).In preparation for the titration, the RNA solution was heated to 95 °C for 5 min and directly snap-cooled on ice for 5 min to ensure proper folding of the RNA. Afterwards, binding buffer was added to a final concentration of 40 mM HEPES, 125 mM KCl, 5 mM MgClY = max*Xh/(KDh + Xh)B, with maxB = maximum binding, h = hill slope, X = concentration of RNA. All measurements were performed at least twice.All measurements were performed with an integration time of 0.5 s at 25 °C. 50 nM CFX or fluoroquinolone-derivative was mixed with increasing amounts of folded RNA and the fluorescence intensity was measured. Between each step of RNA addition, the solution was stirred for 1 min and equilibrated for 1 min. Curve fitting was done using Prism (GraphPad Software) and nonlinear regression analysis with following equation by least squares fitting: 2 squares onto the paper-carriers. The filter paper Whatman Grade 1  was chosen for its weak autofluorescence at the excitation and emission peaks of CFX [2. When combined with 5 µL 10 µM CFX solution, a ratio of aptamer to CFX can be calculated. The parameters used for the experiments are given in The printing experiment consisted of five steps: Aptamer printing, drying, CFX application, redrying, and rehydration. The printing system Autodrop  was used for printing varying amounts  of 26.67 µM RNA into 1 cms of CFX . The depAfter printing, the samples were dried at room temperature for at least one day. One set was dried and stored for eight days before applying CFX. The quenched fluorescence of the applied CFX onto the printed aptamer squares was measured using the imager Fusion FX Edge by Vilber Lourmat . The best fitting excitation illumination (peak at 312 nm) and absorption filter (470–590 nm) were close to the excitation and emission peaks of the analyzed fluoroquinolones. As the UV-transilluminator is fitted with parallel tubes that produce inhomogeneous illumination, each sample set was introduced separately at the same spot along the center tube.Afterwards, the samples were stored again and re-dried at room temperature. After one week, the spots were rehydrated with 5 µL drops of Millipore Milli-Q (MQ) water and pictures were taken with the imager before and afterwards.The 103 nt long CFX-binding RNA aptamer R10K6 was previously selected . R10K6 sThe secondary structure of the aptamer was predicted by mfold ,20,21, F and confThe chemical structure of CFX is shown in Fluorescence titration spectroscopy was then performed with different fluoroquinolone derivatives to analyze the influence of the different side groups on the binding behavior. In The maximum residue limit (MRL) for CFX in food samples, e.g., milk, is approximately 300 nM (100 ppb) ,24 mark. Thus, tFor robust application as a biosensor, it is advantageous if the aptamer is present in a minimal and compact structure and areas that are not necessary for binding are removed. We therefore designed mutants in which different regions of the aptamer had been deleted and determined their remaining binding capacity. The results are summarized in The complete removal of stem P5 and P5a (mutation V4) resulted in a complete loss of CFX binding. In all likelihood, parts of the CFX binding pocket are destroyed with this mutation, especially nucleotide U61. Previously, U61 was identified as part of the major binding pocket by in-line probing A,B. HoweFinally, the R10K6_V11 was revealed as the most functional mutant. It represents a shorter version of the aptamer R10K6 without loss of ligand binding affinity. In comparison to R10K6, R10K6_V11 is shortened by 19 nts and has a final length of 84 nts. The determined dissociation constant of 36 nM for V11 is similar to the original full-length aptamer R10K6 (31 nM).In the next step, we performed inkjet-printing experiments to build a paper-based detector platform for CFX. The sensor principle relies on quenching of the autofluorescence of CFX after being bound by the aptamer. A sensor usually produces a signal when the target is detected. In this case, the detection of the target was associated with a decrease of the signal. Hence, a washing step would be counterproductive as our detection was based on measuring the fluorescence of the unbound CFX.The aptamer solution was printed onto a defined location and the CFX solution was pipetted on top. As unbound CFX will fluoresce, the ratio between aptamer and CFX molecules was varied by printing increasing amounts of the aptamer solution and keeping the CFX concentration and amount constant. This way allowed us to find the ideal ratio, where most of the CFX molecules are bound by the aptamer. For evaluation, we considered four different scenarios. The first scenario represented the ideal situation: the fluorescence intensity was measured directly after CFX application in a wet state. But we were also interested in investigating storage options after application, for example when there is a need to mail the used sensor to a lab for further analysis. There, it might be investigated in its dried state or after rehydration. After storage of seven days, the used sensor is in a dry state. We expected that the evaluation would yield incorrect results on a dried sensor and wanted to try to rehydrate the spots by applying 5 µL of water to each spot in the third scenario. A fourth test took the storability of the printed aptamer into account by storing a printed sample for eight days and then testing its CFX-binding in a wet state.2 squares onto filter paper. By using different distances between printed drops and hence different numbers of drops for each square, 0.25, 0.5, 1, 2, and 5 µL of the aptamer solution were added  did not have a major impact on ligand binding. Moreover, the experiments revealed that especially the carboxyl-group (dCFX) on C3 is important for CFX binding.Since the European MRL for CFX in milk is about 300 nM (100 ppb) ,24, the In addition, we were able to build a paper-based detector platform. We found that the aptamers, which were printed onto filter paper and dried overnight, still retained their functionality of quenching the fluorescence of CFX. After repeated drying of the sample, fluorescence faded slightly and increased again after rehydration of the spots with water. Evidently, fluorescence is strongest while wet and should be measured in a wet state. Another sample, which was stored for a week after printing, showed the strongest fluorescence after CFX application. This indicates that some functionality of the aptamers is lost during storage on paper at room temperature. Different storing conditions should be investigated in the future and could improve the functional integrity.In sum, inkjet printing of RNA-based aptamers allowed us to build a simple detector platform for the detection of CFX on paper-based support material that makes use of a high affinity CFX-binding aptamer. The results demonstrate that an RNA-based aptamer can be printed and dried without loss of functionality. This proof of concept forms the basis for a wide range of potential RNA-aptamer based biosensors on paper-based support material.


Co-transcriptionally folding RNA nanostructures have great potential as biomolecular scaffolds, which can be used to organize small molecules or proteins into spatially ordered assemblies. Here, we develop an RNA tile composed of three parallel RNA double helices, which can associate into small hexagonal assemblies via kissing loop interactions between its two outer helices. The inner RNA helix is modified with an RNA motif found in the internal ribosome entry site (IRES) of the hepatitis C virus (HCV), which provides a 90° bend. This modification is used to functionalize the RNA structures with aptamers pointing perpendicularly away from the tile plane. We demonstrate modifications with the fluorogenic malachite green and Spinach aptamers as well with the protein-binding PP7 and streptavidin aptamers. The modified structures retain the ability to associate into larger assemblies, representing a step towards RNA hybrid nanostructures extending in three dimensions. Over the past two decades, RNA has been found to be involved in many essential cellular processes other than the conventional roles it fulfils as mRNA, tRNA, or rRNA. Non-coding RNAs (ncRNAs) and many RNA–protein complexes are involved in regulatory functions at the transcriptional and translational levels, and have roles in scaffolding , genome-Most naturally occurring RNAs are single-stranded and contain self-complementary sequences that facilitate intramolecular folding into distinct secondary structures. In addition, rigid structural motifs consisting of canonical or noncanonical base-pairing, kissing interactions, and stacking of helices play a significant role in RNA folding, resulting in complex 3D structures exhibiting helices, loops, junctions, bulges, stems, hairpins, and pseudoknots ,5,6. SeeA technique dubbed cotranscriptional ssRNA origami has been developed, in which a single RNA strand folds into a predefined RNA tile that further assembles into hexagonal and rectilinear lattices while the RNA is produced by the RNA polymerase . It emplRationally designed RNA nanostructures and devices have great potential for applications in synthetic biology, metabolic engineering, and nanomedicine ,17,18,19In the present work, we construct a novel three-helix “antiparallel even” RNA tile (3H-AE) based on the cotranscriptional ssRNA origami approach. In our design, the outer two helices of the 3H-AE tile provide a rigid RNA scaffold that can interact with other tiles via kissing loop (KL) interactions. The central RNA double-helical extensions, however, are conceived as modular plug-in modules, which can be modified at will without interfering with two-dimensional tile assembly. Specifically, we modified the plug-in helices with subdomain IIa of the internal ribosomal entry site (IRES) of the Hepatitis C virus (HCV) genome , which az-direction.Our results demonstrate the modularity of the RNA origami tile approach and represent a step towards multifunctional RNA assemblies extending in three dimensions. The most salient feature of the three-helix tile structure introduced in this work is the modular extension of the middle helix with aptamer functions. This allows for connecting the tiles in 2D while presenting separate binding modalities on the two sides of the 2D assembly. Furthermore, it is conceivable to create 3D lattices from such structures by also polymerizing along the 3H-AE and its subsequent modifications were designed by the cotranscriptional ssRNA origami method that has been previously described in detail ,29. BrieAssemble [After the initial modeling, the 3D structures were ligated with a Perl script  and an Assemble . The seqPrimers were generated specific to DNA sequence generated by Nupack and their melting temperature was calculated using the NEB Tm calculator .Genetic templates for all RNA tiles were amplified from “custom dsDNA gBlocks” from Integrated DNA Technologies (IDT) using the polymerase chain reaction (PCR). RNA tiles were then prepared using in vitro transcription from these templates as described in detail in the The formation of the RNA tiles was characterized using gel electrophoresis and atomic force microscopy (AFM). The transcription of RNA tiles modified with fluorogenic aptamers was followed using fluorescence spectroscopy. RNA tiles containing streptavidin aptamers were further investigated using streptavidin-coated microbeads. Detailed experimental procedures are found in the three RNA double helices placed over one another connected via double crossovers, and converted into a cotranscriptionally folding continuous ssRNA using one 180° KLs, four 120° KLs and four tetraloops  RNA tile as described in detail in the Methods section above. In contrast to previously described RNA tiles , it was traloops a. The ceCustom dsDNA segments containing the sequence for the 3H-AE RNA tile and its subsequent modifications were ordered from a gene synthesis supplier and PCR amplified using a Phusion High-Fidelity PCR Master mix (with HF or GC buffer). The purity and amplification of the samples was checked using agarose gel electrophoresis . TranscrWe next designed modified versions of the 3H-AE RNA tile with one or more fluorescent RNA aptamers. The first version included the replacement of one of the two interior tetraloops  by two additional RNA motifs—subdomain IIa of the internal ribosomal entry site (IRES) of the HCV genome (PDB ID: 2PN4) and the malachite green aptamer (MGA) a. SubdomThis modification resulted in an increase in the number of nucleotides in the tile from 248 to 297 nt, which was verified by denaturing PAGE b. The prWe also created a variation of 3H-AE-MGA V1 by replacing the connecting motif subdomain IIa (PDB ID: 2PN4) by the slightly larger HCV IRES domain IIa (PDB ID: 1P5M), which includes a few additional unpaired bases. As a result, the length of the tile was increased by six bases from 297 nt to 303 nt (3H-AE-MGA V2), and the angle of MGA with respect to the tile was expected to tilt slightly . CharactNext, the complexity of the MGA-functionalized 3H-AE tiles was further increased by the addition of another aptamer via an additional subdomain IIa connected to the second internal tetraloop of the tile structure. As shown in As before, transcription of the double-modified RNA tile was monitored via the fluorescence signal generated by the two aptamers in their respective emission channels , indicatWe also designed a modification of 3H-AE-MGA-Spinach-KL, in which the 120° KLs were replaced by tetraloops to avoid inter-tile interactions, keeping all the other features unaltered. The non-interacting tile displayed similar behavior in native gel electrophoresis and fluorescence experiments . An enhaFor specific applications, the fluorogenic aptamer modifications can be easily replaced with other functional RNA sequences. As a proof of concept, two variants of 3H-AE-MGA-spinach-Tetraloop structure were designed and constructed with the MGA replaced by the PP7 aptamer  or a strIn order to indicate one potential application for double-functionalized 3H-AE tiles, we utilized the streptavidin aptamer module to specifically arrange RNA tiles on the surface of streptavidin-coated polystyrene microbeads. 3H-AE tiles double-functionalized with the Spinach and streptavidin aptamer are expected to present the Spinach aptamer in an orientation pointing away from the surface of the beads a. IndeedThe results demonstrated here represent initial efforts towards the design and synthesis of multifunctional tile-based RNA nanostructures extending in three dimensions. Three-helix RNA tiles were folded from a single RNA strand, which was composed of a variety of naturally occurring RNA motifs that assist in reaching the desired target shape. The main focus of the present work was put on domain IIa and subdomain IIa, which are RNA motifs found in the HCV IRES. Both these motifs contain a 90° bend that allows positioning of other RNA modules such as fluorogenic RNA aptamers perpendicular to the RNA tile structure. As examples, we created 3H-AE tile structures either modified with a single malachite green aptamer or with MGA and a Spinach aptamer pointing in opposite directions. Importantly, the modified 3H-AE-tiles retained the ability to associate into hexagonal assemblies via kissing loop interactions.Among the most promising applications of such multi-functionalized RNA tile structures, we envision the creation of artificial ribonucleoprotein complexes, in which RNA structures scaffold the co-localization and arrangement of proteins, e.g., for membrane-less compartmentalization or for the creation of multienzyme structures. As the RNA tiles can be made to polymerize in 2D, it is also conceivable to create extended RNA sheets or “membranes”, which are functionalized with aptamers on either side, which could be used to further spatially organize proteins in order to enhance their structural or enzymatic functions. From these, in turn, one could generate RNA/protein covered surfaces  via self-assembly of such structures. Importantly, such self-assembly processes could all be controlled via gene expression from a plasmid. From a nanotechnology point of view, another interesting opportunity lies in the possibility to stack several such RNA sheets on top of each other—mediated by the perpendicular RNA extensions, resulting in multilayered RNA nanostructures extending in 3D.


Interferometric Photo-Activation-Localization-Microscopy (iPALM) localizes single fluorescent molecules with 20 nm lateral and 10 nm axial resolution. We present a method utilizing glass coverslip lithography for correlative imaging between iPALM and scanning electron microscopy (SEM). Using iPALM on HIV Gag-Dendra virus-like particles (VLPs) we localized the position of HIV Gag proteins. Based on these localizations we reconstructed the central cavity of the VLPs along with imperfections within the HIV Gag lattice. The SEM images and iPALM images overlap and show imaging from single VLPs immobilized on glass coverslips. The localization of many HIV proteins including accessory proteins and Gag-Pol remains unknown, we discuss how the specificity of iPALM coupled with SEM has the potential for resolving more of HIV proteins.The online version of this article (10.1007/s00249-018-1324-0) contains supplementary material, which is available to authorized users. Resolution of fluorescence microscopy is limited by diffraction; however, the advantage of fluorescence is that fluorescent tags are very specific. The position of a single fluorescent molecule can be determined based on the center of its diffraction limited image with nanometer precision  (similar diameter to full length HIV ~ 120 nm in diameter)  for 45 s and the mask cleaning took place using a spin rinse dryer (SRD). The mask design pattern and sizes were verified by optical microscopy. The mask was placed in a chromium etch 1020AC  for 2.5 min, cleaned in DI water for 2 min, and then cleaned in an SRD.2. The second one was dipping the glass in BOE (buffered oxide etch) for few seconds followed by DI water rinse and dry with N2. The final result of fabrication with the first cleaning method was more successful as it produced better contrast between the pattern and the glass while imaging.The first approach was using acetone/isopropanol (IPA) sonication for 5 min each and drying with NAfter cleaning steps the glasses were heated on hot plate at 120 °C for 10 min and then they cool down to room temperature after few minutes.2 and post baked using a hot plate at 110 °C for 2 min.Coverslips were spin-coated with hexamethyldisilazane (HMDS)/xylene (20:80) at 3000 rpm for 60 s to improve adhesion of photoresist to surface. After this step positive photoresist was spin coat S1813  at 3000 rpm for 60 s followed by soft bake using a hot plate at 110 °C for 1 min. The coverslip alignment with lithography mask was done using a Suss MA1006 aligner in hard contact mode. The coverslips were then exposed to UV light for 10 s and dipped into AZ developer 1:1  for 45 s. Coverslips were then rinsed in DI water and dried with NA BOE (buffered oxide etch or buffered HF) was used to etch the pattern for 3 min. This step was followed by thorough rinse with DI water. The etch quality was checked using an optical microscope and a profilometer to measure etch depth of 250 nm. Finally, the photoresist was removed by acetone and isopropanol (IPA) sonication for few minutes.4 precipitation technique. Both cells and media were collected for analysis. VLPs were pelleted from cell supernatants by centrifugation for 2 h through 10% (w/v) sucrose cushion at 15,000×g. Final VLP samples were re-suspended in PBS.Humanized Gag was produced as previously described  using two Nikon 60× TIRF objectives, NA 1.46 and a custom three-way beam splitter as previously described . At each of the planes the fluorescence of the fiducial passes through a three-phase beam splitter. Monitoring the intensity change of these fiducials during the z stack reveals three separate interferograms with 120° phase shift on the three cameras. The raw data of this calibration were used to generate a look-up table for data post processing of the blinking events of the fluorescent proteins of the sample during an iPALM experiment.Raw data acquisition of iPALM consisted of two steps: Calibration of the setup using permanent fluorescent fiducials (20 nm Gold nanoparticles), and the experimental run detecting fluorescent events for 3D localization. During calibration the photons from the permanent fluorescing Gold nanoparticles were collected from the top and bottom objectives. The calibration procedure contained a well defined stepwise axial movement of the sample in between the two objectives  and used to identify regions of interest on a Helios SEM (Thermo Scientific). For fine alignment between reconstructed iPALM images and SEM images Gold fiducials were used as markers in Maps. The final SEM image was reconstructed by creating a high resolution carpet image from six independent images with 10% overlapping scans as shown in Fig. Z-stacks recorded from 20 nm Gold nanoparticles (Nanopartz) located in the observed region. Gold nanoparticles were also used to correct for drift in X, Y and Z.iPALM data were rendered in 3D using PeakSelector  as described in  in SEM without relying on specific markers etched on the coverslip surface. As shown in Fig. iPALM data were acquired for 10,000 frames in which Dendra molecules were localized in each frame and their 3D position recorded. Activated Dendra molecules photobleach within a few frames and therefore their localization is limited to the few frames in which the Dendra signal appears. The Gold nanoparticles, however, fluoresce with similar intensity in all acquired frames. Therefore, as shown in Fig. SEM images and iPALM images are overlapped so the Gold nanoparticles identified in both imaging modules overlap as shown in Fig. X, Y and Z coordinates, to compare with SEM image; during rendering the data are pixelated. The iPALM image obtained after rendering was given a 180° flip about the horizontal axis in the plane (about the x axis).To generate the overlap, the co-ordinates of the Gold nanoparticles were registered for both SEM The transformation equations are:The above equation can be written in the matrix form as:The Thus the β matrix contains the transform coefficients.The coefficients are obtained by running a minimization routine (linear regression):X and 16 nm along Y across the full 30 µm × 30 µm image as shown in Fig. We were able to produce overlap of the two images through translation and rotation operations without introducing warp operations to within 22 nm along As shown in the zoom areas in Fig. Once a VLP is identified in SEM along with its corresponding iPALM data, the iPALM data are plotted in 3D and represent the Gag-Dendra arrangement inside of the virus particle. Figure Given the 15 nm deposition of Gold Cadmium between iPALM imaging and SEM we estimate that the reported sizes of the virions by the SEM should be corrected by the thickness of Gold Cadmium and the inevitable slight collapse of the VLPs and therefore our reported average of VLP diameters of 180 nm is an overestimate of VLP diameters before SEM.The iPALM gallery of Dendra localization within the VLPs, shows clear areas within the virions that are void of any Dendra molecules and represent virion cavities and defects in the Gag lattice.When middle section of the virions are imaged in iPALM, both cavity and lattice spaces void of Gag-Dendra are visible. The VLPs deposited on glass were purified from transfection of Gag-Dendra only and therefore all Gag molecules are expected to carry Dendra. However, approximately only half of the expressed Dendra molecules are actually fluorescent . The probability of having no Dendra molecule within this voxel is therefore 0.997 out of one. Given there are 700 Dendra molecules, having a 10 nm3 voxel void of any Dendra is (0.997)700 = 0.12 which is 12%. All voids found in the cavity of the virions as shown in the gallery in Fig. 3 voxels. The probability of a void composed of n (10 nm3) voxels can be calculated as (0.12)n, therefore any void exceeding a volume encompassing two or more 10 nm3 voxels has a probability below 1% to be due to random positioning of Dendra molecules. This calculation is based on most generous distribution of Dendra within the volume, in reality the Dendra molecules are confined to a shell within the lattice and therefore the probability of having a void 10 nm3 on this shell is significantly lower.As shown below however, based on the statistical calculations, the contribution of dark Dendra to observed voids is insignificant. In average we detect about 700 individual Dendra molecules within each VLP. It is helpful to calculate what would be the probability of detecting a void assuming random distribution of these molecules. The probability of having a Dendra molecule within a 10 nmOptical imaging on HIV budding and recruitment of cellular proteins has shed light on assembly kinetics of virions  Fig. S1: Lithography of Glass coverslips. The images show (A) zoom-out view of the mask showing the general pattern, (B) area imaged around one set of islands, (C) detailed zoom, showing the position of 5 × 5 µm islands, which are etched 250 nm deepSupplementary material 2 (TIFF 402 kb) Fig. S2: Spatial distribution of errors in CLEM. After performing the regression algorithm and finding the best translation, rotation and scale matrix components, the position of Gold nanoparticles was analyzed and the residual difference in the position of Gold particles between SEM and iPALM is plotted as indication of error. As shown in the graph, the error in aligning SEM and iPALM is nonlinear and is more pronounced at certain positions in the sample than others .Below is the link to the electronic supplementary material.


Dairy cow welfare has become a significant topic in recent years. Lying (down) behavior is considered a useful indicator for dairy cow health, welfare, reproductive and productive status. The study evaluated the interaction of climatic conditions on the lying behavior of a group of dairy cows. The developed model seems helpful to identify and predict this important indicator of the welfare of the herd. The prediction model developed, with automatic monitoring of cow behavior, could be a valid early warning system to identify any deviation from the expected behavior, and could be also used to evaluate the goodness of management and to evaluate the heat stress mitigation strategy.p < 0.001) and the r2 was 0.84. All of the effects in the model resulted in being highly significant (p < 0.001). This model, if validated properly, could be a valid early warning system to identify any deviation from the expected behavior, and to assess the effectiveness of thermal stress mitigation strategies.Currently, lying behavior can be assessed using continuous observations from sensors . The analysis of digital data deriving from accelerometers is an effective tool for studying livestock behaviors. Despite the large interest in the lying behavior of dairy cows, no reference was found in literature regarding the prediction of lying behavior as a function of the interaction of environmental parameters. The present study aimed to evaluate the influence of climatic conditions  on the lying behavior of a group of primiparous dairy cows, using data from accelerometers, and develop a prediction model to identify and predict the lying behavior of dairy cows as a function of the effects of environmental conditions. Results from the. GLM Procedure (SAS) showed that the model was highly significant ( Dairy cow welfare has become a significant topic in recent years. Time spent lying down, the frequency and the duration of lying bouts, are considered to be useful indicators for dairy cow health, welfare, reproductive and productive status ,2. DairyOver the course of the day, this time spent lying down varies, with a peak before morning milking, and in the middle of the day between the two daily milkings . In AMS Lying down and resting behaviors are important for the cow, and often, better welfare is associated with longer lying periods . ChangesCurrently, lying behavior can be assessed using continuous observations from video recordings or data from sensors  ,13,14. TLikewise, accelerometers and bio-logger have the great potential of providing large amounts of behavioral data for days or months, and their use is spreading compared to video analysis due to reduced size, weight, low cost and embedded algorithms that provide valuable information regarding individual behaviors and dynamic processes within the herd . Use of Lying behavior in free stall barns is affected by barn design and management factors ,19, sociFurthermore, the lying time can be used as a measure of a cow’s welfare, also in the case of heat stress , that seIn particular, reduced milk productivity and reproductive disorders, such as silent estrus and calving difficulty, can seriously affect the economic balance of the farm .Heat stress is an important threat to cattle breeding, especially in the Mediterranean basin , where tExperiencing uncomfortable thermal conditions  affects the capacity of cattle to dissipate heat, and leads to an increase in body temperature over the physiological limits .Several authors evaluated the effects of heat stress in this species using the temperature-humidity index (THI), an index that combines the simultaneous effect of temperature and humidity, finding a good relationship between this index and the welfare status of cows ,29,32,33Recently, a predictive model for physiological responses, which incorporated air temperature, relative humidity, air velocity and solar radiation and their interactions, was developed and the results were mildly correlated with skin and body temperature and respiration rate .However, despite the large interest in the lying behavior of dairy cows, no reference was found in literature regarding the description of lying behavior as a function of the interaction of environmental parameters.The present study aimed to evaluate the influence of climatic conditions  on the lying behavior of a group of primiparous dairy cows, using data from accelerometers and developing a model to describe the lying behavior of dairy cows as a function of the effects of environmental conditions.Data were collected at the experimental farm A. Menozzi  of the University of Milan, between October 2013 and August of 2014. Dairy Cows were housed in a free-stall pen in a loose-housing layout with a total of 90 cubicles having rubber mats and 60 feeding places .In the study a total of 21 primiparous Holstein dairy cows  were monitored. For each cow, the monitoring period started from the first days after calving and lasted around 150 days. Parturition period ranged from 3 September 2013 to 27 May 2014. The primiparous group belonged to the same feeding group, the proportion between the number of cows and the number of pens and feeding places was 1.5 and 0.9, respectively.A total mixed ration (TMR) was delivered once daily beginning at approximately 10:00. Cows had ad libitum access to six water troughs and were fed a TMR consisting of 33.6% maize silage, 18.5% high moisture corn 12.5% concentrate, 8.4% soybean meal, 6.7% maize meal, 6.2% alfalfa hay, 5.4% cotton seeds, 4.7% grass hay, 2.6% molasses and 1.6% mineral supplement by dry weight. Cows were milked two times daily at approximately 08:30 and 21:00. Stalls were cleaned manually once daily at approximately 09:00.Feed bunk had sprinkler nozzles spaced 1 m apart; these were mounted about 2 m high and angled to avoid wetting the feed, creating a spray radius extending approximately 1.5 m from the feed bunk. The cooling system has been activated when the temperature-humidity index (THI) reached a value of 68.x-axis of the data logger was perpendicular to the ground. The degree of vertical tilt of the x- and z-axis was used to determine the standing and lying behavior of the animal [Lying behavior patterns of 21 cows were automatically recorded for 342 days using HOBO Pendant G Data Loggers . In the first week cows were equipped with an individual HOBO Data Logger, and their activities recorded continuously for 150 days. These devices measured leg orientation at 1-min intervals, and allowed all the standing and lying behavior data to be collected electronically . The deve animal . Data coData collection for this study was performed at a commercial farm, and all the monitoring actions and procedures did not affect the behavior of the cows, and did not change the comfort or welfare of the animals monitored. All the used sensors are widely used in dairy husbandry, since they are not invasive for cows.T is the temperature in °C, and RH is the relative humidity [Four data loggers were used to measure the air temperature and relative humidity continuously during the trial duration . The data loggers were placed in four separate locations at a height of about 2 m above the floor . The rechumidity .An average THI was determined from the calculated THI for each position in the barn.https://www.dati.lombardia.it/Ambiente/Stazioni-Meteorologiche/nf78-nj6b) also provided information relative to temperature (°C), relative humidity (%), global radiation (W/m2), precipitation (mm) and wind speed (m/s).A weather station located in the farm , and part of the regional meteorological network  was applied . In the model, the mean daily lying time (LY) was modeled as a function of the week of the year and of the interaction of THI, solar radiation (RAD), air velocity (WIND) and precipitation (RAIN) (LY = WEEK + (THI × RAD × WIND × RAIN)).Despite the presence of THI, that takes in account the air moisture, precipitation was included in the model to evaluate the effect of this parameter on lying behavior. Indeed, as reported by Bohmanova et al.  heat strIn this study all these data were available and were all included in the model to make the model more robust. Furthermore, all the variables in the data set were grouped into classes, to avoid the inhomogeneity of the sample. The classes included in the model were: The week of the year , the THI (five classes), the solar radiation , the air velocity  and precipitation . The division in classes was performed according the classification of winds for the WIND parameter, and according the Notes on the Temperature-Humidity Index  for the THI parameter. The RAD was classified according the distribution of data, and the precipitation parameter was divided in the presence or absence of rain .2, WIND < 1 m/s and no rain, in a specific week of the year.This model allows us to describe the expected mean lying time based on the combination of each class of each effect with the other classes of the other effects. As an example, it allows us to estimate the mean daily lying time when the THI is in the <50 class combined with a RAD < 40 W/mAt the end of the analysis, predicted values resulting from the GLM procedure were then compared with observed values with the PROC CORR .Mean daily lying time reported in Daily variation of the Temperature Humidity Index (THI), precipitation (RAIN), solar radiation (RAD) and air velocity (WIND) during the trial is reported in p < 0.001) and the r2 was 0.84. All of the effects in the model resulted in being highly significant (p < 0.001). In Results from the PROC GLM showed that the model was highly significant . During rainy days cows tend to spend more time lying respect to clear days (11.4 h vs 9.2 h in average). In this range of THI the lying time range is 9.6–11.7 h, slightly lower than the previous class (THI > 50). During sunny days (RAD above 121 W/m2), dairy cows increased the time spent lying as the WIND increased, reaching and exceeding the 11 h in the cubicle.Regarding THI between 50 and 57, that corresponds to a temperature of 8–13 °C , there iWith THI values included in the range of 57–62 . Also, in this case (THI = 57–62) the time spent lying increased with increasing RAD and WIND.The general tendency for the lying time is much lower than the previous classes, and, except for a couple of values, the expected lying time is lower than 10 h. What is worthy to note is that at the same RAD, what affects the behavior more is the WIND. The highest value (more than 14 h) is expected when the RAD is in a sunny day, between 121 and 240 W/m2 the lying time exceeded 10 h, increasing with the increase of WIND.When the THI increased between 62 and 69, (temperatures between 19 and 24 °C), the expected lying time ranged from 6.5 to 13 h . The maxOver a value of 70 for THI, the lying time was in general lower than previous classes , rangingp < 0.001) between the predicted values and the observed showed the high correspondence between the two datasets.The correlation coefficient of 0.92  represent the quota of data variability explained by the model. The high significance of the interaction of THI, RAD, RAIN and WIND, make evident the strong impact of all these factors on dairy cows’ lying behavior. Although it is already known the effect of each individual factor on cow behavior [The explanatory model developed in this study through the GLM procedure highlights the importance of climatic conditions on dairy cow behavior. Indeed, GLM results and the rbehavior ,38,39, tAs it is possible to see from All the changes in lying behavior are the result of the combination of climatic conditions, and they should be evaluated as the expected daily lying duration. In general, climatic conditions can induce a strong thermoregulatory response in cows , and aniWhat emerges first is the fact that with low THI, low RAD, high WIND and the presence of RAIN, cows tend to stay more in the cubicle.Longer time in the cubicle (more than 12 h) is sometimes associated with higher temperature, high WIND and high RAD, as in Furthermore, several authors found that the effect of RAD has a great impact on dairy cows, affecting how they behave in terms of barn usage and physiological changes ,39,44. IAs expected, high levels of THI (higher than 70) were associated to lower periods spent in the cubicle , and eveIt is quite evident that the effect of heat stress has been well mitigated by the cooling system of the barn, except for the middle of June where the THI increased suddenly, and the cows reacted to the THI change, decreasing the lying time.In literature, since the mid-1960s, a lot has been done regarding the influence of THI on dairy cows, especially to evaluate the effects of heat stress. Lately, the THI and its effect on cows’ behavior was investigated. Indeed, modification in the lying behavior is one of the most common effects of heat stress in dairy cows ,30,45. IRegarding the effect of the rain, only in a few cases did behaviors seemed to vary from situations without it. As reported in RAIN alone cannot explain the changes in lying behavior in a roofed barn, but the combination with the other parameters could represent a discomfort situation for dairy cows ,42,47,48p < 0.001), and this was confirmed by the high correlation coefficients between the expected and the observed values.All the effects included in the model affected significantly the lying behavior  on the lying behavior of a group of dairy cows. The developed model seems helpful into identifying the changes in lying behavior of dairy cows according to climatic conditions. This explanatory model, if validated with the data collected in other facilities with an appropriate sample of animals, associated with the use of accelerometers, could be a valid early warning system to identify soon any deviation from the expected behavior. Potentially, this model could be also used to evaluate the goodness of barn management, and to evaluate the heat stress mitigation strategy.


Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3′ untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts. Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3′ untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were originally proposed to be enhancers of viral replication. Analysis of viruses obtained from infected mosquitoes showed selection of mutations that mapped in DB2. Recombinant viruses carrying the identified variations confirmed that these mutations greatly increase viral replication in mosquito cells, with low or no impact in human cells. Use of viruses lacking each of the DB structures revealed opposite viral phenotypes. While deletion of DB1 reduced viral replication about 10-fold, viruses lacking DB2 displayed a great increase of fitness in mosquitoes, confirming a functional diversification of these similar RNA elements. Mechanistic analysis indicated that DB1 and DB2 differentially modulate viral genome cyclization and RNA replication. We found that a pseudoknot formed within DB2 competes with long-range RNA-RNA interactions that are necessary for minus-strand RNA synthesis. Our results support a model in which a functional diversification of duplicated RNA elements in the viral 3′ UTR is driven by host-specific requirements. This study provides new ideas for understanding molecular aspects of the evolution of RNA viruses that naturally jump between different species. Flavivirus genus includes a large number of emerging and reemerging human pathogens that are transmitted by arthropods, including dengue (DENV), Zika (ZIKV), yellow fever (YFV), and West Nile (WNV) viruses. Dengue is the most prevalent arthropod-borne viral disease around the world. It is endemic in more than 100 countries, with about 390 million infections each year  includes two essential elements for genome replication: (i) the promoter for RNA synthesis, known as stem-loop A (SLA), and (ii) cyclization sequences that mediate long-range RNA-RNA interactions . The gen15–25–The biological significance of maintaining two almost identical RNA structures in the 3′ UTR of flaviviruses is enigmatic. Redundant functions, as replication enhancers, were proposed for the two DB elements , 32. HowPrevious studies have determined other requirements for different viral RNA structures for DENV replication in the two hosts , 34–36. 34–37–Here, using DENV as a model, we found that each of the duplicated DB RNA structures in the viral 3' UTR is under different selective pressures in adult mosquitoes. Sequence analysis of DENV RNA populations showed adaptive mutations mapping in only one of the two DB structures. Using recombinant viruses with the identified mutations, a great advantage for viral replication in mosquito cells was associated with alterations in the DB2 sequence. Mechanistic analysis indicated that the two DB elements differentially regulate genome cyclization, which is a conformation required for viral RNA synthesis. We previously showed that sequences within DB1 hybridize with a region present in the capsid coding sequence, promoting genome cyclization . Now, weAedes aegypti and Aedes albopictus mosquitoes. Extracts from pools of five A. albopictus mosquitoes 14 days postinfection (dpi) were used for titrations and to infect fresh mosquitoes (passage 1 [P1])  . Viruses 1 [P1]) . These m 1 [P1]) . Althoug 1 [P1]) . Because 1 [P1]) , 42, we To evaluate the relevance of the variants selected in mosquitoes, recombinant viruses carrying the identified point mutations were designed in the context of a reporter DENV infectious cDNA clone, and replication was tested in a mosquito cell line. Mutant viral RNAs were transfected along with a wild-type (WT) RNA and a replication-defective control with a mutation in the polymerase NS5. Viral replication was assessed by measuring luciferase activity as a function of time . The virWe conclude that mutations positively selected in adult mosquitoes that affect DB2 structure result in enhanced RNA replication in mosquito cells. Therefore, we used this model to understand mechanistic aspects and functions of the adaptive mutations.Based on the selection of advantageous mutations in mosquitoes in one of the two DBs, we examined the evolutionary relationship between the duplicated RNA structures in different DENV serotypes. To this end, we employed a tree alignment model algorithm, RNAforester , to compThe sequence conservation of the two DBs was also analyzed using different genotypes of all four DENV serotypes. A conservation analysis per site for each structure showed 75% and 63% identity for DB1 and DB2, respectively. For example, in the case of DENV2, a higher sequence variability of DB2 was observed when genotypes from different geographic regions were compared . In thisThe finding that DB2 displays more natural sequence variation than DB1 in different DENV isolates and that adaptive mutations in mosquitoes mapped to DB2 suggested a possible diversification of functions of the duplicated RNA structures. To examine this possibility, we designed deletions of each or both DB structures in mutated genomes and evaluated viral replication in mosquito cells. Viral RNA from the mutants  or contcis-acting signal that is duplicated appears to be required in both hosts.To evaluate whether the phenotype observed with DENVs carrying DB deletions was host specific, the mutant viral RNAs and controls were transfected into A549 human cells. The deletion mutants DENVΔDB1 and DENVΔDB2 showed a subtle but significant reduction or increase of viral replication, respectively . AlthougThe duplicated SL and DB structures in the 3' UTR of flaviviruses are capable of stalling genome degradation by the 5′-exonuclease XRN1 that results in sfRNA accumulation . To evalA deletion within DB2 is an attenuating mutation of a live vaccine candidate, DENV delta30 (del30), which is currently in advanced clinical trials . BecauseAedes sp. mosquitoes  . This CS+ or Mut Cyc−, increase or decrease complementarity between the ends of the viral genome, respectively  reduced by 1 bp the upstream AUG region (UAR) complementarity (reducing cyclization) and (ii) increased stability of the 3′ SL by 1 bp (increasing stability of the linear form) (−-transfected cells had two point mutations that (i) reformed a base pair in UAR, increasing cyclization, and (ii) introduced one mutation in the top loop of DB2, disrupting the PK interaction that competes with cyclization  . Intereslization . This psIn order to confirm our hypothesis that formation of the PK of DB2 competes with genome cyclization and negatively regulates viral RNA replication, a recombinant virus was designed impairing formation of the PK (Mut-PK ). This mAedes aegypti mosquitoes infected with DENV1 has recently been reported. This work investigated viral population expansions after the initial infection, shown to be randomly founded by a few tens of viruses , Japanese encephalitis group (JEVG), YFV group (YFVG), Kokobera virus group (KOKVG), Aroa virus group (AROVG), Ntaya virus group (NTAVG), and Spondweni virus group (SPOVG) . The 3′ In these studies, we found that paralogous viral RNA structures originating from duplications in the 3′ UTR of flavivirus genomes are under different selective pressures in different hosts. The data support the hypothesis that duplicated DB elements, present in most MBFV genomes, conserve redundant functions, but also have evolved divergent host-specific activities that modulate viral RNA replication. Our work provides mechanistic details by which DB structures regulate viral genome conformation by either enhancing or competing with long-range RNA-RNA interactions that promote viral RNA synthesis. We propose a model in which different requirements for viral replication in mosquito and human cells may explain the conservation of duplicated RNA structures in flavivirus 3' UTRs.cis-acting elements at the 5′ end of the genome that could alter DB functions , which is present in both DBs , 54. PerThe DENV genome functions as a dynamic molecule, and changes in the equilibrium between linear and circular forms of the viral RNA have been previously demonstrated to have a great impact on viral infectivity , 55–57. 55–58–Interestingly, the windows of tolerance for alterations of the equilibrium between circular and linear forms of the genome for DENV viability were found to be different in infected mosquito or human cells. Mutations that displace this equilibrium that were tolerated for replication in human cells were detrimental in mosquito cells , 35. DB It has been shown recently that DENV genetic variations in mosquitoes alter the transmission kinetics . Our datThe structure of DB2 has been a target for viral attenuation in different flaviviruses , 66. In MBFV contain two pairs of duplicated RNA structures in the 3′ UTR: two SLs and two DBs. We previously investigated the function of the two SLs in mosquitoes and mosquito and human cultured cells and found that RNA duplication supports efficient DENV host switching . In thisMost MBFVs contain duplications of DB elements in their 3′ UTRs. However, RNA structures containing conserved DB elements are also found in flaviviruses with no known vectors (NKVFs), such as Yokose virus (YOKV), Entebbe bat virus (ENTV) and Modoc virus (MODV), and in insect-specific flaviviruses (ISFs) related to MBFVs, such as Chaoyang virus (CHAOV) and Nhumirim virus (NHUV) (for review see reference The mechanisms by which the two pairs of duplicated RNA elements (SLs and DBs) participate in the viral transmission cycle appear to be different; however, both are under host-specific selective pressures. Sequence and RNA structure duplications at the viral 3′ UTR were also reported to be relevant for host adaptation in other arboviruses. In this regard, an interesting report using chikungunya virus (CHIKV) described the process of viral 3′-UTR evolution in different hosts . In thisIn summary, our findings provide new insights about functions and host-specific requirements of duplicated RNA structures in flavivirus genomes. We believe that understanding mechanisms that enable these viruses to replicate in multiple hosts will help to elucidate factors that govern the geographic expansion of these important viral pathogens.Aedes albopictus cell line, adapted to grow at 33°C) were cultured in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.3% tryptose phosphate broth, 0.02% glutamine, 1% minimal essential medium (MEM) nonessential amino acids solution, and 0.25 µg/ml amphotericin B (Fungizone). A549 cells  were cultured in Dulbecco's modified Eagle’s medium with nutrient mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.C6/36HT cells  inoculated with approximately 70 nl of cell culture medium with a total titer of 200 PFU, using a Nanojet II (Drummond Scientific Company). Mosquitoes were maintained for 14 days, and a pool of five mosquitoes was used for RNA extraction and sequencing (P1). In addition, two batches of 10 mosquitoes each were homogenized in 1 ml of grinding medium and virus titer was determined. New females were injected with 70 nl of P1 extracts. After 14 days, RNA from groups of five mosquitoes was extracted and used for sequencing (P2).Viral stocks were prepared in BHK cells. Supernatants were harvested at different times posttransfection, and viruses were quantified by plaque assays. in vitro-transcribed viral RNA, the DNA plasmid template, and the viral RNA input from BHK supernatants were also sequenced. Libraries were sequenced using a 454 Genome Sequencer FLX Titanium XLR70 system (Roche), and raw data were analyzed as describe below. Reads covering 400 nucleotides of the 3′ and 5' UTRs were aligned to the reference sequence. Positions of alignments from low-complexity regions with repetitions major to four nucleotides were discarded from the analysis. To define selected viral variants, a sequence-to-vector approach was applied  from homogenized mosquitoes and used for reverse transcription-PCRs (RT-PCRs) in duplicates with tagged primers specifically designed to amplify the last 600 nucleotides of both genome ends, using SuperScript III reverse transcriptase (Invitrogen) and Platinum Pfx DNA polymerase (Invitrogen). As a reference, the  applied . For eacViral RNA was TRIzol extracted from clarified supernatants. RNA was treated with tobacco acid pyrophosphatase (Epicenter) to remove caps, and 3′ and 5′ ends of the RNA were ligated with T4 RNA ligase (Epicenter) as previously described . After pFor RNA structure similarity analysis, the RNAforester software was used . Models Renilla luciferase (Rluc) gene previously described .The construction of recombinant DENV cDNAs  was performed as previously described . For theescribed , was modin vitro transcription using T7 RNA polymerase (Ambion) in the presence of m7GpppA cap analog (New England Biolabs). RNA integrity was confirmed in 1% agarose RNase-free gels. RNA transcripts were transfected into C6/36HT and A549 cells grown in 24-well plates using Lipofectamine 2000 and Opti-MEM media (Invitrogen). The Rluc activity in transfected cells was analyzed using the Promega Renilla luciferase assay system according to the manufacturer’s instructions. Data were analyzed by one-way analysis of variance (ANOVA) with Bonferroni’s or Dunnett’s correction for multiple comparisons.Infectious viral cDNA plasmids were linearized with XbaI (New England Biolabs) and used as the templates for For quantification by real-time RT-PCR, total viral RNA was TRIzol extracted at various time points posttransfection. RNA samples were reverse transcribed in 20-µl reaction volumes, as previously described . For rea2 coverslips. At various times posttransfection, coverslips were removed, and cells were fixed with methanol for 15 min at 20°C. For detection of viral antigens, a specific anti-E monoclonal antibody (MAb), E18, was used. Alexa Fluor 488-conjugated rabbit anti-mouse immunoglobulin G (Molecular Probes) was employed to detect the primary antibody under the same conditions.C6/36HT cells transfected with WT or mutated DENV RNAs were grown in 24-well plates containing 1-cmViral titers were determined by PFU in BHK-21 cells.32P-labeled RNA probe complementary to the complete 3′ UTR (nucleotides 10269 to 10723) was used.For DENV sfRNA detection, total RNA was obtained from transfected cells using TRIzol reagent (Invitrogen) at different times posttransfection or postinfection and separated on 5% polyacrylamide–7 M urea gel, as previously described . For det32P-labeled RNA probe complementary to a region of NS2B sequence was used. Samples were loaded into a slot blot apparatus using nylon membrane .For detection of genomic RNA (gRNA), a uniformly Genome sequences of representative viruses corresponding to different subgroups of mosquito-borne flaviviruses were used . The RNAfold package software was used to detect thermodynamically stable and evolutionarily conserved RNA secondary structures. To characterize possible tertiary interactions, complementary base pairs, and covariations, Biostring functions and the RNAaliduplex software were used together with predicted secondary structure models. For each long-range interaction, we calculated the Z-score using a methodology previously proposed . Detaile10.1128/mBio.02506-18.1TABLE S1Table S1, PDF file, 0.5 MB.Accession numbers. Download Copyright © 2019 de Borba et al.2019de Borba et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low‐melting‐point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high‐quality RNA suitable for down‐stream applications such as quantitative reverse transcriptase–polymerase chain reactions or RNA‐sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded  Not only does multicellularity solve size and lifespan limitations caused by diffusion and aging of individual cells, respectively, it also allows increased complexity through the differentiation of cell types that become specialized for particular functions. Thus, to understand how a multicellular organism is built and then its structures maintained, analysis of its constituent cell types is desirable.4 leaf anatomy, bundle sheath strands can be separated from the adjoining mesophyll by differential grinding  was germinated and grown in 1:1 mixture of top soil and sand for 2 weeks in a controlled environment growth room set at 28°C day 25°C night, a relative humidity of 60%, a photoperiod of 12 hr light and 12 hr dark, and a photon flux density of 300 μmol m−2 s−1.Seeds of 2.23 blocks and stored in 50‐ml falcon tubes with self‐indicating silica gel at −80°C. Steedman's wax was prepared as described  acetone or Farmer's fixative (75% (v/v) ethanol, 25% (v/v) acetic acid) on ice for 2 hr and 4 hr, respectively, prior to immediate RNA extraction. To conduct laser capture microdissection, rice leaves were cut into 5–8 mm pieces with RNAZap treated scissors and fixed under vacuum for two 10 min periods in ice‐cold 100% (v/v) acetone and then left with gentle stirring for 3 hr. Arabidopsis leaves were treated in the same way, but to maintain tissue structure, they were not subjected to vacuum infiltration. Leaf tissue was then dehydrated through an ice‐cold series of 70%, 85%, 95%, and 100% (v/v) ethanol for 1 hr each. Samples were incubated in 100% (v/v) ethanol overnight at 4°C, prior to being placed in 25%, 50%, 75% and then 100% Steedman's wax at 37°C for 2 hr. This final solution of 100% Steedman's wax was replaced twice at 2 hr intervals. Tissue was embedded in a 9 cm petri‐dish, and after wax had solidified, it was cut into 1‐cm2.3™ RNA Isolation Kit with on‐column DNaseI treatment. To quantify yields, 1.5‐μl samples of eluted RNA were denatured in 0.2‐ml RNase‐free tubes at 70°C for 2 min, and 1 μl was then analyzed using an Agilent Bioanalyser RNA 6000 Pico assay. Electropherograms were assessed qualitatively for background signal and the appearance of cytosolic and chloroplastic ribosomal RNA peaks, and assessed quantitatively using the common metrics of the 25S to 18S ribosomal RNA ratio, and the RNA Integrity Number (RIN)  and from microdissected cells using the PicoPure2.4Paradermal sections were prepared using a microtome. Paraffin‐embedded sections were placed onto a dry polyethylene naphthalate (PEN) membrane slide (Arcturus) and then floated on diethyl pyrocarbonate (DEPC)‐treated water at 42°C to expand the sections and ensure they were flat. Water was then removed and slides dried at 42°C for 20 min. Steedman's wax‐embedded sections were similarly expanded on DEPC‐treated water at room temperature on a PEN membrane slide, before the slide was dried using tissue paper at room temperature. Before laser capture microdissection, paraffin wax was removed by incubating slides in 100% (v/v) Histoclear for 5 min, whilst Steedman's wax was removed by incubating slides in 100% (v/v) acetone for 1 min at room temperature. Laser capture microdissection was performed on an Arcturus Laser Capture Microdissection system using CapSure Macro Caps to collect bundle sheath strands and mesophyll cells.33.1To limit RNA degradation, tissue fixation needs to be rapid. Compared with cross‐linking fixatives, precipitative fixatives such as acetone and Farmer's fixative have commonly been used for laser capture microdissection sample preparation because they retain good histological detail as well as reasonable RNA yields  and the yield and integrity compared with that recovered from equivalent paraffin‐embedded sections. RNA isolated from tissue in Steedman's wax showed less elevated baselines and clearer peaks representing the cytosolic and chloroplastic ribosomal RNAs Figure a‐d. Both3.3Subsequent to wax embedding, but prior to laser capture microdissection, there are further opportunities for RNA to be degraded. For example, during slide preparation, sections are typically expanded by floating on warm RNase‐free water at 42°C to ensure they lie flat on the microscope slides. Water is then removed and samples dried at 42°C for 20–30 min. Consistent with RNA degradation during this process, after slide preparation from paraffin‐embedded rice tissue, the cytosolic and chloroplastic ribosomal RNA peaks were less defined, and the 28S to 18S ribosomal RNA ratio was lower compared with that isolated from freshly cut sections Figure . This ra3.4To assess the combined impact of the modifications documented above on the final RNA quality obtained from laser capture microdissection, we compared RNA quality from microdissected tissues embedded in either paraffin or Steedman's wax. For this purpose, bundle sheath strands and mesophyll cell sections were captured from both Arabidopsis and rice leaf tissues. RNA isolated by laser capture microdissection from paraffin‐embedded sections of either species showed either no clear or compromised ribosomal RNA peaks Figure a‐d. ThisWith advances and reduced cost of next‐generation sequencing, RNA‐SEQ has become a common tool for profiling transcript abundance. However, a high‐quality RNA input is important for reliable and reproducible results. For example, it has been reported that RNA degradation can have a broad effect on quality of RNA‐SEQ data, including 3’ bias in read coverage, quantitation of transcript abundance, increased variation between replicates, and reductions in library complexity (Chen et al., The authors declare no conflict of interest associated with the work described in this manuscript.Conceptualization, Lei Hua and Julian M Hibberd; Data curation, Lei Hua and Julian M Hibberd; Formal analysis, Lei Hua; Funding acquisition, Julian M Hibberd; Investigation, Lei Hua; Methodology, Lei Hua; Project administration, Julian M Hibberd; Supervision, Julian M Hibberd; Writing—original draft, Lei Hua; Writing—review & editing, Lei Hua and Julian M Hibberd. Click here for additional data file. Click here for additional data file.


The Convention on the Rights of Persons with Disabilities came into place in 2006, as the main instrument for advancing the human rights of persons with disabilities. For many African states, the Convention came amidst ubiquitous marginalisation and discrimination of persons with disabilities. As expected, the Convention has been hailed as a landmark in the struggle to reframe the needs and concerns of persons with disabilities.This article reviews the implementation of the Convention by the Zimbabwean government.The study relies on reviews of extant literature on disability rights. Reviewed documents include the Convention, constitution and other related national laws, policies and measures pertaining to disability rights.This article lauds the state for promulgating a disability-friendly constitution that resembles the Convention to effectuate a human rights approach to disability issues. Relatedly, the state came up with institutions that collaborate with research institutes and disability organisations to conduct research, provide services to persons with disabilities, raise awareness and advocacy and litigate for disability rights.In spite of these efforts, this article shows that Zimbabwe has yet to close the gap on the ideals of the Convention, mainly because of limited resources amongst state-funded institutions for advancing disability issues. The government of Zimbabwe is challenged to domesticate all provisions of the Convention and to provide resources to institutions for progressive realisation of the rights of persons with disabilities. On 13 August 2006, the United Nations General Assembly adopted the Convention on the Rights of Persons with Disabilities (CRPD) and its Optional Protocol to promote and protect the rights of people with disabilities (PWDs) . The CRPD further defines ‘disability’ in a broad and inclusive manner, which indicates a model shift in approaches to disability. For this reason the CRPD:disabling environment and, in its stead, create an enabling environment which is inclusive and accommodates all human beings in their diversity. , which defines terms and explains the purpose of the Convention. Specific political, social, economic and cultural rights of PWDs are explained in detail in articles 10–30. This part is followed with implementation and monitoring mechanisms (articles 31–40). The Convention ends with articles 41–50, which govern the operations of the CRPD. A closer look at this synopsis shows the extent to which the CRPD recognises, reaffirms or guarantees existing rights in the Universal Declaration of Human Rights of 1948. By so doing, the CRPD did not create a new set of rights for PWDs but rather confirmed that PWDs are human beings equal to others, as their rights under the CRPD are the same as other instruments. As such, the ideals of the CRPD become compelling norms to be implemented by every state. Principles of humanity are indeed fundamental and must be respected, promoted and fulfilled by every state actor and non-state actor in the Global South.For the aforementioned reasons, the CRPD came as a beacon of hope for millions of PWDs in Africa, who are the most stigmatised, poorest and least educated citizens It remains to be seen how Zimbabwe has fared with the obligation to implement the CRPD’s provisions for progressive realisation of PWDs’ rights. Accordingly, this article:takes stock of the implementation of the CRPD in Zimbabwe, focusing on how the 2013 Constitution domesticated the CRPD and the best practices by state institutions to implement provisions of the CRPD,unearths challenges faced by state actors in implementing provisions of the CRPD,proffers recommendations for action by state actors toward effective implementation of the CRPD in Zimbabwe.The article is based on a literature review of the CRPD, the constitution of Zimbabwe, journal articles and reports on disability and development in Zimbabwe. The article comprises four sections, including the Introduction and background. This is followed with a review of the constitutional reform and how it domesticated the CRPD. The third section comprises a review of efforts to implement the CRPD by national institutions. The last section concludes the discussion and presents recommendations for action in Zimbabwe.In 2013, Zimbabwe promulgated a new constitution to replace the 1979 Lancaster Constitution. Whilst the 1979 Constitution condemned discrimination against PWDs, it only recognised physical disability, to the express exclusion of all other forms of disability affecting people in society. Unlike the Lancaster Constitution, the 2013 Constitution includes disability as one of the grounds prohibited for discrimination under section 56. Although the provisions of the new constitution are an improvement, in part, the new constitution follows in the footsteps of the old constitution by deploring the discrimination of persons with physical and mental disabilities whilst leaving out persons with intellectual and sensory disabilities. There is therefore a need for Zimbabwe to adopt the meaning of disability as proffered by the CRPD. However, by enacting the new constitution, the government of Zimbabwe has in part implemented the provisions of the CRPD. That is so because, under article 4(a), the CRPD directs the adoption of appropriate legislative measures for the implementation of the rights recognised in the CRPD, and in article 4(b) it directs state parties to take all appropriate measures to modify or abolish existing laws that discriminate against PWDs.The constitution further recognises the dignity, equality and rights of all human beings, including PWDs, under its founding provisions. The constitution thus confers the duty of every human being to respect the rights of everyone, including PWDs. According to Mandipa , the recThe constitution  under seThe constitution  under seAnother important constitutional clause for ensuring access to information is section 16. This clause makes sign language one of the sixteen official languages for communication in Zimbabwe. The inclusion of sign language by the constitution is commendable, as Zimbabwe’s policy regarding official languages always excluded a huge portion of persons with hearing and speech disabilities  that contradicts the spirit of the CRPD for adequate resources and commitment to advancement of issues that concern PWDs. The section in question calls upon the state to come up with appropriate measures, within the limits of the resources available to it, to ensure that PWDs realise their full mental and physical potential (Constitution 2013:39). Apparently this clause only addresses the rights of PWDs and the elderly (section 82) and does not apply for other vulnerable groups such as women (section 80) and children (section 81). This clause limits the effective implementation of PWDs’ rights. Amidst serious economic challenges in Zimbabwe, it is highly expected that the clause on section 83 will become an excuse for non-implementation of the ideals of the CRPD by government agencies, citing financial problems and lack of resources.Whereas articles 6 and 7 of the CRPD prioritise the rights of children and women with disabilities, the constitution  failed tDisability Persons Act, which views PWDs using the damaging medical and charity models of disability. Even with the presence of senators with disabilities, the ministry responsible for PWDs received paltry budgets in 2014 (d), two out of the 80 senatorial positions are reserved for PWDs, who are elected by PWDs through their various formations. The senators are expected to influence policy and law making that protects and takes into consideration the challenges of PWDs in society. However, the inclusion of senators with disabilities has had a limited impact in enhancing the lives of the people they represent. It is a rare case when one finds a motion moved by these senators for the plight of PWDs. Since their appointment in 2013, the senators have not done enough to lobby for a disability policy. In addition, they have failed to lobby for quick alignment of the 1992 Urban Councils Act (2008) implements the CRPD by allowing for the appointment of special councillors, which may bring in councillors with disabilities in local governance appointed by the minister responsible for local government. The inclusion of special needs councillors is expected to ensure adequate promotion of PWDs’ rights and welfare in local governance. Unlike the constitution, which clearly stipulates the election of two senators, there is no specific number of PWDs who should make up the 25% ceiling of special councillors to be appointed into local governance. The failure to specifically mention the number of councillors with disabilities amongst the 25% special councillors may also result in the exclusion of this disadvantaged group. Special groups are numerous, meaning the minister may select special councillors based on age, gender; linguistic, ethnic and religious grounds whilst paying no attention to PWDs. Therefore, the Urban Councils Act must be amended to clearly state the number of councillors with disabilities to be appointed amongst the special councillors. Also important is the amendment of the Rural District Councils Act to introduce special appointments for PWDs in rural councils. Adequate representation of PWDs in both rural and urban councils can go a long way to ensuring that local government policies and service delivery become sensitive to the needs of this disadvantaged group.Similarly, section 4A of the ZHRC Act to help the commission in protecting, promoting and enforcing the human rights of vulnerable groups in society, including PWDs. Together with other departments within the ZHRC, the working group implements articles 8 and 31(1) of the CRPD through research on and raising awareness of PWD issues.The CRPD under article 33(1–2) calls for the establishment of independent national institutions to advance PWDs’ issues. In line with this provision, the constitution of Zimbabwe established the Zimbabwe Human Rights Commission (ZHRC) in terms of section 242 to promote awareness and respect for human rights and freedoms of all human beings, including PWDs. The commission is empowered under section 243(k)(ii) of the constitution to visit and inspect places where PWDs are kept or stay and to inspect the human rights situation in such places. Upon its operationalisation in 2004, the ZHRC established a Special Interest Thematic Working Group in accordance with the In 2015, the ZHRC commissioned a baseline study on the human rights situation in Zimbabwe, which revealed negative societal perceptions and attitudes towards PWDs , have also been crucial in implementing the CRPD through ensuring that PWDs can access welfare and basic needs. This has seen the implementation of article 28 of the CRPD, which mandates that stakeholders ensure comprehensive social protection mechanisms for PWDs. In performing this role, the MoPSLSW works with research institutes, disabled person organisations, NGOs and other government ministries such as the Ministry of Primary and Secondary Education, which strives to ensure access to education for children with disabilities in line with article 24 of the CRPD, which provides for PWDs’ equal access to education. Together, the two ministries administer the Basic Education Assistance Module (BEAM), which is meant to ensure access to education for vulnerable children, including those with disabilities and those with parents with disabilities. Through BEAM, many beneficiaries realise their lifetime dream of accessing education in Zimbabwe. Important as the scheme has become, it only provides bursaries for students in special schools, as opposed to those in inclusive schools. This is promoting the sending of children with disabilities to special institutions as opposed to the inclusive education system advocated by the CRPD. Moreover, the scheme is also affected by underfunding, which results in beneficiaries sometimes being sent back home from schools because of unpaid school fees. Without adequate funding for BEAM, many children with disabilities drop out as a result of failure to raise money for fees.Furthermore, the Ministry of Health and Child Welfare provides assistive devices to PWDs, including wheelchairs, spectacles, crutches, artificial limbs for those PWDs in need of them and treatment creams for Albinism conditions. Like other ministries, the Ministry of Health and Child Welfare is under-resourced and normally fails to adequately provide for these requirements by PWDs. As noted by Eide et al. , only a The government of Zimbabwe also created the Office of the Special Advisor in 2007 to advise the President and cabinet on disability issues. The Special Advisor’s office is the focal point that mainstreams and implements disability-related issues within the government. Although questions have been asked regarding its mandate and appointment criterion Mandipa , the offThe government of Zimbabwe also implements the CRPD through research and documentation of disability issues. In 2013, the Ministry of Health and Child Welfare commissioned a survey in all 10 provinces of Zimbabwe entitled ‘Living Conditions among Persons with Disability’ to provide a comprehensive mapping for the lives of PWDs. Together with the national census of 2012, the studies revealed the various challenges faced by PWDs to participate in socio-economic and political development in society. However, it is concerning to note that the national census of 2012 did not bother to reveal the numbers of PWDs, their disability features and geographical location. Consequently, the available statistics on the prevalence of disability are outdated, and from past studies conducted before 2005. The failure by the Zimbabwe Statistical Agency to collect up-to-date statistics on disability issues contradicts the spirit of article 31 of the CRPD, which obligates state parties to ensure the collection of appropriate information, including statistical data about PWDs. In addition, non-prioritisation of disability issues during national censuses makes it difficult for policymakers to get information about this disadvantaged group of society and may result in their marginalisation when it comes to social protection mechanisms.The government of Zimbabwe also works with Non-Governmental Organisations and state universities to conduct disability research to inform policy formulation and implementation as stated under articles 4(f–g) and 31 of the CRPD. State universities, including the University of Zimbabwe and Midlands State University, have established specialised departments to teach and research disability issues. In 2015, the Great Zimbabwe University also established the Jairos Jiri Centre for Special Needs Education to spearhead disability-related studies and research. The centre aspires to be the hub for transferring cutting-edge research and knowledge in special needs education. Through teaching, holding of research conferences and publication of results, the centre implements articles 4(f–g) and 31 of the CRPD.On the other hand, Midlands State University through its faculty of law established the Disability Legal Aid Clinic in 2012 to advance disability rights. With financial support from university management and the Open Society Initiative of Southern Africa, the legal clinic aspires to become a citadel of disability advocacy and litigation through awareness raising, empirical research and publication of disability issues (MSU Website By ratifying the CRPD, Zimbabwe committed herself to advancing PWDs’ rights and gave impetus to implementation of the provisions and to holding government accountable for compliance with the Convention. Against this background, this article reviewed the implementation of the CRPD in Zimbabwe, through evaluating the extent to which the 2013 Constitution incorporated provisions of the CRPD and the extent to which government ministries addressed the provisions of the Convention on the ground. Notably, the 2013 Constitution is an improvement from the 1979 Constitution in terms of disability rights protection. In the same human rights spirit of the CRPD, section 83 of the constitution confers human rights on PWDs like anyone else in society. To some extent the constitution strives to domesticate provisions of the CRPD and at least recognises, promotes and protects the rights of PWDs as called for by the CRPD. The study also applauds the creation of the ZHRC and the office of special advisor to the president and cabinet on disability for the purpose of advancing human rights issues, including those of PWDs. Within the limits of the resources available to these institutions, the ZHRC together with government ministries have made strides in advancing PWDs’ issues.de jure steps to realise its CRPD commitments, there are major challenges in terms of realising these commitments de facto. Laws, policies and institutional frameworks are strong foundational instruments for realisation of PWDs rights if they are exhaustive of this disadvantaged group’s critical needs and are followed with effective implementation mechanisms. This is because of the existence of vague and weak clauses in the constitution in relation to PWDs’ rights, which is something that limits effective policy enforcement. There is also the challenge of resource constraints and the aforementioned lack of will by the government to support PWDs’ issues. As such, government and the donor community must show commitment to PWDs’ issues and fully support the cause.Although Zimbabwe has taken This article calls for the urgent domestication of the CRPD, as well as review and alignment of all disability related laws that predate the constitution and the CRPD. Constitutional bodies and other institutions advancing the rights of PWDs should be strengthened through adequate budgets. By so doing, the institutions will be able to effectively execute their mandate and bring about change on the ground. It is clear that the CRPD places the primary obligation for implementation on state actors. Hence, the government should make efforts to strengthen coordination of these tasks and to work hand in glove with other interested players to meet the obligations of the CRPD, through awareness raising, service provision and research. The researchers of this article believe that if government works together in good spirit and faith with non-state actors, Zimbabwe will go a long way towards ensuring that the CRPD is effectively implemented to ensure that the rights of PWDs are protected, promoted and enforced.


Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1–10 ng of intact total RNA and 100–500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues. Ribosomal RNAs (rRNAs) constitute >90% of the total RNA mass within cells –2. To enSeveral studies have compared RNA-seq protocols representing the two commercial kits or similar non-commercial protocols –15. The Universal Human Reference (UHR) total RNA  was quantified using the RNA 6000 Nano Kit . The External RNA Controls Consortium (ERCC) spike-in mix 1  was added to UHR total RNA to allow for accuracy and sensitivity assessments. 0.02 μL of the spike-in mix was used per 1 μg UHR total RNA.2 in size (in 2–5 scrolls of 10 μm thickness). Total nucleic acids (DNA and RNA) were extracted from FFPE tissue scrolls using the Agencourt FormaPure (Beckman Coulter) protocol or a combined AllPrep (Qiagen) and High Pure (Roche) protocol, as previously reported )). Library diversity was also lower for the lower input libraries . Despite these differences, correlation of levels of UHR transcripts among the different input libraries was high (r = 0.969–0.997), although there was a trend for the lower input libraries to show slightly lower correlations with the higher input libraries  libraries (87–95%) were lower than for those obtained for the higher input (5–500 ng) libraries (94–97%) . Libraryibraries . Despiteibraries . Similaribraries .Overall, the data described above indicate that the RNase H-based and Ribozero Gold rRNA depletion protocols can be used for as little as 10 ng intact total RNA input amount without significant loss of sequencing data quality. We further show that, using the RNase H protocol, libraries of acceptable data quality can be generated from as low as 1 ng of intact total RNA, and that the RNAse H protocol appears to have generally superior performance for the analysis of partially degraded RNA.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.