Accordingly, an efficient concentration method is necessary for large‐scale production or high‐throughput isolation of EVs from bulk liquid samples, including culture medium and body fluids, to achieve their clinical application. However, current EV concentration methods, including ultrafiltration, are limited with respect to cost, efficiency, and centrifugation time. In this study, we developed the first single‐step, equipment‐free EV concentration method using super absorbent polymer (SAP) beads. SAP beads absorb small molecules, including water, via nano‐sized channels but expel and thereby concentrate EVs. Consequently, the beads drastically enrich EVs by reducing the solution volume in a single step, without affecting EV characteristics. Moreover, the purity of the concentrated EV solution was high due to the absorption of protein impurities by SAP beads. To further demonstrate the versatility of the method, we showed that SAP beads successfully enrich EVs in human urine samples and culture medium, enabling better isolation performance than conventional ultrafiltration. We believe the newly developed approach and insight gained in this study will facilitate the use of EVs as prominent biomaterials for disease diagnosis and therapy.Extracellular vesicles (EVs) contain useful biomarkers for disease diagnosis and are promising biomaterials for the delivery of therapeutic molecules  For the production of EVs, HeLa cells were grown in a medium containing EV depleted FBS, generated by the centrifugation of FBS at 120,000 × g for 10 h at 4°C using a TLA‐100.3 fixed‐angle rotor with clearing factor (k factor) 44 in an ultracentrifuge . The supernatant was collected and filtered using a 0.22‐μm cellulose acetate syringe‐filter  and stored at ‐80°C until further use. EV isolation was performed using ExoQuick‐TC EV precipitation solution  according to the manufacturer's instructions. Cell culture media were centrifuged at 3000 × g for 15 min at 4°C. After centrifugation, the supernatant was filtered using a 0.22‐μm cellulose acetate syringe‐filter and mixed with ExoQuick‐TC solution. The mixture was kept at 4°C overnight and then centrifuged at 1500 × g for 30 min at 4°C. The EV pellet was dissolved in 1× PBS. EVs were stored at ‐80°C until further use.HeLa cells were cultured at a humidified atmosphere of 5% CO2.2www.tpy21c.co.kr). Before use, the beads were washed three times continuously with ethanol and dried in an oven at 50°C. The average diameter and weight of single SAP bead investigated for EV concentration were 2.8 mm and 18 mg, respectively. SAP beads with average outer particle diameters were immersed in 1 mL of 1× PBS for up to 90 min to measure the absorbency of SAP beads. Poly(acrylamide‐co‐itaconic acid) SAP beads were synthesized using a milli‐fluidic system   in deionized water. The synthesis of poly(acrylamide‐co‐itaconic acid) SAP beads includes chain initiation, propagation, and crosslinking. First, ammonium persulfate is decomposed at a high temperature (80°C) to produce sulfate anion radicals. Then, these anion radicals abstract the double bond of a monomer, which, in turn, reacts with another monomer to form a trimer. Finally, N,N'‐methylenebis(acrylamide) leads to the formation of cross‐linked poly(acrylamide‐co‐itaconic acid). To synthesize the beads, mineral oil  was used as the oil phase. The water and oil phase flow in a T‐junction  with a 2 and 4.5 mm inner and outer diameter, respectively, were injected into a PFA tube with a 1/16‐inch inner diameter. The water‐in‐oil droplets were generated. Two high‐precision syringe pumps  were used to inject the oil and water phases at 5.0 mL/min and 10.0 mL/min, respectively, to generate 25 μL water phase droplets. The monomers inside the droplets were polymerized in the tube for 4 h at 80°C. Synthesized beads were separated from the mineral oil and washed using deionized water and ethanol, followed by drying under a vacuum at 40°C overnight. The surface morphology of SAP beads was examined under scanning electron microscope (SEM). SAP beads were coated with a thin layer of pure platinum and imaged in SEM , with an operating voltage of 15.0 kV. The EV concentration in the PBS solution was evaluated by immersing 450 mg/mL SAP beads in 1 mL of EV solution with 1.0 × 1010 EV particles.Two types of SAP beads were used. Poly(acrylamide‐co‐acrylic acid) SAP beads  were purchased via TPY Co., Ltd., Korea  using the Nanosight NS300 system . Camera focus was adjusted to visualize the sharp individual dots. The total protein concentration was measured by a BCA assay  or Bradford assay . The BCA working reagent was prepared following the manufacturer's protocol. Each unknown sample and standard were diluted and mixed with the reagent solution, respectively, and incubated at 37°C for 30 min. The absorbance was measured at 562 nm using a spectrophotometer. The same experimental settings were used for measuring the EV and protein concentrations among the batches. The Bradford working reagent was prepared following the manufacturer's protocol. Each unknown sample and standard were diluted and mixed with the reagent solution, respectively, and incubated at room temperature for 10 min. The absorbance was measured at 595 nm using a spectrophotometer. The same experimental settings were used for measuring the EV and protein concentrations among the batches.2.49 particles) was used for the comparison. A western blot analysis was performed with primary antibodies including mouse anti‐TSG101  at 1:1000, mouse anti‐CD63  at 1:1000, rabbit anti‐Syntenin  at 1:2000, rabbit anti‐Hsc70  at 1:500, rabbit anti‐GM130  at 1:1000, rabbit anti‐Calnexin  at 1:1000, and rabbit anti‐THP  at 1:1000. For the detection, horseradish peroxidase‐conjugated anti‐mouse  at 1:2000 and anti‐rabbit IgG  at 1:1000, and an enhanced chemiluminescence detection system  were used. Images were captured using a ChemiDoc XRS+ imaging system . For ELISA, EV concentrations were measured by ExoELISA Complete Kit  using CD63 antibody following the manufacturer's instructions. For TEM, the sample was applied to the copper grids coated only with a thin carbon foil . After allowing the sample to absorb for 2 min and blotting off buffer solution onto Whatman paper, the sample on the grids were stained with 2% (w/v) uranyl acetate (UrAc) for 1 min. Then, distilled water was added for 1 min to blot off UrAc followed by drying for 15 min. The result was recorded with Bio‐High voltage EM system  at the Korea Basic Science Institute in the Republic of Korea. The zeta potential and polydispersity index (PDI) of the EVs were measured by DLS  at 25°C. The same EV concentrations were adjusted to the equal volume in disposable folded capillary cell  and solvent resistant micro cuvette  for measuring zeta potential and PDI, respectively.SDS‐PAGE was performed with reducing  or non‐reducing ) conditions. The same number of EV particles . Urine samples were collected in a sterilized specimen cup  and stored at ‐80°C until further use. The characterizations of liquid samples including salt concentration and pH are listed in Table 2.6g for 15 min to remove the debris and filtered using 0.22‐μm cellulose acetate syringe‐filter. The Amicon Ultra‐15 Centrifugal Filter Unit with a 10 kDa pore  was used for ultrafiltration, and cell culture media grown in EV‐free FBS were centrifuged at 4000 × g for 120 min at 4°C (90 min for pretreated urine). Additionally, SAP beads were used for concentrating cell culture media or urine samples at 250 mg/mL for 30 min. The supernatant containing EVs was collected. For SEC, 0.5 mL of the concentrated sample was coated on a qEV size exclusion column  and eluted with PBS; 0.5 mL of each fraction was collected in a microcentrifuge tube , and isolated EVs were used immediately or stored at ‐80°C until further use.To compare the concentration performance of ultrafiltration and SAP beads, cell culture media or pretreated human urine samples were concentrated first by ultrafiltration or SAP beads, followed by concentrated EV isolation by SEC. HeLa cells were grown in an EV‐free medium containing FBS. The medium (12 mL) was centrifuged at 3000 × 2.7An MB was designed to target mature miR‐21 (MB‐21). The MB‐21 sequence used in this study was 5′‐Cy3‐GCGCGTCAACATCAGTCTGATAAGCTACGCGC‐BHQ2‐3′  to assess differences between two groups. Shapiro‐Wilk test was performed to assess normality distribution before using paired t‐tests.Paired 33.1Water absorption by SAP beads in a solution causes bead swelling. As shown in Figure 3.210 particles/mL), the final EV concentration after SAP incubation was 8.27 × 1010 particles/mL. With a single‐step and simple incubation process, the EV concentration reached approximately 720% as compared to that of original solution . Thus, the method can be applied for the concentration of EVs regardless of the isolation method.To demonstrate that SAP beads absorb small molecules, including water, but not EVs, EVs were isolated from the culture medium by ExoQuick‐TC and spiked into PBS. The medium was filtered with 0.2‐μm filters before EV isolation because the primary purpose of this method is to concentrate EVs with a size less than 200 nm that are hard to concentrate. Then, the EV‐containing solution was incubated with 200 mg/mL SAP beads (11 SAP beads) for 30 min and the EV concentration and volume were measured. As shown in Figure n Figure . This can Figure . Exosomen Figure . The yien Figure  and inven Figure . NTA  can be used for concentrating EVs. However, they require a centrifuge and filters that increase the time, labour and the cost of production. Thus, these methods are inappropriate for large‐scale production or high throughput analyses of EVs. In contrast, our SAP bead‐based concentration method does not require any equipment for concentration and is extremely low cost. Thus, the SAP bead‐based EV concentration method offers great advantages over conventional concentration methods, including UF.3.3Many EV isolation methods are limited by impurities, including proteins, originating from the samples. This is a major issue because it results in a low purity of the isolated EV solution. For instance, EV isolation from cell culture medium results in a high concentration of protein impurities, including bovine serum albumin, from foetal bovine serum  followed by SAP bead addition. After 30 min of concentration, volume, EV concentration and protein concentration were measured. As shown in Figure Although the main purpose of our method is EV enrichment and not EV purification, we hypothesized that SAP beads might also absorb protein impurities from the solution because many proteins are within several nanometers in size. To evaluate this hypothesis, protein concentrations before and after EV enrichment were measured. The same experimental conditions used for EV concentration were used for the analysis. First, the protein content increased 333% after SAP bead concentration, as expected based on the substantial reduction in solution volume by SAP beads Figure . Howevery Figure . To direTo explore the size‐dependent absorption of molecules by SAP beads, we tested if SAP beads can absorb doxorubicin, polystyrene microspheres and gold nanoparticles with the diameters of 1.5, 30 and 45 nm, respectively. Each material was incubated with the same amount of SAP beads for 30 min, and the SAP beads were separated and observed. As shown in Figure 3.4To determine whether other types of SAP beads with different chemical compositions have a similar EV concentration ability, poly(acrylamide‐co‐itaconic acid) SAP beads were synthesized Figure . The fab3.5For therapeutic or diagnostic applications of EVs concentrated by SAP beads, the concentration method should not affect the biochemical and biophysical properties of EVs. To evaluate whether the SAP bead‐based EV concentration method alters the physiological properties of EVs, the size, morphology, PDI, zeta potential and protein marker content of EVs were analyzed before and after enrichment. Based on NTA, there was no detectable difference in the size distribution of EVs before and after SAP bead concentration, despite a large difference in the EV concentration due to enrichment by SAP beads Figure . The mea3.6P < 0.001; n = 5). The concentrated urine was further used for EV purification by injecting into an SEC column  was drastically reduced. At the same time, there was a slight decrease in the absolute amount of CD63 (EVs), indicating that SAP beads removed a substantial amount of THP in the urine during EV concentration Figure . Overall3.7For practical applications of EVs, large‐scale cell culture is necessary to achieve a high quantities of EVs from culture medium   Figure . The res) Figure . Additio) Figure . Conside3.8in situ detection of miRNAs in exosomes in a single detection reaction using nano‐sized oligonucleotide probes, molecular beacons (MBs)  was constructed and used for miRNA detection in EVs. The specificity of MB‐21 was evaluated against synthetic miR‐21 and miR‐21 in EVs Figure . As shows Figure . In cont3.9Despite their broad and intensive uses in many fields, including agriculture and food industry, few studies have been conducted examining the application of SAP beads for biological purposes. Considering the potential use of SAP beads for the concentration of EVs shown in the study, further investigations should be conducted to overcome the current limitations. First, the SAP beads need to be carefully characterized when used primarily for the therapeutic EV preparations. Moreover, since the SAP beads used in this study were not originally constructed for the concentration of EVs in the solution, it was necessary to optimize their composition and synthetic conditions to adjust their absorption properties. Different mechanisms for the absorption of impurities by SAP beads may likely be involved. For instance, electrostatic interactions can occur between the SAP bead surface and proteins owing to the negative charges of the SAP beads. However, since the beads also absorbed the small oligonucleotide DNA, the electrostatic force was not the only way to introduce and absorb the impurities by beads. Based on this, it is speculated that the mechanism of impurity absorption depends on the type of impurities. Hence, depending on the type and size of impurities to be reduced during the concentration of EVs, SAP beads can be modified to accommodate a high level of impurities. For more practical and comfortable use of SAP beads for the concentration of EVs, the concentrated solution should be fully and easily recovered from the tube. In this context, the construction of an automated device that ensures easy separation of the concentrated solution from SAP beads will ensure convenient and user‐friendly operation. The EV‐containing solutions, including culture media and body fluids, are heterogeneous. No single method measures the full spectrum of EV characteristics, including an EV concentration, in those complex solutions. Currently used NTA measures all types of particles, including EVs, in the solutions. Thus, developing an advanced method or device that enables the specific and accurate quantification of EVs is required to quantify EV numbers in the concentration process using the SAP beads.4EVs are novel biomaterials widely applied in disease diagnosis and therapy. In accordance with their broad applications, remarkable EV isolation methods have been developed. However, methods for efficient EV concentration are lacking. Conventional ultrafiltration is costly and requires a long operation time and specialized equipment for EV concentration, emphasizing the importance of the development of a novel EV concentration method for large‐scale production and high‐throughput analyses. We demonstrated that SAP beads successfully enriched EVs in various solution types, including culture medium and human urine samples, and removed protein and nucleic acid impurities. The approach is simple, efficient, cost‐effective, scalable, and equipment‐free and can be widely used for therapeutic or diagnostic purposes requiring EV enrichment. This method may thus improve our ability to harness EVs for a variety of applications in diagnosis and therapy.The authors declare that they have no conflict of interest.Supporting InformationClick here for additional data file.


The simplicity of homogeneous immunoassays makes them suitable for diagnostics of acute conditions. Indeed, the absence of washing steps reduces the binding reaction duration and favors a rapid and compact device, a critical asset for patients experiencing life-threatening diseases. In order to maximize analytical performance, standard systems employed in clinical laboratories rely largely on the use of high surface-to-volume ratio suspended moieties, such as microbeads, which provide at the same time a fast and efficient collection of analytes from the sample and controlled aggregation of collected material for improved readout. Here, we introduce an integrated microfluidic system that can perform analyte detection on antibody-decorated beads and their accumulation in confined regions within 15 min. We employed the system to the concomitant analysis of clinical concentrations of Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Cystatin C in serum, two acute kidney injury (AKI) biomarkers. To this end, high-aspect-ratio, three-dimensional electrodes were integrated within a microfluidic channel to impart a controlled trajectory to antibody-decorated microbeads through the application of dielectrophoretic (DEP) forces. Beads were efficiently retained against the fluid flow of reagents, granting an efficient on-chip analyte-to-bead binding. Electrokinetic forces specific to the beads’ size were generated in the same channel, leading differently decorated beads to different readout regions of the chip. Therefore, this microfluidic multianalyte immunoassay was demonstrated as a powerful tool for the rapid detection of acute life-threatening conditions. Acute kidney injury (AKI) is a life-threatening condition characterized by a rapid loss of kidney function . In deveCurrently, AKI is diagnosed through monitoring of the patient’s urine output volume and measurement of the level of serum creatinine in blood . Creatin® system of Alere Inc. for the detection of NGAL through a lateral flow assay [® of Eurolyser for Cystatin C levels’ quantification. We aimed to concomitantly detect Cystatin C and NGAL using an immunoassay approach that could be engineered into a PoC device. Such multiple analysis would be an effective mean to obtain an accurate diagnosis of AKI in the first day following the injury. In order to minimize the number of assay steps, our device performed both the binding of the analyte molecules from the sample and the readout phase. We implemented such detection on antibodies-decorated beads to perform sandwich immunoassays. Indeed, functionalized microbeads suspended in a microfluidic channel provided a fast and effective collection of analytes in-flow. However, the gathering of beads into large clusters was required to obtain a high readout fluorescent signal. The spatial handling of beads is commonly performed either through magnetic forces [The critical requirements of timeliness in the diagnosis of AKI call for fast analytical devices to perform the analysis of the relevant biomarkers directly within intensive care units or emergency facilities. The detection of such markers in clinical settings relies on immunoassays carried out by means of bulky analyzers, such as Abbot Architect i1000SR and c4000, used, respectively, for the quantification of NGAL and Cystatin C. Commercial point-of-care (PoC) systems for the individual detection of these targets are also available on the market, namely, the Triageow assay  and the c forces  or by mec forces . These aDielectrophoresis (DEP) is a physical phenomenon appearing when placing a polarizable particle into a non-uniform electric field. It has been successfully employed to sort particles according to their size, shape, and dielectric properties ,15. It aHere, we introduce high-aspect-ratio vertical electrodes in the microfluidic channel to enable highly effective manipulation of microbeads by electrokinetics in minimally diluted buffers (5x). In our system, microbeads decorated with specific antibodies were incubated with analytes on-chip along DEP trapping regions in presence of flow, drastically reducing mass transport issues. They were later moved and concentrated to detection regions, where the optical fluorescence signal of the markers was amplified by clustering multiple beads. Our device allowed carrying out both analyte binding and signal readout under the continuous flow of the same reagents, avoiding rinsing steps.In this paper, we present the first microfluidic multianalyte platform for rapid detection of AKI. The simultaneous assessment of Cystatin C and NGAL levels we achieved allows for rapid and accurate diagnosis of AKI within a large temporal window after the injury and with a matching performance compared with a state of the art ELISA kit.The microfluidic system consists of a 4-mm-wide main channel, spanned by six rows of vertical electrodes. Two inlets allow for sequential injection of beads and reagents, subsequently disposed through a single outlet. The height of both the microfluidic channel and the vertical electrodes is 50 µm.The platform is fabricated through an additive process on a glass substrate . A detaiDielectrophoresis (DEP) is a phenomenon that occurs to a polarizable particle placed in a non-uniform electric field. The presence of an electric field triggers the formation of a dipole in a polarizable particle. The dipole experiences a net force if the electric field is spatially non-uniform, which leads the particle to move. The direction of the force induced by DEP on the particle depends on the relative polarizability of the particle and of the surrounding medium. If the particle is more polarizable than the surrounding medium, the particle dipole will be oriented along the electric field, while a particle less polarizable than the surrounding medium results in a dipole oriented against the electric field. The force experienced by a spheroid particle with a radius R placed in an electric field E can be written as:g medium .η is the fluid viscosity, R the sphere radius, and V0 the fluid constant velocity at infinity.Furthermore, a spherical particle injected into the microfluidic channel is dragged by the flow according to the following equation:In our device, the electric field is generated by linearly arranged vertical electrodes. If the line of electrodes is normal to the flow, the beads flowing through the channel experience a DEP force directed against the drag force a. ProvidEquation (1) establishes the dependence between the size of the beads and the DEP force they will experience. Each line of electrodes can generate a different DEP force and can consequently trap and displace specific beads depending on their size. In this way, beads of different sizes can be driven to distinct regions on the chip f. If beaThe microfluidic chip for DEP-based immunoassays is shown in w/v) in 1 mL of diluted FBS, was delivered from one inlet at 2 µL/min, maximal flow that could be applied without having beads escaping the incubation region, while the incubation line exerted a holding force . The obtained small beads’ aggregates were spatially confined by the DEP force, while kept in slight agitation by the flow: This turbulent motion favors the convective transport of target molecules. (2) After a sufficient number of beads was collected (1–2 ms), the beads–cAbs solution flow was stopped and the solution of antigen–dAbs complexes was immediately dispensed from the other inlet at 2 µL/min. The antigen–dAbs solution was flushed in the chip for 15 min. During this incubation step on-chip, the binding between the analyte–dAbs and the cAbs led to the formation of the complete sandwich assay on the beads’ surface. (3) After incubation, the beads were released by turning off the electrical signal. Concomitantly, the concentration line was activated  and the beads were led to “surf” in-flow along the diagonal concentration lines until they reached a region where they were trapped against the microfluidic wall, as shown in 2 provided that their footprint was equal or larger than 50 µm2. (5) Finally, the beads were released from this concentration region by deactivation of the electrodes’ line.Bead-based sandwich immunoassays were performed on our platform as illustrated in w/v) 2-µm beads and 10 µL 0.5% (w/v) 6-µm beads in 1 mL 5-fold diluted FBS. The incubation step was performed on two different lines  in presence of a 0.4 µL/min flow, maximal flow that was applied while holding the small 2-µm beads against the flow. The line upstream was set to exert a weaker electric field so that beads of larger size were trapped by the first line, while the smaller ones could pass through, to be successively trapped by the second line, with a trapping efficiency of 90% and 70% for the 6-µm and the 2-µm beads, respectively. After beads’ clustering, incubation was performed flushing a solution containing the analyte–dAbs complexes of both Cystatin C and NGAL, with the dAbs labeled with CY3 and CY5 fluorophores, respectively. Following incubation, the two beads’ populations carrying the two distinct sandwich assays were released and concentrated at different locations on-chip. To do so, the two accumulation lines were activated , and the flow was set to 0.8 µL/min, maximal flow allowing accumulation of 2-µm beads. The two distinct clusters were imaged in CY3 and CY5 fluorescent channels with 5000-ms exposure times. On average 20 experiments  could be performed before the appearance of clogging issues preventing the chip from further use.A similar protocol can be applied to run two distinct immunoassays in parallel on the same channel. Two different beads’ populations (2-µm and 6-µm diameter) were functionalized with cAbs for Cystatin C and NGAL, respectively. The beads–cAbs solution was obtained by adding 10 µL 0.5% , a TG5012A function generator , an optical fluorescence microscope (Leica DM2500M) with Y3 and Y5 filter cubes , and a camera . The electric signal was provided to the chip through a homemade printed circuit board (PCB), depicted in Polystyrene beads covalently coated in Streptavidin (Spherotec Inc.) were purchased at sizes of 2-µm diameter (binding capacity = 0.42 nmol/mg) and 6-µm diameter (binding capacity = 0.14 nmol/mg). The 2-µm beads were incubated with biotinylated Cystatin C monoclonal capture antibodies  and the 6 µm-beads were incubated with biotinylated NGAL polyclonal capture antibodies  for at least four hours. They were then resuspended and incubated for 1–2 h in 1% bovine serum albumin  in 0.1 M phosphate buffer  for blocking. After blocking, beads were washed four times through a procedure of centrifugation, supernatant removal, and resuspension in a solution of 0.05% Tween-20  in 0.1 M PB. After the final washing steps, beads were stored in 0.1 M PB at 4 °C. Both antigens for Cystatin C  and NGAL  were acquired and used as is. Monoclonal detection antibodies for Cystatin C  were purchased with a Dylight 550 fluorophore, while monoclonal detection antibodies for NGAL  were labeled with a fluorophore using a Lightning-Link Rapid Alexa Fluor 647 antibody labeling kit . Prior to experiments, antigen and detection antibodies were incubated together for 15 min in fetal bovine serum  diluted five times in MilliQ water.All images and videos acquired in the scope of this paper were analyzed using ImageJ software (Fiji). For each cluster, the neighboring background signal was calculated and subtracted from the mean intensity in the regions of interest, to account for possible variations of the background intensity. With this approach, we quantified the normalized fluorescence signals displayed in The on-chip incubation of antibody-functionalized beads with antigens and detection antibodies took place on dedicated horizontal incubation lines. We maintained beads decorated with cAbs against the flow and concentrated them in small clusters of about 10 to 20 beads. Significant variability was observed in the size of the growing clusters, reflecting inhomogeneities in the flow lines within the microfluidic channel. Such inhomogeneities were mainly caused by microfabrication defects or debris accumulation at the entrance of the chip. Then, a solution containing the dAb–NGAL complexes was injected at a flow rate of 2 µL/min for 15 min. The binding of the dAb–NGAL complexes to the cAb-decorated beads was observed and quantified in real time by fluorescence measurements of the CY5 channel . Figure We designed the DEP holding action at the horizontal incubation lines in a way that the size of the clusters would be maintained sufficiently small to keep the accumulated beads in slight agitation. This approach allows for uniform binding of analytes in-flow and access to the whole surface offered by the beads. In fact, in the case of large clusters, the reagents would be depleted at the downstream portion of the aggregate, an issue that can be noticed in previous works . This liMoreover, previous approaches employing DEP force to immobilize beads and expose them to reagents relied on the use of planar electrodes to generate electric fields . This apAnother advantage deriving from the three-dimensional electrodes employed to generate DEP forces is the use of minimally diluted solutions. Currently, as DEP forces are weakened in high ionic strength solutions , most DEFurthermore, the integration of the incubation step on-chip appears as a key milestone in the process of embedding our technology in a point-of-care device (PoC). In fact, it would limit the variance and errors in the concentration readout that might derive from additional manipulations .incubation lines with the antigen–dAbs complex, as described in the previous section, or off-chip, by placing the Eppendorf containing the beads and the complexes in a rotating mixer. The obtained beads were then either clustered on-chip, as shown in The step directly following on-chip incubation is the accumulation of beads in dedicated areas, to obtain a larger signal and, thus, increase the sensitivity of detection. This section aimed to assess the impact of carrying out beads’ incubation and aggregation in our microfluidic system, comparing our approach both to a bulk process of incubation of the reagents and to the detection of the signal from single beads. To do so, the experimental plan described in The comparison between plots 1 and 2 of On the other hand, the influence of incubation conditions on the attained signal could be analyzed comparing plots 2 and 3 of The integration of the incubation step within the microfluidic system is a key step in the design of autonomous lab-on-chip platforms. Recent approaches emphasize the need to maintain a certain level of agitation for beads during incubation, in order to maximize the interaction between beads and target analytes in the solution ,31,32. IThe NGAL concentrations that could be detected with our device ranged from 1 to 100 ng/mL, with a limit of detection of 1 ng/mL, calculated using a three-times standard deviation approach. However, as the concentrations expressed in the calibration curve refer to the five times diluted serum, this interval can be translated into a 5–500-ng/mL detection range in non-diluted serum. This interval covers the clinical NGAL concentration values observed in healthy patients (around 80 ng/mL) and patients suffering AKI (above 300 ng/mL) . FurtherAll experiments carried out within this publication were run in 5 times diluted serum sample . As none of the previous studies employing DEP to perform immunoassay on beads relies on serum ,20, we aThis section aimed to investigate the possibility to concomitantly quantify the concentration of two biomarkers on a single chip. The two chosen analytes, NGAL and Cystatin C, spike into serum at different stages of the kidney injury . Three hIn order to describe the efficiency of beads’ separation, NGAL and Cystatin C detection antibodies see b were laFor the CY5 channel b, red, cRegarding the CY3 channel b, green,Our platform could achieve the differentiation of healthy, “early AKI” stage and “late AKI” patient by detecting both NGAL and Cystatin C in clinically relevant ranges ,36,37,38Previous approaches aiming to simultaneously carry out multiple biomarkers’ detection mainly relied on the use of fluorophores embedded in the beads ,40. ThisIn this paper, we presented a fully integrated system for fast and efficient acute kidney injury diagnostics. The introduction of high-aspect-ratio vertical electrodes within the microfluidic channel permitted an accurate manipulation of antibodies-decorated beads through a novel method named “DEP surfing”. The incubation step was conducted in a dedicated area under a high flow rate, ensuring an effective collection of analytes on the surface of beads, an important feature for the detection of analytes at low concentration. We introduced the possibility to perform in the same microchamber, while in two separate phases, sample beads’ incubation and beads’ accumulation, controlled solely by electrical signals. The amplified fluorescent signal acquired with our method proved to be as bright as the one obtained with turbulent mixing. We employed this approach to the concomitant detection of two analytes, thanks to a size-based beads’ separation technique: NGAL and Cystatin C could be simultaneously detected within 15 min in a minimally diluted matrix, and the detection performance matches the one of a commercial ELISA kit. The combined detection of both biomarkers allows for the diagnosis of AKI conditions at different stages, which could be greatly beneficial to patients in intensive care units.Currently, our device is designed for the detection of two biomarkers. Nonetheless, the technology could be easily readjusted for the detection of more analytes through the use of beads of different sizes. Furthermore, since this system relies on the largely established biochemistry of antibody–beads’ conjugation and on-sandwich assays, it can be easily translated to the analysis of other acute conditions or infectious diseases.In order to miniaturize the platform and promote its integration in a one-step, point-of-care device, the active fluidic components would need to be replaced by passive fluidic structures, such as capillary pumps, while the beads could be dried in the channels prior to exposure to the patient sample ,44. An aThis work resulted in the deposition of the following patent: Thiriet, P.-E.; Medagoda, D.; Guiducci, C. Dielectrophoresis detection device. European Patent (EP) priority: 5 June 2020 no. 20178445.1


Eptesicus fuscus) is widely distributed and common. Big brown bats are a known reservoir for rabies virus, which, combined with their propensity to roost in human structures, necessitates testing for rabies virus following human exposure. The current pandemic caused by severe acute respiratory syndrome coronavirus 2, likely of bat origin, illustrates the need for continued surveillance of wildlife and bats for potentially emerging zoonotic viruses. Viral metagenomic sequencing was performed on 39 big brown bats and one hoary bat submitted for rabies testing due to human exposure in South Dakota. A new genotype of American bat vesiculovirus was identified in seven of 17 (41%) heart and lung homogenates at high levels in addition to two of 23 viscera pools. A second rhabdovirus, Sodak rhabdovirus 1 (SDRV1), was identified in four of 23 (17%) viscera pools. Phylogenetic analysis placed SDRV1 in the genus Alphanemrhavirus, which includes two recognized species that were identified in nematodes. Finally, a highly divergent rhabdovirus, Sodak rhabdovirus 2 (SDRV2), was identified in two of 23 (8.7%) big brown bats. Phylogenetic analysis placed SDRV2 as ancestral to the dimarhabdovirus supergroup and Lyssavirus. Intracranial inoculation of mouse pups with rhabdovirus-positive tissue homogenates failed to elicit clinical disease. Further research is needed to determine the zoonotic potential of these non-rabies rhabdoviruses.Bats are a host and reservoir for a large number of viruses, many of which are zoonotic. In North America, the big brown bat ( Long recognized as a reservoir of rabies virus, bats are the host to an ever-expanding cadre of zoonotic viruses . RepeateRhabdoviridae is comprised of 20 genera and 144 recognized species identified from a wide range of hosts including plants, insects, amphibians, fish, and mammals [Lyssavirus, which includes rabies virus, exist in bat or wildlife reservoirs and are zoonotic, causing encephalitis. Members of the genera Ledantevirus (Le Dantec virus) and Vesiculovirus , and possibly Tibrovirus, are largely arboviruses that can variably infect humans and cause febrile illness or encephalitis [The family  mammals . A numbephalitis ,7.Rhabdoviridae are found in bats. The most studied genus, Lyssavirus, includes numerous species with a bat reservoir that can infect a wide array of vertebrates [Lyssavirus, several members of Ledantevirus have been identified in bats, including Le Dantec virus, Kern Canyon virus, Mount Elgon bat virus, Vaprio virus, and Kumasi rhabdovirus [Vesiculovirus species, as well as divergent strains phylogenetically distinct from established genera [Members of multiple genera of tebrates . Besidesbdovirus ,9,10,11.d genera ,13.Eptesicus fuscus) and one hoary bat (Aeorestes cinereus) submitted to the South Dakota State University Animal Disease Research and Diagnostic Laboratory (SDSU ADRDL) for rabies testing due to human exposure between March and June 2020. Thirty-nine of the bats were collected in South Dakota, and one was collected in Minnesota (n = 23), a tissue homogenate was prepared from all viscera.To identify novel, possibly zoonotic viruses in bats with human exposure, metagenomic sequencing was performed on 39 big brown bats  reads were generated per sample. Contigs were assembled de novo using CLC Genomics and analyzed by BLASTX using the BLAST2Go plugin and the non-redundant protein sequence database in May 2020.Rhabdoviridae [Phylogenetic analysis was performed on conserved regions (Gblocks) of the L protein from 135 members of oviridae ,15. SequE. fuscus, a Taqman assay was designed to target the conserved L gene . Similarly, Taqman assays were designed to detect Sodak rhabdovirus 2  and Sodak rhabdovirus 1 . Real-time RT-PCR was performed using the Taqman FAST virus 1-step master mix following the manufacturer’s suggested protocol.To assess the prevalence of American bat vesiculovirus (ABVV) in Eptesicus fuscus kidney (Efk3) cells [2. Confluent monolayers in 12-well plates were inoculated with 100 µL of 0.22 µm filtered tissue homogenate and incubated for seven days before passaging to new cells. Cell culture supernatants were passaged one time to a new monolayer of cells and incubated for seven days before being considered negative when no cytopathic effects were apparent.Virus isolation was attempted for samples positive for ABVV, SDRV1, and SDRV2 on Vero and immortalized 3) cells . Cells wPregnant CD-1 mice were purchased from Charles River Laboratories. A single female was caged with her litter. Following parturition, three-day-old suckling mice were injected intracranially with 10 µL of 0.22 µm sterile filtered bat tissue homogenate . A singlMouse studies were approved by the South Dakota State University Institutional Animal Use and Care Committee . Bat specimens were submitted to the SDSU ADRDL for diagnostic testing, and as such, no specific approval was necessary.An approximately 10.7 kbp contig was assembled de novo each in four heart and lung tissue homogenates from big brown bats submitted for rabies testing . BLASTX Sequence analysis of a viscera pool identified a 11,221 bp contig that was 46–54% identical to nematode rhabdovirus genomes spanning the L protein . Open reAlmendravirus encodes a class 1a viroporin of a similar size between the G and L genes; it is unknown if a similar function is encoded by SDRV2 U1 [Analysis of a second viscera pool found a 12,600 bp contig that had ~33–45% identity to rhabdovirus L proteins over the 3′-half of the contig . BLASTP SDRV2 U1 .Alphanemrhavirus. Sodak rhabdovirus 2 formed a distinct lineage in a basal position to Almendravirus, Lyssavirus and the dimarhabdovirus  supergroup.The four strains of ABVV were closely related to the single previously identified strain of ABVV that together formed a sister clade to one comprised of Radi vesiculovirus and Yug Bogdanovac vesiculovirus, both identified from sandflies . TogetheCt values 11–33, indicating that ABVV infection of bats in South Dakota is common and includes a high titer viremia. Only three out of 23 (13%) viscera pools were positive for ABVV.Seven out of 17 (41%) heart and lung pools were positive for ABVV with Sodak rhabdovirus 1 was only detected in four of the 23 viscera pools (17%). Similarly, SDRV2 was only identified in two of the 23 viscera pools (8.7%). Cycle threshold values for SDRV1 were 24–32 and were 25–34 for SDRV2.Intracranial inoculation of mouse pups with ABVV, SDRV1, and SDRV2 was conducted to evaluate potential virus pathogenicity. A complete litter comprised of 13, 11, or 12 pups was inoculated with each virus, respectively, and observed for 18 days. No clinical signs of disease or mortality were observed.Rhabdovirus-positive tissue homogenates were used to inoculate Vero and Efk3 cells. No cytopathic effects (CPE) were evident following seven days of incubation. Likewise, no CPE was evident after seven days following passage of cell culture supernatants to a new monolayer of Vero and Efk3 cells.Rhabdoviruses from multiple genera cause disease in humans, including rabies virus, which is responsible for an excess of 50,000 human fatalities annually . Bats arE. fuscus, was found in 23% of bats [Ct values were found in viscera pools. These results suggest that ABVV infects bats as opposed to detection due to environmental or dietary contamination. While the zoonotic potential of ABVV is unknown, several members of Vesiculovirus cause disease in humans. Chandipura virus, for example, has caused outbreaks of encephalitis in children in India [Vesiculovirus are arboviruses, suggesting a possible insect reservoir for ABVV. The high titer of ABVV in bat cardiac and pulmonary tissue also raises questions on possible adverse health effects on bats.American bat vesiculovirus, previously identified in  of bats . While tin India ,21. AnotAlphanemrhavirus is comprised of two species, Xingshan alphanemrhavirus and Xinzhou alphanemrhavirus, both identified by metagenomic sequencing of nematodes [Eptesicus [Ledantevirus were identified both in Nycteribiid bat flies and in the Angolan soft-furred fruit bats that they parasitize [ematodes . Nematodematodes , includiptesicus ,25. Nemaptesicus . Sodak rrasitize .Almendravirus, Lyssavirus, and the dimarhabdovirus supergroup. Almendraviruses are only known to infect mosquitoes, while lyssaviruses exist in a bat reservoir and infect other mammals [Phylogenetic analysis suggests that SDRV2 is derived from an ancestral virus precursor of  mammals . DimarhaVirus isolation using immortalized cell lines and suckling mice were unsuccessful. Suckling mice have long been used for rhabdovirus research, as they are highly susceptible to virus infection that results in high viral titers and clinical disease. While ABVV, SDRV1, and SDRV2 all failed to cause disease here, further studies, including serosurveys, are needed to evaluate the potential host range of these viruses.Rhinolophidae bats. These viruses met the criteria for representing a distinct species. Additionally, a highly divergent virus, Yangjiang bat virus (YJBV), was identified in multiple Hipposideridae bats. While only a partial L gene sequence was determined, phylogenetic analysis placed YJBV in a sister clade relationship to dimarhabdoviruses similar to SDRV2. Detection of novel vesiculoviruses and divergent unclassified rhabdoviruses in multiple bat genera in different countries suggests bats may constitute an under-appreciated reservoir for diverse Rhabdoviridae.A viral metagenomic survey in China likewise reported diverse rhabdoviruses in bats . Two ves


Ciona intestinalis type A (Ciona robusta), exhibit strict self-sterility. In a previous study, we found that the candidate genes responsible for self-sterility in Ciona reside in chromosome 2q (locus A) and chromosome 7q (locus B). Two pairs of multi-allelic genes, named s(sperm)-Themis-A and v(vitelline-coat)-Themis-A in locus A and s-Themis-B and v-Themis-B in locus B, are responsible for self-sterility. In this study, we identified a third multi-allelic gene pair, s-Themis-B2 and v-Themis-B2, within locus B that is also involved in this system. Genetic analysis revealed that the haplotypes of s/v-Themis-A, s/v-Themis-B and s/v-Themis-B2 play essential roles in self-sterility. When three haplotypes were matched between s-Themis and v-Themis, fertilization never occurred even in nonself crossing. Interestingly, gene targeting of either s/v-Themis-B/B2 or s/v-Themis-A by genome editing enabled self-fertilization. These results indicate that s/v-Themis-A, -B and -B2 are S-determinant genes responsible for self-sterility in the ascidian C. intestinalis type A.Many hermaphroditic organisms possess a self-incompatibility system to avoid inbreeding. Although the mechanisms of self-incompatibility in flowering plants are well known, little is known about the mechanisms of self-sterility in hermaphroditic marine invertebrates. Ascidians are hermaphroditic sessile marine invertebrates that release sperm and eggs into the surrounding seawater. Several species, including  In particular, Halocynthia roretzi and Ciona intestinalis type A (recently proposed to be renamed Ciona robusta3) exhibit strict self-sterility, although their mechanisms for prohibiting self-fertilization appear to be very different5.Most living organisms produce offspring by sexual reproduction to create genetic diversity in the next generation. However, hermaphroditic marine invertebrates such as ascidians  potentially allow self-fertilization. Although compound ascidians appear to release sperm and eggs at different times to avoid self-fertilization, several solitary ascidians are known to release sperm and eggs almost simultaneously into the surrounding seawater during the spawning season. However, even such ascidians show self-sterilityH. roretzi, HrVC70, a 70-kDa protein that is a main component of the vitelline coat (VC), appears to be responsible for self-sterility; HrVC70 attaches to the VC during oocyte maturation, wherein self-sterility is acquired, and HrVC70 can be specifically extracted from the isolated VC by 1–5 mM HCl, which is in agreement with the results showing that self-sterile eggs become self-fertile following short treatment with acidic seawater (pH 2.5–3.0). Importantly, nonself-sperm, rather than self-sperm, efficiently bound to HrVC70-immobilized beads, and HrVC70 from nonself-eggs more efficiently inhibited fertilization than did HrVC70 from self-eggs. In addition, HrVC70 is composed of 12 EGF-like repeats showing a polymorphism in restricted regions among individuals. These biological and biochemical data led us to propose that HrVC70 is a candidate protein involved in self-sterility in H. roretzi (see reviews5).In Ciona intestinalis type A, a HrVC70 orthologue, CiVC57, is present in the VC; however, it is not known whether CiVC57 is responsible for self-sterility, although CiVC57 appears to be involved in gamete interaction5. Thus, we attempted to identify S (Self-sterility or Self-incompatibility)-determinant gene(s) in C. intestinalis type A by a genetic approach. Thomas Hunt Morgan first studied the genetic mode of self-sterility in Ciona approximately a century ago (beginning in 1910)9. He reported that self-sterility is genetically controlled because artificially self-fertilized F1 siblings showed one-way or reciprocal sterility following crossing; this sterility was scarcely observed in natural crossing between nonself-fertilized siblings9. The mode of self-sterility was later studied by Murabe and Hoshi, who showed that at least two multi-allelic loci are involved in self-sterility10. Rosati and De Santis11 and Kawamura and his colleagues12 investigated the mechanisms of self-sterility in Ciona. These authors showed that self/nonself recognition in gamete interaction takes place on the VC and that nonself-sperm, but not self-sperm, are capable of binding tightly to the VC of glycerinated eggs12. However, the molecular mechanism of self/nonself recognition during fertilization is not known.In contrast, in C. intestinalis type A by positional cloning and proteomic analysis13. To explain one-way cross-sterility, Morgan proposed a “haploid sperm hypothesis”, according to which self-sterility is achieved by haploid expression of the S-determinant gene in spermatozoa and diploid expression in eggs . According to his hypothesis, there are two populations in heterozygous (A/a) sperm (A-sperm and a-sperm), either of which can fertilize homozygous eggs because A/A-eggs (or a/a-eggs) are recognized by a-sperm (or A-sperm) as nonself eggs  and locus B (chromosome 7q). Each locus contained a pair of similar genes despite there being no overall synteny, i.e., fibrinogen-like proteins expressed in the VC, designated v(vitelline coat)-Themis-A (in locus A) and v-Themis-B (in locus B), and sperm polycystin 1-like proteins named s(sperm)-Themis-A (in locus A) and s-Themis-B (in locus B)  channel) domain in addition to the domains shared with s-Themis-A, including HVR, receptor for egg jelly (REJ), G protein-coupled receptor proteolysis site (GPS), and lipoxygenase homology 2 (LH2) domains13 , which is followed by detachment from the VC or by the sperm entering a quiescent state on the VC15. However, it is still unclear for the reasons stated below whether s/v-Themis-A and s/v-Themis-B gene pairs alone are sufficient for execution of self-sterility in C. intestinalis type A.With this approach, Harada  B) Fig. . The entnes Fig. . This fiC13 Fig. . We prop 13 Fig. . s-Themis13 Fig. . We prevs/v-Themis gene pair may exist near the s/v-Themis-B gene in locus B, since three alleles of B-type v-Themis proteins were detected in the VC by mass spectrometry14; further, one additional s/v-Themis-B gene pair has been proposed to exist in locus B on the basis of the Ghost database 16. In addition, it was recently reported that only the s/v-Themis-B gene region showed heterogeneity in an inbred strain and that the copy number of s/v-Themis-B gene pairs is variable among individuals17. These unexpected results led us to further investigate the gene structures and their roles in the self-sterility of B-type s/v-Themis. Since the DNA sequence around s/v-Themis-B has not yet been completely determined, we first cloned and sequenced the genomic DNA in locus B. We identified a new gene pair, designated s-Themis-B2 and v-Themis-B2, which resides approximately 70 kbp away from the s/v-Themis-B gene pair. We also showed that three gene pairs, s/v-Themis-A, -B and -B2, are S-determinant genes responsible for self-sterility in C. intestinalis type A.We previously reported that one additional B-type s/v-Themis-B, we isolated and sequenced a 35-Mb genomic DNA clone containing s/v-Themis-B from the BAC library of C. intestinalis type A genomic DNA, and we compared the genomic DNA sequences in the Ghost database16 and the JGI database18. Our newly determined BAC clone sequence, designated JP1 haplotype (or B-6 haplotype), was compared with DNA sequences in the two databases by dot matrix plot analysis  were located at the s/v-Themis gene pair in locus B by primer walking, and we identified a novel s/v-Themis gene pair, designated s/v-Themis-B2. These genes were approximately 70 kbp away from the locus of s/v-Themis-B  for s/v-Themis-B2 have been identified  for s/v-Themis-A, -B, and -B2 are S-determinant genes, we performed genetic analysis by cross-fertilizing selfed F1 and F2 siblings, whose s/v-Themis-A, -B, and -B2 alleles had been identified using allele-specific PCR primers. According to the “haploid sperm hypothesis”9, there must be haploid expression in s-Themis-A, -B, and -B2 alleles and diploid expression in v-Themis-A, -B, and -B2 alleles. When sperm s-Themis-A, -B, and -B2 recognize either of two alleles of v-Themis-A, -B, and -B2 as the same haplotype, respectively, fertilization is thought to be blocked  haplotypes were not functional, as discussed later. These allele analysis experiments clearly demonstrated that s/v-Themis-A, -B, and -B2 are S-determinants that play a pivotal role in self-sterility in C. intestinalis type A.As summarized in Fig. s-Themis-A and s-Themis-B/B2 using TALEN (transcription activator-like effector nuclease). Fertilized eggs of individuals with known alleles were subjected to electroporation with TALEN plasmids or mRNAs. After electroporation, embryos were cultured for 3 months in the laboratory, and gametes were collected from sexually mature individuals. As shown in Fig. s-Themis-B/B2 CR (conserved region)-targeted individuals. However, when the s-Themis-A  gene was targeted, self-fertilization was allowed but required a longer time  showed a small or null fertilization ratio Figs.  and S5. s/v-Themis-B2, it is notable that a certain haplotype in s/v-Themis-B2 may not function as an S-determinant. Among the three exceptional cases in s/v-Themis-B2, in two exceptional cases shown in trial 1  genes are non-functionable pseudogenes.Concerning ORF Fig. . In addis-Themis-B/B2 CR-targeted individuals. In contrast, when the s-Themis-A  gene was targeted, self-fertilization was observed but required a longer time  cells in a haploid manner, which is different from how they are expressed in germ cells , and the latter utilizes pollen PrpP and stigmatic PrpS22. On the other hand, Solanaceae and Rosaceae utilize nonself-recognition systems, where stigmatic S-RNase and pollen tube SLF (S-locus F-box protein), a ubiquitin ligase involved in the degradation of nonself-S-RNase that has entered a pollen tube, are nonself-recognition partners. From this viewpoint, it is not surprising that Ciona (order Phlebobranch) and Halocynthia (order Stolidobranch) utilize different S-determinant genes. It is not known whether S-determinant genes are genus-, family-, or order-specific. It also remains elusive whether s/v-Themis play a key role in Stolidobranch ascidians, although s/v-Themis genes are present in the genome database of Halocynthia.It is notable that the mechanism of self-sterility in S-determinant genes are highly polymorphic and tightly linked in the same or contiguous loci, and the other is that male and female S-determinants are a single haplotype that make up a recognition partner. In particular, the self-incompatibility system in Papaveraceae is very similar to the self-sterility system in Ciona intestinalis type A: calcium influx takes place, resulting in apoptosis in pollen to prohibit self-fertilization, after pollen PrpP recognizes the stigmatic PrsP as self25, while calcium influx takes place in C. intestinalis type A sperm after sperm binding to the VC of self-eggs15.In any case, it should be emphasized that there are two similar points in animal and plant self-incompatibility (or self-sterility) systems: one is that male and female 26. On the other hand, it has been reported that human sperm PKDREJ, a candidate sperm-born receptor27 and a homologue of ascidian sperm s-Themis-B/B2, exhibits amino acid polymorphisms among 14 primates and 48 human individuals27, and it has been proposed to have evolved by positive selection28. Therefore, it is interesting to speculate that allogeneic polymorphisms in PKDREJ might be related to the efficiency of human fertility, similar to how s-Themis-B/B2 functions in ascidians.Our results provide new insights into the molecular mechanism of self-sterility in ascidians, which is very similar to the mechanisms of self-incompatibility in flowering plants. This is a good example of convergent evolution between plants and animalsCiona intestinalis type A (recently proposed to be renamed Ciona robusta), that were used in this study were collected in Gamagori, Mikawa Bay, and some of the ascidians were supplied by National BioResource Project (NBRP) from Tokyo and Maizuru Bay. Spermatozoa and eggs were collected as described previously29. Fertilization and embryo culture were carried out as described previously13. To determine the genotype of the animals, DNA was extracted from the testis, and direct sequencing of the HVRs of s/v-Themis-A, -B, and -B2 was carried out using primers listed in Table The ascidians, C. intestinalis type A genome databases: the Ghost database 30, which is linked to the Ascidian Network for In Situ Expression and Embryological Data (ANISEED) containing 13 ascidian databases (http://www.aniseed.cnrs.fr)31, and the JGI (DOE Joint Genome Institute) database (http://genome.jgi-psf.org/Cioin2/Cioin2.home.html)18.The DNA sequence in locus B was searched for with two 32 encoding s/v-Themis-B as a template and P1(TBg) and P2(TBg) primers  and P4(TBg), which correspond to the conserved region of s-Themis-B2 and to the KH.C7.700.v1.C.ND1-1 that is located near s/v-Themis-B2. The amplified genomic DNA fragments were used as a template, and DNA sequencing was carried out by primer walking.PCR was carried out using a BAC DNA clone (GECi44_m19)s-Themis-B, which resulted in amplification of the 5′-region of s-Themis-B. Next, using the reverse primer P1(sTBc) (conserved region of s-Themis-B) and haplotype-specific forward primers (P2(sTBc) - P6(sTBc)) located in the first exon of s-Themis-B, PCRs were carried out for the region around v-Themis-B using genomic DNA as a template. Since there are one or more exons in v-Themis-B, v-Themis-B sequences were determined by using ovary cDNA as a template and two primers P7 and P12 that corresponded to the first and second exons, respectively 33.DNA dot matrix plot analysis was carried out according to DNA DotPlot v1.0  2.0 program and GenomeScanZFN to avoid off-target effects. TALEN was constructed using a Golden Gate TALEN and TAL Effector Kit 2.039. The TALEN mRNA was synthesized with mMASSAGE mMACHINE T3 (Ambion) and a Poly(A) Tailing kit (Ambion) using pHTB-Ci-s-Themis-A(A-2) and pHTB-Ci-s-Themis-B/B2(CR) plasmids39 as templates after treatment with XhoI. We also constructed TALENs targeting the genes s-Themis-A(A-2) and s-Themis-B/B2(CR) by inserting them into Ci-EF1α TALEN expression vectors39.TALEN target sites within the C. intestinalis type A eggs were removed by gentle pipetting after two additions of 5 ml of filtered seawater (FSW) containing 0.2 g of sodium thioglycolate and 0.02 g of actinase E to an equal volume of egg suspension. After the VC had been removed by this treatment, the VC-free eggs were gently washed with FSW 3 times. The eggs were placed on a gelatine-coated Petri dish filled with FSW and were then inseminated. After 20 min, the eggs were washed twice with 5 ml of FSW containing 0.7 M mannitol and were put into a cuvette for electroporation. TALEN mRNA (60 µg) and TALEN expression vectors (pEF1a > TALENs) (60 µg) were added to the egg suspension and subjected to electroporation with a Gene Pulser Xcell electroporation system (BIO-RAD). The eggs were transferred to a new gelatine-coated Petri dish filled with seawater, washed with FSW 3 times, and then kept at 16–18 °C until the hatched larval stage.The VCs of 30. The results are shown in Fig. DNA was isolated from hatched larvae after electroporation. To distinguish between s/v-Themis-B and s/v-Themis-B2, each locus was amplified using the primers that were used for genotyping listed in Table 4, 4 × 104, or 103. One hour after insemination, the number of cleaved eggs was counted, and the number was divided by the total number of eggs in each well to calculate the fertilization ratio (%). In the case of animals in which s-Themis-A(A-2) had been mutagenized by TALEN, the number of tadpole larvae was counted 18 hours after insemination to calculate the fertilization ratio (%) again.TALEN mRNAs were injected into embryos by electroporation. The injected embryos were cultured in a water tank until sexual maturation. Gravid animals were dissected for egg and sperm collection from the gonoduct and vas deference, respectively. Eggs were held in FSW for one hour at 20°C, and sperm were kept on ice until use. Nine hundred microliters of the egg-containing FSW was added to each well in a 24-well plate, and 100 μl of sperm diluted in FSW was added to each well so that the final numbers of sperm were 10Supplementary Information


Different countries have set different policies to control and decrease the costs of cardiovascular diseases (CVDs). Iran was aiming to reduce the economic burden of different disease by a recent reform from named as health transformation plan (HTP). This study aimed to examine the economic burden of CVDs before and after of HTP.This cross-sectional study was conducted on 600 patients with CVDs, who were randomly selected from a specialized cardiovascular hospital in the north-west of Iran. Direct and indirect costs of CVDs were calculated using the cost of illness and human capital approaches. Data were collected using a researcher-made checklist obtained from several sources including structured interviews, the Statistical Center of Iran, Iran’s Ministry of Cooperatives, Labor, and Social Welfare, the central bank of Iran, and the data of global burden of disease obtained from the Institute for Health Metrics and Evaluation to estimate direct and mortality costs. All costs were calculated in Iranian Rials (IRR).Total costs of CVDs were about 5571 and 6700 billion IRR before and after the HTP, respectively. More than 62% of the total costs of CVDs accounted for premature death before (64.89%) and after (62.01%) the HTP. The total hospitalization costs of CVDs was significantly increased after the HTP (p = 0.038). In both times, surgical services and visiting had the highest and lowest share of hospitalization costs, respectively. The OOP expenditure decreased significantly and reached from 54.2 to 36.7%. All hospitalization costs, except patients’ OOP expenditure, were significantly increased after the HTP about 1.3 times. Direct non-medical costs reached from 2.4 to 3.3 billion before and after the HTP, respectively.Economic burden of CVDs increased in the north-west of Iran after the HTP due to the increase of all direct and indirect costs, except the OOP expenditure. Non-allocation of defined resources, which coincided with the international and national political and economic challenges in Iran, led to unsustainable resources of the HTP. So, no results of this study can be attributed solely to the HTP. Therefore, more detailed studies should be carried out on the reasons for the significant increase in CVDs costs in the region. Cardiovascular diseases (CVDs) are among the leading causes of death and the essential reason for premature death (before 65 years old). In 2017, CVDs were responsible for 17.7 million death around the world, which is around 31.8% of the total deaths in the world . IncreasCardiovascular diseases costs are very high, so that in the European Union (EU) in 2016, the direct and indirect costs (premature death and disability) of CVDs were estimated to be about 25 and 18 billion euros, respectively , 8. It hDifferent countries, especially low-income and developing countries, have implemented several reforms in their health system framework to prepare financial protection to patients by decreasing the economic burden of diseases , 14. To Therefore, due to the high rate of CVDs in Iran, especially in Ardabil, as well as the implementation of the HTP as the essential equity-based intervention of the health system, this study was conducted to estimate the economic burden of CVDs before and after the HTP in the north-west of Iran.This cross-sectional study was conducted during September 2019 to February 2020, to evaluate the economic burden of CVDs in two periods: before (2013) and after (2016) the HTP implementation. The bottom-up approach, the cost of disease, and the human capital approach (HCA) were used to calculate the total costs.This cross-sectional study was conducted before and after the HTP implementation in the north-west of Iran. A 550-bed cardiovascular specialized hospital affiliated with MoHME was selected as the study site. At both time points (before and after the HTP), data on direct, indirect, and non-medical costs were collected from hospital-based cardiovascular inpatients records and compared. Baseline data were collected from households with CVDs at two intervals.Patients with one of the CVDs  in two periods, before (March 2012 to March 2013) and after (March 2016 to March 2017) the HTP, were eligible to participate in the study. Given that most hospitalization costs occur in the early years of the diagnosis and treatment of CVDs, the diagnosis of the disease over the past year was the basis of inclusion.Samples were collected from a specialized cardiovascular hospital the most extensive and only reference for patients with CVDs using a purposive random sampling method. Initially, a list of all patients admitted to the study during the two intervals was prepared, and data were collected from inpatients medical records through a checklist. If necessary, patients who were eligible to participate in the study were contacted, and additional information was obtained via telephone interview.Four types of CVDs with high frequency  were selected to estimate sample size. Then, the medical records of 40 discharged patients were randomly selected and evaluated for cost. Based on the mean and variance of hospital costs and using Cohen’s sampling method, the sample size was estimated to be 253 (n = 800) before and 284 (n = 1300) after the HTP.d is simply a measure of the distance between two means, measured in standard deviations [M1 and M2 are the means for the 1st and 2nd samples, and SDpooled is the pooled standard deviation for the samples. SDpooled is appropriately calculated using the following equation:Cohen’s viations . It is cAccordingly, the total number of hospitalizations for CVDs was extracted from the hospital information system (HIS), then, 600 patients for both intervals (300 before and 300 after the HTP) were randomly selected using random number table.A pilot study was conducted a month before (August 2019) on 30 inpatients with CVDs by a face-to-face interview to estimate indirect costs in the last month. The data were extracted using a checklist. Data were multiplied by 12 to estimate the total costs in the last year. Based on the mean and variance of hospital costs and using Cohen’s sampling method, the sample size was estimated to be 180 for both time points, before and after the HTP implementation. By telephone follow-up, patients were selected from the direct costs calculation phase (n = 300). Contact with all selected patients continued until the designated sample size was obtained.Data were collected using a researcher-made checklist included age, sex, length of hospital stay, hospitalization costs, and other related costs , surgery, and medication), which were extracted from the patients’ medical records. A checklist used in other similar international studies was used to collect non-hospital and non-medical costs , 25. TheIn this study, multiple data sources were used to estimate the economic burden of the disease. The most important data source used to estimate direct medical costs were the information extracted from the medical records of the patients with CVDs.This type of costs is directly attributable to patients admitted to various types of health care facilities , as well as treatment costs, including different services, such as diagnostic, medical, and rehabilitation services . To estiIndirect costs are a combination of the costs incurred to the patients and their families, such as the cost of medical travel, cost of time lost, and cost of patient in-home care .Time costs are calculated based on the mean number of days lost due to receiving health care services and hospitalization in the hospital. In this study, time costs were calculated by multiplying the number of hospitalization days by the average daily wage. Data for an average daily wage were obtained from the report of the Ministry of Cooperatives, Labor, and Social Welfare of Iran in 2018.The human capital approach was used as the primary method for the estimation of premature death costs in this study. In previous studies, HCA was used for the estimation of premature death due to non-communicable diseases, such as CVDs , 29. TotPopulation, employment rate, and life expectancy were extracted from the latest report provided by the Iranian Statistic Center (ISC).CVDs morbidity and mortality data were extracted from the National and Subnational Burden of Diseases, Injuries, and Risk Factors (NASBAD) survey.The final report by the Central Bank of the Islamic Republic of Iran was used to identify and determine the exchange rate by year.The average daily cost (the daily minimum wage lost) was calculated according to the principles presented by the Ministry of Cooperatives, Labor, and Social Welfare of Iran.The difference between payments by patients (patient’s share) and the insurance liability was calculated to determine the OOP expenditure.Indirect costs identified by data extracting through interviews with patients and their relatives who were aware of patients’ diseases using the designed checklist.Given that, there were no precise statistics on the level of household income, their income was estimated based on the minimum declared incomes of the Ministry of Labor, Cooperatives and Social Welfare.Other information, such as disease incidence, occupancy rate, payroll, GDP, national health expenditures, and standardized life expectancy were extracted from previous studies, treatment guidelines, World Bank databases, Iranian Ministry of Health (MoHME), and Iranian Statistic Center (ISC).All costs were equalled and presented in Iranian Rials (IRR) in two periods .Data were collected using structured interviews with patients, medical records, and valid data sources.The human capital approach considers the valuation of waste resulting from premature death for the whole economy . IndividIn the present study, the discount rate of 3% was used to obtain the present value of the lost production. The Present Discounted Value of Lifetime Earnings (PVLE) model developed by Max et al. (2000) was also used to calculate and estimate the present value of the patients’ income .Data were analyzed using SPSS version 16   and ExceThe prevalence of CVDs in Ardabil before and after the HTP was 44.12% and 50.11%, respectively. The number of deaths due to CVDs in Ardabil before and after the HTP was 2627 and 2940, respectively that accounts for 42.3% and 45.4% of the total deaths in the province, respectively.The results showed that 68.8% and 65.3% of the participants were men before and after the HTP, respectively. The mean age of patients in the two intervals was 62.4 ± 9.2 and 62.6 ± 8.6 years, respectively. The minimum and maximum hospital stay were one to 11 days, while the mean length of hospitalization in the two periods (before and after the HTP) was 1.5 ± 1.3 and 1.5 ± 3.9 days, respectively  and medication (18.3% and 19.1%). The ratio of indirect costs to the total hospitalization costs was reduced by 33%. In terms of all costs associated with providing services to patients with CVDs, except OOP expenditure, there was an increase in the costs after the HTP, while the highest rate belonged to the cost of open-heart surgery and the lowest one was related to angioplasty and angiography  were identified and calculated by telephone interviews with the patients before and after the HTP.Before and after the HTP, 60.6% and 57.1% of the patients were employed  and others were unemployed , respectively. The mean total days lost due to the diseases for both groups of patients (employed and unemployed) were estimated to be 61 days . The average cost per patient per day was calculated based on the mean income. For this purpose, the average monthly income of employed persons was calculated and divided into 30 days, and one-day income was calculated, which was 0.28 and 0.33 million IRR before and after the HTP. The average daily cost of unemployed patients was also calculated based on the minimum wage, which was declared by the Ministry of Cooperatives, Labor, and Social Welfare in the two time periods. Accordingly, the average daily cost of unemployed patients (minimum wage lost daily) was estimated to be 0.16 and 0.27 million IRR before and after the HTP.The total cost of time lost due to medical travelling by employed patients was estimated to be 4.80 and 5.63 million IRR before and after the HTP. The total cost of time lost due to disability was estimated to be 12.42 and 14.59 million IRR before and after the HTP. The total cost of travel and disability of employed patients before and after the HTP was 1.87 and 2.08 billion IRR, respectively, and for unemployed patients, it was estimated to be 541.94 and 734.35 million IRR before and after the HTP, respectively. The average cost of time lost due to medical travel of unemployed patients was 2.76 and 4.60 million IRR before and after the HTP, respectively. The cost of time lost due to disability in the two time periods was estimated at 7.14 and 11.91 million IRR, respectively. The total cost of time lost for the unemployed patients was estimated to be 0.526 and 1.27 billion IRR before and after the HTP, respectively.Indirect costs were estimated in terms of premature death and mean life expectancy for both sexes (75.15 years in 2013 and 75.95 in 2016). In Ardabil province, 880 people with ages of 35 to 75 years died due to CVDs in 2013, which was reduced to 782 in 2016. The total cost of premature death costs was 3615.3 and 4155.4 billion IRR before and after the HTP, respectively  before and after the HTP was 5571.06 and 6700.58 billion IRR, respectively. Overall, the results showed a significant difference in the hospitalization and indirect costs  of patients before and after the HTP (P < 0.05). However, despite the relative increase, the indirect and indirect mortality costs were not significantly different (P < 0.05).Cardiovascular diseases (CVDs) are among costly diseases for patients, their families, and health systems around the world. Different health systems trying to control and decrease the costs of CVDs by different reforms and restructures with a non-communicable disease control approach . Health This study aimed to evaluate the economic burden of CVDs in the north-west of Iran, an area with the highest incidence and prevalence of CVDs , before This study showed that mortality cost is one of the main components of CVDs economic burden in the north-west of Iran in both periods (before and after the HTP) so that more than 62% of the total costs of CVDs can be attributed to the mortality costs. The study also showed that despite a reduction in the percentage of the CVDs mortality cost after the HTP, this reduction was not statistically significant compared to that before the HTP, which is not consistent with the findings of most studies in developed countries. In European countries, more than 60% of the total costs of CVDs attributed to the hospitalization , 36, whiFurthermore, it was revealed that surgical services accounted for the highest proportion of costs compared to the other components of hospitalization costs before and after the HTP, which means that the total costs of open surgery and angiography/angioplasty increased more than other hospitalization costs after the HTP. In the UK, hospital inpatient care was estimated about 63% of the total CVDs costs, and surgical services were introduced as the largest share of hospitalization costs , which cThe proportion of hospitalization costs have been increased significantly after the HTP. One of the leading causes of this increase could be increasing hospitalization rate after the HTP. Karami et al. (2017) reported that the hospitalization rate was increased after the HTP in Iran . Tabari-Increasing hospitalization rate and the average length of stay: the increase of the average length of stay can be due to the increase of patients satisfaction and decrease of personal discharge rate .Lack of initiatives, such as failure to fully implement the referral system, and increasing referrals to specialized levels of health care system led to system failure to achieve Universal Health Coverage (UHC) , 49.Lack of timely and appropriate allocation of defined resources after the HTP caused a failure to fully adhere to the HTP guidelines , 50.The increase of outpatient OOP, where Rad et al. (2017) reported a significant increase of OOP payments for outpatient care services increased after the HTP, which influenced the total OOP . Also, dFailure to fully implement the HTP and its related laws, as Sajadi et al. (2019) reported that unsustainable resource allocation is one of the main challenges of Iran’s health system before and after the HTP . This haFinally in developing countries, the increase of the costs of health care services with a decrease of patients OOP expenditures can mean the provision of the costs to the public budgets, which can be interpreted as an increase in the government subsidies to the health sector . TherefoThe main objective of the first phase of the HTP was decreasing OOP expenditures of inpatients based on the patients’ insurance type  by allocating subsidies to increase the share of government health expenditures , 44, 45.However, this study had several strengths and limitations related to the study design and costs measurement. First, a province from the north-west of Iran (Ardabil), which has the highest level of CVDs, was selected, and the economic burden of CVDs that has not been studied before, was investigated. In addition, this study, for the first time, investigated the HTP and its effect on the economic burden of CVDs in Iran. In this regard, the evaluation of the economic burden of CVDs before and after the implementation of the main reform in Iran’s health sector, which may help policy makers to reorder the existing policies incorporated in the HTP in terms of affordability of the use of valid databases and robust methods with acceptable sample size to identify and capture the expenditures, is another strength of this study. Classification of the costs into three groups in general, non-calculation of outpatient costs and informal payments, and the possibility of recall bias in the patients during the study period, especially regarding indirect and non-medical costs, and excluding some demographic data because of high rate of missing can be considered as the main limitations of the present study.According to the results, after the implementation of the HTP in Iran, the economic burden of CVDs increased significantly (about 1.2 times), especially in the mortality costs and inpatient surgical services costs. Therefore, it can be concluded that the HTP, despite the decline in OOP expenditures and patients’ share of the total health expenditures, the economic burdens on the health system because of several reasons, including failure to fully implement the referral system and increased referrals to the specialized levels of the health system, increased while the OOP expenditures decreased. As a result, the total costs of the health system increased and imposed pressure on the country’s general budget. Finally, based on the HTP objectives in terms of equity in health care financing and the results of this study, it can be concluded that the costs of CVDs in Iran are significantly high. The HTP has not touched its all goals about CVDs costs. It could be because of the non-allocation of financial resources defined to the HTP, one per cent value-added tax, and 10% of targeted subsidies, after the implementation of the plan, as well as the political and economic challenges occurred during the years following the HTP implementation in Iran. Further studies are needed to find ways to modify the challenges and reduce the economic burden of CVDs by the HTP in Iran.


Picorna-like plant viruses are non-enveloped RNA spherical viruses of ~30 nm. Part of the survival of these viruses depends on their capsid being stable enough to harbour the viral genome and yet malleable enough to allow its release. However, molecular mechanisms remain obscure. Here, we report a structure of a picorna-like plant virus, apple latent spherical virus, at 2.87 Å resolution by single-particle cryo-electron microscopy (cryo-EM) with a cold-field emission beam. The cryo-EM map reveals a unique structure composed of three capsid proteins Vp25, Vp20, and Vp24. Strikingly Vp25 has a long N-terminal extension, which substantially stabilises the capsid frame of Vp25 and Vp20 subunits. Cryo-EM images also resolve RNA genome leaking from a pentameric protrusion of Vp24 subunits. The structures and observations suggest that genome release occurs through occasional opening of the Vp24 subunits, possibly suppressed to a low frequency by the rigid frame of the other subunits. Naitow et al. report the structure of a picorna-like plant virus, apple latent spherical virus at 2.87 Å resolution by single particle cryo-EM. The cryo-EM map reveals unique structures that stabilize the capsid frame, and images reveal genome leakage from the protrusion of subunits, giving insight into the proliferation of this virus. This tubular structure is modified by a viral protein called movement protein (MP2) to facilitate the entry of viral replication complexes  or virus particles themselves . Then, the viral genome is released again, bound for new destinations. In most cases, no specific host receptors are thought to be involved in infection and proliferation. Thus, capsid design for the survival of these viruses should require some basic functions, such as retention of the viral genome inside the virion, genome release, and then its encapsidation with newly synthesized capsid proteins. In addition, the capsid protein of viruses that form a virion during transport through the modified plasmodesma needs to be able to interact appropriately with MP8.Plant viruses invade host cells through a surface wound in the cell wall, often expedited by transmitting insects, and promptly release their genome for translation of their own proteins. Newly proliferating viruses spread to neighboring cells through so-called plasmodesmata, which are channels traversing the cell walls normally for the transport of chemicals and even small proteins between the cells 14. Because of this, biotechnological applications have been explored, such as virus-induced gene silencing16, virus-induced flowering19, epitope presentation on the surface of virus particles20, and development of plant vaccines23.Apple-latent spherical virus (ALSV) is a picorna-like plant virus found in apple leavesSecoviridae. This family contains approximately eight genera, and only a few virion structures in the family have been reported, i.e., cowpea mosaic virus (CPMV24), broad bean stain virus (BBSV25), bean pod mottle virus (BPMV26), and red clover mottle virus (RCMV27) in the genus Comovirus and tobacco ringspot virus (TRSV28) and grapevine fanleaf virus (GFLV29) in the genus Nepovirus. The virions of the genus Comovirus are composed of two proteins, and those of Nepovirus only a single protein. The virions of ALSV consist of three proteins, Vp25, Vp20, and Vp24, and they show only a weak sequence homology to other virus capsid proteins. In fact, ALSV was initially classified separately in the family Sequiviridae30, but is now placed in the genus Cheravirus, family Secoviridae, after the latter was formed from an amalgamation of families Sequiviridae and Comoviridae.Such picorna-like plant viruses are classified in the family We have determined the structure of ALSV at 2.87-Å resolution by single-particle cryo-EM with a cold-field emission beam, which allowed de novo modeling of atomic coordinates on the cryo-EM map from a relatively smaller number of particle images with an improved B factor for map sharpening. The structure reveals that the three capsid proteins have characteristic jellyroll folds that contribute to capsid stabilization. Uniquely among the known structures in this virus family, a long N-terminal extension of Vp25 interacts with all other subunits, including a neighboring Vp25 through the inner surface of the capsid, as well as the genomic RNA. Image analysis resolves RNA leaking from protrusions of the virus particles. Our results suggest how capsid stabilization is achieved, and we propose a mechanism for genome release.31  of each Vp25, Vp20, and Vp24 monomer among the surrounding subunits in the virion Table . The bur33 as representative of the genus Comovirus  and small (S). The L subunit contains two domains that correspond to Vp25 and Vp20 of ALSV, and the S subunit corresponds to Vp24—the overall structures and folding are similar .Here we compare the structure of ALSV with those of other picorna-like plant viruses. We take CPMV (PDB ID: 5MS1)rus Fig. . Structu28 and grapevine fanleaf virus (GFLV29) from the genus Nepovirus in the family Secoviridae show the same protein fold 37, obtained in the same way as above using the Dali server for alignment of 3D structures35.The long N-terminal extension in Vp25 of ALSV is unique among picorna-like viruses, except that some insection Fig. , e. The ion Fig. , e, and 30. There are concentric shells of density inside the capsid and humps of density beneath the Vp24 pentameric protrusions . The original map showed no clear interaction of capsid protein density with the genome density. However, after applying the autosharpening algorithm implemented in the Phenix suite38 to the map  encodes proteins for replication, such as helicase, protease, and RNA polymerase, and the latter two (RNA2s) encode a MP and the capsid proteinsons Fig. . The denmap Fig. , d, we fmap Fig. . These r40. We cut out images that included RNA and part of the virion, and applied 2D classification. Thereby, the 2D-class averages resolved that RNA chains extrude from one of the vertices of the virion, which likely corresponds to the tip of a pentameric protrusion  are unlikely as genome release would be too facile, and the virion would not be suitable for carrying the genome to nearby cells. Then, the defect should only appear at a single defined site rather than at random sites or solid parts of the capsid. The pentameric protrusion in the capsid of ALSV appears eminently suitable to be a release site.Recent reports proposed that geometric defects found in spherical viruses by single-particle analysis without symmetry enforcement might be a site for infection and genome release. However, 2D-class averages of ALSV images , 0.1 M NaCl, and 5 mM MgCl, concentrated to about 10 mg/ml, and kept at −80 °C before use. The sample was negatively stained and checked with a JEM-2100 electron microscope . It revealed virus particles of round shape with ~300-Å diameter.ALSV was isolated and purified from infected 45, when the samples are exposed to the electron beam. Three microliters of sample solution containing virus particles at a concentration of 1–2 mg/ml were applied onto the grid, blotted manually with filter paper from the reverse side of the grid, and rapidly frozen in liquid ethane with a homemade plunger in a cold chamber with a humidifier.To prepare frozen–hydrated samples, gold was first sputtered on holey carbon film-coated copper grids . This reduces beam-induced movement46. The distribution of ALSV particles in ice appeared to be quite uneven, and most images collected by automated data collection47 showed no virus particles or very few. Hence, we manually collected images of ALSV particles at a nominal magnification of ×40,000. Dose-fractionated frames were recorded on a Gatan K2 summit in superresolution mode using Gatan DigitalMicrograph. The setting of critical lenses and deflector coils was monitored with ParallEM48. The physical pixel size of the K2 detector corresponded to 1.24 Å, and one image view included ~100 virus particles, each with a diameter of ~300 Å. Thus, a total of 279 movies were collected. Imaging parameters are shown in Table The samples were examined at a specimen temperature of ~96 K with a JEOL CRYO ARM 300 electron microscope. A parallel electron beam with a diameter of ~2.3 μm illuminated the sample with a condenser aperture of 150 μm, a spot size of 4, and a convergence angle of 2. Inelastically scattered electrons were removed through an in-column energy filter with an energy slit width of 20 eV. Beam tilt was adjusted to the coma-free axis, and twofold astigmatism was removed by SerialEM31. Image stacks were first drift-corrected, dose-weighted, and summed with the MotionCor2 algorithm49 implemented in RELION-3. Contrast- transfer function (CTF) parameters were estimated with CTFFIND50, and particles were manually picked up through the image display of RELION for initial templates. Then, automatic particle picking and reference-free 2D classification were carried out by following the standard protocol of RELION-331. Here, picked particles were edited by visual inspection before the 2D classification. Creation of a de novo initial 3D map, 3D classification based on the initial map, and 3D auto-refinement were all done with icosahedral symmetry enforcement. After applying refinement of defocus and Bayesian polishing for motion correction of individual particles51, the final 3D structure was reconstructed from particle images  recorded on 110 movie stacks. Beam tilts were also corrected for each micrograph  values against the cryo-EM map , 3–168 (Vp20), and 6–190 (Vp24), and residues 41–55, 100–116, and 146–151 in Vp24 are missing on the map.38 for visualization of finer structures. This revealed residues contributing to interactions with the internal densities of the symmetrized average RNA genome (Fig. The reconstructed volume of ALSV was sharpened by phenix.auto_sharpenome Fig.  d.Images, including leaked RNA and part of the virion, were cut out (Supplementary Fig. 47, although the yield rate was not good due to the uneven distribution of viruses in ice as above. Particle images were processed together with manually collected ones, and 2D classification was applied to a total of 37,328 particles from 1,296 movie stacks with an option of “Ignore CTFs until first peak,” the setting of which is expected to be suitable for detection of finer features. A 3D map was reconstructed without icosahedral symmetry from a total of 28,633 particles. Icosahedral symmetrization of this combined dataset did not improve the resolution of the structure probably due to an increased heterogeneity among the datasets.We picked up more ALSV particles from images taken with automated data collection software JADASThe parameters and statistics of data collection, image analysis, and de novo atomic model building are shown in Table Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Data 1Peer Review FileReporting Summary