Crinivirus (Closteroviridae) is transmitted in a semi-persistent manner by the greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). Although several approaches exist for modeling insect population growth, modeling vector-born virus spread remains difficult because fundamental knowledge on the relationship between virus transmission and temperature is lacking for most vector transmitted viruses. To address this challenge, we initially developed a temperature-dependent phenology model for the whitefly vector using the Insect Life Cycle Modeling (ILCYM) software. In the present study, the effect of temperature on the efficiency of virus transmission by the whitefly was determined through controlled laboratory experiments at 8 constant temperatures in the range from 10 to 25 °C. The vector capacity to transmit the virus was highest at 15 °C (about 70 % probability of infection) but decreased radically as temperature deviated from this optimum temperature to <10 % at temperatures of 10 and 20 °C, respectively. The temperature-dependent probability of virus transmission by a single adult whitefly could be described by a nonlinear function, which was validated by transmission frequencies observed at fluctuating temperatures. This function combined with life table parameters calculated from previously established temperature-dependent phenology model for the vector provided a full temperature-responsive model for predicting PYVV spread potential and transmission probabilities. For spatial risk predictions, we devised two virus transmission risk indexes and tested their performance in correctly predicting virus presence/absence with field survey data. The best performing risk index was used to generate risk maps, which reflected well the current  occurrence of the virus but also predicted areas at high risk, where the virus has not previously been reported. One of them in western Panama was targeted for surveillance and resulted in identification of the virus in the country, where it was not previously known to occur. Simulated risk maps for the year 2050 revealed that climate change may significantly affect, the risk of distribution, generally reducing in tropical areas of the world, but increasing in the temperate regions.Management of viral plant diseases can be improved by using models to predict disease spread. Potato yellow vein virus (PYVV) of the genus  The crop is affected by various pest and disease, among which viruses are one of the most important due to their gradual accumulation over generations of clonal propagation, eventually leading to yield decreases of 50 % or more. Global trade and a changing climate affect the distribution and impact of pests and diseases leading to emergence in new areas. Potato yellow vein disease, caused by Potato yellow vein virus (PYVV), a member in genus Crinivirus (family Closteroviridae) is an important disease of the crop, in Colombia, Ecuador and northern Peru, causing up to 50 % yield reductions   can act as reservoirs for PYVV (PYVV is considered a quarantine pathogen by various agencies such as the European and Mediterranean Plant Protection Organization (EPPO) and the Animal and Plant Health Inspection Service (APHIS)  . The model, which was implemented in Insect Life Cycle Modeling (ILCYM) software  . A tempesoftware , 2017, psoftware . Also fosoftware , althouga) to assess the effect of temperature on the PYVV transmission rates by its vector, the greenhouse whitefly, b) establish a temperature-dependent model for PYVV transmission probability by its vector and c) combine this model with the previously established T. vaporariorum phenology model for predicting the risk of virus spread based on temperature on global, regional, or national scales. The virus transmission model was implemented as an additional module in the ILCYM software. Thus, we demonstrate an approach that extends the knowledge gained from insect phenology models to assess risks of diseases transmitted by insect species in cultivated crop.The objectives of this study were 22.1Virus–free potato plants (Clon W.A 077: CIP397077.16 and cultivar Canchan INIA: CIP380389.1) were obtained from the International Potato Center (CIP) germplasm bank in Lima, Peru.PYVV was obtained from the CIP potato virus collection and was propagated and maintained in potato. Plants grown from infected tubers served as source plants for the transmission experiments.For virus acquisition in the transmission experiments, two PYVV-infected source potato plants (grown from infected tubers) of the cultivar ‘Canchan INIA’ were planted in a temperature-controlled growth chamber per temperature tested. The temperature in the growth chamber was maintained at the same temperatures as used in the transmission experiments described below. Virus infection was confirmed approximately 25 days after planting by RT-PCR. In addition, properly sprouted healthy potatoes to be used as targets in inoculation experiments, were planted 10 days before virus transmission testing in growth chambers at the same temperature as used later in each transmission experiment.T. vaporariorum originating from Lantana camara, but maintained on potato plants for 5 years at the institutes own rearing facility (greenhouse) at a temperature between 20−23 °C, 70–95 % RH and a photoperiod of 12:12 h light (L): dark (D), was used in the experiments.A colony of Additional plant samples were collected from fields in Peru, Ecuador and western Panama and are described in section 2.2T. vaporariorum was determined at 8 constant temperatures of 10°,12°, 14°, 15°, 16°, 18°, 20°, and 25 °C (±0.5 °C). For each temperature, 50 single whitefly transmissions (described below) were performed per replication and replicated 2 times per temperature initially. Based on results, 2 more replications were performed to assess the variation in results at key temperatures . Whitefly transmissions were performed with an acquisition access period (AAP) to acquire the virus from the infected plants and inoculation access period (IAP) to transmit acquired virus to target plants . During the AAP, PYVV-infected potato plants (1 plant per 100 insects) were placed inside an acrylic box, covered with nylon mesh, in a growth chamber under each of the 8 predetermined temperatures. Adult whiteflies were caged with a clip-cage (17 × 13 × 13 cm) covered with nylon mesh to two leaves per plant  consisted of 50 + 2 plants; 50 plants were exposed individually to a single whitefly that had previously an AAP as described above and 2 plants were controls in which the AAP was performed on uninfected plants. After the 72-h IAP, the insects were removed from the cages and the plants treated with the insecticide Applaud (Buprofezin) (Bayer) at 0.1 % to ensure that no insects remained. The inoculated plants and were subsequently maintained in a greenhouse at a photoperiod of 12:12 h L: D and temperature of 18° and 23 °C during the light and dark period, respectively for 35 days. After this 35-d incubation period, virus presence was assessed taking systemic leaves (non-inoculated leaves) submitted to RT-PCR analysis section .Separately, to validate the predictions of the temperature dependent transmission function section  below we2.3Total RNA was extracted from sampled leaves using Trizol® reagent  according to the manufacturer's instructions. The cDNA for RT-qPCR was synthesized as follows: 7 μl of nuclease-free water (NFW), 2 μl of Random primers (250 ng/μl), 1 μl of dNTPs (10 mM) and 2 μl of total RNA (500 ng/μl) were mixed and denatured in a thermocycler  at 65 °C for 10 min and then cooled to 10 °C for 5 min.Subsequently, RT Mix  was added and then incubated at 37 °C for 50 min, followed by 70 °C for 15 min and finally cooled to 10 °C. The final volume obtained was a 20 μl cDNA, which was diluted with 80 μl of NFW to obtain a dilution of 1:5 for subsequent PCR.Relative PYVV concentrations in source leaves from each temperature were assessed to determine if temperature and leaf position had any effect on virus titres, that could influence transmission results. The PYVV primers  targeted the capsid region of the virus and the Cytochrome oxidase I (COX) gene was used as a reference gene (Supplementary Table 1).Ct values were determined using Applied Biosystem StepOne version 2.3 software. Samples were considered positive when they reached the Ct value before the 35th cycle. Each sample was performed in three technical replicates, including infected and healthy controls. Blanks were included in each separate PCR run. Data was analyzed using the 2(−ΔΔCt) method , 0.4μM of each primer, and 5μl PCR Master Mix Power SYBR Green .The following PCR program was used: PCR: 95 °C for 10 min, and then 35 cycles at 95 °C for 10 s, 50 °C for 45 s, 60 °C for 1 min, and finalized with a melting curve analysis. Fluorescence was measured after each amplification cycle. ) method  to deterTo verify successful virus-transmission by each individual whitefly to healthy potato plants as well as field samples from Panama (see section 2.6), regular RT-PCR was used, with primers PYVV CP1_F and PYVV CP2_R that amplified a 760 base pair fragment of the CP gene (Supplementary Table 1). The reaction conditions were as follows: 4 μL of PCR 5X Buffer (Promega), 1 μl of MgCl2 (25 mM), 0.5 μl of dNTPs (10 mM), 0.5 μL of Primers (CP1_F / CP2_R) both at 10 μM, 0.5 μl of the Taq polymerase (Promega), 8 μl of nuclease-free water (NFW) and 5 μl of diluted cDNA. The mixture was placed in a thermal cycler with the following conditions: one cycle at 94 °C for 5 min, followed by 35 cycles at 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min followed by 10 min at 72 °C. Amplified bands were visualized in a 1% agarose gel stained with Gelred (Invitrogen) under UV light.Samples from field surveys in Peru and Ecuador were tested for PYVV by nucleic acid spot hybridization (NASH). Two ½ leaflets were macerated in 2 ml, of 5x SSC  buffer in 100 μm polyethylene plastic bags (4 × 6 cm) and 30 μl of crude leaf sap was blotted on a nylon membrane  dried and cross-linked by UV-irradiation (50 mJ) in a cross-linking oven (Stratagene). Each membrane also included extract from a positive PYVV infected and healthy control plant of cv. Canchan INIA. Digoxigenin (DIG)-labelled probes encompassing the coat protein gene region (CP) of PYVV were synthesized by PCR as described above using primers PYVV CP1 and PYVV CP2 , and DIG2.4jth temperature yij, with values of 1 for success and 0 for failure, for the Ith insect at the jth temperature to obtain the absolute frequency, or total number of insects that transmitted the virus for every temperature The transmission as evaluated for each adult in a We tested about 20 models available in ILCYM that were developed for describing insect development rates, and the best models were selected according to AICc. Parameters for each of these models are automatically estimated by ILCYM according to the Levenberg-Marquardt algorithm  using thAll candidate models were implemented as optional functions in a new tool within the ILCYM package for analyzing temperature-dependent vector virus transmission probability (efficiency) in insect vectors. All statistical calculations and coding for model implementation were made in R-3.4.1 .2.5T. vaporariorum temperature-dependent life table parameters–namely net reproduction rate (0R), mean generation time (T), intrinsic rate of natural increase (mr), finite rate of increase (λ) and doubling time (tD), based on monthly minimum and maximum temperature data. They provide useful information about the expected vector abundance in a given environment. The two indices were developed combining the information on virus transmission efficiency and the vectors life table parameters.We tested two alternative methods to establishing a PYVV risk index based on the temperature-dependent virus transmission efficiency combined with the phenology model established previously for the vector . The temperature based phenology model predicts λ, defines the average per-capita multiplication factor per time-step (in this study per month); it means, populations are expected to grow–assumed that the population has reached a stable age-stage distribution–by the factor λ each month. Thus, during a given time interval of x months a population of the size iN grows by the factor λx.The first index, “virus transmission index (VTI) 1″, is based on the virus transmission probability and the whitefly’s finite rate of population increase. The finite rate of increase, λ seems an adequate factor for projecting relative spatial or seasonal differences in field densities (abundance) of pest populations, assuming that the initial population size is equal among space or at the beginning of a season. The temperature-dependent vector transmission efficiency factor, p, as described in the previous section, defines the probability that a single virus-carrying vector transmits the virus to the plant after attacking the plant. Since knowledge about the biological processes for successful virus transmission and disease development in virus-inoculated potatoes is lacking, we favored a non-additive model, in which each vector that has acquired the virus has the same chance of causing an infection in the plant after feeding for a given time interval on the host plant. The attack (contact) rate is directly proportional to the density of the vector. If p is the vector’s probability of inducing a virus infection after a given feeding period, the probability that the plant will be not infected at vector densities of x vectors per plant is:P, isTherefore, the growth rate λ, as a useful indicator for a reasonably representation of relative differences in vector densities among different locations or between specific seasons in a given location in regard to environmental temperature. Accordingly, for our risk index we exchanged the parameter for vector densities in eq. [1] with the temperature-dependent life table parameter λ. The risk index for a given month in each location is calculated as follows:iVTI is the virus transmission risk index for the ith month, ip is the temperature-dependent virus transmission efficiency rate during the ith month (as described in the previous section), iλ is the temperature-dependent finite rate of increase of the vector population, and ω is a shape factor . The index values are bound between 0 and 1. The annual virus transmission risk index for given location is calculated as:We considered the vectors life table parameter, R0, multiplied with the temperature-specific virus transmission efficiency, p, over a year. The equation was:R0i and pi are the net reproduction rates and the virus transmission probability, respectively, predicted for the ith month.The second index VTI2 is a simple index, calculated by averaging the whitefly’s monthly net reproduction rate, To evaluate the predictive capacity of VTI1 and VTI2 we used a dataset containing 817 geo-referenced virus presence/absence data derived from a field surveys performed in Colombia, Ecuador and Peru during 2007–2010 (see section 2.6T. vaporariorum and PYVV in potato crops in Peru (1788 fields) and Ecuador (3 fields) between 2007–2010 . Depending on the size of the fields, a number of 10–20 plants were evaluated. The presence or absence of PYVV symptoms were recorded for each plant on fully expanded leaves. Collected leaves were stored in a Styrofoam box filled with ice and later (at the same day) plant sap blotted on nylon membranes and air dried for virus-detection tests using NASH as described in section Geo-referenced field surveys were performed to assess the presence of Subsequently, to evaluate the usefulness of the model to guide surveillance, we identified an area with significant risk, based on the selected VTI, for establishment and spread of PYVV in western Panama and surveyed the area for presence of whiteflies and symptoms corresponding to virus infection during 2016. Leaves from 26 potato plants were collected in 3 potato fields to verify the presence of PYVV, and identify the whitefly species from nymphs. The fields were located at Cerro Punta: ; Las Nuves  and La Gavidia . The samples were tested with RT-PCR using primers specific for PYVV as described above.2.7www.worldclim.org)and converted to .flt (float type) data format. For future predictions the temperature database with the RCP 6.0 scenario and the CCSM model were used. The extracted temperature data were organized in 12 × 2 matrices using the longitude as columns and latitude as rows representing 12 matrices each for the minimum and maximum temperatures. Thereafter, a point object was created for each geographical coordinate (longitude and latitude) in form of a two-column table (the first column included the minimum temperature and the second the maximum temperature for each point) that was used directly for spatial phenological simulation . These temperature data served as input data for calculating the Virus Transmission index (VTI) as, described in section Maps were generated using ILCYM, which works with monthly maximum and minimum temperature data sets for one year. Twelve months data sets with their respective geographical coordinates were obtained at a resolution of 10 min for world prediction and 2.5 min for the region between South America and Central America from the WorldClim database  at the optimum temperature (optT), and k is an empirical constant determining the shape of the function. The function explained >81 % of the variation in the data  causing 50 % reduction in the transmission rate (T½) can be written in terms of the decay constants as: T½ = lowk * ln (2) and T½ = lowk * ln (2), respectively.high1k into a state at which their activity (probability to be transmitted and cause infection in the plant) is reduced to γ, and 2) virus transmission of all states are reduced by a slower decay constant determined by high2k. Based on the conditions of the model and its application in terms of a function, the Sporleder 2 model can be defined as follows:where The Sporleder 2 function was 277 million times better than the modified Janish according to AICc evidence ratios To reveal if a relationship exists between virus transmission and virus titres in the source plantsat different temperatures, we performed qRT-PCR analysis from all source leaves used in the transmission experiments.The transmission efficiency had no correlation with virus concentration (correlation coefficient = -0.31) in the plant as determined by qRT-PCR, as virus titres did not vary much between 10° to 18 °C, but then increased significantly at temperatures above 20 °C , where transmission was inefficient . The relThe screenhouse experiment in La Molina for testing virus transmissions at naturally fluctuating temperatures during 3 seasons: February (summer), May (autumn), July (winter) were in agreement with the established virus transmission model. Only during July, that was the period in which temperature frequently fell below 17 °C at which efficient virus transmission was expected, plant exposure to vectors resulted in successful virus transmission. During the other season, temperature remained >17 °C, which was above the expected temperature limit for efficient virus transmission, and no transmission was observed (Supplementary Table 2).3.2Jand VTIS respectively, whereas VTI applies to the risk indices in general. The differences between the indices VTI1 and VTI2 is based on the expected temperature-dependent virus transmission efficiency and the vector’s two life table parameters, either the finite rate of population increase or the net reproduction rate are illustrated in J and VTIS indices was the narrower range of optimal temperatures in VTIS and a lower but broader secondary peak at higher temperatures in VTIS  of 0.71, while the highest AUC of 0.67 was obtained using VTIJ1 with ω = 35  vs specificity (true negative rate) plot for the field survey data for VTIJ2 and VTIS2 are illustrated in J2 and VTIS2 respectively and resulted in the best overall prediction accuracy of 70.4 % for VTIS2  and 72.1 % of the true absence (specificity) were correctly predicted, revealing a precision  of 29.0 %, while the negative predictive value was 93.9 %. The low positive predictive value is due to the unbalanced occurrence of the virus in the sampled locations (PYVV was detected in 10.2 % of the surveyed locations only).VTI2 performed better than VTI1 at any scale parameter setting when tested against the real virus presence/absence data sampled during the survey . VTIh ω = 35 d. Therefor VTIS2  whereas S2 threshold of 2.23. In contrast, VTI1 with variable parameter settings of ω revealed low F1 scores (not shown).The F1 score, as a measure of the threshold accuracy considering the tradeoff between precision and the true positive rate, (sensitivity) revealed a value of 0.516 for a VTI3.3J2 and year 2000 climate data (the most recent data available in 2015) and identified a region in western Panama that was predicted to have high VTIJ2 (red circle in T.vaporariorum was present in two of the three fields sampled (Station IDIAP and La Gavidia).To test the effectiveness of using the VTI2 maps for guiding surveillance, we used VTIS2 maps were generated. VTIJ2 and VTIS2 maps were very similar except that areas with higher annual average temperature (between 18−30 °C) increased in their risk predicted by VTIS2, but were still low, and areas of the highest risk were reduced .Subsequent to this effort the new Sporleder 2 function, which better fit the transmission data, was developed based on which new VTIS2 indices using current and future predicted annual climate data, revealing current and future areas at significant risk of PYVV epidemics , Colombia, Venezuela, Ecuador and Peru, that there are areas at risk of further spread and establishment in Costa Rica, Bolivia, Chile and Argentina as well  it can be a highly efficient vector with a transmission rate of 0.58−0.66 per individual insect. Strikingly, the transmission efficiency had no correlation with virus concentration in the plant as determined by qRT-PCR, as virus titres did not vary much between 10° to 18 °C, but then increased significantly at temperatures above 20 °C , where transmission was inefficient  for VTI1 and 2, always resulted in the simpler VTI2 function generating better predictions based on testing with the field survey data. VTI2 generates risk indices with values ranging from 0 to ∼11. The maximum accuracy of correctly predicting virus presence and absence using our field survey data was 70.4 % % using a VTIS2 of 2.23 as a cutoff  resulting risk maps may inform about potential establishment, but they do not provide information on the population growth and damage potential or temporal population change within a cropping season or year in a given region.T. vaporariorum using the RCP 6.0 climate scenario for 2050, reveals a general reduction of transmission risk in most tropical areas of the world , also showed a large temperature dependency with inoculation temperature having a larger effect than acquisition temperature  by its aphid vector perature . Similarperature , Barley perature  and Pea perature  by severT. vaporariorum and PYVV as an example. Risk maps generated can be used to guide surveillance, as demonstrated in this study, or pest risk analyses. Bases on the VTI2 maps for PYVV, the virus is predicted to have potential (VTIS2 > 2.23) to establish in southern regions of Peru, northern Bolivia, parts of Chile and Argentina, and this potential will increase at larger latitudes in the future, whereas it will decrease in tropical regions. The modeling described in this study was performed using ILCYM v4.0, which has a user-friendly “Shiny” (developed with Shiny software) interface making development of insect phenology models and virus transmission curves extremely easy once the data is entered. Generating maps is equally easy and it should thus provide a versatile tool for researchers to rapidly generate risk maps for insect pest or insect vectored diseases and could be particularly useful to prepare for changes in disease pressure during El Niño years for example. Whereas ILCYM is currently geared at generating risk maps based on monthly weather data, it should relatively easy be modified for local epidemiological simulation and forecasting to provide decision support, even miniaturizing it for smartphone use by farmers and extension workers alike.In Conclusion, our results show that the combination of insect life parameters and virus transmission functions can be used to effectively identify areas at risk for vector based spread, using H. Gamarra: Data curation, Investigation, Project administration, Supervision, Writing - original draft, Writing - review & editing. P. Carhuapoma: Data curation, Formal analysis, Software, Visualization. L. Cumapa: Investigation. G. González: Investigation. J. Muñoz: Investigation. M. Sporleder: Formal analysis, Methodology, Validation, Writing - review & editing. J. Kreuze: Conceptualization, Funding acquisition, Methodology, Project administration, Writing - original draft, Writing - review & editing.


Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the  Reoviridae undergo a complex assembly pathway in the host cell. Here the authors use cryo-electron tomography to visualize the assembly stages of mammalian orthoreovirus revealing a single shelled intermediate with gross similarity to an early assembly stage of a family of prokaryotic dsRNA viruses. Reoviridae, which infect vertebrate, invertebrate and plant hosts and are responsible for economically and medically important diseases1. The mature virus particles are ~850 Å in diameter and are approximately spherical, with icosahedral symmetry. The particles harbour 10-separate genome segments and comprise two principle protein layers, the innermost composed of 120 copies of protein λ1 clamped together by 150 copies of σ2 . The outer layer consists of 600 copies each of μ1 and σ3. The array of μ1 and σ3 is interrupted at the fivefold axes by pentameric λ2 turrets which enzymatically cap the mRNA transcripts produced by the polymerase on egress from the particle. Finally, trimers of the fibrous cell adhesion protein σ1 emerge from the centres of the turrets. Unlike most RNA viruses these particles do not completely uncoat during cell entry, instead μ1, σ3 and σ1 are stripped off, maintaining in the cytoplasm a protein shell (known as the core) secluding the dsRNA genome from cellular pathogen recognition receptors. These particles are transcription-competent and, in the ribonucleoside triphosphate rich environment of the cytoplasm, they synthesise and extrude capped mRNAs, derived from the ten genome segments. These are translated to make the viral proteins which not only build new particles but also re-organise the volumes of the cytoplasm devoted to the manufacture of new virus (virus factories). These early stages of infection are relatively more understood than the later stages of assembly and egress2. Single-stranded gene segments are thought to interact with each other, facilitating the encapsidation of the complete viral genome3, but how the multi-layered particles assemble, how the RNAs and replication enzymes are incorporated, what the signal for production of double stranded gene segments is, how assembly is completed by attachment of the final proteins and how particles leave the cell remain largely open questions.Orthoreoviruses belong to a large family of viruses, the Here we demonstrate that vitrification of infected cells followed by cryo-focussed ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) allows high resolution reconstruction of assembly intermediates of a mammalian reovirus, allowing atomic models to be constructed which throw light on some of these fundamental questions.Reoviridae family were determined by X-ray crystallography7, and advances in single particle cryo-electron microscopy (cryo-EM) have recently yielded structures at comparable or higher resolution for several members of the family11. Structural information is available for two types of purified orthoreovirus particles: the intact virus (we term similar particles that we see inside infected cells virion-like particles) and partially disassembled transcriptionally competent cores12. Cryo-EM has also allowed lower symmetry structures within the particle to be deconvoluted from the icosahedral structure of the protein shell, yielding insight into the spatial organisation of the polymerase and genome segments14. To link such atomic descriptions of stable purified particles to the sequential processes of virus assembly occurring inside infected cells, we grew MA104 cells on gold EM grids (Methods) and infected them with a mammalian orthoreovirus. Twelve-hours post infection the grids were plunged into liquid ethane and inspected by scanning electron microscopy  yielded a reconstruction at 6.6 Å. For the empty virion-like particles, reconstructions focused on the fivefold regions and pseudo sixfold regions yielded reconstructions at 6.5 and 5.6 Å respectively . These classes were then subjected to sub-tomogram alignment and averaging, applying icosahedral symmetry, initially using PEET and subsequently emClarity12, which fit the density for the in situ structures extremely well . It is possible that these termini play a role in interactions with, and possibly organising, the genome in the mature virus.The virion-like structures can be directly compared to known structures of purified virions18 (Methods). The quality of the fit was convincing, with principle secondary structural elements occupying strong density features . There is a 10° flex inwards of the fivefold adjacent part of molecule A and a similar 15° flex in molecule B . In addition the density level in the tomogram is far lower than that in the full virion-like particles , or not found at every vertex, or be displaced away from the fivefold, which would lead to it being washed out due to fivefold averaging, or it could be simply not present at all. The interior volume of the SLP is 59% of that of the virion-like particle, thus the dsRNA genome segments cannot fit inside  it has been proposed that they share a common ancestor19. The Cystoviridae have acted as a model system for understanding assembly and RNA packaging in dsRNA viruses22 and the first stage in the assembly of these viruses is the formation of a highly indented SLP23. There is a striking similarity between the indented SLPs of phage Phi8 (representative of the Cystoviridae) and reovirus .MA104 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% (v/v) foetal bovine serum at 37 °C in 5% COGlow-discharged carbon-coated gold TEM grids (Quantifoil R2/1) were seeded with 33,000 MA104 cells and allowed to adhere for 4 h at 37 °C. Cells were infected at a multiplicity of infection of 10 and incubated at 37 °C. At 12-h post-infection the grids were washed three times in PBS, blotted from the back side for 6–8 s and flash frozen in liquid ethane using a manual plunger.15. Grids were clipped into autogrid rims (Thermo Scientific) and loaded into a Scios DualBeam system (Thermo Scientific) using a Quorum cryo transfer station (PP3010T) and a custom built cryo stage cooled to −165 °C. Prior to milling, grids were coated with a ~2 μm layer of platinum using the gas injection system. Milling was performed in a step-wise fashion, using a 30-kV Ga ion beam, and beam currents of 300, 100, 49, and 30 pA to generate a lamella with a final thickness of ~150–200 nm. Milling progress was monitored with the scanning electron microscope using 3–5 kV beam and 25 pA beam current.The method was based on a previous report29 under low-dose conditions using SerialEM software30. Each tilt series was collected with a nominal defocus value between 4.3 and 5.9 µm. Each tilt was acquired as movies (containing 5 frames) in counting mode using a dose of 2 e−/Å2 per tilt. The total cumulative dose for each tilt series was 82 e−/Å2, with calibrated pixel size of 1.8 Å.Tilt series were collected on several positions from three different lamellae using a Thermo Scientific Krios electron microscope (eBIC Krios III) operating at 300 keV and equipped with a Gatan post-column energy filter (selecting a 20 eV window) on a K2 Summit direct electron detector (Gatan). Data acquisition was performed covering an angular range from −40° to +40° with 2° angular increments recorded automatically using the dose-symmetric tilting scheme31, but without dose weighting since this is implemented in emClarity16 at the final sub-tomogram averaging step. Initial analysis used IMOD/PEET33, and these preliminary results are shown in Fig. 34 using Protomo35. The five tilt series of best quality  were selected and subjected to sub-tomogram alignment and averaging using emClarity. Tomograms were also reconstructed at binning 4 using SIRT for visualization. A total of 242 SLPs were picked from five tomograms and were subsequently aligned and averaged. The 12 fivefold vertices were identified in IMOD isosurface view from the averaged particle map allowing icosahedral symmetry to be applied to the whole SLP, resulting in an averaged map of SLP at 7.8 Å resolution. The fivefolds were then extracted from the symmetrized particle map and used as a reference for emClarity sub-tomogram particle picking by template matching. For emClarity processing, a total of 2683 SLP fivefolds were extracted. The 3D alignment procedures of SLP fivefolds were carried out gradually with binning of 4, 3, 2 and 1, each with 3 iterations. No manual mask was applied during the refinement. Dose-weighted filtering was applied at the final step. The FSC was calculated by the gold-standard method from even and odd data sets. The resolution of the SLP fivefolds is 6.6 Å at the 0.143 FSC cut off. The same process was carried out for a total of 65 empty virion-like particles, from those a total of 625 fivefolds and 3039 pseudo sixfolds were extracted, iteratively aligned and averaged. The resolution of these fivefold and pseudo sixfold reconstructions at the 0.143 FSC cut-off is 6.5 Å and 5.6 Å, respectively. A summary of cryoET data acquisition and data processing are presented in Supplementary Table A total of eight tilt series were recorded from three lamellae, subjected to movie-frame alignment using UnblurPDB 1EJ6, 2CSE, 1JMU and 3S6X. Initial fitting was performed by placing by hand into the density followed by Chimera36 rigid body fitting. For the virion-like particle this was followed by molecular dynamics flexible fitting implemented in Namdinator37. For the SLP individual proteins (A and B) were fitted in Chimera and then each λ1 divided into two portions which were further rigid body fitted. Finally the fitting was optimised using Namdinator. Representative FSC curves for the fit of the models to the map are shown in Supplementary Fig. 40 and are presented in Supplementary Table Models were derived from 41 program Areaimol43. Since contact surface calculations are very sensitive to coordinate error we enlarged the probe radius to 5.0 Å to reduce sensitivity and allow values to be compared with greater confidence.Contact areas between protein subunits were calculated using the CCP4Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


The 2.0 Å resolution A2AAR structure model is presented and compared with previous A2AAR structures determined in a vacuum and/or at cryogenic temperatures.Serial femtosecond crystallography of adenosine A 2A receptor (A2AAR) at 2.0 Å resolution is determined and compared with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A So far, only a few GPCR structures have been resolved to better than 2.0 Å resolution using synchrotron radiation  of its full power  using the Cornell–SLAC pixel array detector (CSPAD)  using SFX data collected in a helium environment under atmospheric pressure and at room temperature. We compare this model with the 1.8 Å synchrotron structure  instead of the commonly used conventional vacuum chamber for SFX experiments at CXI. A Rayonix MX170-HS detector was used to collect the SFX data at a 2 × 2 binning mode with a data-acquisition rate of 10 Hz. A representative diffraction image at ≃1 mJ recorded to 2.0 Å at the edge of the detector is shown in Fig. 1b).Microcrystal samples of human A2.2.2.50 and Ser913.39 . Overall, all the models were found to be in agreement with each other without any significant observable differences between the synchrotron single-crystal diffraction method, SMX and SFX structures. We observed similar crystallographic statistics between the models, with higher B factors for structures determined at room temperature compared with cryogenic conditions (4eiy), as expected  and used to build the model presented here. After molecular replacement (MR) and refinement, the electron-density maps revealed three clear densities, corresponding to cholesterol molecules near the receptor, and a density for the ligand ZM241385 consistent with previous structures Fig. 2. Densitid Table 1. Our fins Table 1.3.mFo − DFc maps between our model and previously published A2AAR structures . Analysis of structural characteristics such as disulfide bonds, the sodium binding pocket, ligand binding residues and cholesterol molecules showed similar quality between the 2mFo − DFc maps . We observed slightly larger RMSD values in regions including ICL2, the intracellular portion of TM6 and ECL2 [Fig. 3a)]. Moreover, weaker densities were observed across all the models for ICL2 and ECL2, potentially indicative of the dynamic nature of these loops. These regions of weaker density and higher RMSD also correlate to higher B factors from the model [Fig. 3b)]. Nonetheless, all the structure models generated using different diffraction methods are comparable, with important structural characteristics clearly resolved  shows a ribbon representation of all the structure models aligned to our current model.After refinement, we observed no significant differences in the 2ed Fig. 2. Fig. 3 et al., 2015et al., 2013−1; 167 × 167 mm), can count single photons, with a maximum signal of 2700 photons (8 keV) pixel−1, and has a fast 120 Hz data-acquisition rate, all of which are amenable for XFEL SFX experiments. The results reported here were recorded using the Rayonix MX170-HS detector. Beyond the technical differences (CCD versus pixel arrays), which are outside the scope of this study, we mainly focus on the advantages the MX170-HS detector offers. During our SFX experiments, the MX170-HS detector was recording in a 2 × 2 binning mode  which has a capacity for recording a max signal of 50 000 photons (12 keV) pixel−1  pixel−1 at the high-gain mode, significantly less than that of the MX170-HS detector. Additionally, the lower dynamic range of the CSPAD limits the amount of tolerable background noise since a detector readout must include a full LCLS pulse, an elevated background can utilize all of the dynamic range of the detector and lead to signals above background saturating the detector. Performing experiments in vacuum, as regularly carried out at XFELs, alleviates this problem. In contrast, our experiment was performed at ambient pressure in a helium atmosphere. Utilizing the high dynamic range of the MX170-HS detector allowed us to overcome the background scattering effects contributed by helium atoms.At the time of writing, the standard detector installed in the primary chamber of the LCLS CXI instrument is the CSPAD, capable of high-speed readout at 120 Hz   are capable of repetition rates at or above 100 kHz. For new detectors to be able to match the repetition rates of these new machines, the physical dimensions of the detectors and the total amount of pixels could be diminished while increasing the pixel size 〉 is lower than that of 5k2d (Table 15k2d (72 753).An additional benefit to our method is the better measurement of low-resolution diffraction spots from better intensity estimates owing to the aforementioned detector characteristics. We observed that the indexing rate from our experiment was 63.5% while the other Ad Table 1, possibld Table 1. Furtherd Table 1. In our et al., 2019et al., 2015et al., 2015et al., 2015et al., 2017et al., 2019A central problem around SFX is the scarcity of XFEL facilities and the difficulty in obtaining experimental beam time. Enabling the use of the full power of the XFEL source, as demonstrated in this work, along with a high dynamic range and high repetition rate detector should greatly increase the efficiency of SFX experiments for membrane protein microcrystals grown and delivered in LCP matrix. Additionally, simultaneous data collection in the primary vacuum sample chamber at CXI as well as in the secondary helium-filled chamber using the refocused beam should further increase the number of experiments conducted at the facility. The methods and hardware setup presented here have led to the development of the macromolecular femtosecond crystallography (MFX) instrument , 1.0 mg ml−1 iodoacetamide (Sigma), EDTA-free cOmplete protease inhibitor cocktail (Roche), 1%(w/v) n-dodecyl-β-d-maltopyranoside  and 0.2%(w/v) cholesteryl hemisuccinate  for 3 h at 4°C. The insoluble material was removed by centrifugation at 250 000g for 45 min and the supernatant was incubated with TALON affinity chromatography resin (Takara–Clontech) overnight in the presence of 20 mM imidazole. The resin was washed using successive volumes of buffers containing 100 µM of ZM241385  with increasing concentrations of imidazole. A2AAR was eluted in the elution buffer  and subsequently concentrated to ∼40 mg ml−1 using an Amicon centrifugation concentrator .Receptor purification and crystallization followed previously published protocols (Liu w/w) monoolein and 10%(w/w) cholesterol at a ratio of 2 parts protein to 3 parts lipid by volume using a lipid syringe mixer . After removing precipitant solutions, samples from 3–4 syringes were combined together and titrated with a few microlitres of monoolein to absorb the residual precipitant and ensure that the sample remains in cubic phase. The final sample was loaded in the reservoir of an LCP injector  was used for indexing and integration  based on the peaks found by Cheetah , a saturation cutoff of 10 000 detector intensity units and one scaling/merging iteration. Using data up to a resolution of 2.0 Å, an initial model was generated by MR phasing using the 1.9 Å XFEL structure (PDB entry 5k2d) modified to a polyalanine model, as the search model in the Phaser MR module [Phenix version 1.17  refinement parameters in five TLS groups. Manual inspection and model modifications in Coot (Emsley & Cowtan, 2004et al., 2010Coot. Data-collection and refinement statistics are presented in Table 1PyMol (Schrödinger, 2015The SFX data were first processed with 10.1107/S2052252520012701/it5021sup1.pdfSupporting information. DOI:


Nod2 protects from high fat diet (HFD)-dependent hepatic cancer. We used an obesity-dependent hepatic tumor model. WT and Nod2−/− mice were treated with the carcinogen dimethylbenz[a]anthracene (DMBA) and maintained on HFD. Nod2−/− mice treated with DMBA and maintained on HFD gain significantly more weight and develop more liver tumors than similarly treated WT mice. Livers of Nod2−/− tumorigenic mice had increased expression of genes involved in cell proliferation, immune responses, and cholesterol biosynthesis, increased infiltration of neutrophils, inflammatory monocytes, and T cells, and increased activation of STAT3 and ERK during the later stages of tumorigenesis. Bioinformatic analyses of genes with differential expression predicted an increase in cancer, immune, and cholesterol biosynthesis pathways. In summary, we have identified a novel role for Nod2 and demonstrate that Nod2 protects from HFD-dependent liver malignancy and this protection is accompanied by decreased cell proliferation, inflammation, steroid biosynthesis, neutrophils and macrophages infiltration, and STAT3 and MAPK signaling in the liver.Nod2 is a pattern recognition receptor that modulates host innate immune responses and protects from inflammation, steatosis, and obesity. Obesity and inflammation are risk factors for hepatocellular carcinoma, however, the role of Nod2 in obesity-dependent hepatic tumorigenesis is not known. Here we tested the hypothesis that  Obesity is a significant risk factor for liver cancer, and the rising epidemic in obesity parallels increasing incidence in hepatic malignancy2. The development of liver cancer in obese individuals is accompanied by chronic inflammation, which implicates a role for the immune system in hepatic tumorigenesis in these individuals6. However, specific host immune factors and molecular mechanisms that contribute to the development of liver cancer remain poorly understood7.Liver cancer is the fifth most frequent malignancy and one of the leading causes of cancer-related deaths worldwide. There are approximately 700,000 deaths worldwide each year, and the 5-year survival rate in patients with liver cancer is a dismaying 18%10. Activation of Nod2 by peptidoglycan results in the activation of NF-κB and MAP kinase-signaling cascades and production of inflammatory molecules and anti-microbial peptides12. In addition to bacterial peptidoglycan, there is increasing evidence that Nod2 is likely activated by other ligands, including cellular ligands released during stress16. In vivo, Nod2 has a critical role in maintaining intestinal homeostasis and is a major susceptibility gene for inflammatory bowel disease18. Inflammation is a risk factor for some cancers, and polymorphisms in Nod2 are associated with increased risk for colorectal cancer21 and may be associated with increased risk for lymphoma, gastric, breast, ovarian, lung, and laryngeal cancers21. In experimental models, Nod2 deficiency was shown to promote the development of colitis and colorectal cancer23. Nod2-dependent protection from colorectal cancer was dependent on the inhibition of TLR-mediated activation of NF-κB and MAPK signaling23. Recently, Nod2 was shown to protect from the development of hepatocellular carcinoma (HCC) using N-nitrosodiethylamine (DEN)/carbon tetrachloride (CCl4) and xenograft tumor animal models24. In vitro, molecular changes associated with HCC were dependent on the Nod2-dependent activation of adenosine 5′-monophosphate activated protein kinase (AMPK) and increased apoptosis24. Dysregulation of Nod2 is also linked to metabolic and endoplasmic reticulum stress25. However, the functional role of Nod2 in these different pathologies remains poorly understood.The Nod-like receptors (NLRs) are intracellular innate immunity proteins. Nod2, one of the members of the NLR family, is a sensor for bacterial peptidoglycan fragments and has a critical role in innate immune responses to bacteriaNod2 protects from the development of diet-dependent obesity15. Nod2−/− mice on high fat diet (HFD) become obese, develop steatosis, and exhibit increased expression of genes for a large number of inflammatory molecules, compared with wild-type (WT) mice on HFD15. We also demonstrated that the Nod2−/− HFD microbiome contributes to the development of obesity and steatosis in these mice15. However, the role of Nod2 in the development of diet-dependent hepatic malignancy is unknown. In the current study, we tested the hypothesis that increased obesity in the absence of Nod2 would result in increased tumorigenesis. We used the dimethylbenz[a]anthracene (DMBA)-HFD mouse model, which is an established system for the development of obesity-dependent liver tumors26. We show that Nod2 inhibits hepatic tumorigenesis and that the development of liver tumors in Nod2−/− mice is dependent on HFD and involves increased expression of genes involved in cell proliferation, immune responses, and cholesterol biosynthesis. We further demonstrate that the development of liver tumors in Nod2−/− mice is associated with increased infiltration of neutrophils, inflammatory monocytes, and T cells in the liver, and with increased activation of STAT3 and ERK signaling molecules.We previously demonstrated that Nod2−/− mice increases sensitivity to liver cancer, we treated WT and Nod2−/− 4–5 day old pups one-time with the carcinogen dimethylbenz[a]anthracene (DMBA) and 3 days later placed their nursing mothers on HFD. The DMBA-treated mice were continued on HFD after they were weaned and until the end of the experiment. This is an established method for the development of obesity-induced liver cancer in mice26. DMBA causes an oncogenic Ras mutation affecting a pathway that is frequently activated in human cancers, but by itself is not sufficient to cause liver cancer in this mouse model26. Control WT and Nod2−/− mice were not treated with DMBA, but were started and maintained on HFD similar to the DMBA + HFD group or were treated with DMBA and maintained on chow.To test our hypothesis that development of diet-dependent obesity in Nod2−/− mice compared with male WT mice in both HFD and HFD + DMBA groups . However, the Nod2−/− HFD + DMBA mice started losing weight after 23 weeks, and at 27 and 31 weeks there was a significant difference between Nod2−/− HFD and Nod2−/− HFD + DMBA mice. The loss in weight in Nod2−/− HFD + DMBA mice at these later time points is likely due to the development of tumors in these mice, which is described below. At 31 weeks, 94% of male Nod2−/− HFD + DMBA mice had liver tumors, whereas only 36% of male WT HFD + DMBA mice developed liver tumors  and only one mouse had a large tumor . In contrast, Nod2−/− HFD + DMBA mice had significantly higher numbers of both low and high-volume tumors compared with WT mice  did not develop liver tumors.Thus, our data indicate that Nod2−/− mice in a unbiased manner, we performed RNAseq using total RNA from the livers of male WT and Nod2−/− mice treated with DMBA and maintained on HFD for 31 weeks. We focused on male mice because males are more susceptible to the development of liver tumors compared with female mice  , organization of the mitotic spindle , activation of cells  extracellular matrix remodeling, migration, and adhesion , and apoptosis   Fig. . The majNod2−/− DMBA + HFD mice had significantly different expression of more than 100 genes associated with immune cells and immune responses , effector molecules , chemotaxis and adhesion , receptors , and ROS production . Using the IPA software, we identified that about 30 of the upregulated immune genes are also associated with the development and progression of hepatocellular carcinoma , peroxisome proliferator activated receptor alpha  and SCAP (P = 3.18E−15). POR transfers electrons to all microsomal P450 enzymes, including enzymes in steroid biosynthesis. PPARA, is a transcription factor that regulates the expression of genes in lipid metabolism, cell proliferation and differentiation, and immune responses. SCAP is a protease that cleaves SREBP, a key transcription factor that induces expression of several enzymes involved in cholesterol biosynthesis. Based on the changes in these regulators, Nod2−/− DMBA + HFD mice are predicted to have increased cholesterol biosynthesis. We also demonstrate that many genes involved in steroid biosynthesis including Hmgcs1, Idi1, Msmo1, Mvk, Sqle and in phospholipid biosynthesis, including Chka, Lpcat2, Plpp2 were elevated in Nod2−/− mice. Increased cholesterol and phospholipids are essential for rapidly dividing cells27, and metabolites of cholesterol, including oxysterols, promote tumor development28. These results also correlate with our previous results demonstrating hypercholesterolemia in Nod2−/− mice on HFD15. Genes involved in the biosynthesis of asparagine (Asns) and serine (Phgdh) and in folate metabolism (Mthfd1l and Mthfd2) were also elevated in Nod2−/− DMBA + HFD mice and are associated with the development of hepatocellular carcinoma. Differentially expressed genes associated with the development of liver cancer based on IPA are marked with an asterisk in Fig. ism Fig. . In our ism Fig. . Additio29 for differentially expressed genes in Nod2−/− mice treated with DMBA and maintained on HFD for 31 weeks compared with similarly treated WT mice. We performed the analysis with all genes that had a fold change of ≥ 2 and 5% FDR and identified 423 interactions that have a combined score of 0.7 or higher  in Nod2−/− mice , kinetochore localized astrin binding protein (Knstrn), and nucleolar and spindle associated protein 1 (Nusap1) were the top nodes in the cell proliferation group and formed a tight cluster with several other proteins involved in the regulation of the cell cycle or in the assembly of the mitotic spindle Fig. . All thewth Fig. . The topsis Fig. .Nod2−/− DMBA/HFD mice have reduced expression of Cish and increased expression of Lepr, Il2rg, and Osm  of STAT3 (p-STAT3) was significantly lower at 5 weeks of HFD but significantly higher at 20 and 31 weeks of HFD  at 5 weeks, no difference at 20 weeks, and higher level of phosphorylated ERK at 31 weeks compared with WT mice  at 5 weeks, and there was no difference at 20 or 31 weeks of HFD compared with WT mice. There was no difference in the levels of phosphorylated p38 (p-p38) or NF-κB (p-p65) between WT and Nod2−/− mice at any of the time points , and NF-κB, in HFD Fig. . Nod2−/−ice Fig. . Nod2−/−NF-κB p-p5 betweenNod2−/− mice. Furthermore, Nod2 is directly involved in innate immune responses, indirectly regulates adaptive immune responses, and we have previously demonstrated that the development of obesity in Nod2−/− mice on HFD is accompanied by a strong immune response in the liver and adipose tissue15. Thus, we next determined changes in the number of different immune cells in WT and Nod2−/− mice treated with DMBA and maintained on HFD for 5, 20, or 31 weeks or maintained on chow (Ctr). We determined numbers of neutrophils, macrophages and T-cells in the liver, blood, spleen, and bone marrow by flow cytometry. All samples were first gated on CD45+ followed by gating for singlets and then viable cells were selected for further analysis  and inflammatory monocytes (CD45+CD11b+Ly6c++) compared with similarly treated WT mice  were significantly higher in Nod2−/− mice at all time points as well as in the control mice. However, the total number of neutrophils and the fold increase compared with WT mice for the same group was higher at 31 weeks than at the other time points or in the control mice . Inflammatory monocytes (CD45+CD11b+Ly6c++) were significantly higher in the liver of Nod2−/− mice at 31 weeks but not at 5 or 20 weeks or in the control mice compared with similarly treated WT mice .In the spleen, Nod2−/− mice were treated with DMBA and maintained on HFD for 5 or 20 weeks or maintained on chow without DMBA treatment, and liver RNA was analyzed by qRT-PCR. Nod2−/− mice had significantly increased expression of proinflammatory genes Ccr1, Mmp9, and Osm starting at 5 , a marker for neutrophils, was significantly increased at 20 weeks  and at 31 weeks . These results further correlate with the decreased activation of signaling molecules at earlier, but enhanced at later time points in Nod2−/− mice , however, the mechanism behind this difference is not understood.Here we demonstrate that Nod2−/− mice treated with DMBA and maintained on HFD for 31 weeks had altered expression of genes in pathways associated with the development of cancer, liver hyperplasia, liver carcinoma, liver steatosis, hepatocellular carcinoma, inflammation, and cholesterol biosynthesis. We analyzed the gene expression data for changes in GO pathways and identified increased expression of genes involved in cell division, immune responses, and lipid metabolism. Similar molecular changes have been identified as core transcriptional hallmarks of hepatocellular carcinoma7. Our STRING analysis confirmed an enrichment of pathways involved in immune responses, cell cycle, cholesterol biosynthesis, and carcinogenesis in tumorigenic liver of Nod2−/− mice, and we also predicted specific protein–protein interactions in these pathways. We further demonstrate that during the later stages of tumor development in Nod2−/− mice, STAT3 and ERK are activated, and there are increased numbers of neutrophils, inflammatory monocytes, and CD4+ T cells in the blood and liver. However, during the earlier stages there is decreased activation of STAT3 and MAPK in Nod2−/− mice compared with WT mice. These results suggest that during the early stages of tumorigenesis Nod2 is required for the activation of these signaling pathways, however, at later stages of tumorigenesis Nod2 is required for suppressing the activation of these pathways. These changes may be direct or indirect effects of Nod2. Our data further suggest that before tumors appear Nod2 is primarily responsible for the changes in the signaling pathways and differences in immune cell infiltration, and once tumors appear, both Nod2 and tumors could be responsible for the modulation of these responses.We demonstrate that Nod2 protects from liver malignancy by maintaining optimal levels of cell growth and division, immune responses, and lipid metabolism, and that in the absence of Nod2 and exposure to a carcinogen, combined with long-term HFD, many of these critical pathways are enhanced and Nod2−/− mice become susceptible to liver tumorigenesis.Altogether, our results demonstrate that Nod2 to inflammatory diseases, primarily inflammatory bowel disease (IBD), was demonstrated almost two decades ago18 and have been followed by numerous studies12, but the mechanism of the effect of Nod2 on colitis remains obscure. Polymorphisms in Nod2 are also linked to colorectal cancer21, and in experimental models, Nod2-deficiency was shown to enhance sensitivity to colorectal cancer with hyperinflammation in the gut23. Additionally, we and others have demonstrated a novel role for Nod2 in metabolic dysfunction. In experimental models, Nod2 deficiency is associated with increased sensitivity to diet-dependent obesity, steatosis, and hepatic and adipose tissue inflammation15. These results further demonstrate that Nod2 deficiency combined with different kinds of stress (infection or high fat diet) results in increased inflammation and other diseases including obesity and cancer. However, the mechanism of Nod2-mediated pathology remains poorly understood and controversial. Activation of Nod2 results in increased production of inflammatory molecules and anti-microbial peptides9. The Nod2 mutations associated with IBD are mainly missense30, which would suggest decreased inflammation in these patients. However, IBD patients have increased inflammation in the colon31.The relevance of Nod2 deficiency is that Nod2 suppresses signaling through TLRs, which are major contributors to inflammation. In the absence of Nod2, TLR activation is unchecked, which results in hyperinflammation. This hypothesis is supported by several studies, which demonstrate that Nod2 inhibits TLR-mediated activation of immune cells and cytokine production34 and increased intestinal permeability35. A recent study demonstrates that Nod2, in a colorectal cancer model, induces activation of NF-κB and MAPKs during the early stages of tumorigenesis, yet suppresses TLR-mediated activation of these signaling molecules during the later stages23. Our previous and current results demonstrate that the development of obesity and diet-dependent hepatic malignancy in Nod2−/− mice is accompanied by increased expression of many immune response genes in the liver and adipose tissue. Here, we demonstrate that Nod2−/− mice with hepatic tumors have enhanced molecular and cellular signals in inflammatory pathways. These mice have increased expression of many genes involved in the immune responses in the liver, increased numbers of neutrophils, monocytes, and CD4+ T cells in the liver and blood, and increased activation of STAT3 and ERKs in the liver. Thus, one likely explanation for our results is that the development of hyperinflammation in Nod2−/− mice maintained on HFD or treated with DMBA and HFD is due to the loss of Nod2-mediated suppression of inflammation, possibly triggered by TLR signaling.One proposed explanation for increased inflammation associated with Nod2−/− mice may be the recently described Nod2-mediated activation of AMPK24. In that study, the authors demonstrate a novel role for Nod2 in protecting from N-nitrosodiethylamine/carbon tetrachloride-induced hepatocarcinogenesis24. Using hepatic tumor cell lines, they further demonstrated that Nod2 binds to and activates AMPK signaling, which inhibits mTORC1 resulting in an anti-tumor effect24. However, these in vitro results have not been confirmed in vivo. AMPK is a master regulator of metabolism, regulates cell proliferation, and activation of AMPK in the liver reduces liver steatosis and inflammation37. Thus, one likely explanation for our data demonstrating the development of HFD-dependent hyperlipidemia, steatosis, and inflammation in Nod2−/− mice could be due the loss of Nod2-mediated activation of AMPK. Currently, our data only indirectly support these hypotheses and will be confirmed in future.A second parallel mechanism that may contribute to the development of diet-dependent obesity and hepatic malignancy in Nod2−/− mice are more susceptible than WT mice to diet-dependent obesity and metabolic disease, and that this susceptibility was in part due the Nod2−/− HFD microbiome (15). Thus, it is possible that both the microbiota and metabolic disease contribute to the development of hepatic malignancy, which we will investigate in the future. However, all the observed changes in tumorigenic Nod2−/− mice are due to the deficiency of only one gene—Nod2, but there may be multiple downstream pathways affected. We have taken precautions in breeding and maintaining our mice (described in the Methods section) to avoid random differences in the environment and the microbiome that may influence disease development.Nod2 has multiple direct and indirect effects in the body, including modulation of the immune systems, metabolism, and the gut microbiota, and it is likely that multiple mechanisms influence the development of liver tumorigenesis in our current model. In our previous work, we have demonstrated that Nod2 protects from hepatic malignancy by maintaining optimal levels of cell proliferation, immune responses, and steroid metabolism. These results further support the increasing evidence that Nod2 possess other functions in addition to the immune responses to bacterial peptidoglycan and that Nod2 is required for maintaining cellular homeostasis in response to different stresses.In conclusion, we have demonstrated that the pattern recognition receptor Nod2−/− mice on BALB/c background were described previously and deletion of the Nod2 genes was confirmed by PCR analysis of genomic DNA39. The original founder WT BALB/c mice were obtained from Harlan–Sprague–Dawly. All WT and Nod2−/− mice were bred and kept under conventional pathogen-free conditions in the same room in our facility to minimize the influence of differences in the environment. For each experiment, mice from several different cages and breeder pairs were used. Some investigators use homozygous knockout and WT littermates from heterozygous breeding pairs to minimize parent-to-parent and cage-to-cage variations and equalize the microbiomes. We do not use this strategy for two reasons: first, this strategy may skew the results to the particular microbiota present only in this breeding pair. Second and most importantly, we noticed that the effect of innate immunity genes on the composition of microbiota is not instantaneous, but takes time, and stabilization of microbiota characteristic of a given knockout may take more than one generation. We consciously did not want to eliminate or minimize the role of microbiota in our experiments, because the effect of Nod2 on microbiota composition could be one of the possible mechanisms responsible for the observed increased incidence of tumors, which will be determined in the future. The BALB/c background of knockout mice and their negative status for all common viral and bacterial pathogens and parasites (including negative PCR stool tests for mouse Norovirus) were confirmed as previously described38. All experiments with mice were approved by the Indiana University School of Medicine-Northwest Institutional Animal Care and Use Committee. All animal work was performed in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and regulations.Nod2−/− 4–5 day old pups were treated with 50 µl of 0.5% DMBA, which was applied on their backs26 and after 3 days, nursing mothers and pups were placed on HFD  for the entire length of the experiment. Mice were placed on HFD diet for 5, 20, or 31 weeks. Control mice were treated with DMBA and maintained on chow for the length of the experiment (up to 35 weeks) or were not treated with DMBA and placed on HFD for the length of the experiment. The pups were weaned at 4 weeks of age and female and male mice were kept in separate cages. Mice were weighed every other week. At the time of sacrifice, tumors were measured using Mitutoyo Vernier caliper for height, weight, and length. In cases when the height of the tumor could not be measured the smallest measurement was taken as the height. The formula V = (π.W.H.L)/6 was used to calculate the volume of each tumor. Blood, liver, spleen, and bone marrow was collected for biochemical and molecular assays. Wherever possible tumor samples were collected separately. Liver samples from WT and Nod2−/− mice treated with DMBA and maintained on HFD for 31 weeks were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin15.WT and Nod2−/− and WT mice treated with DMBA and maintained on HFD for 31 weeks, we analyzed the total RNA population in the liver by RNA sequencing. RNA was isolated from the liver of individual mice using the TRIZOL method (InVitrogen), followed by purification on RNeasy spin columns using the Qiagen RNeasy Mini Kit40. RNA sequencing and initial analysis were performed at the Center for Genomics and Bioinformatics, Indiana University Bloomington. Reads were adapter trimmed and quality filtered using Trimmomatic ver. 0.33 (http://www.usadellab.org/cms/?page=trimmomatic), with the cutoff threshold for average base quality score set at 20 over a window of 3 bases. Reads shorter than 20 bases post-trimming were excluded. More than 98% of the reads (on average 22.8 million read pairs per library) passed the quality filters. Cleaned reads were mapped to mouse reference genome GRCm38 (gencode M16), using TopHat2 ver. 2.1.1 (https://doi.org/10.1186/gb-2013-14-4-r36). Approximately 94.7% of the total cleaned reads mapped to the reference and more than 95.5% of those mapped uniquely. RNA sequencing data will be deposited in NCBI.For identification of genes that were differentially expressed between 41. The fold change was computed for Nod2−/− DMBA + HFD with respect to WT DMBA + HFD and genes that had a fold change of ≥ 2 (increased and decreased) and P ≤ 0.05 and with a 5% FDR were considered as biologically and significantly different between the 2 groups. Gene products identified as differentially expressed were analyzed to identify biological processes that were altered between the two groups of mice.Differential expression with statistical analysis was calculated using the software DESeq2Nod2−/− and WT mice at earlier time points (treated with DMBA and maintained on HFD for 5 or 20 weeks) and between control mice (not treated with DMBA and maintained on chow) we performed qRT-PCR. Total RNA was isolated, converted to cDNA using Applied Biosystems™ High Capacity cDNA Reverse Transcription Kit and real-time PCR was performed using Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix and the Applied Biosystems™ QuantStudio 7 Flex Real-Time PCR system43. All primers used are listed in Table To identify genes that were differentially expressed between http://string-db.org)29 with the combined score set at ≥ 0.7. STRING uses genomic context information text mining, experimental data, and database searches as sources for interaction criteria29.Protein–protein interactions amongst the differentially expressed genes were predicted using Search Tool for the Retrieval of Interacting Genes (STRING) software v11.0 , 1 mM sodium orthovanadate, and 10 mM sodium fluoride. Tissue debris was removed by centrifugation at 20,000×2+ and Mg2+ free HBSS with 0.5 mM EDTA, pH 8.0)44. The medium was pumped through until the liver swelled and then followed by an incision to the portal vein to release the fluid from the liver. Perfusion medium was switched to digestion medium  after the liver lost color. After six to seven min the liver was removed and torn into fine pieces with forceps in 10 ml DMEM. Cells were filtered through 100 µm cell strainer and pelleted. Any remaining red blood cells were lysed using 0.2% NaCl and hepatocytes were pelleted with a 50 g spin. Cells in the supernatant, which included the immune cells, were centrifuged at 450 g for 9 min and resuspended in 2 ml DMEM and 1 × 106 cells were used for flow staining44. For single cells from the spleen, the spleen was homogenized in 2 ml 1X BioLegend Red Blood Cell lysis buffer through a 70 µm cell strainer. After 5 min at room temperature, lysis was stopped with 5 ml of wash medium (RPMI with 5% FCS) and the cells were pelleted. Cells were resuspended in wash medium and ~ 1 × 106 cells were used for flow staining42. For bone marrow cells, femurs and tibia were cut and cells were flushed using cold RPMI with 10% FBS and 2 mM EDTA. Cells were filtered through a 100 µm cell strainer and pelleted. Red blood cells were lysed using 0.2% NaCl, immune cells were pelleted and washed with cold RPMI with 10% FBS and 2 mM EDTA. Cells were resuspended in 1 ml cold HBSS and 1 × 106 cells were used for flow staining45.Mice were sacrificed, blood was collected in EDTA-coated tubes, EDTA was added to a final concentration of 1.5 mg/ml, and 8 µl was used for the flow staining (Miltenyi protocol). The liver was perfused through the inferior vena cava cannulated with a 25 G needle and perfusion medium , CD11b-PE/Vio770 (Cat. # 130-113-246), Ly6g-APC/Vio770 (Cat. # 130-118-949), Ly6c-VioGreen (cat. # 130-111-784), CD3-FITC (Cat. # 130-119-758), and CD4-APC (Cat. # 130-116-526) from Milteny and CD14-PE (Cat. # 130-119-758) from BioLegend for 10 min at 4 °C, and analyzed by flow cytometry using MACSQuant (Miltenyi) cytometer29. The N and P values are indicated in the Figures and Tables; P ≤ 0.05 was considered significant. The heatmaps were generated using Java TreeView and represent individual and mean fold changes in Nod2−/− DMBA + HFD mice relative to WT DMBA + HFD mice, after converting < 1 ratios to negative fold difference using the formula: (− 1)/ ratio in the heatmap. N = 6–7 mice/group. The numerical data for RNA levels in individual WT and Nod2−/− mice and the fold increase in individual and the average for all Nod2−/− mice are shown in Supplementary Table Differential expression of genes with statistical analysis was calculated using the software DESeq2 and is described in the RNA sequencing section. The significance of differences for other quantitative results are presented as means ± SEM and were determined by the Student’s t-test using Microsoft Excel or by Chi square. The significance of differences in pathway analysis was determined using the Ingenuity Pathway Analysis (Qiagen) or STRING softwareSupplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.Supplementary Figures.


The third Sustainable Development Goal for 2030 development agenda aims to reduce maternal and newborn deaths. Pregnant women’s understanding of danger signs is an important factor in seeking timely care during emergencies. We assessed knowledge of obstetric danger signs using both recall and understanding of appropriate action required during obstetric emergencies.p < 0.05 were considered significant.This was a cross-sectional study among pregnant women attending antenatal clinic at Bhutan’s largest hospital in Thimphu. Recall was assessed against seven obstetric danger signs outlined in the Mother and Child Health Handbook (7 points). Understanding of danger signs was tested using 13 multiple choice questions (13 points). Knowledge was scored out of 20 points and reported as ‘good’ (≥80%), ‘satisfactory’ (60–79%) and ‘poor’ (< 60%). Correlation between participant characteristics and knowledge score as well as number of danger signs recalled was tested using Pearson’s correlation coefficient. Association between knowledge score and participant characteristics was tested using t-tests  for numeric variables. Socio-demographic and clinical characteristics associated with the level of knowledge ('good’ versus ‘satisfactory’ and ‘poor’ combined) were assessed with odds ratios using a log-binomial regression model. All results with Four hundred and twenty-two women responded to the survey (response rate = 96.0%). Mean (±SD) knowledge score was 12 (±2.5). Twenty women (4.7%) had ‘good’ knowledge, 245 (58.1%) had ‘satisfactory’ knowledge and 157 (37.2%) had ‘poor’ knowledge. The median number of danger signs recalled was 2  while 68 women (20.3%) could not recall any danger signs. Most women were knowledgeable about pre-labour rupture of membranes (96.0%) while very few women were knowledgeable about spotting during pregnancy (19.9%). Both knowledge score and number of danger signs recalled had significant correlation with the period of gestation. Women with previous surgery on the reproductive tract had higher odds of having ‘good’ level of knowledge.Most pregnant women had ‘satisfactory’ knowledge score with poor explicit recall of danger signs. However, women recognized obstetric emergencies and identified the appropriate action warranted.The online version contains supplementary material available at 10.1186/s12884-021-03580-4. The Sustainable Development Goals highlight the interconnectedness of maternal and neonatal health with other aspects of development . The thiWhile many countries struggled to achieve the Millennium Development Goal related to reducing maternal mortality , 4, BhutOver the years, the antenatal care uptake in Bhutan increased from 18.9% in 2000 to 97.9% in 2012 . SkilledIn order to reduce the consequences of ‘first delay’ in seeking care during obstetric emergencies, a pregnant woman’s knowledge and understanding of danger signs during pregnancy is vital in seeking timely care . StudiesThis was a cross-sectional study among pregnant women attending antenatal clinic at Bhutan’s largest hospital, Jigme Dorji Wangchuck National Referral Hospital, between May 2019 and July 2020.Bhutan is situated in the eastern Himalayas with a population of 0.7 million; Thimphu is its capital and largest city . In 2017The government provides free healthcare services across all levels – primary, secondary, and tertiary . MCH serThe MCH Handbook is a recording tool as well as an information booklet. The handbook records parents’ demographic details, mother’s antenatal records, birth preparedness plan, birth details of the child, maternal and neonatal records, child’s growth charts, and vaccination records. It provides information on breastfeeding and nutrition, vaccination schedules, obstetric danger signs, monitoring developmental milestones, dental care, and general advice on antenatal and postnatal care .Druk Health Management and Information System [A pregnant woman is issued the MCH Handbook during her ‘booking’ visit. Each Handbook has a unique identification number that tracks the pregnancy through the n System . Health The MCH Handbook of Bhutan outlines seven key obstetric danger signs: 1) vaginal bleeding, 2) high fever, 3) preterm labour, 4) severe abdominal pain or vomiting, 5) severe headache, blurred vision or convulsions, 6) fast or difficult breathing, and 7) reduced or absent foetal movements.This study was conducted at the Gyaltsuen Jetsun Pema Mother and Child Hospital, which is a part of the Jigme Dorji Wangchuck National Referral Hospital complex in Thimphu.Pregnant women aged ≥18 years and attending ANC clinic at Gyaltsuen Jetsun Pema Mother and Child Hospital were eligible for the study. Participants were selected using systematic random sampling: every third pregnant woman registering for their routine ANC visit for the day was invited to participate in the study; those who consented were interviewed. Repetition of study participants was avoided by careful assessment of their MCH tracking number.Data were collected through interviewer-administered questionnaire that was designed for this study . The quePregnant women were asked to recall the danger signs outlined in the MCH Handbook (7 points). Their understanding of the danger signs was evaluated with 13 multiple choice questions – each question presented an obstetric emergency situation related to the danger signs outlined in the MCH Handbook (13 points). A knowledge score was calculated out of 20 points by adding the number of danger signs recalled and the number of correct responses to the 13 questions.Additional variables that might influence women’s level of knowledge were collected. These included demographic characteristics , clinical characteristics , and whether pregnant woman had read the MCH Handbook.In the absence of a baseline level of knowledge on obstetric danger signs among Bhutanese women, we calculated a sample size of 441 for a finite population of 24,846 based on the following assumptions: 50% probability for good knowledge, 5% margin of error, and 15% drop out rate.Data were entered into EpiData Entry version 3.1 and analyzed using EpiData Analysis version 2.2.3  and STATA version 13.1 (StataCorp LP USA). Numeric variables were reported as mean, standard deviations, median and interquartile range. Categorical variables were reported as frequencies and percentages. Normality for continuous variables was tested using Shapiro-Wilk test.Knowledge was categorized as ‘good’ , ‘satisfactory’  and ‘poor’ . Given the high importance attached to increasing pregnant women’s level of knowledge on obstetric danger signs to reduce ‘first delay', we decided on a higher threshold to categorize knowledge as ‘good'.Pearson’s correlation coefficient was calculated between knowledge score as well as number of danger signs recalled and woman’s age, period of gestation, gravidity and parity. Association between knowledge score and participant characteristics were tested using t-tests  for numeric variables .p-value < 0.1 during the unadjusted analysis were included in the final adjusted analysis. All results with p < 0.05 were considered significant.Associations between level of knowledge ('good’ versus ‘satisfactory’ and ‘poor’ combined) and socio-demographic characteristics  and clinical characteristics  were assessed with odds ratios (OR) using a log-binomial regression model. Those factors with a Ethics clearance was obtained from the Research Ethics Board of Health, Ministry of Health, Bhutan. Informed written consent was taken from each participant prior to the interview.th – 10th ANC visits.A total of 422 pregnant women were interviewed (response rate = 96.0%). The mean age of participants (±SD) was 27.9 (±4.8) years. Almost half of the women  were in their first pregnancy and the median gestational age was 34 weeks . Three-quarters of the women  were in the third trimester of their pregnancy and a quarter of them  had come for their 7In their past obstetric history, one woman was admitted for gestational hypertension or pre-eclampsia, three women had experienced stillbirths and 13 women reported at least one child who had died under 5 years of age. Among multigravid women, none had a “bad obstetric history” . Table Over three-quarters of women  had heard about obstetric danger signs. Most women cited ‘Nurse/midwife’ as their source of information on danger signs . Among 375 literate women, 308 82.1%) reported having read the MCH Handbook and 165 (44.0%) reported the MCH Handbook as a source of information on obstetric danger signs. Figure % reporteThe mean knowledge score (±SD) was 12.0 (±2.5) out of 20. Twenty women (4.7%) had ‘good’ knowledge on obstetric danger signs. Most women  had ‘satisfactory’ knowledge while a third of them  had ‘poor’ knowledge. The median number of danger signs recalled was 2 . Among the 335 women who reported having heard of danger signs previously, 68 (20.3%) could not recall any danger sign. Most women recalled vaginal bleeding  while very few women recalled symptoms of pulmonary embolism  among the seven danger signs.Of the 13 questions, over 90% of women chose the correct response to five questions related to: pre-labour rupture of membranes (96.0%), vaginal bleeding with fleshy parts (95.5%), preterm labour pain 92.7%), puerperal sepsis (91.0%) and reduced foetal movements (90.5%). Three questions with most incorrect answers were related to: spotting during pregnancy (19.9%), postpartum haemorrhage (75.8%) and symptoms of pulmonary embolism (78.6%). The details of knowledge assessment using the 20 items are shown in Fig. .7%, puerp < 0.001) and number of danger signs recalled  had significant correlation with the period of gestation. There was a significant association, based on Kruskal-Wallis test, between the number of danger signs recalled with mother’s level of education (p < 0.001), father’s level of education (p = 0.011) and having read the MCH handbook (p < 0.001). Level of knowledge had significant association, based on t-test, with number of ANC visits (p = 0.002) and gestational age (p = 0.033).Knowledge score , previous surgery on the reproductive tract  and father’s level of education  had significant association with ‘good’ level of knowledge. However, in the adjusted analysis, only one factor – ‘previous surgery on the reproductive tract’ – had significant association  with ‘good’ level of knowledge. Table In the unadjusted analyses, having read the MCH Handbook  for home-based records for maternal, newborn and child health reported that home-based records empower women to make decisions by improving their knowledge . WHO alsIn our study, having undergone surgery in the past  had five-times higher odds of having ‘good’ knowledge on obstetric danger signs, a finding similar to that reported in Uganda . Both knThe level of education of both the mother and father influenced the number of danger signs recalled but did not impact their overall knowledge score. Other studies have reported increased odds of knowing and recalling danger signs with higher levels of mother’s education , 21, 28.Knowledge of danger signs during pregnancy is one of the components of birth preparedness and complication readiness . Those wDisseminating information through the media could improve knowledge of danger signs as only half of women in our study reported media as a source of information on danger signs. However, ‘good’ knowledge of danger signs alone without easy access to health facilities would hinder receiving timely services. Geographical inaccessibility has been reported as a barrier to using ANC services in Bhutan  with womAchieving the Sustainable Development Goals requires an integrated approach as health shares an intricate and reciprocal relationship with other socioeconomic and environmental factors . Income The high rate of correct responses to most questions could have been due to respondent’s social desirability bias where the respondents may have felt compelled to choose “seek care from a hospital” or simply “guessed” it to be the expected response.Most pregnant women had ‘satisfactory’ knowledge of obstetric danger signs. Although explicit recall of danger signs was poor, women recognized obstetric emergencies and identified the appropriate action warranted.Additional file 1.


We exploit this effect to trigger and measure the associate change of resistance in tunnel devices. We show that engineering the device structure by inserting an auxiliary dielectric layer, the electroresistance increases by a factor near 2 × 103%, and a robust electric and optic cycling of the device can be obtained mimicking the operation of a memory device under dual control of light and electric fields.In the quest for energy efficient and fast memory elements, optically controlled ferroelectric memories are promising candidates. Here, we show that, by taking advantage of the imprint electric field existing in the nanometric BaTiO Designing energy efficient and fast optoelectric neuromorphic systems remains a challenge. Long et al. report that the combined optical-electric stimulus enables switching the ferroelectric polarization and cycling the resistance state of BaTiO3 tunnel barriers, showing that the optical control of resistance is non-volatile. Ferroelectric tunnel junctions (FTJs) are prime memristor candidates12. In FTJs, voltage-induced polarization switching produces a change in the electrostatic energy landscape13 resulting in important changes of resistance [called electroresistance (ER)], that can be induced by modest electric fields14. Dual optical and electrical manipulation of resistance state is of interest for neuromorphic vision sensors or optoelectric neuromorphic devices15 and the optical control of resistance states in FTJ has been proposed as the ultimate energy efficient and ultrafast control mechanism16.With rapid development of information technology and artificial intelligence, building energy efficient memory devices is one of the main objectives of the scientific community. Among the different strategies, memristor are attracting attention because they can act as artificial synapses able to simulate the excitation/depression process, fundamental to build bionic-like energy-efficient neural networks17 and the electric control of photovoltaic response18, have attracted interest. For instance, photostriction in ferroelectric/magnetic heterostructures has been used to change magnetic properties by light20 and memory cells based on electrically controlled and polarization-dependent photoconductance have been designed23. Among them the optical control of polarization, namely, optical polarization switching, would be of the highest interest to achieve the fast control of resistance in memristive components based on ferroelectric materials16. The control of ferroelectric polarization by the photoabsorption and concomitant carriers generation in ferroelectrics was studied in the 1980s24, and investigations of integration of ferroelectric materials in photo-storage applications were reported26. Nowadays, the interest on light-control of ferroic interaction is undergoing a renaissance27. Recently, it has been shown that the population of polar domains with different orientations and concomitant domain walls, in ferroelectric multidomain single crystals , can be modulated by suitable coherent illumination29 or ultrafast light pulses30. Polarization switching via light absorption at semiconducting electrodes 32 or subsidiary devices34 has been reported, and it has been used to control device resistance by light and electric field in a three terminal ferroelectric field-effect device36. Exploitation of polarization reversal by light absorption within the ferroelectric remains rather unexplored38.Exclusive phenomena related to the presence of ferroelectric polarization and its interaction with light, such as large photostriction2)36, whose integration with ferroelectric films is challenging. We shall exploit a radically different approach, that relies on two concepts. First, the amplitude of the polarization (P) of a ferroelectric semiconducting single crystalline epitaxial film shall depend on the amount of available free charges24 and consequently it can be modulated or eventually suppressed by internally or externally  generated photocharges40. Second, growth conditions43 and electrostatic boundary conditions45 during thin film deposition may lead to the existence of a so-called imprint electric field (EIMP) that favors a particular direction of the emerging polarization P of the films. For instance, in Fig. EIMP downwards favors PDOWN ferroelectric domains, and a therefore a macroscopic PDOWN polarization. Still, the polarization can be switched from PDOWN to PUP by an applied electric field (E) larger than EC and EIMP   of FTJs, avoiding to rely on the photoabsorption in 2D systems  Fig. . By the 2/3Sr1/3MnO3 (LSMO) and platinum (Pt), respectively. This heterostructure is used to elucidate the presence of EIMP and its direction, and to demonstrate the light-induced (blue light) polarization reversal. Next, the ER is measured. Data analysis allows to disentangle different contributions to ER, namely the contribution related to the ferroelectric polarization reversal, which is prevalent at low-voltage and high writing speed, superimposed to the ionic motion contribution, which is dominant in the opposite limits. Measurements under illumination clearly reveal that the resistive switching associated to polarization reversal is very sensitive to photon absorption. Finally, we show that the electric and optical responses of the device are radically improved by engineering the interface between BTO and the LSMO bottom electrode. Indeed, by inserting a nanometric dielectric layer  between the bottom LSMO electrode and the ferroelectric BTO barrier, a larger ER (increased by a factor near 2 × 103%) and a robust light-induced ON to OFF (LRS to HRS) resistance switch is observed allowing to demonstrate a dual electric-optical control of resistance in a FTJ memory element.Here, we report on the polarization and resistance switch of a ferroelectric tunnel junction by visible light and we show how the device can be cycled to store information, which is subsequently read by measuring the tunnel current across the device. In a first realization, an ultrathin epitaxial ferroelectric BTO layer is sandwiched between bottom and top metallic electrodes, La46. The BTO layer is deposited on a LSMO (30 nm) conducting film grown on a (001) STO substrate in a single process (see the “Methods” section). X-ray diffraction θ−2θ scans of the samples, confirm the excellent quality of the films and its [001] texture n on a 00 STO subsn on a 00 STO subsPDOWN, indicating the presence of EIMP pointing downwards. It will be shown below that EIMP ≈ −2.8 MV cm−1, which roughly corresponds to voltage imprint (VIMP) ≈ −1.0 V. By application of bias voltage (V = ±8 V) to the microscope tip (Supplementary Note PUP (black) and PDOWN (yellow) domains are defined . As shown by data in Fig. PUP switching to PDOWN. Control experiments indicated that, in dark, PUP states remain stable, without any obvious change in PFM contrast in at least 6 h . In the following, we shall exploit this phenomenon to demonstrate optical control of resistance in BTO FTJs.The polarization direction of the as-grown film, inferred by piezoelectric force microscopy (PFM) Fig.  is foundned Fig. , showingned Fig. , 4 stronns λ = 40 nm. As sns λ = 40 nm. As sI(V) curve recorded in dark (black line). It can be appreciated that the I(V) curve displays two current peaks indicated by dashed lines for increasing positive applied voltage at: V+C-LOW ≈ 3 V  curves were measured by applying triangular V(t) pulses with a maximum amplitude of 15 V. ER loops were collected by extracting the resistance at 0.5 V from I(V) curves collected in the 0.5 V range measured after applying trapezoidal voltage pulses of amplitude VW and τwrite, were τwrite is the rise time, plateau time and decay time of the trapezoidal signal  and I(V) loops were performed by antiserial connection of two ferroelectric capacitors in the so-called top–top configuration  asymmetry is canceled-out. Needles directly in contact with the surface to minimize the contact area and thus the presence of leakage current added to polarization switching were used. The effective contact area was ≈80 μm2. The leakage was subtracted following the procedure described in Supplementary Note Ferroelectric n-type) cantilevers with Pt coating (ANSCM-PT-50). Scanned areas were 30 × 30 μm2 and the electrically written regions were 10 × 10 μm2.PFM measurements were performed with an MFP-3D ASYLUM RESEARCH microscope (Oxford Instrument Co.), using the AppNano silicon  driven by a CPX400SA DC power (AimTTi Co.). At λ = 405 nm the optical transparency of the used Pt electrodes is ~15%66. Incidence angle ≈ 45° is fixed for all the measurements. In PFM and electrical measurements performed after illumination and shown in Figs. −2 and exposure time of 10 min. PFM experiments were conducted either before or after illumination.Optical illumination during or between the electrical measurement was performed with blue laser (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary