We found that light signals are transmitted from the LOV core to the effector bZIP domain via the hydrophobic residues on the β-sheet. The light-induced conformational change possibly deforms the hydrophobic regions of the LOV core and induces the detachment of the A’α helix to expose the dimerization surface, likely activating the bZIP domain in a light-dependent manner.Light-Oxygen-Voltage (LOV) domains are responsible for detecting blue light (BL) and regulating the activities of effector domains in various organisms. Photozipper (PZ), an N-terminally truncated aureochrome-1 protein, contains a LOV domain and a basic leucin zipper (bZIP) domain and plays a role as a light-activatable transcription factor. PZ is monomeric in the dark state and undergoes non-covalent dimerization upon illumination with BL, subsequently increasing its affinity for the target DNA. To clarify the molecular mechanism of aureochromes, we prepared site-directed mutants of PZ and performed quantitative analyses in the dark and light states. Although the amino acid substitutions in the hinge region between the LOV core and A’ The LOV domain consists of ~ 100 amino acids and a chromophore represented by flavin mononucleotide (FMN). FMN in the LOV domain absorbs blue light (BL), forming a covalent adduct at the C4 position with a highly conserved cysteine residue and inducing protonation at the N5 position6. FMN subsequently induces the LOV core to undergo a conformational change to the light activated state. The cysteinyl adduct spontaneously dissociates, and the LOV domain returns to the dark ground state (dark regeneration). Most LOV domains are conjugated with effector domains that are generally located at the C-terminal of the LOV domain, and light information received by these domains is used for biological activities3. For example, plant phototropins have a kinase domain at the C-terminal, and BL induces the detachment of the Jα helix at the C-terminal of the LOV core, increasing the activity of the kinase domain10.Many organisms on earth use light as an information carrier and have evolved various photoreceptor molecules. The light-oxygen-voltage (LOV) domain is one of the photoreceptor domains found in various organisms 12. They have a basic leucin zipper (bZIP) domain, which is an α-helical DNA-binding motif at the N-terminal of the LOV domain. In a previous study, aureochrome-1 of Vaucheria frigida (VfAUREO1) was found to be responsible for the BL-induced branching response through transcriptional regulation11. We previously reported a synthetic gene (opZL) encoding the N-terminally truncated VfAUREO1 called “Photozipper” (PZ)  at 447 nm. Dynamic light scattering (DLS) and size-exclusion chromatography (SEC) have been used to indicate that PZ exists as a monomer in the dark state, and BL induces the non-covalent dimerization of PZ14. We previously developed the quantitative measurements to evaluate the affinity of PZ for target DNA sequences through electrophoretic mobility shift assay (EMSA) and quartz crystal microbalance (QCM), and clarified that PZ increases its affinity for the target DNA upon illumination17. To elucidate the intramolecular signaling mechanism, we quantitatively measured the monomer–dimer equilibrium and DNA binding of PZ mutants with amino acid substitutions of F298 and Q31717. Since all mutants increased the dimer fractions and affinities for DNA upon illumination, we concluded that there is an equilibrium between “closed” and “open” conformations of the LOV domain and suggested that the BL-regulated switching of VfAUREO1 is achieved by the shift in this equilibrium driven by a synergistic interaction between the chromophore and protein moiety. However, the LOV core signaling pathway that activates the bZIP domain is still elusive.Aureochromes constitute a family of LOV domain-containing proteins (LOV proteins) found among stramenopilesVfAUREO1-LOV by analyzing the crystal prepared in the dark and suggested that F298 of the β-sheet displays distinct side-chain conformations in the dark and light states18. Heintz and Schlichting successfully produced the crystals of the aureochrome-1a of the diatom Phaeodactylum tricornutum (PtAUREO1a) LOV domain under both dark and light conditions and proposed a model in which BL induces the undocking of Jα from the β-sheet of the LOV core and releases the A’α helix from the hydrophobic dimerization site19. In contrast, Kalvaitis et al. recently suggested from their studies on the AUREO1a-LOV of Ochromonas danica (OdAUREO1a-LOV) that the formation of a new hydrogen bond network creates a route for the conformation of Gln293 to be communicated to the A’α helix through Asn194 (corresponding to N218 of VfAUREO1)20. Therefore, two hypothetic LOV core-signaling mechanisms that activate the bZIP domain have been proposed: (1) reconstruction of a hydrogen bond network in the hinge region between the LOV core and A’α helix; and (2) deformation of the hydrophobic region on the β-sheet of the LOV core.Mitra et al. reported the tertiary structure of β-sheet of the LOV core in most LOV domains. However, the roles of these hydrophobic regions remain unknown, probably because hydrophobic interactions are more delicate than hydrogen bonds. In this study, we prepared site-directed mutants, in which amino acids conserved among aureochromes were substituted for smaller residues , and performed quantitative evaluations of dimer formation and DNA binding. Amino acid substitutions are in the hinge region between the A’α helix and LOV core , in the β-sheet of the LOV core , and in the A’α helix   and primer sets: Q216A-F and Q216A-R, Q217A-F and Q217A-R, N218A-F and N218A-R, F219V-F and F219V-R, F299V-F and F299V-R, L303A-F and L303A-R, V314A-F and V314A-R, V314I-F and V314I-R, and V316A-F and V316-R  at the λmax of each mutant. Each regeneration curve was fitted to a single exponential formula, ∆Amax (1-ekt-), where k is the rate constant of dark regeneration.Recombinant proteins were diluted to 4 µM in a loading buffer  containing 1 mM DTT. UV–visible absorption spectra were measured using a V550 spectrophotometer  as previously reportedRH(app)) in solution were determined using Zetasizer software (version 7.12) as previously described16. After removing the aggregates by ultra-centrifugation, DLS analyses of the recombinant proteins (30–60 µM) were conducted several times in the dark (D state), immediately after the termination of BL illumination for 1 min (L state), and after regeneration in the dark (LD state). RH (app) of the samples was plotted against the concentration, and hydrodynamic radii (RH) values were obtained from the extrapolated data at 0 µM protein concentration23.DLS of the protein solutions were measured using a Zetasizer µV system  in automatic mode at 25 °C, and the z-average molecular sizes expressed as the apparent hydrodynamic radii  were incubated for 20 min at 25 °C in the dark, and 200-µL aliquots were subjected to SEC. To analyze proteins in the L state, reaction mixtures were illuminated for 2 min with BL and applied to the column under continuous irradiation with an LED. The molecular weight (MW) was calculated from elution peak volumes using the following formula23:SEC was performed using an ÄKTA purifier column chromatography system  with a Superdex 75 30/10 column in a loading buffer at a temperature of 25 ± 1 °C and flow rate of 0.5 mL/min17. To investigate DNA binding in the L state, each reaction mixture was irradiated with BL and separated by electrophoresis under gel illumination with BL (5 W/m2). The fluorescence signal from the Alexa Fluor647-labeled oligonucleotide of each DNA band was quantified using a Fluor-S MAX image analyzer  and ImageJ software24. The fraction of dsApo bound to PZ mutants was determined from the ratio of free dsApo to total dsApo, normalized by the amount of free ssApo, and plotted against the monomer concentrations of PZ mutants applied to the mixture ([monomer]0). EC50 was estimated by curve fitting to the Hill equation using the maximum of the bound fraction (Bmax).Alexa Fluor647-labeled double-stranded palindromic oligonucleotides with (dsApo) or without (dsCpo) the AUREO1 target sequence  were added to the solutions in the chamber for each injection. In the L state, protein solutions were irradiated for 2 min with BL and applied to the solutions in the chamber while being continuously illuminated with a blue LED. The frequency change due to the DNA binding to PZ mutants was plotted against [monomer]0. In each case, EC50 was estimated by curve fitting to the Hill equation using the maximum of the frequency change (∆Fmax).The DNA binding of each PZ mutant was detected using a 27 MHz-QCM system  as described previouslyβ-sheet of the LOV core . The absorption spectra in the D state were almost identical to that of wild-type PZ (wtPZ), showing triplet vibrational structures characteristic of LOV domains  of dark regeneration were distributed from 5.7 to 12 min  and in the RH(app) measured by DLS depends on the protein concentration23, the RH(app) values were plotted against the monomer concentration subjected to each measurement ([monomer]0) in the dark , immediately after BL illumination , and after dark regeneration   mutants with RH of ~ 3.7 nm irrespective of light conditions (F219V and V314I); (2) mutants with similar RH in the D state but smaller RH (~ 3.4 nm) in the L state (F299V and L303A); and (3) mutants with greater RH (~ 3.2 nm) in the D state but similar RH (~ 3.8 nm) in the L state (V314A and V316A).Since ro Table . The RH RH3 value of each mutant at both 8 and 50 µM concentrations  under the DLS conditions. Therefore, we conclude that the variation of RH and peak volumes of PZ mutants are caused by the shift in the monomer–dimer equilibrium.The sizes of PZ mutants were further investigated by SEC at 8 µM Fig. a and 50 β-sheet mutants showed significant variation. The peak volume of the F219V mutant in the D state was significantly left-shifted but similar to that in the L state, indicating that most of F219V is dimeric irrespective of the light conditions. Likewise, the V314I mutant showed elution profiles that were similar in the D and L states, suggesting that the substitution of V314 for a larger residue (Ile) completely suppresses BL-induced dimerization. The peak volumes of the F299V and L303A mutants were significantly greater in the L state than in the D state, indicating that F299V and L303A substitutions decrease dimer formation in the L state. The elution profiles of the V314A and V316A mutants suggest that the substitutions involved slightly induce the destabilization of monomer and dimer forms in the D and L states, respectively. At 50 µM, the peak volume of the V314A mutant appeared to be smaller than that of wtPZ in the L state, probably due to the oligomerization of this mutant at high concentrations. The elution peak volume of each PZ mutant is consistent with the RH value of each mutant obtained by DLS measurements.The peak volumes of the Q216A and Q217A mutants showed minimal right-shifts compared to that of wtPZ in the L state, suggesting that the substitutions slightly destabilize the dimer form. However, the peak volumes of the N218A mutant were similar to those of wtPZ in both the D and L states, clearly indicating that the N218A substitution has a minor effect on the monomer–dimer equilibrium of PZ. On the other hand, the elution profiles of the monomer]0. The maximum of the bound fraction  and EC50 were estimated by fitting the data to the Hill equation. The specificities of DNA binding were confirmed using the control dsCpo  due to the formation of the PZ-dsApo complex. Supplementary Figure ∆Feq/∆Fmax against [monomer]0 of each PZ mutant. The EC50 values of each PZ mutant estimated using EMSA and QCM showed good agreement with each other in the D , assuming that these Leu residues interact with F219, V314, and V316 on the β-sheet. The absorption spectra and dark regeneration kinetics of the A'α mutants were similar to those of wtPZ  in the A'α helix and β-sheet are well conserved among the LOV domains of aureochromes   are located at the hydrophobic interface of a dimer  and Arabidopsis phototropin1 LOV2 (Atphot1LOV2)35. For aureochromes, the crystal structures of PtAUREO1a-LOV indicated that the A'α helix is in contact with the hydrophobic surface of the β-sheet in the dark state but is detached from the LOV core in the L state  the bZIP domain in aureochromes.Molecular mechanisms that involve the regulation of effector activity by LOV domains in phototropins have been studied. Harper et al. produced a series of point mutations along the JOdAUREO1a-LOV, Kalvaitis et al. proposed a model in which the rotation of the side chain of Asn194 (corresponding to N218 of VfAUREO1) due to a rearrangement of hydrogen bonds leads to conformational changes in the A'α helix20. Through simulation analyses, Tian et al. reported that A248, Q249, Q250, and N251 in the hinge region are the residues important for signal transduction in PtAUREO1a38. The rearrangement of hydrogen bonds in the N-terminal region was also suggested for Neurospora VVD27. Zoltovskii and Crane surmised that the flavin protonation state is influenced by a series of compensatory conformational and hydrogen bonding changes from Q182 and C71 to Ncap39. In another simulation study, Gangly et al. also suggested that the hinge-bβ is directly affected by the new hydrogen bond to Ala72 upon Gln182 rearrangement40. However, our data clearly demonstrates that amino acid substitutions in the hinge region have minor effects on the dimer formation and DNA binding of PZ. In VfAUREO1, the displacement of the hinge region unlikely drives the detachment of the A'α helix upon BL-illumination.Through the structural analysis of β-sheet of the LOV core, the roles of each residue remain to be clarified, probably because hydrophobic interactions are not that simple to investigate compared with hydrogen bonds. In the present study, we produced a series of PZ mutants in which the hydrophobic amino acids in the β-sheet were substituted for smaller residues and quantitatively measured the monomer–dimer equilibria and DNA-binding activities. Substitutions of F299 and L303 for smaller residues appeared to destabilize the dimer form in the L state; such finding is consistent with structural data showing that the F299 and L303 residues are located beside the A’α helix in the D state and at the peripheral region of the dimerization surface in the L state19. When V314 was replaced by a larger residue (Ile), the mutant forms dimer and binds DNA irrespective of light irradiation, probably because the V314I substitution caused a large deformation of the hydrophobic regions in both the D and L states. In contrast, the F219V, V314A, and V316A mutants, similar to the L201A and L208A mutants, exhibited altered RH and EC50 values in the D state, suggesting that the substitutions involved in these mutants induce the partial detachment of the A’α helix in the D state.Although hydrophobic residues are often found in the RH and EC50 values of the F298 and Q317 mutants, due to the existence of the closed-open conformational equilibrium of the LOV core that regulates the monomer–dimer equilibrium of PZ17. In the present study, the correlations between the RH3 and EC50 values of the hinge and β-sheet mutants were similar to those of the F298 and Q317 mutants (Supplementary Fig. β-sheet and induces the detachment of the A’α helix to expose the dimerization surface (Fig. α helix, stabilizing the monomeric form. In the L state, the shift in the conformational equilibrium induces the detachment of the A’α helix to expose the dimerization surface composed of F219, F299, L303, V314, and V316, stabilizing dimer formation and resulting in the increased affinity of PZ for the target DNA sequence. The deformation of the hydrophobic regions in the β-sheet may be a general mechanism for signal transduction in the LOV domains that lack the BL-induced rearrangement of a hydrogen bonding network.We previously reported the correlation between the α helix and the LOV core, and in the hydrophobic regions of the β-sheet have minor effects on the spectroscopic properties of PZ. Although the BL-induced dimerization and DNA binding properties of the hinge mutants were similar to those of wtPZ, those of the β-sheet mutants were significantly different, indicating that light signals are transmitted via the hydrophobic residues in the β-sheet. Moreover, the substitutions of Leu201 and Leu208 for Ala in the A’α helix affected the dimerization and DNA binding similar to those in the β-sheet mutants, suggesting that the BL-induced deformation of the hydrophobic regions causes the detachment of the A’α helix and results in the activation of the effector bZIP domain in aureochrome-1. The deformation of the hydrophobic regions may be a general mechanism for the transmission of light signals in the LOV domains that lack the BL-induced rearrangement of a hydrogen bonding network.To elucidate the molecular mechanism of the LOV domain in aureochromes, we prepared site-directed mutants of PZ. Amino acid substitutions in the hinge region between the A’Supplementary Information.


The collision warning system (CWS) plays an essential role in vehicle active safety. However, traditional distance-measuring solutions, e.g., millimeter-wave radars, ultrasonic radars, and lidars, fail to reflect vehicles’ relative attitude and motion trends. In this paper, we proposed a vehicle-to-vehicle (V2V) cooperative collision warning system (CCWS) consisting of an ultra-wideband (UWB) relative positioning/directing module and a dead reckoning (DR) module with wheel-speed sensors. Each vehicle has four UWB modules on the body corners and two wheel-speed sensors on the rear wheels in the presented configuration. An over-constrained localization method is proposed to calculate the relative position and orientation with the UWB data more accurately. Vehicle velocities and yaw rates are measured by wheel-speed sensors. An extended Kalman filter (EKF) is applied based on the relative kinematic model to combine the UWB and DR data. Finally, the time to collision (TTC) is estimated based on the predicted vehicle collision position. Furthermore, through UWB signals, vehicles can simultaneously communicate with each other and share information, e.g., velocity, yaw rate, which brings the potential for enhanced real-time performance. Simulation and experimental results show that the proposed method significantly improves the positioning, directing, and velocity estimating accuracy, and the proposed system can efficiently provide collision warning. The global status report on road safety 2018, launched by the WHO in December 2018, highlighted that the number of annual road traffic deaths had reached 1.35 million T can be derived asAfter v, yaw rate ω, and tuning radius r can be derived as shown in Equation (9).rv denotes the speed of the right wheel, lv represents the speed of the left wheel, and L indicates the rear wheelbase.The proposed system consists of four wheel-speed sensors, which are installed on the rear wheels of two vehicles. According to the Ackerman steering model shown in tv, and tyam at time t as shown in Equation (10).Then, the position kX as the state vector at time k. It contains the relative position/orientation We define t as the time period from time k − 1 to time k. kX can be predicted by kX−1 based on the relative kinematics model shown in We define ΔA can be derived as Equation (13).Then, the transition matrix of the state vector Similarly, the transition matrix of process noise is:Q of process noise consists of error covariances of speeds and yaw rates, that is:The error covariance matrix Thus, the predicting process of the model is:kZ as the observation vector, containing the relative position and orientation of vehicle 2 measured by the UWB system, four wheel-speeds measured by the DR system, and the observation noise kV. Then, the observation equation can be expressed as Equation (17).We define Referring to Equation (9), the velocities and yaw rates of the two vehicles can be expressed by the velocities measured by wheel-speed sensors as Equation (18).H is obtained as Equation (19).Then, the Jacobian matrix The estimating process is:R represents the error covariance matrix of kZ. It can be divided into the error covariance matrix of the UWB system UWBR and the error covariance matrix of the DR system DRR. That is:In Equation (20), DRR is decided by measurement errors of the wheel-speed sensors directly, whereas UWBR is decided by positioning and directing errors, which is indirectly decided by the ranging error of UWB modules. Define D = . On the basis of Equation (5), we can derive the relationship between the deviation D and the deviation of UWB modules’ position MX and NX as Equation (22). d5 is ignored because it is not a measurement but a constant, which means DF can be derived as Equation (23).UWBX =  and the deviation of the UWB modules’ position MX and NX as Equation (24).From Equation (8), we can get the relationship between the deviation of the vehicle position and orientation UWBR can be expressed as Equation (26).Then, CWS mainly works in two ways, headway measurement warning (HMW) and TTC-based warning . Both ofThe TTC-based system takes relative velocity into account, so it provides a more accurate collision warning. In this paper, the proposed system allows vehicles to share information through UWB, such as velocities. The TTC method is apparently the better choice.Two vehicles driving on the road have the probability of collisions in various types, such as head-to-head collision, rear-end collision, and side collision. Different kinds of collisions may happen at different times. That means all cases need to be taken into account in order to obtain the exact TTC. Before establishing the collision warning model, we simplified the shape of a vehicle as a rectangle. With this assumption, all kinds of collisions can be described as point-to-edge collisions. Edge-to-edges collisions and point-to-point collisions are also covered by point-to-edge collisions, as shown in iR  represents the four corners of vehicle 1. iF  represents the four corners of vehicle 2. Therefore, the coordinate of X = T and the relative orientation β are estimated by the UWB/DR system. Then, the coordinates of vehicle 2′s corners in the axis system of vehicle 1 can be derived as Equation (28).After unifying different collision types, TTC can be calculated in the same way. We take the collision type shown in kR at time We define all the points at the collision time as iF collides on the edge between jR and kR means Point t of Equation (30) is the collision time under the condition that corners of vehicle 2 collide on edges of vehicle 1, including 16 different conditions altogether. In the other 16 cases in which the corners of vehicle 1 collide on the edges of vehicle 2, the collision times can be calculated similarly. Thirty-two collision times can be calculated in total. Ignoring negative values, the minimum of the rest value is TCC. That is:Solution TTC → ∞ or TTC < 0, there is no risk of collision.When d5. Secondly, the accuracy of the UWB/DR fusion model based on EKF is compared to the accuracy of UWB and DR separately. Finally, plenty of driving scenarios are generated to evaluate the success rate of the CWS.In this section, simulation is conducted to evaluate our algorithm. Firstly, the accuracy of the UWB positioning and directing system is validated by comparing the algorithm with and without the constraint of dσ = 0.05 m, and the wheel speed error is set to vσ = 0.2 m/s referring to the sensors we will use in experiments. Calculating results of our algorithm are compared to the real values exported by the model.In d5 are compared in d5 is very intuitive, especially for x and β. In Solutions of the algorithm with and without the constraint of We also take the scenario in In this section, we generate plenty of driving scenarios with different velocities, relative positions, and relative orientations, as shown in realTTC is certain when a scenario is established, and estTTC estimated by CWS is calculated every 10 ms. The collision warning threshold is set to 3.0 s. It means that when estTTC ≤ 3.0 s, the CWS will send an alert. errTTC = estTTC – realTTC denotes the TTC error at the warning time as errTTC can be divided into three conditions corresponding to three evaluations of collision warning as Equation (32).In order to guarantee driving safety, we set 2.7 s as the latest warning time. If the system does not work when the vehicle is colliding within 2.7 s, the collision warning evaluation is failed. In addition, in order not to disturb the driver too much, if the system sends alerts when vehicles have no risk of collision within 4 s, we regard the warning as false. Then, “Failed” denotes warning too late or not warning;“Correct” denotes warning in the proper time period;“False” denotes warning too early or warning by mistake.The scenario marked with gray background is the typical rear-end collision scenario, which is the most critical function of a collision warning system. One hundred and ninety-six rear-end collision scenarios are generated, and realTTC less than 3 s will not be considered. In the remaining 10,823 scenarios, 10,593 of them perform correctly. The collision warning success rate is 97.9%.Then, we emulate other scenarios in which two vehicles drive in any lanes from any positions to any directions defined in In this section, experiments are divided into two parts: straight driving experiments and curved driving experiments. The straight driving experiments are conducted referring to JT/T883-2014, which describes the standard experiments for FCWS, published by the Ministry of Transport of the People’s Republic of China (MOT). As JT/T883-2014 only regulates straight driving experiments, to further validate the performance of our system, curved driving experiments are conducted in addition. Since the CWS is implemented based on the UWB/DR relative positioning/directing system, the positioning/directing accuracy can reflect the performance of the CWS. Therefore, in the curved experiments, we drive through complex routes and compare the positioning/directing accuracy to the parameters of a commercial millimeter-wave radar (MMWR) used for collision warning.According to JT/T883-2014 , experimTest 1 is designed as shown in Test 2 is designed as shown in Test 3 is designed as realTTC, which is derived from the RTK-GPS/INS information; (2) CWSTTC, which is estimated using the UWB/DR measurements. Since the terminating conditions in the three experiments are different, to satisfy all the three tests and reserve some margin, we set the warning CWSTTC to 3.0 s. The software in realTTC or CWSTTC reaches its marginal value. If realTTC reaches the regulated marginal value when CWSTTC is still greater than 3.0 s, the test is terminated and evaluated as failed. In the standard, only the minimum threshold of the collision warning time is regulated, whereas the maximum threshold is not. In other words, the standard only cares about “how safe” the warning is, with no consideration of “how accurate” it is. However, as we explained in realTTC > 4.0 s, we also regard the test as failed. According to JT/T883-2014, each test needs repeating seven times. During each test, two TTC values are calculated: (1) According to ρ, θ, and v represent the relative distance, azimuth angle, and velocity, respectively. The MMWR has two working modes, middle-distance mode and long-distance mode, and the accuracies are different.JT/T883-2014 only regulates the straight driving experiments but does not request or give advice to curved driving experiments. However, to further validate the superiority of our system, we conduct curved driving experiments and compare its accuracy to a commercial MMWR, Aptiv  ESR 2.5, which is used in CWS. The MMWR measures the relative distance, relative azimuth, and relative velocity. x, y] are converted to the polar coordinate , and the velocities of the two vehicles  are converted to the relative velocity v, as shown in β cannot be measured by MMWR directly.In order to facilitate comparison, the curved experiments are also divided into a middle-distance experiment under vehicle distances within 50 m and a long-distance experiment under vehicle distances within 100 m. The estimated values of the relative position  in the simulation but as polar coordinate  in the experiments. Both x and y improve after fusion as shown in θ without ρ improves after fusion according to θ can contribute to better accuracy of both x and y, as According to The distance accuracy of the proposed system is always much better than the MMWR, no matter with or without fusion. The azimuth accuracy without fusion is about 0.76° in both experiments, which is better than the middle-distance MMWR but is inferior to the long-distance MMWR. However, velocity accuracy without fusion is worse than the MMWR in both modes. As for the fusion system, the accuracy of relative distance and azimuth performs significantly better than the MMWR, and the relative velocity accuracy also improves to a similar level as the MMWR in both middle and long-distance modes. In addition, the proposed system can provide the relative orientation, which is not available directly in the MMWR system.The proposed relative positioning/directing algorithm with an additional distance constraint significantly improves the relative positioning/directing accuracy, especially the directing accuracy, as shown in The fusion method significantly improves the relative positioning/directing accuracy and slightly improves the velocity accuracy according to the simulation and experiment results.The proposed CWS passes the regulated tests in JT/T883-2014 published by MOT, which proves the feasibility of the proposed system.In middle-distance mode up to 50 m, compared to the MMWR, the proposed system improves the relative positioning/directing accuracy by 44%, 69%, and 8%, respectively, in the relative distance, azimuth angle, and velocity. As for in long-distance mode, the enhanced rate is 66% and 38%, respectively, for the relative distance and azimuth angle. The relative velocity accuracy of the proposed system is similar to the MMWR.In both middle and long-distance modes, the proposed system can provide relative orientations with errors no more than 0.4° RMSE, which is not available directly in MMWR systems, but it is very beneficial to the CWS.In this paper, we proposed a CWS combining UWB and DR. An improved relative positioning/directing algorithm based on UWB is presented, and a DR model based on the speeds of the rear wheels is established. Then, a fusion algorithm using EKF is proposed to improve the accuracy of relative position, orientation, and velocity. Afterwards, the advantage of the proposed system is preliminarily verified by simulation. Finally, experiments are conducted to further validate the performance of our system, and the experiment results are compared to a commercial MMWR used in CWS. The main conclusions are summarized as follows:The inadequacy of the proposed system is the velocity accuracy. Although it performs at the same level as MMWR in terms of velocity accuracy, it can be further improved. To facilitate comparison of the proposed system and the MMWR, the velocity data of the experiments are shown as relative velocity. We also analyze the accuracy of absolute velocities of two vehicles. In the middle-distance experiment,


The enzyme soluble guanylate cyclase (sGC) is the prototypical nitric oxide (NO) receptor in humans and other higher eukaryotes and is responsible for transducing the initial NO signal to the secondary messenger cyclic guanosine monophosphate (cGMP). Generation of cGMP in turn leads to diverse physiological effects in the cardiopulmonary, vascular, and neurological systems. Given these important downstream effects, sGC has been biochemically characterized in great detail in the four decades since its discovery. Structures of full-length sGC, however, have proven elusive until very recently. In 2019, advances in single particle cryo–electron microscopy (cryo-EM) enabled visualization of full-length sGC for the first time. This review will summarize insights revealed by the structures of sGC in the unactivated and activated states and discuss their implications in the mechanism of sGC activation. The prototypical sGC stimulator is the benzylindazol derivative YC-1 (3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole), which was first identified in a screen of compounds for platelet inhibition . Since irtension ,33,34,35rtension ,36.Kd in the low picomolar range, or lower [The existence of various activity states for sGC in vitro begs the question of physiological relevance—which activity states of sGC are relevant in vivo? In cells, the concentration of NO has been estimated to be in the range of 100–5000 pM , whereasor lower . Based oor lower ,39. The Although more than four decades of work have revealed much about the biochemistry of sGC, obtaining a structure of the full-length enzyme proved elusive. In lieu of such information, structural insights on sGC came from crystal structures of individual sGC domains and their homologs, as summarized below.2-sensitive H-NOX domains fused to C-terminal effector domains, generally a methyl-accepting chemotaxis protein [2-binding H-NOX domains have been studied in detail structurally and spectroscopically to provide insight into the function of this domain in sGC. The first H-NOX structure to be solved was of the O2-binding H-NOX domain from the thermophilic anaerobe Caldanaerobacter subterraneous (Cs) (previously known as Thermoanaerobacter tengcongensis) [b-type heme cofactor is found sandwiched between the N- and C-terminal subdomains of the protein and is ligated by a conserved histidine residue, termed the proximal histidine, which is located on the αF helix of the protein. The N- and C-terminal H-NOX subdomains are termed distal and proximal, respectively, with the proximal subdomain taking its name from the presence of the proximal histidine residue within the subdomain. The distal subdomain consists of α-helices A–E and the proximal subdomain is composed of helices αF and αG and the four-stranded β-sheet.Efforts to better understand the effect of NO binding to sGC turned to bacterial homologs of the β H-NOX domain. In aerobic and facultative anaerobic bacteria, NO-sensing H-NOXs are found as standalone proteins that interact with cognate signaling partners, usually a histidine kinase or a diguanylate cyclase ,41. In c protein ,42. Bothngensis) ,44. The Shewanella oneidensis (So) in the unliganded and NO-bound states revealed the structural effects of gas binding to the protein [2-binding Cs H-NOX, and it has been suggested that this rotation of the distal subdomain may be a general feature of H-NOXs which could play a key role in signaling [Subsequent crystal structures of the NO-binding H-NOX from  protein . Upon NO protein ,46,47. ASo H-NOX, NO was observed bound to the proximal side of the heme, rather than to the distal side as had been expected. Subsequent spectroscopic work, however, revealed this phenomenon to be a result of the large excess of NO used in the crystallization conditions and that under standard solution conditions NO primarily binds to the distal side of the heme [In addition to the overall conformational change, the structures also show NO-induced rupture of the iron–histidine bond between the heme cofactor and the protein, consistent with previous spectroscopic data . This bothe heme .Manduca sexta (moth) sGC was determined [Rattus norvegicus (rat) sGC, consistent with the idea that perturbations in this region occur upon activation [PAS and CC domains are ubiquitous and versatile structural motifs that are found across domains of life and that often function together in a signaling capacity. PAS domains are named for the proteins Per, Arnt, and Sim, the three proteins in which homologous sequence regions, now recognized as the PAS domain, were first identified . There atermined . How thetermined . Hydrogetivation .As the name implies, CC domains are composed of two or more α-helices that intertwine, forming a coil of coils. These domains are found in a wide range of proteins and serve in a variety of functions. As in sGC, CC domains are often found C-terminal to PAS domains, where they are thought to serve as an intermediary signal transduction module between the PAS domain and a connected effector domain, this effector domain being the CAT domain in sGC . PreviouThe first nucleotide cyclase CAT domains to be structurally characterized were those of adenylate cyclases (ACs) ,58,59. TFor many years, AC CAT domain structures served as a basis for understanding the structure of GCs, given their high sequence similarities. Indeed, homology models of the bovine and rat sGC CAT domains that were made based on AC CAT domains were used to identify key residues involved in catalysis and substrate specificity ,61. UsinTo date, all GC CAT domain crystal structures represent inactive conformations in which the active sites are not appropriately oriented for substrate binding and subsequent catalysis ,20,21,22The first direct insight into the structure of full-length sGC came in 2014 with the report of low-resolution negative stain electron microscopy (EM) envelopes of the rat enzyme . Three-dThe single-particle classification and alignment in this study yielded a total of 94 3D reconstructions of the unliganded rat sGC that suggested significant conformational flexibility . The recThis low-resolution analysis of full-length sGC unveiled for the first time the overall shape and general domain organization of the enzyme, however, the mechanism of allosteric activity regulation remained unclear. It was not until the determination of higher resolution structures that these details began to become more apparent.Manduca sexta (moth) in the unactivated, Fe2+-unliganded state and in the excess-NO and YC-1-bound activated state to 5.1 and 5.8 Å resolution, respectively [Homo sapiens (human) sGC in different states: the Fe2+-unliganded state (to 4.0 Å resolution), the Fe3+-unliganded state (3.9 Å resolution), the excess-NO activated state with bound Mg2+ and GMPCPP (3.8 Å resolution), and the β H105C variant of the enzyme (6.8 Å resolution) [3+-unliganded form of human sGC was found to be structurally similar to the Fe2+-unliganded form and the β H105C variant, in which the heme-ligating histidine is replaced with cysteine, was found to lack the heme cofactor and to be similar in both structure and activity to the activated state of the enzyme [Within the last decade, the so-called “Resolution Revolution”  in singlectively . The secolution) . These tolution) ,66,67. Ce enzyme .Consistent with the low-resolution, negative stain EM reconstructions, the high-resolution cryo-EM data reveal an oblong particle composed of two distinct lobes connected through a stalk-like density. The N-terminal regulatory lobe consists of the α and β H-NOX domains and the α/β PAS dimer . The twoIn the basal, unactivated state, sGC adopts a contracted conformation in which the β H-NOX domain is in close proximity to, although not directly interacting with, the β CAT domain A. This bInterestingly, the structures do not indicate that there is any direct contact between the regulatory and catalytic lobes. Such direct contact had previously been suggested as the means of activity regulation in sGC, with the β H-NOX domain interacting with the CAT domains in an inhibitory fashion that was relieved upon NO binding ,68. InstThe high-resolution structures of full-length sGC in both the unactivated and activated states provide a first exciting look at the structural details of this important enzyme. They also set the stage for future experiments aimed at further understanding the mechanism of allosteric, intramolecular signal transduction. Aspects of individual domains that have been revealed by these new structures will be discussed in more detail below. The higher resolution of the human sGC structures allowed visualization of many amino acid side chains throughout the electron density maps, and so references to residue numbers will reflect the numbering of the human sGC sequence.The α H-NOX domain of sGC has long been an enigma. It has previously been shown to be unnecessary for dimerization or activity regulation, and yet is present in all known sGCs . DespiteSo structures in So H-NOX. Also in contrast to the So H-NOX structures is the relative positioning of β-His105 (His103 in So H-NOX) in the unactivated (unliganded) and activated (NO-bound) structures. Rather than the histidine side chain flipping away from the heme, the rotamer conformation of β-His105 is maintained following iron–histidine bond cleavage, while the Cα atom of the residue shifts by 2.1 Å  to generate a constitutive break in one or the other of the CC helices . Both vaIn addition to straightening, the CC helices also rotate about one another upon activation A. When t® [To stabilize the fully active form of moth sGC, Horst et al. included the small molecule sGC stimulator YC-1 in their EM grid preparation . Electro® , may fun3 [KM(GTP) upon sGC activation [As predicted from the crystal structures of isolated GC CAT domains, the CAT domains in full-length sGC form a wreath-like heterodimer that undergoes a conformational change upon activation ,20,21,223 . Togethetivation .The conformational changes in the sGC CAT domains appear to be driven by upstream rearrangements within the protein. In particular, rotation of the CC helices brings the two C-terminal CC residues into closer proximity B, thus sKd of NO release from the heme, as discussed above [The cryo-EM structures presented by Horst et al.  and Kanged above ,38. To ped above . The anaThe structures reported by Horst et al. and Kang et al. represent a major advance in our understanding of sGC and provide a foundation for elucidating the biochemical details of sGC activation. In particular, the rearrangements that occur in the CC domains upon activation appear to be critical in communicating the ligation state of the protein to the CAT domains . Other hAdditionally important is developing a better understanding of the 1-NO state of sGC, as this state is likely to be the physiological resting state of the enzyme. The SEC-SAXS analysis by Horst et al. suggested that the 1-NO state contains multiple conformations. Future work to characterize these conformations could include additional advanced SAXS techniques such as ligand titration SAXS coupled to singular value decomposition to identify structural transitions involved in forming the 1-NO state and in transition of the 1-NO state to the excess-NO state . Additio


Hemp flour from Dacia Secuieni and Zenit varieties was added to bread in different proportions  to improve its nutritional properties. The purpose of this paper was to present the advanced nutritional characteristics of these bread samples. The selected varieties of hemp, accepted for human consumption, met the requirements for the maximum accepted level of THC in seeds. The protein content of new products increased from 8.76 to 11.48%, lipids increased from 0.59 to 5.41%, mineral content from 1.33 to 1.62%, and fiber content from 1.17 to 5.84%. Elasticity and porosity decreased from 95.51 to 80% and 78.65 to 72.24%, respectively. K, Mg, Ca, P, Mn and Fe are the main mineral substances in bread with addition of hemp flour from the Dacia Secuieni and Zenit varieties. The total amount of unsaturated fatty acids in the bread samples with hemp flour ranged from 67.93 g/100 g and 69.82 g/100 g. Eight amino acids were identified, of which three were essential . Lysine, the deficient amino acid in wheat bread, increased from 0.003 to 0.101 g/100 g. Sucrose and fructose decreased with the addition of hemp flour, and glucose has not been identified. The amount of yeasts and molds decreased in the first 3 days of storage. Regarding the textural profile, the best results were obtained for the samples with 5% addition. In conclusion, bread with the addition of hemp flour has been shown to have superior nutritional properties to wheat bread. Bread is one of the most consumed foods on the planet. Its importance in the development of mankind, in terms of food, is undeniable. It is a staple food product, dating back to Mesopotamia, reminiscent of its consumption during the construction of the great pyramids of Egypt ,2.Bread is consumed in various forms around the world, and the average global consumption is 70 kg per year per capita, and in Europe it is 59 kg of bread per year per capita .The success behind the high consumption of bread is given by the simplicity in terms of accessibility of ingredients, preparation, and low cost, and last but not least the multitude of cereals that can be used to bake them . Ragycentron canadum) [Cumin cyminum) and caraway seeds (Carum carvi) [Given the growing interest in “redefined” healthy foods, the enrichment of bread with various bioactive compounds, micro- and macroelements is becoming more widespread ,6. Amongcanadum) , proteinm carvi) , soybeanm carvi) , or glutm carvi) .The concept of “enriching” or “fortifying” a food, according to the Codex Alimentarius, is the addition of one or more nutrients, whether or not they are found in that food. Fortification aims to prevent or correct a nutrient deficiency, in relation to the entire population or to certain groups held  . FortifiSvetlana Popel states that the most affordable and effective way to provide various nutrients to the population is to fortify daily foods, especially bakery products. Also, these enrichments should not diminish the shelf life, but neither the sensory nor nutritional qualities of the products . Food foFortified products are increasingly preferred because consumer needs and preferences have changed greatly in recent years. The contemporary consumer no longer considers bread just an energy source, wants bread to be a product with adequate nutritional and functional value for the daily diet .Cannabis sativa L., belongs to the Cannabaceae family and is one of the oldest plants originally cultivated in Central Asia, and from the Bronze Age and in Europe [Hemp, n Europe ,19. For n Europe .C. sativa L., approved and regulated by the European Union Plant Variety Database, for human consumption [Although some studies show that the two components present in hemp with psychotropic activity, delta-9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD), are not found in seeds, only in the leaves, stems, and inflorescence of the female plant ,22,23,24sumption ,27,28. Hsumption . Thehempsumption ,30,31. Psumption ,36,37,38sumption . The amosumption ,36. A grsumption . Mineralsumption ,33,36,37sumption ,41 and asumption ,43,44,45As the awareness of the nutritional value of hemp seeds began to increase, there was a growing interest in their food use. Thus, there have been more and more recommendations for the introduction of hemp seeds and hemp seed foods in the diet. Although there are several studies on the analysis of hemp seeds in various foods such as bread, biscuits, cookies ,46,47,48The results presented in The bread samples studied had a moisture between 35.98% and 25.63%. As the amount of hemp flour in the bread increase, a decrease in the moisture content was observed. This decrease is due to the low humidity of the hemp flour used 4.49% for hemp flour variety Dacia Secuieni, respectively 4.98% for hemp flour variety Zenit . The samThe quality of proteins from vegetal sources is an increasingly promoted topic in the field of nutrition. Due to the importance of consuming essential amino acids in a balanced diet, nutritionists recommend the use of vegetable protein sources. Due to the low protein content of wheat flour , the addIn addition to a significant increase in protein content, bread with the addition of hemp flour also came with a significant lipid intake. There wasan increase from 0.59% in the case of the control sample to 5.31%, respectively 5.41% for the samples with the addition of 20%. A tendency to increase the lipid content wasreported by several authors, but the differences are quite large and are justified by the variety of hemp seeds chosen. Apostol et al. report increase of up to 3.11% in the case of bread with the addition of 20% hemp flour and 2.02% in the case of samples with 10% . For samThe ash content reported in the bread samples with hemp flour shows a slight increase of 0.27% for hemp flour variety Dacia Secuieni, respectively 0.29% for hemp flour variety Zenit, compared to the control sample. This increase is due to the relatively high content of mineral substances in hemp flour. Apostol et al. , report Similar to the results obtained in the case of proteins and lipids, the fiber content shows an increase from 1.17% in the case of the control sample, up to 5.39%, respectively 5.84%. In the case of samples who 10% addition of hemp flour, an increase of 2.37% is observed, respectively 2.12% compared to the control sample. Our results are lower than those reported by Lukin et al., who reported increase of 3.48% or 4.31% reported by Apostol et al., reporting the same percentage of the addition. In the case of total fibers, higher results were reported than those obtained for the Dacia Secuini and Zenit varieties ,57. ThesIn the case of acidity, there wasa slight increase in the case of samples with added flour from the Dacia Secuinei variety and a higher increase in the case of samples with added flour from the Zenit variety, compared to those mentioned above. The increasewith a maximum of 0.4% in the case of samples coming from the Dacia Secuieni variety, and a maximum of 0.9% in the case of those coming from the Zenit variety. Similar results were reported by Bădărău et al.  and AposAnother advantage of adding hemp flour to bread is the increase in energy value from 253.71 kcal/100 g to 311.95 kcal/100 g, respectively, 316.81 kcal/100 g, the date of high-quality nutrients. The same trend is reported in the case of fortifying bread with mushroom powder [The results presented in Hemp flour is rich in fiber and protein, and its addition to the mix with wheat flour has a significant influence on rheological parameters, such as elasticity and porosity, reflected in the quality of the finished product. The addition of hemp flour to bakery products had as a side effect the decrease of their elasticity and porosity. The influence of hemp flour on the porosity and elasticity of the bread is presented in The mineral elements determined in the bread samples were Cu, Cd, Cr, Ni, Pb, Zn, Fe, Mn, Ca, Mg, K, and P. Of these, in all samples a significantly higher Ca content was identified and of K. The bread obtained by adding 20% hemp flour from the Dacia Secuinei variety had the highest percentage in Ca, and the one with the addition of 20% hemp flour Zenit variety had the highest percentage in the case of K. However, it was the quantity of Mg which reached up to 394.33 mg/100 g. The smallest amounts of micro and macroelements were recorded in the case of Cu, Cd, Cr, Ni, Pb, Zn, Fe, Mn .Enrichment with micro and macro elements of bread samples with the addition of hemp flour was also reported in the literature. Lukin et al.  reportedThe highest values were given by linoleic acid. It can be seen that it a decrease can be observed with the addition of hemp flour. Most studies performed on hemp seeds or flours obtained from hemp seeds reported the presence of an unsaturated fatty acid content of up to 90% ,29,61. IAmong the unsaturated fatty acids, linoleic acid was followed by oleic acid, gamma-linoleic acid, which was constantly increasing with the addition of hemp flour, and palmitoleic acid. The total amount of unsaturated fatty acids in the bread samples with hemp flour were between 67.93 g/100 g and 69.82 g/100 g. They can be monounsaturated (MUFA) and polyunsaturated (PUFA), the polyunsaturated ones being found in proportion of about 50%. Linoleic acid  and gamma-linoleic acid , the main PUFA acids, belong to the category of essential fatty acids (EFA). These acids are essential because they cannot be synthesized by the human body and must be assimilated from the diet, they are indispensable for the normal functioning of the human body ,29,30. CIn terms of the amount of saturated fatty acids, they were found in quantities of less than 30% in fortified bread samples and were constantly decreasing with the addition of hemp seed flour.The results on the amounts of saturated and unsaturated fatty acids were reported by Apostol et al., who reported a higher percentage than reported by us of unsaturated fatty acids of up to 84.77% in the case of bread with the addition of hemp flour of 20% . It shouAccording to the data in The quality of proteins was given by the composition of amino acids and their availability. Due to the promotion by nutritionists of the quality of vegetable proteins, plant fortified products are in great demand. Due to the importance of essential amino acids in the body, many nutritionists recommend the consumption of proteins of vegetable origin. The low content of wheat protein, especially in essential amino acids such as lysine, makes wheat flour protein considered inferior .A total of 8 amino acids were identified in the bread samples, of which 3 are essential  and 5 non-essential , according to In the case of non-essential amino acids, we observed a constant increase for all, directly proportional to the addition of hemp flour. The most significant increase being reported for arginine and glutamic acid.The above results are justified by the high amounts of amino acids present in the flours obtained from the seeds of the hemp varieties Dacia Secuinei and Zenit .Regarding the Pearson correlations for amino acids of bread samples presented in Sucrose and fructose were quantified in the bread samples. Glucose was not found. It was observed according to Decreased concentrations of carbohydrates , fermentable carbohydrates, were constantly changing throughout the technological process, both due to the amylolytic process and the consumption of yeast during the process of fermentation and bread production .Glucose was not detected in the wheat bread or hemp flour fortified samples. Similar results were reported by Carocho et al.  for wholRegarding the Pearson correlations for carbohydrate of of bread samples  semiposiThe amount of yeasts and molds in the bread samples had a decreasing tendency with the addition of hemp flour, regardless of variety . They haThis decrease during storage was due to the low moisture content of the bread samples . This coThe inhibition of yeasts, molds and the development of gram-positive and negative bacteria was also due to the presence of lactic acid in bread or other fermented products ,68.Regarding the Pearson correlations for the microbiological parameters) of bread samples , there wOne of the main selection parameters of the quality of a food product is given by its texture, which is the analysis from different points of view such as sensory, total appearance, shelf life, or nutritional value .According to Through these analyses, the hardness, cohesiveness, viscosity, and chewability of the bread with the addition of hemp flour in different proportions from the Dacia Secuieni and Zenit varieties were evaluated. Firmness was accepted as a measure of the freshness and quality of bread, and hardness is the main mechanical characteristic for the consumer when consuming solid foods, being defined as the force needed to achieve a certain deformation of the finished product .The apparent increase in the hardness of cycle 1, but also of cycle 2 was due either to the reduction of the amount of gluten or to the emulsifying property of hemp flour which helps to improve the hardness parameter, even if the addition of hemp flour reaches 20%. The Zenit 20% hemp flour bread sample was the one that behaves best in terms of hardness, recording the lowest value of 387.A possible explanation for the decrease in hardness as the hemp flour content increase is its oil content, an oil that can improve the texture of the finished product . CohesioThe degree of cohesiveness around 0.67 is due to the addition of water. Both the control sample bread and the PFCDS 10% recorded the same value 0.69 being the highest value recorded. Regarding the viscosity parameter, it can be observed that the highest percentage is presented by P PFCDS 5% (612.67) compared to PFCDS 10% with the lowest percentage (221.67).Chewability is defined as the energy required to perform the chewing process until the beginning of the swallowing process . The samThe influence of the addition of hemp flour in bread on hardness has also been reported by Švec and Hrušková  and KoruCannabis Sativa L.) from which hemp flour (obtained by grinding) was obtained belong to the Dacia Secuieni and Zenit varieties. They were purchased from the Secuieni Agricultural Research–Development Station, Neamț. These two varieties of hemp (Cannabis Sativa L.) are among the 70 varieties accepted in Europe. They were purchased with certificates of analysis stating that these seeds have a delta-9-tetrahydrocannabinol (THC) content <0.2%. The advanced characterization of these varieties is presented by Rusu et al. [All ingredients used to make the bread were purchased from the local market in Cluj Napoca, Romania. The flour usedwas type 550 wheat. The type of flour depends on the ash content, according to the Romanian Standardization Association . Hulled u et al. .The first step was to make flour mixes according to v/v) ethyl alcohol method and the crude fiber content was determined according to ISO 5498: 1981 [Proximate composition  were determined according to Romanian official methods SR 91: 2007 . The nit98: 1981 . The eneDetermination of the content of micro and macroelements by atomic absorption spectrometry were determined according to SR EN 14082:2003 ,80.m/z, the ion source temperature was 210 °C the interface temperature was 255 °C, the solvent was interrupted in 3 min, the scanning speed was 2500 units/s, and the actuation time was 0.2 s. The peaks of the fatty acid methyl esters were determined by comparing the equivalent chain length with the retention time. FAME standards from Supelco Inc., Bellefonte, PA, USA (Supelco 37 component FAME Mix) and other reagents from Merck, Germany were used. A total of 3 determinations were performed.An application used was the GC—MS system (gas—chromatograph coupled with mass spectrophotometer) of a sensitive analytical method to be able to identify fatty acids from composite flour samples. With the help of the BF3-MeOH method, the methyl esterification of the samples used in the analysis was performed, thattakes place after the alkaline hydrolysis process. For a 20 mg oil sample 2 mL 0.5 mol/L methanol solution was added, the mixture being heated for 7 min at 100 °C. After cooling, 3 mL of BF3-MeOH having a concentration of 14% is added, and the vessel is heated for 5 min at 100 °C. After cooling, 7 mL of saturated NaCl solution is added, after which the solution is extracted. The resulting hexane layer (2 mL) was used as a solution for gas chromatography . The FAMDetermination of amino acids using high performance liquid chromatography (HPLC) involves their acid hydrolysis in the presence of 6 M HCL except for sulfur amino acids, identification and chromatographic dosing where the DIONEX ICS—3000 amino acid analyzer is used. Take 0.5 g of the sample and hydrolyze with 10 mL of 6 M HCl at 100 °C for 24 h. The sample is filtered through a 0.2 μm Milipore filter and the sample is diluted in a ratio of 1:10 or 1:1000 with 0.1 N HCl depending on the nature of the sample, finally injected into the chromatograph. Chromatographic conditions: chromatographic column Pre-column AMINOPAC PA10 . Mobile phases: E1: water; E2: 250 mM NaOH and E3: 1 M NaAc, gradient, Mobile phase flow  with CARBOSep 87C cartridge and CARBOSep COREGel 87C cartridge. The mobile phase was represented by ultrapure Milipore water, the flow rate was 0.5 mL/min., For the determination of glucose, sucrose, and fructose, the injector volume was 20 µL. The process of extracting carbohydrates from the samples studied was performed in water. Three g of the sample was weighed and grinded well, then 8 mL of extraction solvent water was added. The mixture was ultrasonified for 15 min and then centrifuged for a period of 20 min at 4500 rpm. The supernatant was filtered through a 0.45 μm filter and injected into HPLC. Carbohydrate amounts were expressed in mg carbohydrate/100 g sample. The following materials were used in the determination of carbohydrates: acetonitrile (HPLC purity), purchased from Merck ; the standards of resveratrol, fructose, sucrose, and glucose were purchased from the United States, from Milwaukee, Aldrich, Milipore ultrapure water (18.2 MΩcm). The equipment used consisted of Jasco HPLC chromatograph (Japan) for analysis, it is equipped with an HPLC pump (Model PU-980), a column thermostat Model CO-2060 Plus, ternary gradient unit Model LG-980-02, UV-VIS detector (Model UV-975), injection valve equipped with a 20 µL test loop (Rheodyne) and detector with refractive index Model RI-2031 Plus. The samples were injected with a Hamilton Rheodyne syringe (50 mL). The HPLC system was controlled, and the experimental data was performed using ChromPass software. For the construction of the calibration curve a standard mixture of carbohydrates with different dilutions (5 concentrations) was used, with a quality between 333 µg/ml and 2000 µg/mL. This HPLC-RI method developed by Filip et al. , was impISO 21527-2:2008 standard method was used . An amouBriefly, 0.1 mL of the diluted sample (10−1 and 10−2) was transferred into a sterile Petri dish covered with Dichloran Glycerol Agar  and spread using a Drigalsky spatula. The plates were incubated for 5 days h at 25°C. Typical colonies of yeasts and molds were counted after 5 days using a colony counter .The main objective of the method was to measure the firmness of sliced bread as a result of its storage (freshness vs. aging). The test was based on the theory that an increase in load and the decrease in compressibility occurs with the storage of bread. This adaptation of the method of compressing the bread samples not only provides valuable information on the storage conditions of the product but is also a valuable indication of the differences in texture caused by the production process and the recipe. A slice of bread 25 mm thick or two slices 12.5 mm thick are placed under a cylindrical probe with a diameter of 38.1 mm. The bread is compressed 3 mm, and the tip of the texture curve is used as an indication of freshness. The sample can also be compressed to a target load. The distance traveled to reach the load is defined bycompressibility (the ability to compress), being a recommendation of freshness. The bread sample is placed centrally, horizontally on the table of the texture analyzer. During the analysis, the treatment performed on the compression load provided data that was continuously recorded on the computer using software.Texture analyzer settings: target distance: 3.0 mm; target standby time: 0 s; minimum detectable load: 4 g; probe speed during test: 1.00 mm/s; probe speed after test: 4.5 mm/s; number of test cycles: 1.0; probe speed before the start of the test: 0.5 mm/s; device used: Brookfield CT3 Texture Analyzer; probe used: cylinder TA4/1000 with the following characteristics: 38.1 mm diameter cylinder probe, clear acrylic 26 g. 20 mm long.Statistical data analysis was performed using statistical interpretation programs R Studio version 3  and StatPearson correlations. The correlation in the broadest sense is defined as a measure of an association between variables. Changing the size of one variable in correlated data is associated with changing the size of another variable, either in the same direction (positive correlation) or in the opposite direction (negative correlation). The term correlation is used in the context of a linear relationship between two continuous variables and expressed as a Pearson product-moment correlation .Given the growing interest in fortified products and at the same time for the use of hemp seeds in human consumption, this study showed that the addition of flour from hemp seeds in bread improves its nutritional properties. It is very important to note that these properties can be influenced by the hemp variety. This bread fortification process has led to a high content of proteins and essential amino acids, lipids and unsaturated fatty acids, fiber, and minerals, not significantly affecting the rheological properties of the finished product. The results of this study showed the significance the use of hemp seed flour in a product used daily. So, when hemp seed flour was used as a source of nutrients in fortified bread making, it added value in diversifying the assortment range and obtaining new nutritional assortments.


Dioscorea alata, L). The addition of 0, 10, 15, 20, and 30% purple yam flour aims to increase the functional value of plain bread as a source of natural antioxidants. The bread produced with two baking temperatures (170°C and 180°C) was tested for anthocyanin levels, total phenol, antioxidant activity , volume expansion, color, and preference test. The results showed that the temperatures of the bread baking did not affect anthocyanin levels, total phenol, antioxidant activity, volume expansion, and bread color (p > 0.05). The substitution of purple yam flour had a significant effect on anthocyanin levels, total phenol, antioxidant activity, volume, and bread color (p < 0.05). The levels of anthocyanins, total phenol, antioxidant activity, and darker color increased with the addition of purple yam flower whereas the rate of expansion reduced. However, the addition of purple yam flour did not affect the level of preference for the bread produced. Purple yam flour can be added to the manufacture of bread made from wheat flour as much as 30% with a baking temperature of 180°C. The resulting bread contained total anthocyanins 54.62 mg/100 g db, total phenol 391.14 mg EAG/100 g db, antioxidant activity 48.53% and volume expansion 64.85%, color L∗ = 53.06, a∗ = 6.52, b∗ = 13.32, C = 14.87, H = 64.06, and sensory score = 3.24 (preferred).In this study, plain bread was made by substituting wheat flour with purple yam flour ( The bakery industry in Indonesia is developing rapidly. This is triggered by an increasing middle-class population, high income of young people, and changing consumption patterns of people who adopt an urban lifestyle. The consumption of plain bread has increased gradually (0.062 packs/cap/year in 2014 to 0.37 packs/cap/year in 2017) .Dioscorea alata L.) and wheat flour with high protein content were obtained from Bantul, Yogyakarta . The chemicals diethyl ether , DPPH , ethanol , methanol , KCl, sodium nitrate, buffer Na-acetic acid, sodium hydroxide. , sodium acetate, HCl, gallic acid , and Folin-Ciocalteu reagent were purchased from Sigma.The purple yam tubers , left to stand for 10 min, and referred to as dough A. Wheat flour, eggs, sugar, full cream milk, bread improver, and yam flour were mixed at various ratios and referred to as dough B. Dough A and dough B were mixed and added with margarine and salt. The dough mixture was carried out by the mixing process in order to obtain a smooth-textured dough and let stand for 40 min. Next, the dough was put into the mould, before baking, let stand for 60 min. Baking was carried out for 25 min with temperature variations of 170°C and 180°C. The bread produced was tested for moisture content, anthocyanin content, total phenolic content, antioxidant activity, color, volume expansion, and sensory evaluation.A = (A520 − A700) pH 1 − (A520 − A700) pH 4.5. The anthocyanin concentration was calculated as cyanidin-3-glycoside using a molar extinction coefficient of 26,900 L cm−1 and a molecular weight of 484.82. Anthocyanin concentration (mg/L) = (A × BM × FP × 1000)/(e × 1), where A is the absorbance, BM is the molecular weight (484.82), FP is the dilution factor (3 mL/0.4 mL), and e is the molar extinction coefficient .Total anthocyanins were determined by the method proposed by Giusti and Wrolstad . A totalμL of sample was added with 250 μL of Folin-Ciocalteu solution, then let stand for 1 min and added 750 μL of 20% NaCO3, then pulverized, and added aquades to a volume of 5 mL. After incubation for 5 min at room temperature, the absorbance was measured at λ 760 nm . Gallic acid was used as a standard, and a calibration curve was made with gallic acid 31.875 to 510 mg/L with r = 0.99. The calculation result of total phenolic content was expressed as mg gallic acid equivalent (EAG) per gram of dry extract.Total phenolic levels were determined by the Folin-Ciocalteu method proposed by Roy et al.  using gaThe antioxidant activity test was carried out by knowing the capacity of DPPH free radical scavenging. Around 0.2 mL of the sample plus 3.8 mL of 0.1 mM DPPH solution was vortex  for 3 min and observed the absorbance at 30 minutes with a spectrophotometer  at a wavelength of 517 . The calMeasurement of bread expansion was carried out using the rape seed displacement method proposed by Matz . In thisThe sensory test was carried out using the Hedonic scale method proposed by Mitiku et al. . The assThe experimental design used was a two-way design with the addition of purple yam flour  and baking temperatures (170°C and 180°C). All data analyses were carried out in duplicate. Data were expressed as mean ± SD. The data obtained were tested statistically by the SPSS method and if significantly different, then continued with the “Duncan new multiple range test” (DMRT) at the 5% degree of confidence.p > 0.05), while the treatment of adding the purple yam flour had an effect on the anthocyanin levels of the fresh bread (p < 0.05). A study of Tamaroh et al. [p < 0.05). The anthocyanin levels of plain bread in this study can be seen in The statistical test results of anthocyanin levels showed that the baking temperature had no effect on anthocyanin levels  was found in plain bread with the addition of 30% purple yam flour at baking temperature of 170°C which was not statistically different from the anthocyanin levels of 54.62 mg/100 g at baking temperature of 180°C. Another latest study by Bartl et al.  showed tp > 0.05), while the addition of purple yam flour had an effect on the phenol content (p < 0.05). Results for the total phenol content of plain bread can be seen in The results of the phenol content in the formulated plain bread showed that the baking temperature treatment had no effect on the phenol content , while the addition of purple yam flour had a significant effect (p < 0.05). The results for the antioxidant activity of plain bread can be seen in The results for antioxidant activity of formulated bread showed that the baking temperature treatment had no effect on the antioxidant activity , but the baking temperature had no effect (p > 0.05). Bread without purple yam flour and the addition of 10% purple yam flour which was baked at 180°C shows the expansion of bread greater than baking at 170°C. This can be explained that at a baking temperature of 180°C, there has been completed gelatinization of the gluten dough, so that the gluten network can hold the gas formed during baking. The temperature of baking bread is in accordance with the finding reported by Miñarro et al. [The results of statistical tests on the expansion volume of plain bread showed that the addition of purple yam flour had a significant effect on expansion  puree in brownie formulations significantly decreased lightness (L∗).The results for the color of the plain bread are shown in ightness . The addi et al. , who madSouripet , they ren et al. , they rea∗ value indicates the chromatic color for red. In this study, the a∗ value did not show a significant difference (p > 0.05). The value of b∗ represents the chromatic blue color, decreased with the addition of purple yam flour indicative of the increasing blueness of plain bread. The increasing blueness of plain bread shows an increase anthocyanins and total phenol. The results of the present study corroborate with the findings of Liu et al. [b∗ values of breads decreased with increasing amounts of purple yam flour in bread. In a study by Azni et al. [b∗ color indicates the presence of anthocyanin and phenol components.The u et al.  which rei et al. , they reC value or the chroma values indicates the color intensity of samples perceived by humans. The higher the chroma values, the higher the color intensity of samples [C value in this study showed a decrease with the addition of purple yam flour in the plain bread, which means that the intensity of the bread produced decreased in color. This can be explained that the bread substituted with purple yam flour shows the presence of anthocyanins and phenol components which cause a decrease in color intensity.The  samples . The C vH), considered the qualitative attribute of color, is the attribute according to which colors have been traditionally defined as reddish or greenish [H value shows a decrease with the addition of purple yam flour in the plain bread. This shows that the addition of purple yam flour will cause the bread to be more purple which indicates it is the presence of anthocyanin and phenol components.Hue angle . There was a significant difference observed in texture acceptance which indicates that the addition of purple yam flour reduced the acceptance at 170°C. However, at baking temperature of 180°C, the acceptance was not significantly different. Bread substituted with purple yam flour and baking temperature did not give any significant difference in taste acceptance (p > 0.05). The overall acceptance value showed that the addition of purple yam flour and baking temperature was not significantly different (p > 0.05). The average value of acceptance of the bread produced was 3.28 which indicates the panelists like the bread. The overall acceptance of plain bread supports acceptable values for color, aroma, texture, and taste.L∗ = 53.06, a∗ = 6.52, b∗ = 13.32, C = 14.87, H = 64.06, and Hedonic score = 3.24 (preferred). Based on the results obtained in this study, substitution of yam flour can be done up to 30%.The results of this study indicate that plain bread made with wheat flour substituted with purple yam flour increased levels of anthocyanins, total phenol, and antioxidant activity whereas decreased the volume expansion rate. Baking temperature generally does not affect several chemical and physical parameters of bread. At the baking temperature of 180°C, there has been completed gelatinization of the gluten dough, so that the gluten network can hold the gas formed during baking. Bread with the addition of 30% purple yam flour, roasting at 180°C, resulted in good bread volume development and high antioxidant activity. The characteristics of bread with 30% yam flour substitution and 180°C baking temperature were found to have total anthocyanin 54.62 mg/100 g, total phenol 391.14 mg EAG/100 g, antioxidant activity 48.53%  and the percentage of volume expansion 64.85%, color


By combining voltage-dependent spectroscopicmeasurements on macroscopic and local scales, we demonstrate a pronouncednonlinear response at the electrode-wall junction, which correlateswith the domain-wall charge state. The dependence on the ac drivevoltage enables reversible switching between uni- and bipolar outputsignals, providing conceptually new opportunities for the applicationof charged walls as functional nanoelements in ac circuitry.The direct current(dc) conductivity and emergent functionalitiesat ferroelectric domain walls are closely linked to the local polarizationcharges. Depending on the charge state, the walls can exhibit unusualdc conduction ranging from insulating to metallic-like, which is leveragedin domain-wall-based memory, multilevel data storage, and synapticdevices. In contrast to the functional dc behaviors at charged walls,their response to alternating currents (ac) remains to be resolved.Here, we reveal ac characteristics at positively and negatively chargedwalls in ErMnO The approach is intriguing as it breaks themold of classical device architectures, taking full advantage of theultrasmall feature size of ferroelectric domain walls. Compared tomore than a decade of research on domain-wall devices that operatebased on the injection and deletion of domain walls,15 little is known about the technological potential of stationarywalls. Only recently, it was shown that ferroelectric domain wallscan be used to emulate the behavior of electronic components at thenanoscale, acting as binary switches12 andhalf-wave rectifiers.13 First insight intothe electronic properties of domain walls under alternating currents(ac) was obtained for neutral domain walls in the gigahertz regime20 and applications as tunable microwave devices and acoustic wavefilters have been suggested.21 In contrast,charged domain walls, which exhibit unusual conduction propertiesunder direct current (dc), have been found to be electronically inactiveat high frequencies in the gigahertz regime.22Ferroelectric domainwalls are excellent candidates for the developmentof next-generation nanoelectronics, exhibiting a thickness that approachesthe unit cell level.3, we observe domain-wallspecific cutoff frequencies, fc, at whichthe current-voltage characteristic of the electrode-wall junctionchanges from asymmetric to symmetric. By varying the ac voltage amplitudeapplied to negatively charged walls, we show that the cutoff frequencycan readily be tuned by about 1 order of magnitude. This tunabilityenables reversible switching between uni- and bipolar output signals,facilitating active signal conversion in ac circuits at the nanoscale.In this Letter, we study the electronic response at positivelyand negatively charged ferroelectric domain walls at intermediatefrequencies in the kilo- and megahertz regime. Performing nanoscalespectroscopic measurements on ErMnO3 is a ferroelectricnarrow band gap semiconductor.25 The spontaneous polarization is parallel to the c-axis (P ≈ 6 μC/cm2)26 and originates from a structural lattice-trimerization,28 leading to explicitly robust ferroelectric domain walls, includingall fundamental types of 180° walls .29 The conduction ofthe neutral walls has been intensively investigated both in the dc31 and ac16 regimes continuously covering frequenciesup to the gigahertz range, and their basic electronic properties arewell understood. In contrast, at charged domain walls only the dctransport behavior32 and the response at high frequencies in the microwave range16 have been studied, whereas their ac propertiesat intermediate frequencies remain to be explored.Hexagonal ErMnO3 single crystal(in-plane polarization) is displayed in the conductive atomic forcemicroscopy (cAFM) map in 29 that is, enhancedconductance (bright) at the tail-to-tail walls and reduced conductance(dark) at the head-to-head walls. In addition, enhanced conductionis observed at nominally neutral domain wall sections, which is consistentwith previous work, where the enhancement was attributed to an accumulationof oxygen interstitials13 and the sub-surfacedomain wall orientation.30 To investigatethe electronic properties of the charged domain walls in the kilo-to megahertz regime, we perform AC-cAFM13 scans at the same position. AC-cAFM is a recent spectroscopy method,that allows for probing the dc response (Idcout) under appliedbipolar voltages (Vacin) as a function of frequency .13Vacin describesthe amplitude of the bipolar voltage. f = 0.5 MHz. In contrast to previous measurements performedunder microwave frequencies,16 a pronouncedresponse to the ac voltage is detected at the charged domain walls,clearly separating them from the surrounding domains. In addition,the scan in f → 0 Hz.The electricaldc transport of a (110)-oriented ErMnOIdcout at charged domain walls asa function of the frequency of the applied ac voltage is presentedin Idcout on a logarithmic frequency scale recorded along the solid line indicatedin f = 0.1MHz, Idcout at the insulating head-to-head domain wall is suppressedin comparison to the domains, whereas an enhancement of Idcout is observedat the tail-to-tail domain wall. With increasing frequency, Idcout reveals a steplike feature indicating a relaxation process  characteristics.Furthermore, forthe conductive tail-to-tail domain wall the cutoff frequency (fc←→ ∼ 4.0 MHz) is about four times higher than for the domains(fcDomain ∼ 1.0 MHz). Consistent with its reduced dc conductance in parallel with a capacitor (with permittivityεbulk). The barrier between tip and sample is describedby a barrier conductivity (σbarrier) connected inparallel with a capacitor (with permittivity εbarrier).36 For f < fc, the transport behavioris dominated by the diode-like tip-sample contact, leading to asymmetriccurrent-voltage characteristics and, hence, a pronounced current signal Idcout in AC-cAFM. The asymmetric current-voltage characteristics originatefrom the contact between the probe tip and the p-type semiconductingErMnO3.31 Because of the different workfunction of the tip and ErMnO3, a Schottky barrier is formedat the tip-sample interface, resulting in rectifying current-voltagebehavior as discussed, for example, by Wu et al. in ref (3. For higher frequencies (f > fc), the Idcout contrast vanishes,indicating that the tip-sample contact gets short-circuited via thebarrier capacitance.37 Within our simpleequivalent circuit model, the cutoff frequency is defined by the bulkconductivity, σbulk.38 Here, it is important to note that the measured widthof the charged domain walls in our local transport measurements isseveral tens of nanometers due to spreading of the tip-injected currentsas discussed in refs ,which accumulate to screen the negative bound charges at these walls.In contrast, hole depletion occurs to screen the positive bound chargesat the head-to-head walls, leading to reduced conductivity relativeto the surrounding domains.To rationalizethe behavior probed at the charged domain walls,we apply the same equivalent circuit model as used in ref , which i. in ref  for the  in refs  and 39)RC elemenquencies 1d, fc←→ urements 1a,29 whefc beyond the simplisticequivalent circuit model40 in fc.To explore the emergence of additionalcontributions to Figure S2 for complementary cAFM and PFM data). Vacin. To avoid possible artifacts caused by repeatedly scanning the samearea,41 the measurements are performedat different positions on selected walls with comparable dc conductance. We observethat fc increases with increasing Vacin, shifting by more than 1 order of magnitude as Vacin is raisedfrom 0.40 to 1.00 V. To systematically analyze the correlation between fc and Vacin, we record frequency-dependentAC-cAFM maps for a wider voltage range from which we calculate fc pixel by pixel as explained in Supporting Information . Vacin. The mean cutoff frequencies obtained for the domains and domainwalls are displayed in The effect of varying drive voltage on the cutofffrequency ispresented in –4 to 2 MHz isshown in Figure S4. The peak in tan δ at f = 5 × 10–2 MHz represents the transition regime between theelectrode-sample interface and the intrinsic bulk properties of ErMnO3.43 Because of the broadness of the peak,44 the electrode-sample interface affects the overalldielectric response even up to much higher frequencies . Analogous tothelocal measurements  compared to the electric fields(E ≈ 0.4 kV/cm) used in the macroscopic measurements.As indicated by the solid lines in Figure S4, the macroscopic dielectric response can be described via fits usingthe equivalent circuit model displayed in the inset of Supporting Information). The analysis shows that σbarrier increases by more than 1 order of magnitude when Vacin is increased from 1 to 20 V, while all other parameters remain almostunchanged.To clarify the originof the additional drive-voltage dependencerevealed by AC-cAFM 2, we perurements 1 and 2, Figure S4),46 analogous to previous macroscopic measurements on CaCu3Ti4O1247 and BiFeO3-based48 materials. Thus, the AC-cAFMdata gained at the charged domain walls expands previous macroscopicstudies on dielectrics to the nanoscale. The voltage dependence of fc  as a function of time,varying the Vacin repeatedly between 0.40 V (orange) and 1.25V (green) while keeping the probe tip stationary at the position ofthe wall. Depending on the applied voltage amplitude, we measure twoqualitatively different responses, switching between asymmetric (Idcout ≠ 0) and symmetric (Idcout = 0). The two-terminal acelement emulated by the electrode-wall junction and the respectiveequivalent circuit model is sketched in the inset in Vacin, whereas an asymmetric responseis detected for high Vacin.The relation between Idcout on both theapplied voltage amplitude and frequency is summarized in Figure S4c and refs (Vacin = 0.4 V and Vacin = 1.25 V (f = 1 MHz) are displayed in The change in voltage allows reversible switching between unipolarand bipolar output. The dependence of and refs  and 48)Idcout on13 In contrast to the neutralwalls, which owe their transport properties to the accumulation and/ordepletion of ionic defects,30 the conduction at chargeddomain walls is driven by bound polarization charges, that is, anintrinsic mechanism. The latter implies that defect migration andeffects from mixed ionic-electric condictivity49 play a less important role compared to neutral domain walls,which is important in order to ensure a reversible and deterministicelectronic response at the electrode-wall junction. Furthermore, thebound polarization charges can be used as quasi-dopants50 to tune the local conductivity and, thereby,engineering the electronic properties of the electrode-wall junctionon demand. Our work introduces charged ferroelectric domain wallsas versatile building blocks for ac nanoelectronics in the kilo- tomegahertz regime, establishing innovative concepts for domain-wallbased nanotechnology and the downscaling of electronic ac componentsin general.The electronic tunabilityof the diode-like properties at the electrode-walljunction represents an additional degree of freedom, enabling thedesign of domain-wall based ac electronic components with ultrasmallfeature size. In particular, the involvement of the domain walls ensuresthat the lateral size is naturally confined with the electronicallyrectifying area defined by the smallest achievable contact. Applicationopportunities range from domain-wall based thyrectors that can bufferripple currents and diodes in transponder circuitry to walls actingas the interconnect between active and passive devices in ac nanoelectronics.In general, the application of charged domain walls in low-frequencynanoelectronics offers several advantages compared to their neutralcounterparts.


Pattern recognition receptors (PRRs) are evolutionarily ancient and crucial components of innate immunity, recognizing danger-associated molecular patterns (DAMPs) and activating host defenses. Basal non-bilaterian animals such as cnidarians must rely solely on innate immunity to defend themselves from pathogens. By investigating cnidarian PRR repertoires we can gain insight into the evolution of innate immunity in these basal animals. Here we utilize the increasing amount of available genomic resources within Cnidaria to survey the PRR repertoires and downstream immune pathway completeness within 15 cnidarian species spanning two major cnidarian clades, Anthozoa and Medusozoa. Overall, we find that anthozoans possess prototypical PRRs, while medusozoans appear to lack these immune proteins. Additionally, anthozoans consistently had higher numbers of PRRs across all four classes relative to medusozoans, a trend largely driven by expansions in NOD-like receptors and C-type lectins. Symbiotic, sessile, and colonial cnidarians also have expanded PRR repertoires relative to their non-symbiotic, mobile, and solitary counterparts. Interestingly, cnidarians seem to lack key components of mammalian innate immune pathways, though similar to PRR numbers, anthozoans possess more complete immune pathways than medusozoans. Together, our data indicate that anthozoans have greater immune specificity than medusozoans, which we hypothesize to be due to life history traits common within Anthozoa. Overall, this investigation reveals important insights into the evolution of innate immune proteins within these basal animals. Animals sense and interact with microbes through their immune system . The majThe four best studied families of PRRs are Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), and C-type lectins (CTLs) , 7. WhilCaenorhabditis elegans or derived in function like the arthropod model Drosophila melanogaster  are transmembrane PRRs that are capable of recognizing a wide variety of ligands including bacterial cell wall components, viral RNA, and developmental cues –23. Canoelopment , 26. Addical TLR .N. vectensis genome (Retinoic acid-inducible gene I-like receptors (RLRs) are cytosolic PRRs that detect intracellular viral RNA –30. Mamms genome .H. vulgaris and two stony coral species have been shown to have large NLR repertoires, containing unique domain combinations that are not seen in mammalian NLRs (Nucleotide-binding oligomerization domain-like receptors (NLRs) are intracellular PRRs capable of recognizing a wide array of DAMPs and MAMPs including reactive oxygen species (ROS) , organelian NLRs , 42, 43.N. vectensis CTLs also found a large repertoire that could not be categorized by the mammalian CTL classification system (C-type lectins (CTLs) are a very diverse protein family that can act as either soluble or transmembrane PRRs , 44. Then system .H. vulgaris and Hydractinia symbiolongicarpus , and two model systems within the medusozoan class Hydrozoa: gicarpus , 47, 48.RR types , 43, 47.Within the past couple of years, a wealth of cnidarian genomic resources has become available, particularly in the medusozoan clade, making it possible to investigate PRRs in a far more diverse set of cnidarian species (nine anthozoans and six medusozoans), with strong potential to provide a more detailed picture of PRR and innate immune evolution –53. TherAcropora millepora   , Hydra vvulgaris , Montipocapitata , Morbakkirulenta , Nematosectensis , Orbicelaveolata , Pocillomicornis , Xenia senia sp. , and sposlandica  were surslandica . A summae genome . The come genome . Transdee genome . Then CDe genome . This ree genome . However-4.9 following the HMMR search were then run through Pfam to predict protein domains using the batch search tool  have expansions in TLRs, specifically TLR-like proteins   were found in C. xamachana and C. hemisphaerica. As with the NLRs, the CTL search yielded surprising domain combinations. Reverse transcriptase domains were found in combination with CTLD in four species, M. virulenta, H. vulgaris, E. daiphana and M. capitata, and integrase and CTLD were found in M. capitata. Additionally, M. capitata and P. damicornis had predicted proteins with the MAC/Perforin domain in addition to CTLD  was the most common domain found in conjunction with CTLD. The concanavalin A-like lectin domain was most commonly found with CTLD in four of the six medusozoans and two of the nine anthozoans. In the remaining medusozoans, N. vectensis, grouping tightly across both PC1 and PC2. The anthozoans excluding N. vectensis group relatively tightly across PC1, driven largely by CTLs and NLRs, but show more variance across PC2. The separation of the anthozoans across PC2 is most likely due to the expansions in prototypical TLRs and TLR-like proteins found in stony corals and the expansion of RIG-I/MDA5-like proteins in A. tenebrosa and E. daiphana , containing the Rel binding domain, Rel dimerization domain, ankyrin repeats, and the death domain  can be found in the article/ME and BD conceived of the project. Data collection and bioinformatic analysis was conducted by ME. ME, BD, and LM wrote the manuscript. All authors contributed to the article and approved the submitted version.Funding was provided by NSF grant no. OCE 1712134 and OCE 1928771 to LM, as well as support from UTA Research Enhancement Program and UTA College of Science for support for ME.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.