The affective Brain-Computer Interface (aBCI) systems, which achieve predictions for individual subjects through training on multiple subjects, often cannot achieve satisfactory results due to the differences in Electroencephalogram (EEG) patterns between subjects. One tried to use Subject-specific classifiers, but there was a lack of sufficient labeled data. To solve this problem, Domain Adaptation (DA) has recently received widespread attention in the field of EEG-based emotion recognition. Domain adaptation (DA) learning aims to solve the problem of inconsistent distributions between training and test datasets and has received extensive attention. Most existing methods use Maximum Mean Discrepancy (MMD) or its variants to minimize the problem of domain distribution inconsistency. However, noisy data in the domain can lead to significant drift in domain means, which can affect the adaptability performance of learning methods based on MMD and its variants to some extent. Therefore, we propose a robust domain adaptation learning method with possibilistic distribution distance measure. Firstly, the traditional MMD criterion is transformed into a novel possibilistic clustering model to weaken the influence of noisy data, thereby constructing a robust possibilistic distribution distance metric (P-DDM) criterion. Then the robust effectiveness of domain distribution alignment is further improved by a fuzzy entropy regularization term. The proposed P-DDM is in theory proved which be an upper bound of the traditional distribution distance measure method MMD criterion under certain conditions. Therefore, minimizing P-DDM can effectively optimize the MMD objective. Secondly, based on the P-DDM criterion, a robust domain adaptation classifier based on P-DDM (C-PDDM) is proposed, which adopts the Laplacian matrix to preserve the geometric consistency of instances in the source domain and target domain for improving the label propagation performance. At the same time, by maximizing the use of source domain discriminative information to minimize domain discrimination error, the generalization performance of the learning model is further improved. Finally, a large number of experiments and analyses on multiple EEG datasets  show that the proposed method has superior or comparable robustness performance  in most cases. To this end, mainstream DA learning methods primarily eliminate distribution biases between different domains by exploring domain-invariant features or samples . In ordeDA theoretical studies have been proposed for domain adaptation generalization error bound  by the fTo address the domain distribution shifting phenomenon, early instance re-weighting methods calculate the probability of each instance belonging to the source or target domain by likelihood ratio estimation . The domain shift problem can be relieved by re-weighting instances based on their membership. MMD  is a widIt is worth noting that existing MMD-based methods did not fully consider the impact of intra-domain noise when measuring domain distribution distance. In real scenarios, noise inherently exists in domains, and intra-domain noise can lead to mean-shift problems in distance measurement for traditional MMD methods and their variants. This phenomenon to some extent is affecting the generalization performance of MMD-based DA methods. As shown in The traditional MMD measurement is transformed into a clustering optimization problem, and a robust possibilistic distribution distance metric criterion (P-DDM) is proposed to solve the domain mean-shift problem in a noisy environment;It is theoretically proven that under certain conditions, P-DDM is an upper bound of the traditional MMD measurement. The minimization of MMD in domain distribution measurement can be effectively achieved by optimizing the P-DDM;A DA classifier mode based on P-DDM is proposed , its consistent convergence is proven, and the DA generalization error bound of the method is proposed based on Rademacher complexity theory;A large number of experiments are conducted on two EEG datasets , demonstrating the robust effectiveness of the method and a certain degree of improvement in the classification accuracy of the model.Existing research  pointed 2.th class, The other elements m data points. However, the distributions of source domain  is the domain adaptation learning loss function that includes the label matrix Y  and the comprehensive learning model W of the source domain and the target domain.where 2.1.2.1.1.In a certain reproducing kernel Hilbert space (RKHS)ϕ:ℝd→H . The cor methods . For the methods  that whe methods . That isf, making the two domain distributions as similar as possible. When the sample size of the domain is sufficiently large (or approaches infinity), the expected difference approximates  the empirical mean difference. Therefore, MMD measure minimizes the expected difference between two domain distributions through the function Theorem 1. The MMD measure can be loosely modeled as a special clustering problem with one cluster center, where the clustering center is To prove the universal connection between the traditional MMD criterion and the mean clustering model, we give the following theorem: Proof: As defined by MMD:From As indicated in 2.1.2.Recently proposed possibility clustering models can effectively overcome the impact of noise on clustering performance . Therefob is the weight exponent of b is set to 2 in the subsequent equations of this paper. The detailed process of different values of b can be found in references  Calculate the difference in distribution between kernel space domains based on the possibility clustering assumption, by measuring the distance between each instance in the domain and the overall domain mean; (2) Measure the matching contribution of each instance. Any instance in the overall domain has a matching contribution value ferences . Ωp, the second inequality in According to the value range of c-th class in the source domain, c-th class in the target domain, and c-th class mean values of the source domain and the target domain, respectively. c-th class in the overall domain .To further improve the robustness and effectiveness of the possibilistic distribution distance metric method on noisy data, we add a fuzzy entropy regularization term related in 2.2.The P-DDM criterion addresses the problems of domain distribution alignment and noise impact. Next, we will achieve the two goals required for the inference of target domain knowledge: (1) to preserve the geometric consistency in the source domain and the target domain, i.e., the label information between adjacent samples should be consistent, and (2) to minimize the structural risk loss of both the source and target domains. Given the description of the objective task, the general form of the objective risk function can be described as:2.2.1.Firstly, In combination with source domain knowledge transfer and graph Laplacian matrix , the objh matrix  with D2.2.2.In our proposed method, the classifier of the source domain (the corresponding target domain classification model) is defined as According to the minimum square loss function, the problem of minimizing structural risk loss in both domains (source domain and target domain) can be described as:W. By using where the first term denotes the structure risk loss and Y and the decision function W to guarantee the reliability of the prediction. The target classification function is combined by The classification task proposed in this method is ensured by the dual prediction of the label matrix 2.3.By combining the semantic alignment P-DDM form i.e.,  and the With all model parameters obtained, target domain knowledge inference can be achieved by maximizing the utilization of source domain discriminative information, linearly fusing the two classifiers 3.The optimization problem of C-PDDM is a non-convex problem with respect to 3.1.Theorem 3. The optimal solution to the primal optimization problem of the objective function As we fix W and Y, the objective function in Proof. By setting the derivative Combining and simplifying the terms in 3.2.c-th class.Theorem 4.The optimal solution to the primal optimization problem of the objective function Since the first and the third terms in Proof. According to Equation (19), let i-th vector of 3.3.Finally, The optimization problem 4.4.1.z-th iteration. In unsupervised domain adaptation learning scenarios , in order to achieve semantic alignment between domains, initial labels of the target domain can be obtained through three strategies : (1) rank-Nearest Neighbor  graph and compute the kernel matrix This article uses Big Before training in Algorithm 1, pre-computing the C-PDDM kernel matrix and Laplacian graph matrix and loading them into memory can further improve the computational efficiency of Algorithm 1. In short, the proposed algorithm is feasible and effective in practical applications.5.5.1.To prove the convergence of Algorithm 1, the following lemma is proposed.Lemma 1  in each iteration and converges to the optimal solution.Then, we prove the convergence of the proposed algorithm through Proof. For expression simply, the updated results of optimization variables According to the definition of matrix Based on Lemma 1, we can obtain the following inequality:Therefore, we can derive:Finally, Theorem 6 is proved.According to the update rule in Algorithm 1 and Theorem 6, it is known that the optimization objective 5.2.Rademacher complexity can effectively measure the ability of a function set to fit noise . Therefoneighborhood distribution The domain distribution difference between the source domain distribution Let Their corresponding expected loss is denoted as The following theorem gives the generalization error bound of the proposed method:Theorem 6  (r Bound) . Let H:Theorem 6 shows that the possibilistic distribution distance measure 5.3.The literature Theorem 7   to overc6.6.1.In order to make a fair comparison with stat-of-the-art (SOTA) methods, a large number of experiments were conducted for effective validation on two well-known open datasets , the corresponding differential entropy (DE) is extracted to represent the logarithmic power spectrum in the specified frequency band-pass, and a total of 310 features (5 frequency bands and 62 channels) are obtained in each EEG segment. Then, all features are smoothed by the Linear Dynamic System (LDS) method, which can utilize the time dependency of emotion transitions and filter out the noise EEG components unrelated to emotions .6.1.1.k neighbors to compute the Laplacian matrix. In the experiment of this paper, it can be observed that the performance slightly varies when k is not large. Therefore, to construct the nearest neighbor graph in C-PDDM, this paper conducts a grid search for the optimal number of nearest k neighbors in The settings of the hyper-parameter for the C-PDDM method are also crucial before analyzing the experimental evaluation results. For all methods, in both the source and target domains, a Gaussian kernel Before presenting the detailed evaluation, it is necessary to explain how the hyper-parameters of C-PDDM are tuned. Based on experience, the parameter Considering that parameter uncertainty is still an open problem in the field of machine learning, we determine these parameters based on previous work experience. Therefore, we evaluate all methods on the dataset by empirically searching the parameter space to obtain the optimal parameter settings and give the best results for each method. Except for special cases, all parameters of all relevant methods are tuned to obtain the optimal results.As unsupervised domain adaptation does not have target labels to guide standard cross-validation, we perform leave-one-subject-out on the two datasets: SEED and SEED-IV (the details of this protocol are shown in Section 6.2). We obtain the optimal parameter values on {6.2.Cross-subject cross-session leave-one-subject-out cross-validation. To fully estimate the robustness of the model on unknown subjects and trials, this paper uses a strict leave-one-out method cross-subject cross-session to evaluate the model. All session data of one subject is used as the target domain, and all sessions of the remaining subjects are used as the source domain. We repeat the training and validation until all sessions of each subject have been used as the target domain once. Due to the differences between subjects and sessions, this evaluation protocol poses a significant challenge to the effectiveness of models in emotion recognition tasks based on EEG.Cross-subject single-session leave-one-subject-out cross-validation. This is the most widely used validation scheme in emotion recognition tasks based on EEG   to compa6.3.6.3.1.For verifying the efficiency and stability of the model under cross-subject and cross-session conditions, we used cross-subject cross-session leave-one-subject-out cross-validation on the SEED and SEED-IV databases to validate the proposed C-PDDM. As shown in 6.3.2.6.3.3.By calculating the mean and standard deviation of the experimental results for each subject, the cross-session cross-validation results for each subject on the different datasets SEED and SEED-IV are shown in 6.3.4.The previous evaluation strategy only considered the first two sessions of the SEED dataset as the source domain for the experiment. The evaluation results of emotion recognition for each subject within each session are presented in For the SEED-IV dataset, we calculated the performance of all three sessions . Our proposed model outperforms the existing latest classical research methods and achieves the highest accuracy of 71.85 and 83.94% in 6.4.For comprehensively study the performance of the model, we evaluated the effects of different settings in C-PDDM. Please note that all the results presented in this section are based on the SEED dataset, using the cross-subject single-session cross-validation evaluation protocol.6.4.1.We conducted ablation studies to systematically explore the effectiveness of different components in the proposed C-PDDM model and their respective contributions to the overall performance of the model. As shown in For the pseudo-labeling method, when the pseudo-labeling method changes from fixed to linear dynamic, the corresponding accuracy increases from 89.95 to 92.19%. When adopting multi-kernel learning, the accuracy further improves to 93.68%. The results indicate that multi-kernel learning helps rationalize the importance of each kernel in different scenarios and enhances the generalization of the model.Next, we analyze the impact of different hyper-parameters on the overall performance of the model. According to the experimental results, it can be seen that the recognition accuracy with 6.4.2.In order to further verify the robustness of the model in the noisy label learning process, we randomly add noise to the source labels at different ratios and test the performance of the corresponding model on unknown target data. Specifically, we replace the corresponding proportion of real labels in 6.4.3.In order to qualitatively study the performance of the model in each emotion category, we analyze the confusion matrix through visualization and compare the results with the latest models . As shown in 6.4.4.The proposed C-PDDM adopts an iterative optimization strategy and uses experiments to prove its convergence. The experiment is completed on the MATLAB platform, and the device configuration used is as follows: 64 GB memory, 2.5 GHz CPU, and 8-core Intel i7-11850H processor. 7.This paper proposes a novel transfer learning framework based on a Clustering-based Probability Distribution Distance Metric (C-PDDM) hypothesis, which uses a probability distribution distance metric criterion and fuzzy entropy technology for EEG data distribution alignment, and introduces the Laplace matrix to preserve the local structural information of source and target domain data. We evaluate the proposed C-PDDM model on two famous emotion databases (SEED and SEED-IV) and compare it with existing state-of-the-art methods under four cross-validation protocols . Our extensive experimental results show that C-PDDM achieves the best results in most of the four cross-validation protocols, demonstrating the advantages of C-PDDM in dealing with individual differences and noisy label issues in aBCI systems.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Manihot esculenta Crantz) is a vital crop for food and economic security in many regions of the world. Despite the economic and social importance of cassava, challenges persist in developing superior varieties that meet the needs of farmers in terms of agronomic performance, nutritional quality, and resistance to pests and diseases. One of the main obstacles for genetic improvement is the lack of synchronization in flowering and the abortion of young flowers, making planned crosses and progeny production difficult. Therefore, the aim of this study was to evaluate the effect of photoperiod, premature pruning, and growth regulators on cassava flowering under low-altitude conditions in Brazil. Eight cassava clones with contrasting flowering capacity were assessed in Cruz das Almas, Bahia, using two photoperiods (ambient condition and extended photoperiod with red light for 12 hours), premature pruning at the first and second branching levels (with and without pruning), and the application of growth regulators: 0.5 mM 6-benzyladenine (BA) and 4.0 mM silver thiosulfate (STS) (with and without). Plots were assessed weekly for the number of female (NFF) and male (NMF) flowers, height of the first branching , number of days to the first branching (ND1B), and the number of branching events up to 240 days after planting (NOB). The extended photoperiod did not promote an increase in the number of flowers but allowed for precocity in cassava flowering, reducing the onset of flowering by up to 35 days, and significantly increasing the number of branches, which is closely related to flowering. The use of pruning and plant growth regulators (PGR) resulted in an increase in NFF from 2.2 (control) to 4.6 and NMF from 8.1 to 21.1 flowers. Therefore, under hot and humid tropical conditions at low altitudes in the Recôncavo of Bahia, manipulating the photoperiod and using premature pruning and plant growth regulators can accelerate cassava flowering, benefiting genetic improvement programs.Cassava ( Manihot esculenta Crantz) is an important source of carbohydrates in the diet of about 800 million people  al al−2 s−1 numbers .Vigna subterranea, as well as a reduction in the number of flowers produced per plant, in response to different photoperiod extension treatments. Furthermore, [While there are reports of successful photoperiod extension inducing flowering in cassava, some studies have also reported the inhibition of flowering when using photoperiod extension strategies.  reportedhermore,  reportedThe low effect of photoperiod extension on male and female flower production observed in the present study may be attributed to the soil water control throughout the experiment, especially during dry periods when no supplemental irrigation was performed. Extended photoperiod increased flowering and fruiting in cassava when soil moisture was maintained through supplemental irrigation . In our In a comparative study conducted in two cities in Nigeria (Ubiaja and Ibadan), it was observed that the rate of flowering in cassava plants increased during periods of higher rainfall . These fPruning had a positive effect on flower production in the first two branching tiers of the plant. Early pruning is known to induce flowering by inhibiting the unrestrained growth of vegetative branches, which in turn can inhibit the growth of inflorescences and lead to their abortion. Furthermore, pruning has been found to increase the expression levels of homologs of IAA16 (a repressor of auxin responses) and TIFY10b (a transcription factor for jasmonic acid regulator) proteins, indicating an interaction between hormone signaling genes and pruning .Pruning in the 2nBT resulted in a higher NFF and NFM. Furthermore, early pruning of the genotype BR17-006-62, which is characterized by low production of flowers, resulted in a 23% increase in the number of male flowers. Similar results have been reported in other studies, indicating that pruning can increase flower production in late-flowering genotypes with low seed production . In pomeLaelia anceps, the use of BA and GA3 significantly reduced the number of days to the beginning of flowering and increased the number of stems and flowers [The results showed that pruning alone did not significantly increase the number of female flowers, but the combination of pruning with PGR resulted in a significant increase from 2.24 (control) to 5.19 female flowers. The flowering process is complex and involves various endogenous and exogenous factors that stimulate changes from vegetative to reproductive state in the apical meristem , 48. Hor flowers . In cass flowers . Additio flowers .The number of flowers in cassava plants tends to increase with the number of branches. A study conducted by  revealedAlthough we did not observe a direct effect of photoperiod extension on cassava’s flowering and fruit set, our study found that extending the photoperiod increased flowering precocity, resulting in a 35-day reduction in the onset of flowering. Additionally, the use of PGR significantly increased the number of female flowers. Future studies investigating the molecular mechanisms responsible for the influence of BA on flower feminization and STS on the overall increase in flower number in cassava could help identify genes involved in this process. A recent study by  found thCombining treatments such as ‘Pruning + PGR’ with extended photoperiods can offer a viable alternative for reducing the initiation of flowering and increasing flower production in cassava. However, it will be necessary to validate our results in blocks of crosses with a greater number of elite genotypes from the breeding program, which generally have varied behavior regarding the initiation of flowering and number of flowers produced.The synchronization of flowering in crossing blocks has been a significant challenge in improving cassava species, particularly when aiming to obtain progenies with wide recombination between specific chromosomal segments or breaking undesirable genetic links between certain traits. Therefore, increasing our understanding of the environmental and endogenous factors that regulate flowering time in cassava and their underlying molecular mechanisms is crucial. This understanding can help accelerate and enable the synchronization and maximization of flowering among cassava genotypes, thus improving the efficiency and effectiveness of cassava breeding programs.Our study demonstrates the potential of innovative approaches such as extending the photoperiod, premature pruning of branches, and using growth regulators to promote flowering induction in cassava. the extended photoperiod not only increases earlier flowering, but also enhances branch production. In contrast, pruning and growth regulators significantly increase the number of flowers produced. Moreover, growth regulators induce feminization, leading to an increase in the number of female flowers. These results offer valuable insights into optimizing the production of flowers required for crosses, allowing for an increase in the production of individuals per progeny.S1 Fig(TIF)Click here for additional data file.S2 Figst branching tier (1stBT); T3: pruning in the 2nd branching tier (2nBT); T4: ‘Pruning + PGR’ (plant growth regulator) in the 1stBT; and T5: ‘Pruning + PGR’ in the 2ndBT.The abbreviations mean the respective treatments, T1: no pruning; T2: pruning in the 1(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


Vitis vinifera L.) is widely cultivated as an important fruit crop destinated for wines, table grapes, and raisins. However, grapevines are plagued by a variety of diseases each year, depending on the causal agents, cultivar susceptibility, and climate location, and these diseases are limiting factors in commercial vineyards and nurseries  and Basidiomycetes fungi species into genera Arambarria, Fomitiporia, Fomitiporella, Inocutis and Inonotus  by 15 authors.The present Research Topic was launched in late 2021 in the section ‘Fungi-Plant Interactions’ of Frontiers in Fungal Biology, including four original research  and California (table grapes) was studied by Phaeomoniella chlamydospora is one of the most prominent trunk pathogens associated with Petri and Esca-like diseases (P. chlamydospora in pruning wounds is fundamental to developing an effective management strategy for Petri and Esca diseases. P. chlamydospora is one of the main GTDs pathogens in Chile. Nevertheless, the duration of the susceptibility of grapevine pruning wounds for this pathogen is still unknown.diseases . SeveralDíaz and Latorre in their article ‘Duration of the susceptibility of pruning wounds of different ages to infections by Phaeomoniella chlamydospora on grapevine cv. Cabernet Sauvignon in Central Chile’ evaluated the period of susceptibility of pruning wounds artificially inoculated by P. chlamydospora in different periods ranging from 1 to 45 days. They evaluated rooted cuttings and spurs from two consecutive seasons in two Cabernet Sauvignon vineyards in Central Chile. Their results showed that pruning wounds remained susceptible to P. chlamydospora for up to 45 days after pruning. These data agreed with those previously reported from other vineyards worldwide. The results highlight that a single fungicide application could not be sufficient to avoid the infection, and further studies evaluating the proper number of applications of fungicides and biocontrol agents are needed.In this framework, All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


The effect of pulsed electric field (PEF) treatment depends mainly on the electric field strength and treatment time. In this study, wheat flour–water suspensions were treated with PEF at an electric field strength of 3 kV/cm for 0 to 1400 pulses to obtain a specific energy input of 0 to 656 kJ/kg. The effect of PEF on the removal or unfolding of proteins from the starch surface, digestibility, starch granule structure, and physicochemical properties of wheat flour was studied. The removal of proteins from the surface and the damage to the internal structure of wheat starch granules after PEF treatment was detected by confocal laser scanning microscopy (CLSM) and FTIR. The damage of the PEF-treated wheat starch granules was observed by scanning electron microscopy (SEM). From CLSM results, penetration of dextran  into starch granules of wheat flour was dependent on the energy input of PEF. The high the energy input showed the intense penetration of the biopolymer. The benefits of the accessibility of biopolymer in starch granules are to increase enzyme digestion, especially rapidly digestible starch (RDS). The RDS of wheat flour treated with PEF at 656 kJ/kg was 41.72%, whereas the RDS of wheat flour control was 27.59%. Pulsed electric field (PEF) is an electroporation method that can create pores in the microbial cell membrane that allow the introduction of DNA fragments, chemicals, or drugs into the cell. A high-intensity pulsed electric field (10–80 kV/cm) is a practical non-thermal sterilization method due to its short time requirement and allows homogeneous treatment. Currently, customers are interested in minimizing food processing or using non-thermal methods that can preserve the freshness and flavor of products [Escherichia coli, Streptococcus faecalis, Bacillus subtilis, Streptococcus cremoris, and Micrococcus radiodurans [Generally, a PEF processing system consists of a high-voltage generator, an energy storage capacitor bank, a charging resistor, a discharging switch, a pulse controller, a treatment chamber (static or continuous), a raw material container, a finished product container, and control devices. The electrical energy from the power supply is collected in the capacitor and then discharged through the treatment chamber to create an electric field strength in the food product. Non-thermal processing at temperatures below 30–40 °C can avoid denaturation of foods containing proteins and vitamins. PEF technology can be applied to liquid, semi-solid, and solid foods, e.g., milk, orange juice, tomato paste, and fruit. PEF technology can extend the shelf life of food by breaking the cell membranes of foodborne pathogens and spoilage microorganisms, such as iodurans . Sale anThe principle of PEF treatment of microbial cell cultures and starch granules is the same. PEF can be used for electroporation by piercing the cell membrane, regardless of the size or shape of the cell . HoweverPEF treatment with electric field strengths of 0.5 to 1 kV/cm and a treatment duration of 102–104 s enabled effective disintegration of cell membranes in beet and potato samples ,13. NatiTriticum aestivum L.) flour is a versatile ingredient and is widely used in the production of homemade and almost all commercial baked goods  and pasta products . It is unique for the presence of proteins that form a gluten network and give the wheat flour dough viscoelastic properties that allow the expansion of the dough and the storage of gas. However, the constant innovation in food products and their higher quality requirements have forced the food industry to search for flours with new specific functions and properties. Chemical, enzymatic, and/or physical modifications have been the alternatives to change the functionalities and properties of wheat flour [Wheat  flour with a protein content of 10.50–11.00% was obtained from United Flour Mill Public Company Ltd. . Wheat gluten was purchased from Verasu company, Bangkok, Thailand. Ethanol was purchased from Sigma-Aldrich  Company Ltd. The ethanol used in this experiment was of analytical grade. Distilled water was used to prepare the starch dispersion.Commercial refined all-purpose wheat (The PEF system consists of an AC 220 V supply line into 3 kV using a high-voltage transformer and then rectifies the AC high voltage into DC high voltage. A pulse controller delivers monopolar pulses of nearly rectangular shape with a constant pulse width of 10 μs and a pulse frequency of 1 Hz. The experiments were performed at ambient temperature. The treatment chamber was rectangular in shape (W × L × H: 4 × 40 × 32 cm) and consisted of two essentially parallel stainless steel plates connected on each side with electrical wires and sealed at the bottom with Teflon as an insulating spacer. The parallel plate treatment chamber with a gap of 40 mm was used in this study because the parallel plate electrodes provide uniform electric field distribution along the gap axes and electrode surfaces.® Tube Mill 100 control, IKA-Werke GmbH & Co. KG, Staufen im Breisgau, Germany) at a speed of 10,000 rpm for 1 min and sieved with a 200 mesh sieve.Wheat flour dispersion (10%) was placed in the batch treatment chamber at 15 °C and treated with PEF at 3 kV/cm for 0–1400 pulses, corresponding to a total specific energy input of 0 to 656 kJ/kg wheat flour. The PEF treatment was adjusted to a wide range from 0 to 656 kJ/kg to investigate the effects of specific energy input on the physical properties of wheat flour at a low electric field strength of 3 kV/cm. The sample temperature was measured after the treatment. The sample temperature did not exceed 35 °C after each treatment. The wheat flour dispersion was cooled in a water ice bath immediately after PEF treatment. The wheat flour was separated from the water by centrifugation, washed with 50% ethanol, and air dried on a tray. The dried wheat flour was ground into powder with a mill  with an accelerating voltage of 5 kV, a working distance of 10 mm, an aperture of 3, and a probe current of 3 × 10Confocal laser scanning microscopy (CLSM) was carried out to visualize the microscopic distributions of protein and starch in the flour samples before and after PEF treatment using the method described by  with sliw/w). Wheat flour was weighed  in an aluminum RVA sample canister, and distilled water was added to obtain a total weight of 25 g (6% flour concentration). The standard 1 profile of the heating and cooling program was used in this study. Samples were held at 50 °C for 1 min, heated from 50 °C to 95 °C for 3 min 42 s, held at 95 °C for 2 min 30 s before decreasing to 50 °C for 3 min 48 s, and held at 50 °C for 2 min. The pasting temperature, peak, trough, breakdown, final, and setback viscosities were determined for wheat flour samples treated with PEF at specific energy inputs ranging from 0 to 656 kJ/kg. Thermocline for Windows (TCW3) software was used for operation and data analysis.A rapid visco analyzer (RVA), model RVA-TecMaster , was used to determine the pasting properties of wheat flour before and after PEF treatment. The moisture content of wheat flour samples was determined using a moisture analyzer . The moisture content of the wheat flour samples was 12–15% . Wheat flour  concentration in distilled water) was hermetically sealed in a 40 μL aluminum pan. Samples were heated from 25 to 100 °C at a heating rate of 10 °C/min. An empty aluminum pan was used as a reference. The onset (To), peak (Tp), and conclusion (Tc) gelatinization temperatures were recorded. The gelatinization enthalpy (ΔH) was analyzed based on the moisture-free and protein-free basis of the wheat flour according to [The thermal properties of the native and PEF-treated wheat flours were analyzed using a DSC1 StarX-ray diffraction analysis was performed using an X-ray diffractometer  equipped with a conventional copper target X-ray tube operating at 30 kV and 30 mA. The wheat flour samples treated with PEF at specific energy inputs of 0–656 kJ/kg were placed on the slides for X-ray scanning. Data were collected from 2θ of 5° to 40°. Crystallinity (%) was defined as the percentage ratio of the diffraction peak area to the total diffraction area. Native starch granules have a crystallinity between 15 and 45%. −1 was scanned at a resolution of 4 cm−1 to obtain the spectra. PerkinElmer FT-IR Spectrum 10 spectroscopy software was used to analyze the Fourier infrared spectrum.Fourier-transform infrared (FTIR) measurements of wheat gluten, the wheat flour control sample, and wheat flour treated with PEF at 656 kJ/kg specific energy inputs were carried out by using a Spectrum One FT-IR spectrometer  equipped with a MCT detector. The samples and chromatographic pure KBr  were mixed in a ratio of 1:100, ground, and pressed into 20 mg thin slices. The range of 0–2500 cmThe starch particle size distribution was detected using a Mastersizer 2000 instrument . The samples were diluted with deionized water and stirred at 2000 rpm. The refractive index of the starch samples was 1.53.v/v, 10 mL) to terminate the enzymatic reaction and then centrifuged at 1500× g for 10 min. The supernatant was used to analyze the amount of glucose released using the GOPOD assay kit . The percentage of starch hydrolysis at different hydrolysis times and starch fractions  were calculated as follows:ht is the glucose content released in each hydrolysis time; G20 and G120 are the glucose content released at 20 and 120 min of hydrolysis, respectively; and TS is total starch content.The digestibility of starch in the flour samples studied was determined in uncooked states according to the procedures of Englyst et al.  with slip ≤ 0.05).All sample analyses were performed in triplicate, and statistical analysis was performed using SPSS v. 22 . Duncan’s multiple range test was used to estimate significant differences among means at a probability level of 5%  and gluten protein (red), respectively. The microscopic distribution of gluten protein and starch granules in the flour samples before and after PEF treatment is shown in The native wheat flour and the wheat flour treated with PEF at specific energy inputs of 188 and 656 kJ/kg were stained with FITC-dextran  to indirectly observe the destruction of the crystallinity of the starch structure after PEF treatment by CLSM. Our results showed that FITC-dextran barely penetrated native wheat starch granules A. MoreovIn addition, wheat starch granules have surface pores and channels that extend into the interior of the granules . Removalp ≤ 0.05) with increasing specific energy input. This could be due to the damage of the surface structure of the wheat starch granules, as shown in the SEM images (p ≤ 0.05) with increasing specific energy input, indicating an improvement in the cold paste stability of the PEF-modified wheat flour. The setback viscosity reflects the retrogradation tendency of amylose in the starch paste and is related to the texture of various food products. Therefore, PEF treatment is considered desirable for improving product quality since retrogradation usually causes quality deterioration characterized by syneresis or increased hardness [p ≤ 0.05) at 469 kJ/kg, remaining almost constant until 656 kJ/kg. At an electric field strength of 3 kV/cm, Qiu et al. [The pasting properties of the native and the various PEF-treated wheat flours are shown in M images  [6,20,21hardness ,21. Compu et al. , who fouTo), peak (Tp), and conclusion (Tc) gelatinization temperatures; the gelatinization temperature range (ΔT = Tc − To); and the gelatinization enthalpy (ΔH) of the native and the different PEF-treated wheat flours calculated from the DSC measurements are listed in To, Tp, and ΔH obtained from DSC analysis of 30% native wheat flour suspension were 57.0 °C, 62.3 °C, and 5.3 kJ/kg, respectively, which agrees well with our results. In addition, the presence of gluten protein in native wheat flour was reported to decrease gelatinization enthalpy and increase gelatinization temperatures of wheat starch. This could be due to the fact that the gluten protein and wheat starch compete for water, resulting in less water available for starch gelatinization [H of the different types of wheat flour ranged from 8.56 to 9.51 kJ/kg, whereas the ΔH of the control native wheat flour in this study was 4.24 kJ/kg. In addition, Tiga et al. [To, Tp, and ΔH of wheat flour were 57.07 °C, 64.12 °C, and 0.93 kJ/kg, respectively.The onset  decreased with increasing specific energy input, while To remained almost unchanged. The gelatinization temperatures and enthalpy could be related to the degree of crystallinity and the amylopectin structure of the starch granules [H values [To, Tp, and ΔH values of maize starch with increasing electric field strength and treatment duration, with electric field strength playing a dominant role in PEF treatments. Qiu et al. [To and ΔH values of glutinous rice flour when the number of PEF pulses was increased from 50 to 300 pulses at a field strength of 3 kV/cm. The gelatinization temperature range (ΔT) of wheat flour also decreased significantly (p ≤ 0.05) with increasing specific energy input. This is usually associated with increased crystallite homogeneity of starch granules. PEF treatment may have facilitated the fusion of weak crystallites, lowering the energy required for melting and ultimately increasing the interactions between the remaining crystallites [In this study, granules . Han et granules  reportedgranules . From thH values ,18. Han H values  also repu et al.  reportedThe X-ray diffraction (XRD) patterns of native and various PEF-treated wheat flours are shown in Abduh et al.  studied −1 and 1047 cm−1 of FTIR spectra were related to the amount of crystalline, while the peak at 1022 cm−1 was related to the content of amorphous structures. The decrease of 1047/1022 and 995/1022 showed the decrease in the short-range molecular structure and the decrease in the double helix in the starch after PEF treatment, especially at high electric field strength [−1 was related to the formation of starch–protein complex by forming the C-N amide II band [−1. However, the shoulder at 1538 cm−1 of wheat flour treated with PEF at 656 kJ/kg showed the lower intensity compared with the wheat flour control and gluten. That showed the decrease in the starch–protein interaction after PEF treatment. FTIT results of overlay spectra (−1) region of FTIR. The wheat flour control sample and wheat flour treated with PEF at 656 kJ/kg showed the decrease in the gluten band at peak 1642 cm−1 after PEF treatment. Qiu et al. [−1 were reported to be related to protein and lipid components in the rice flour . The peGranule disruption of wheat starch granules was revealed in The starch hydrolysis rate of uncooked native and PEF-treated wheat flours is presented in The digestibility, particle size distribution, and physicochemical properties of wheat flour were found to be affected by PEF treatment at various specific energy inputs ranging from 0 to 656 kJ/kg. Most of the protein was removed or unfolded from the surface of the wheat starch granules, and the external and internal structures of the starch granules were damaged, with the extent of damage increasing with increasing specific energy input. The removal of protein in PEF-treated wheat flours facilitated the enzyme penetration and accelerated the starch hydrolysis. This resulted in a significant decrease in the overall viscosity and particle size of starch granules in wheat flour when the specific energy input was increased. The relative crystallinity of wheat flour decreased after PEF treatment, resulting in a significant decrease in gelatinization temperatures and enthalpy with increasing specific energy input. The use of PEF-treated wheat flour in various food products needs to be investigated for food application.


Enterococcus faecalis, which has been shown to play major roles in conjugation, biofilm formation, and importantly also in bacterial virulence. Since prgB orthologs exist on a large number of conjugative plasmids in various different species, this makes PrgB a model protein for this widespread virulence factor. After characterizing the polymer adhesin domain of PrgB previously, we here report the structure for almost the entire remainder of PrgB, which reveals that PrgB contains four immunoglobulin (Ig)-like domains. Based on this new insight, we re-evaluate previously studied variants and present new in vivo data where specific domains or conserved residues have been removed. For the first time, we can show a decoupling of cellular aggregation from biofilm formation and conjugation in prgB mutant phenotypes. Based on the presented data, we propose a new functional model to explain how PrgB mediates its different functions. We hypothesize that the Ig-like domains act as a rigid stalk that presents the polymer adhesin domain at the right distance from the cell wall.Type 4 Secretion Systems are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. In Gram-positives, these secretion systems often rely on surface adhesins to enhance cellular aggregation and mating-pair formation. One of the best studied adhesins is PrgB from the conjugative plasmid pCF10 of  Enterococcus faecalis is one of the leading causes of hospital acquired infections, such as urinary tract infections and endocarditis  motifs . HoweverE. coli, but instead produced and purified PrgB188–1233. This version of the protein only lacks the disordered N-terminal region and the LPXTG anchor and elutes from size exclusion chromatography in two peaks corresponding to dimeric and a monomeric PrgB. PrgB is in a monomer–dimer equilibrium and the dimer has been described as the main biologically functional unit in vivo  as a search model. Surprisingly, the resulting electron density lacked the previously crystallized polymer adhesin domain of PrgB, and instead only contained residues 584–1233. There is also no space in the crystal packing to allow for a flexible polymer adhesin domain, so it was likely cleaved off in the crystallization drop before the crystals were formed. The modeled protein indeed consists of four Ig-like domains: two CSA and two CSC domains  domain, which mediates adherence in these proteins. This BAR domain is absent in PrgB , were collected. This yielded relatively low-resolution volumes . See 584–1233) and the previously solved polymer adhesin domain (PDB code: 6EVU) (584–1233) in vitro. However, neither size-exclusion chromatography nor native polyacrylamide gel electrophoresis (PAGE) indicated any binding of the polymer adhesin domain to the stalk domain in vitro   into thee: 6EVU) . Therefoin vitro .E. faecalis OG1RF pCF10ΔprgB with different prgB mutants (from a nisin-inducible plasmid) and characterizing their phenotypes in cellular aggregation, biofilm formation, and plasmid transfer efficiency. In line with previous experiments  were also found to have a decreased plasmid transfer efficiency in the conjugation assay  and after overnight incubation (corresponding to a time point relevant to the aggregation and biofilm assays). All PrgB variants were present in the cell wall extract after 1-hr induction , indicatprgB mutants were exogenously expressed in the background of pCF10ΔprgB for functional complementation; or in the background of wild-type pCF10 to test for any potential dominant negative effects as previously observed for PrgBΔ246–558 (deletion of the polymer adhesin domain) . The results indicate that the introduced changes in PrgB did not affect its ability to bind eDNA, as the DNA-binding affinities of PrgB188–1233 and PrgB188–1233:S442A,N444A were the same within experimental error  . Notablyn) , and these binding interactions have been shown to be vital for their function . Howeverngivalis . Howeverngivalis , we do nngivalis .583–1233 was complemented by single particle analysis of PrgB188–1233 via cryo-EM  renders the protein incapable to support aggregation, biofilm formation, and conjugation. However, exogenous expression of prgB with single Ig-like domain deletions, prgBΔCSA1 and prgBΔCSC2 in the pCF10ΔprgB background, restore cellular aggregation, while they do not rescue biofilm formation and conjugation , besides mediating cellular aggregation, to further support conjugation and biofilm formation. We therefore propose that PrgB performs at least one, currently unknown, additional function besides binding to eDNA and/or LTA from the cell wall of the recipient cell. It is highly likely that at least one of these additional functions is mediated by the conserved site in the polymer adhesin domain.The conserved Ser–Asn–Glu site in the negatively charged cleft of the polymer adhesin domain intrigued us, as its function is unknown. Any changes that we made in these conserved residues resulted in PrgB variants that did not facilitate biofilm formation or conjugation . SurprisprgB mutants made  could also form an unfolded linker region allowing for the variant to retain its aggregation phenotype. For PrgBΔ993–1138 and PrgBΔ668–1138, unfortunately no biofilm formation or conjugation efficiency were reported, but based on our results it is likely that those capabilities would have been severely compromised.Deletion of residues 993–1138 leads to a loss of about half of both the CSA2 and CSC2 domains. Therefore, we were surprised to find that this mutant was described to behave like wild type in aggregation assays . PossiblTaking past and present results into account combined with our new structural insights, we propose a new mechanistic model for the function of PrgB. Our in vivo data show that removal of one of the Ig-like domains does not largely affect PrgB function. Previous data also indicate that parts of the Ig-like domains can be deleted without affecting the function of PrgB. We therefore hypothesize that the Ig-like domains provide two important features to the protein. First, a rigid stalk that is needed to present the polymer adhesin domain at the correct distance from the cell wall. Second, providing the correct structural positioning for PrgA-mediated cleavage of the polymer adhesin domain, as the potential protease domain in PrgA is also presented away from the cell on a ~40-nm long stalk . The exaBesides providing a structural basis to explain about 30 years of work on PrgB, we here also uncovered that the conserved Ser–Asn–Glu site in the polymer adhesin domain likely provides additional functionality to PrgB that is needed for optimal biofilm formation and conjugation, but that does not affect cellular aggregation. To fully investigate the function of this conserved site in PrgB and other homologous virulence factors from Gram-positive bacteria, remains an exciting question to address in future research.Escherichia coli Top10 was used in molecular cloning and grown in Lysogeny broth (LB). E. coli BL21 (DE3) was used for recombinant protein expression and grown in Terrific Broth (TB). The E. faecalis strains were cultured in Brain–Heart infusion broth (BHI) or Tryptic Soy broth without dextrose (TSB-D) as indicated in each assay. Concentrations of antibiotics for E. coli selection were as follows: ampicillin (100 µg/ml), kanamycin (50 µg/ml), spectinomycin (50 µg/ml), and erythromycin (150 µg/ml). In E. faecalis cultures, antibiotics were used as the following concentrations: tetracycline (10 µg/ml), fusidic acid (25 µg/ml), erythromycin , spectinomycin , and streptomycin (1000 µg/ml). Plasmids were transformed to E. coli by heat-shock transformation, whereas E. faecalis strains were transformed by electroporation (See poration .prgK vector (prgK insert). This was done in a 30 µl ligation mixture with 90 ng of the digested vector and a nine times molar access of insert. 5 µl of this ligation mixture was transformed into Top10 competent cells and plated on LB agar plates with spectinomycin (50 µg/ml) and erythromycin (150 µg/ml). The constructed plasmid was analyzed by restriction digestion and sequenced to confirm the correct insertion of the multiple cloning site and is further called pMSP3545S-MCS.To insert a multiple cloning site in pMSP3545S, DNA oligos of MCS_fwd and MCS_rev were resuspended in milliQ to 100 µM, mixed 1:1, denatured at 95℃ for 15 min and slowly cooled to room temperature. The annealed MCS oligo was ligated into the gel-purified backbone fragment from pMSP3545S-K vector  digestedprgB was constructed by PCR of prgB from pCF10 with the NcoI-prgB-F and BamHI-stop-prgB-R primer pair and placement into the pMSP3545S-MCS vector via NcoI/BamHI restriction, gel purification of the correct DNA fragments and subsequent ligation. The constructed pMSP3545S-prgB was subsequently used as a template for mutagenesis creating pMSP3545S-prgB deletion or point mutation variants. This was carried out with inverse PCR (iPCR) using partially overlapping primer pairs. The iPCR products were gel purified with the DNA clean-up kit and digested with DpnI to remove residual template plasmid DNA. The processed iPCR products were then transformed to Top10 competent cells. For overexpression and protein purification, pET-prgB246–558 and pET-prgB188–1233 were transformed into E. coli BL21 (DE3) as previously described  tag from p7XC3GH-prgB in E. coli BL21 (DE3). The cells were grown at 37°C in TB medium until they reached an OD600 of 1.5. Then the temperature was lowered to 18°C and protein production was induced by adding 0.5 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG). Cells were grown for 16 hr before harvesting by centrifugation. Cells were resuspended in 20 mM HEPES/NaOH (pH 7.0), 300 mM NaCl, 30 mM imidazole, and 0.02 mg/ml DNase I and broken with a Constant cell disruptor at 4°C and 25 kPsi (Constant Systems). The cell lysate was clarified by centrifugation for 30 min at 30,000 × g, 4°C and incubated at 4°C with Ni-NTA (Macherey-Nagel). The Ni-NTA column was washed with 10 column volumes of 20 mM HEPES/NaOH (pH 7.0), 300 mM NaCl, 30–50 mM imidazole, and bound proteins were eluted from the column with the same buffer supplemented with 500 mM imidazole. The histidine affinity tags and the GFP when present were cleaved off from the purified protein fractions by incubation with TEV protease (for pET-prgB) or Prescission protease (for p7XC3GH-prgB) in a 1:100 ratio for 20 hr at 4°C. The cleaved proteins were loaded on a Superdex-200 Increase 10/300 GL column (Cytiva) equilibrated in 20 mM HEPES/NaOH (pH 7.0) and 150 mM NaCl. The elution profile showed two peaks corresponding to a PrgB dimer and monomer, as previously reported (PrgB was produced as previously described . Brieflyreported . These tElution fractions from size-exclusion chromatography were mixed with native gel sample buffer (Invitrogen) and loaded on a Novex 4–20% Tris-Glycine gel (Invitrogen). Staining was carried out with InstantBlue Protein Stain (VWR).188–1233 from the monomeric fraction, with a protein concentration of 15 mg/ml, were thawed and used in crystallization trials. Crystals of PrgB188–1233 were grown in 8–12 weeks, at 20°C by sitting drop vapor diffusion in a condition with 0.2 M CaCl2 and 20% PEG 3350 and a protein to reservoir ratio of 1:1 in the drop. Crystals were flash cooled in liquid nitrogen. X-ray diffraction data of PrgB188–1233 were collected on ID23-1 at the ESRF, France. The data were processed using XDS   . FurtherB: 2WOY) . The strX refine . The finlProbity . Atomic 188–1233 dimer fractions were thawed on ice and loaded on a Superdex 200 10/300 GL gel filtration column  equilibrated in 20 mM HEPES/NaOH pH 7.0 and 150 mM NaCl. Protein peak fractions, corresponding to the dimer, were diluted to 0.1–0.3 mg/ml and for the DNA-bound structures 120-bp ssDNA (PrgBbp ssDNA  was addeCryo-EM data were collected on an FEI Titan Krios transmission electron microscope (Thermo Fisher Scientific), operated at 300 keV that was equipped with a K2 direct electron detector. Data were collected by the AFIS method using the EPU software V2.8.0 (Thermo Scientific) at a nominal magnification of ×165,000 (0.82 Å pixel size). Data collection parameters are listed in Cryo-EM data of apo PrgB and DNA-bound PrgB were processed in the same way, but separately using cryoSPARC (v3.2.0-3.3.1) . Beam-inThe adhesion domain (PDB code: 6EVU)  and stalE. faecalis strains were inoculated in BHI medium with the indicated antibiotics and cultured overnight at 37°C. Overnight cultures were diluted in a 1:100 ratio in TSB-D with the indicated antibiotics, 10 ng/ml cCF10, and 50 ng/ml nisin and dispensed into polystyrene cuvettes (Sarstedt) in 0.9 ml triplicates. These were incubated for 24 hr at 37°C without agitation. Afterwards, the optical density of each sample was determined at 600 nm both before (ODsup) and after (ODmix) vigorously mixing of the bacterial culture by pipetting. The autoaggregation percentage was then calculated as follows: 100 × [1 − (ODsup/ODmix)] (/ODmix)] .E. faecalis strains were inoculated in BHI with the indicated antibiotics and kept 16 hr at 37°C. The next morning, they were diluted in a 1:100 ratio in TSB-D with the indicated antibiotics, 10 ng/ml cCF10, and 50 ng/ml nisin. 200 µl fractions were dispensed into a 96-well microtiter plate (Costar) with 8 replicates per strain. 200 µl TSB-D fractions were used as blanks. The 96-well plate was then incubated aerobically at 37°C without agitation in a humidified chamber for 24 hr. The suspension was transferred to another 96-well plate to determine the optical density at 600 nm (OD600). The plate containing the biofilm was washed with distilled water three times and then left to air dry at room temp for 2.5 hr. The biofilm was stained with 100 µl 0.1% (wt/vol) safranin (Sigma) at room temp for 20 min, then washed three times with distilled water and left to air dry at room temperature. Afterwards the absorbance was determined using a plate reader (BMG Labtech) at 450 nm. Biofilm production was calculated as an index of safranin staining of the cell biomass divided by absorbance of its optical density (OD450/OD600) (0/OD600) .g, 4°C for 5 min. The supernatant, containing the cell wall extract, was mixed with protein loading dye and boiled at 100°C for 12 min. Subsequently, the samples were run on a 8% sodium dodecyl sulfate–PAGE, transferred to western blot and probed with the PrgB antibody produced in rabbit , 25% sucrose, 15 mg/ml lysozyme) for 30 min at 37°C. The lysozyme-treated bacterial cells were spun down at 13,000 × n rabbit .Donor (OG1RF pCF10 pMSP3545S derivative strains) and recipient (OG1ES) strains were inoculated in BHI with the indicated antibiotics and incubated overnight at 37°C with agitation. Overnight cultured strains were refreshed in BHI without antibiotics in a 1:10 ratio, and donor strains were induced with 50 ng/ml nisin (Sigma). All strains were then incubated at 37°C for 1 hr without agitation. Afterwards each of the donor strains was mixed with the recipient cells in ratio of 1:10 and mated at 37°C statically for 3.5 hr. These mixtures were then serially diluted with BHI and plated out in triplicates on BHI agar plates supplemented with tetracycline and spectinomycin (to select for donor cells), or with tetracycline, erythromycin, and streptomycin (to select for transconjugants). Plates were incubated at 37°C for 48 hr, counted and enumerated for colony-forming units (CFU). The plasmid transfer rate was determined as CFU of transconjugant over CFU of donor (Tc’s/Donors) .EMSA was carried out in the same way as previously described . 0.1–20 All in vivo data are from three independent experiments and were plotted and analyzed using GraphPad Prism (version 5.0) (GraphPad Software). The indicated error is the standard deviation over three biologically independent replicates. Statistical significance between the PrgB variants were analyzed with one-way ANOVA analysis of variance, with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.All data generated or analyzed during this study are included in this published article and its supplemental information. All structural data have been deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Base (EMDB) and is publicly available. DOIs are listed in the key resources table. Any additional information required to reanalyze the data reported in this paper, for example bacterial strains, is available from the corresponding author upon request. valuable structural data for the bacterial adhesin PrgB, an atypical microbial cell surface-anchored polypeptide that binds DNA. There is convincing support for the claims regarding the overall function and importance of individual domains, which integrate a wide range of new and previously published experimental data. The structure-based model of PrgB molecular activity will be impactful in the field of bacterial adhesins, conjugation, and biofilm formation, especially because it focuses on a clinically-important Gram-positive pathogen, whereas most work in the field has been focused on Gram-negative model systems.This study presents  E. faecalis strains expressing various PrgB mutants. Generally, the study has been conducted with a good level of rigor, and there is consistency in the findings. Initial concerns about inferences initially made from low-resolution Cryo-EM structures have been addressed experimentally and the manuscript correspondingly updated.Sun and colleagues outline structural and mechanistic studies of the bacterial adhesin PrgB, an atypical microbial cell surface-anchored polypeptide that binds DNA. The manuscript includes a crystal structure of the Ig-like domains of PrgB, cryo-EM structures of the majority of the intact polypeptide in DNA-bound and free forms, and an assessment of the phenotypes of  E. faecalis, the authors looked to build on that work by crystallizing a nearly full-length construct of PrgB. Though they were successful in their crystallization endeavors, the crystal contained only what was previously thought to be two domains with RGD motifs. The authors' high-resolution structure shows that in fact the C-terminal portion of PrgB is made up of four immunoglobulin-like domains. The authors then set out to collect single-particle cryoEM data in a bid to obtain a full-length structure of PrgB, both in the presence and absence of ssDNA. The authors were only able to obtain quite low-resolution data, which they fit their crystal structures into. The authors then used these structures to inform the design of novel deletion mutants and point mutations, as well as to rationalize years of phenotypic data from other published mutants.Having previously solved the X-ray crystallographic structure of the polymer adhesin domain (PAD) of PrgB from The X-ray crystallographic structure is beautiful and in combination with their in vivo data allowed them to propose a model where PrgB positions cells at an appropriate distance for conjugation. The in vivo experiments appear to be done well and the authors' discovery that the Ser-Asn-Glu is not important for generalized aggregation but has an additional yet unknown role in conjugation and biofilm formation is exciting and well supported by their data. The following is the authors’ response to the original reviews.First of all, we would like to again thank the reviewers for their work. We appreciate the constructive review comments and useful suggestions to further improve our article. With those comments in mind, we have now revised our manuscript. Please see below for a point-by-point response (our responses in green) to all comments.Reviewer #1 (Recommendations For The Authors):E. faecalis strains expressing various PrgB mutants.Sun and colleagues outline structural and mechanistic studies of the bacterial adhesin PrgB, an atypical microbial cell surface-anchored polypeptide that binds DNA. The manuscript includes a crystal structure of the Ig-like domains of PrgB, cryo-EM structures of the majority of the intact polypeptide in DNA-bound and free forms, and an assessment of the phenotypes of Generally, the study has been conducted with a good level of rigor, and there is consistency in the findings. However, I do have some specific technical concerns relating to the study that necessitate the undertaking of additional experiments. These are summarized as follows:1. Recombinant PrgB188-1233 produced in the study purifies as a mixture of monomeric and dimeric species separatable by SEC. There is very limited discussion in the text re. the significance and/or implications of this. Is it feasible that the dimeric form is biologically relevant in the context of the in vivo situation? Or alternatively, is this simply an artifact of protein production?Experimental data that we published in 2018 indeed indicates that the dimer is relevant in the in vivo situation. We did not discuss this here since this was discussed in detail in the previous paper: Schmitt et al, 2018. We have now added a bit more information on this in the results section, highlighting this, so that it is clearer to the reader (lines 114-116).1. The authors see no evidence of the adhesive domain of PrgB in their PX structure highlighting that this must have been cleaved during crystallisation. Is this claim supported by an inspection of the crystal packing? It could be that this region of the protein is dynamic within the context of the crystal and is thus not observed. This should be clarified in the text either way.The crystal packing does not provide any space for the PAD. We have added this to the results section. We have added a sentence describing this in lines 122-124.1. The Cryo-EM structures reported are both at ~10-angstrom resolution. Are the authors truly confident in the placement of their crystal structures on these maps? Visual inspection indicates that their positioning of the PrgB domains into the EM envelopes is somewhat questionable. The authors need to provide some quantitative measures of the quality of their domain fitting. The narrative of the manuscript very much hinges on this being correct.This is something that the other reviewer also commented on. The fitting of the crystal structures in the maps are indeed not optimal, but was the best we could do with the available data. In line with point #6, we have now constructed new protein variants of the stalk domain (the four Ig-like domains) alone, and have assayed it’s interaction with the PAD in vitro using native gels and size exclusion chromatography. The outcome of these experiments is that the two domains do not interact in any substantial way on their own. Thus, the added experiments do not support the hypothesis that the PAD interacts with the Ig-like domains, at least not without the local high concentration provided by the linker region in the in vivo situation.To account for these new experiments, we have moved the cryo-EM structure to the supplement, and rewritten this part of the manuscript to say that the cryo-EM data indicated that there might be an interaction, but that we have not been able to verify this in vitro, indicating that if the interaction at all exists it must have a low affinity and is likely not physiologically relevant. In line with this, we have also further modified the text throughout the manuscript to account for this.1. The manuscript would be significantly strengthened if the authors could include confirmatory hydrodynamic data in support of the observed conformational reorganization of PrgB in the presence of DNA. SAXS analysis of the DNA-free and bound complexes would be ideal for this and would also help address the issues raised above in pt 3.To analyze PrgB radius with and without DNA, we tried both SEC-MALS and DLS experiments. It proved difficult to obtain precise and reproducible values, but the initial data indicated that no large changes were observed upon DNA binding. As we could also not measure specific interaction between the PAD and the stalk in vitro, we did not perform SAXS experiments. As mentioned in the response to point #3, we have modified the results and discussion regarding the potential interaction of th PAD and Stalk domains.1. The authors present binding studies of various PrgB mutant-expressing strains. A number of the mutations generated delete significant portions of the polypeptide. Can the authors confirm that these mutant proteins are correctly folded despite the introduced mutations? It could be that loss of function is simply a consequence of mutation-induced misfolding. I would like to see some confirmatory data  in support of the foldedness of the mutant proteins.E. faecalis. However, CD or SEC experiments would only give indications of the contrary if the overall fold had been majorly affected in an in vitro situation where the protein is not anchored to the E. faecalis cell wall.We cannot completely rule out that the folding of some of the variants is affected in E. faecalis producing wild-type or variant PrgB and performed Western blot . The results of the Western blot with cell wall extract largely matches the whole cell experiments that were in the initial manuscript. If a protein variant was largely misfolded, it would likely not be targeted and linked to the cell-wall, nor would it be stable in vivo. We have added this new data as a new fig 3 – figure supplement 1 and on lines 201-214To alleviate this valid concern, we probed if all variants are correctly exported and linked to the cell-wall. Therefore we have now extracted the cell wall of 1. The authors suggest a direct interaction between the PAD and the stalk domains in PrgB. The discussion of this is very generic and no evidence to support this is provided other than the 10-angstrom resolution EM map. If they believe this to be the case, then additional evidence should be provided.Answer: As mentioned previously, we have now performed additional in vitro experiments to probe this potential interaction, but conclude that this indication from the EM data is likely not a real high affinity interaction. In line with this, we have modified the results and discussion regarding this point, see also response to point #3 and 4.Reviewer #2 (Recommendations For The Authors):As currently presented, I don't feel that the cryoEM data support the authors' proposed model, largely because the fit of the crystal structures to the EM volumes does not seem entirely reasonable for the apo- dataset and because the EM volume for the ssDNA bound dataset is not even contiguous. For me to believe the model as it is currently built, I would want to see a dataset with the PAD deleted, showing that its proposed density disappears, or a dataset with a PAD-specific antibody as a fiducial marker. It would be nice to see some goodness of fit metric with a comparison to other crystal structures fit such low-resolution data as well. At the very least, the authors must include the standard cryoEM workflow supplementary figure showing representative micrographs, 2Ds, and 3Ds along with particle numbers.In line with the comments raised by reviewer #1, we have now added more experiments where we have analyzed the potential interaction between PAD and the stalk domain. From this new data, it looks like they do not interact with any substantial affinity, at least not on their own without any linker region holding them together, and that this interaction if it all exist likely is not physiologically relevant. The cryo-EM data has been moved to the supplement as we agree with both reviewers that the resolution, and the fitted model, is not good enough to draw any hard conclusions. The standard table for the cryoEM workflow was present as supplementary table 2, where eg particle numbers etc are described, but we have now also added a new supplementary fig 2 – figure supplement 2 that shows the EM processing workflow, including representative micrographs, 2D and 3D classes. We debated whether we should remove the EM data, but decided against it in line of transparency and to explain why the interaction studies with the PAD and stalk domains were performed.The X-ray crystallographic structure is very nice, but I was a bit surprised by the R factors in Table 1. After downloading the structure factors and coordinates from the PDB (thank you for depositing before submission!) I was able to see quite a few positive peaks in the difference map that could probably use some cleaning up. I realize I may just be a bit of a masochist when it comes to adding/deleting waters and moving around side chains to get things just right, but for such lovely data, I would have liked to see the model polished up a bit more. I was going to say that the isopeptide bond should be modelled, but I can see from a cursory Google that the authors did in fact try to find a way to model this and that it is indeed a bit of a pain.The model refinement proved surprisingly recalcitrant with regards to the remaining difference density, so we took the decision to only model what was solidly there (which leads to slightly higher R factors). We did indeed try to model the isopeptide bond, but we did not find a good way to do so (despite trying quite extensively), and ended up determining them as a linker in the PDB file, so that the bond shows up when one opens the structure in eg. Pymol.For protein production/purification in general I would have liked to see actual traces for the gel filtration and pure protein on a gel in a supplementary figure. I strongly believe that this type of information is so critical for future researchers looking to replicate or build upon published work so that they have some sense that what they are doing is working in the way it should be.We have now added a supplementary figure  that shows SEC and SDS-PAGE for the purification of PrgB188-1233.Finally, I think for the in vivo data it only makes sense to show the reader whether any or all the differences measured across your different mutants are statistically significant. Having done the graphing and analysis in GraphPad this should be a simple thing to achieve.We have now added statistical test (One way Anova) that show the statistical significance between the mutants, and show that in Fig 3 and Fig 4.Overall, I think it's a very nice paper and while I feel that the cryoEM data in its current form doesn't support the model of occlusion from PrgA, I also don't think that removing the cryoEM data and that specific mechanistic idea from the paper detracts from its overall message and impact.Thank you for those comments.


The thermal deformation requirement for wavefront preservation through an X-ray crystal monochromator is found to restrict the standard deviation of the height error to less than 25 pm under certain conditions. By optimizing the effective cooling temperature of liquid-nitrogen-cooled crystals, combined with compensation of the second-order component of the thermal deformation, an approach to reach this unprecedented requirement is described. The wavefront preservation of coherent X-ray free-electron laser beams is pushing the requirement on the quality and performance of X-ray optics to an unprecedented level. The Strehl ratio can be used to quantify this requirement. In this paper, the criteria for thermal deformation of the X-ray optics are formulated, especially for crystal monochromators. To preserve the X-ray wavefront, the standard deviation of the height error should be sub-nm for mirrors and less than 25 pm for crystal monochromators. Cryocooled silicon crystals combined with two techniques can be used to achieve this level of performance for monochromator crystals: (1) using a focusing element to compensate the second-order component of the thermal deformation; (2) introducing a cooling pad between the cooling block and silicon crystal and optimizing the effective cooling temperature. Each of these techniques allows the thermal deformation in standard deviation of the height error to be reduced by an order of magnitude. As an example, for the LCLS-II-HE Dynamic X-ray Scattering instrument, the criteria on thermal deformation of a high-heat-load monochromator crystal can be achieved for a 100 W SASE FEL beam. Wavefront propagation simulations confirm that the reflected beam intensity profile is satisfactory on both the peak power density and focused beam size. The peak XFEL power has reached terawatt levels. The average power from high-repetition-rate XFELs can reach a few hundred watts. The requirement to preserve the wavefront of coherent X-ray beams leads to new engineering challenges in X-ray optics. Minimizing thermal deformation of X-ray optics with tens or hundreds of watts of power load demands increasingly innovative approaches.i.e. the shape variation due to the heat load, the thermal slope error Δθ or height error Δh over the beam footprint on the optics are often used. The thermal slope error for constant material properties can be expressed as  minimizing the geometrical function fgeom by working on the cooling design and geometrical shape of the optics, and (2) minimizing the figure of merit in material properties (1 + ν)α/k by choosing the materials and/or the temperature range when the material properties are strongly temperature dependent. The current state-of-the-art solutions to minimize the thermal deformation of the X-ray optics include:With the advent of third-generation synchrotron light sources back to the late 1980s and 1990s, the X-ray power load on the optics reached several hundred watts. Thermal deformation of the optics can lead to photon flux reduction, divergent X-ray beams, and ultimately a significant reduction of the X-ray beam brilliance. Many engineering efforts and initiatives have been conducted across the world to minimize or compensate the thermal deformation of the X-ray optics. To quantify the thermal deformation, fgeom. Cool the mirror using water along the top sides of the substrate, fully illuminate (overfill) the mirror length, optimize the mirror cross section with notches, use a secondary slit downstream of the mirror to shape the beam to the desired final size  Synchrotron X-ray white-beam mirrors to minimize the geometrical function k of the silicon at LN2 temperature (77 K at 1 atm), is about 1–2% of that at room temperature. Therefore, LN2 cooling can significantly reduce the thermal deformation of the silicon crystal, compared with water cooling at near room temperature. LN2-cooled silicon crystal monochromators have been widely investigated  Cryo- or liquid-nitrogen (LN2) cooling of the silicon crystal monochromator. The figure of merit of material properties, the ratio α/et al., 1992et al., 1995et al., 2015et al., 2020(3) Actively compensated X-ray mirrors (flat or bendable): piezo-actuator dynamic bendable mirror  of the LCLS-II-HE Dynamic X-ray Scattering (DXS) instrument. Wavefront simulation results of such monochromator crystals will also be reported and discussed.2.L12 between the two rays with glancing incident angle θ reflected by an X-ray mirror with a surface shape height error Δh12 . The phase error is then φ12 = 2πΔh12/λ where λ is the beam wavelength. Now let us assume that Δh is the standard deviation  of the height error over the beam footprint on the optic, and φ is the RMS phase error over the cross section of the reflected beam. This phase error is given byh and λ are the same, for instance nm; the units of phase error φ are radians. In the literature, some authors omitted 2π or a factor of 2.The performance requirements of X-ray optics since the advent of third-generation synchrotron light sources are mostly related to the preservation and improvement of the quality of the photon beam, such as the photon flux, beam collimation, beam focusing, harmonic rejection, monochromatization and beam stability. Wavefront preservation becomes an increasing requirement on the X-ray optics for coherent high-power X-ray light sources. It is useful at this stage to define a quantitative measure of this requirement on the optics. One approach is to compare the X-ray beam intensity in the focal plane after reflection from an aberrated optical system with the perfect case. The ratio of the real versus ideal peak intensity is widely known as the Strehl ratio , we can use the following criterion  induced by the photon beam heat load.To minimize the thermal deformation of an X-ray mirror subjected to a photon beam with incidence angle θ and photon energy h for the LCLS-II X-ray mirrors are plotted in Fig. 2h should be smaller. As the mirror grazing incidence angle is small, sinθ ≅ θ, and the threshold value Δh is inversely proportional to the grazing angle θ and photon energy eph. The effects of smaller grazing incidence angle of LCLS HXR mirrors compared with SXR mirrors on the Δh requirement quite offset the effects of higher photon energy with HXR mirrors. The requirements on height error are similar for the SXR and HXR mirrors. The thermal deformation of the X-ray mirrors should be limited to sub-nanometres in RMS height error.For the LCLS-II soft X-ray (SXR) mirrors, incidence angles are between 7 and 30 mrad. For the LCLS-II-HE hard X-ray (HXR) mirrors, grazing incidence angles are between 1.4 and 3 mrad. The RMS height error requirements Δeph), and crystal d-spacing parameter d are governed by Bragg’s law,d are nm. For a crystal diffraction plane (ijk), the d-spacing parameter is dijk = a/(i2 + j2 + k2)1/2, where a is the lattice constant . Note that the crystal monochromator works for HXR, for instance above 2 keV with a silicon crystal. Combining equations (2)h and Strehl ratio for a crystal is the same as for a mirror. But the criterion to limit the thermal deformation or other aberration on the crystal monochromator depends only on the crystal d-spacing parameters. The influences of the beam incident angle and photon energy on this requirement are indirect through the d-spacing parameter as equation (5)h versus Strehl ratio for various silicon crystals is shown in Fig. 3a). To reach a Strehl ratio of higher than 0.8, the distortion of the crystal planes should be limited to smaller than 25 pm in RMS height error for Si(111), and smaller than 10 pm for Si(333). These numbers are about 50 times smaller than the threshold for mirrors. This big difference in requirement between mirror and monochromator crystal is basically related to the beam incident angle. For example, the grazing incident angles for an HXR mirror are about 2 mrad; Bragg angles for the three silicon crystals shown in Fig. 3b) in the photon energy range up to 20 keV are larger than 100 mrad. The beam incident angle of the silicon crystal is about 50 times that of the grazing incident angle of the HXR mirrors.For crystal monochromators, the deformation of the crystal plane should be considered rather than the surface. We use the term ‘height error’ mostly to differentiate from the ‘slope error’ for crystal monochromators, but both refer to the deformation of the crystal plane. The same criterion as for the mirror in equation (3)h and d are nm. For mirrors,h and λ are nm and those of photon energy eph are keV.In latter sections, we will show that the 10–25 pm height error requirements for crystal planes are relevant and appropriate for the coherent wavefront preservation of the X-ray beam. The Strehl ratio can be calculated from the thermal deformation height error as for monochromator crystals,3.The power load on the X-ray optics for the new coherent X-ray light sources (high-repetition-rate FEL and MBA lattice DLSR) reaches a few hundred watts. Minimizing thermal deformation of the X-ray optics becomes critical for the wavefront preservation that is required for coherent beams.We have mentioned some of the state-of-the-art solutions to minimize the thermal deformation of the X-ray optics. For the crystal monochromator, cryo-cooled silicon crystals have been widely used for the third-generation synchrotron light sources. LN2 is the typical coolant used due to its high cooling capacity and better stability compared with pulse tube cryocoolers.The X-ray beam power-load-induced temperature gradient is the driving force for the thermal deformation of the crystal. The thermal mechanical properties of silicon, such as thermal conductivity and thermal expansion coefficient, are temperature dependent. The thermal expansion coefficient of silicon is zero around 125 K. The optimum temperature level of the crystal should be such that the temperature in the beam footprint adjacent volume is around 125 K; the maximum temperature should be higher than 125 K. The objective function of the optimization is the thermal deformation, for instance the standard deviation of the height error over the beam footprint.In this paper, we will present the thermal deformation minimization of the crystal monochromators for the LCLS-II-HE DXS instrument . The key optics components in the DXS instrument  scheme was considered. The first crystal block in the first DCM was composed of three crystals side-by-side with different crystal planes and different asymmetry cut angles. Each of the crystals in this first crystal block is a rectangular shape of dimensions 75 mm in length, 20 mm in width and 30 mm in thickness. Any one of these three crystals could be used by lateral translation of the crystal block. A 0.2 mm-thick indium foil was assumed to be used between the crystals, as well as between the crystal and cooling block. In this study, finite-element modelling is applied only to the silicon crystal block shown in Fig. 5l. 2013b and Lee  al. 2020. Gaussia3.2.hcv is not a convenient controllable parameter. Once the cooling technique is chosen, and the geometry of the cooling block and the interface with the crystal defined, the cooling coefficient hcv is mostly fixed. The effective cooling temperature Tcool can be a controllable parameter. This can be realized by using a pulsed cryocooler cold head, or by an LN2 cryocooler combined with electric heater strategically placed on an intermediate cooling pad between the crystal blocks and cooling pipes, as illustrated for the LCLS REAL cooled mirror  of the crystal surface over the footprint length, denoted stdUy. With dynamic focusing elements (periscope mirrors) on the instrument (beamline), we can compensate the second-order component in the thermal deformation. Denoting the best spherical shape fit of the thermal deformation displacement as Uy-fit, the residual height error is then dUy = Uy − Uy-fit. The standard deviation of the residual height error is noted as stdUyd. Both height error stdUy or residual height error stdUyd can be used as the objective function for thermal deformation minimization.We can optimize the cooling temperature by minimizing the thermal deformation of the crystal for the whole photon energy range 6–25 keV. But we will focus on several photon energies at which most of the science cases are anticipated. Thermal deformation of the crystal can be quantified by the standard deviation of the height error which is the thermal deformation displacement  direction for the vertical oriented crystal.3.3.Tcool is shown in Fig. 6Tcool at which the residual height error stdUyd is minimum. By optimizing the cooling temperature, the residual height error can be reduced by about a factor of ten compared with standard LN2 cooling where the effective cooling temperature Tcool is about 80 K. This optimal cooling temperature depends on the photon energy and crystal Bragg and asymmetry cut angles. The values of this optimal cooling temperature and the maximum temperature in the crystal are given in Table 1The HHLM at the DXS instrument will cover a photon energy range between 6 and 18 keV. For a vertical oriented crystal and 70 W absorbed power (more than 100 W of FEL power from the source), the residual height error of the crystal in the transverse direction  calculated with different effective cooling temperature Uyd in the (beam) horizontal direction is plotted in Fig. 7The minimized residual height error stdFrom the height error shown in Fig. 74.To confirm the expectations based on the Strehl ratios calculated in Section 34.1.Synchrotron Radiation Workshop (SRW) software  ray-tracing package .The wavefront propagation method used here is inspired by that employed by the 4.2.1-1–Si(111) with three degrees asymmetry cut and photon energy at 10 keV. Four cases . Using a bendable focusing mirror (periscope mirror) to compensate the second-order component in the thermal deformation, the residual height error stdUyd can be reduced to 34 pm, and the Strehl ratio reaches 0.62. The beam profile and intensity [Fig. 10b)] are significantly improved compared with the uncompensated case [Fig. 10a)].With 5 W absorbed power and standard LN2 cooling, the height error of the crystal stdUy [case (c)] reaches 444 pm, and the Strehl ratio is close to 0. The beam aberration is greater than for case (a) with the beam intensity at the centre of the beam reduced to nearly 0, and the beam profile is uglier. The compensation by the focusing mirror of the second-order component in the thermal deformation allows the residual height error stdUyd to be reduced to 24 pm, and the Strehl ratio is increased to 0.79. The reflected beam intensity profile is satisfactory: peak intensity reduction of about 20% and a very small tail of less than 5% of peak intensity. Note that when optimizing the temperature based on the compensated height error (stdUyd) versus uncompensated height error (stdUy) there is a 3 K difference.With 50 W absorbed power and optimized cooling temperature, the uncompensated height error stdb) and (d) corresponds well to the value of the Strehl ratio estimations calculated with equation (7)b) and 0.79 for case (d).The ratio of the peak intensity with aberrated crystal to the perfect crystal in the cases  over the footprint is variable; there is always a small region around the centre of the beam footprint where the local thermal deformation is very small.Considering the relevance of the thermal deformation requirement and the Strehl ratio, the ratio of the peak intensity of the reflected beam by the crystal with aberration related to the perfect crystal is close to the Strehl ratio when the ratio of the intensities is larger than the 10%. When the Strehl ratio is small (<0.1), the ratio of the peak intensity is no longer relevant. The Strehl ratio is calculated with a global metric of thermal deformation – the RMS height error Δ5.In this paper, we revisited the state-of-the-art solutions to minimize the thermal deformation of X-ray optics, detailed the optical requirements for wavefront preservation, and formulated the criteria on thermal deformation of X-ray optics, especially for crystal monochromators. These criteria can be used to guide opto-mechanical engineering design decisions. The height error standard deviation requirement is sub-nm for mirrors (surface shape), and smaller than 25 pm for crystal monochromators . Such unprecedented requirements are pushing opto-mechanical engineering efforts to address this challenge. We have demonstrated that the thermal deformation of a crystal monochromator cooled by liquid nitrogen can be minimized by optimizing the effective cooling temperature. A factor of ten in performance can be achieved compared with standard liquid-nitrogen cooling. Using dynamic focusing elements (for instance a focusing mirror) to compensate the second-order component of the thermal deformation, we can reduce the residual height error by another order of magnitude. As an example, for the LCLS-II-HE DXS instrument the criteria on thermal deformation of the HHLM crystal can be achieved for a 100 W SASE FEL power load. The wavefront propagation simulations confirm that the reflected beam intensity profile is satisfactory both in the peak power density and focused beam size.The path forward includes the detailed engineering design to implement the proposed solution for crystal monochromators: optimization of effective cooling temperature, compensation of the second-order component in the thermal deformation, and minimization of the fabrication/mounting/cooling-induced deformation.