To achieve this purpose, we developed a scanning probe microscopy-based measurement technique, termed direct piezoelectric response microscopy (DPRM), to observe the domain structure of BiFeO3 films via the direct piezoelectric response. Quantitative analysis of the direct piezoelectric response obtained by DPRM, detailed analysis of the domain structure by conventional piezoelectric force microscopy, and microscopic characterisation of the direct piezoelectric properties of BiFeO3 films with different domain structures revealed that their direct piezoelectric response is enhanced by the walls between the domains of spontaneous polarisation in the same out-of-plane direction.Polarisation domain structure is a microstructure specific to ferroelectrics and plays a role in their various fascinating characteristics. The piezoelectric properties of ferroelectrics are influenced by the domain wall contribution. This study provides a direct observation of the contribution of domain walls to the direct piezoelectric response of bismuth ferrite (BiFeO In addition to practical applications such as inkjet printer heads and gyro-sensors, various piezoelectric MEMS devices, including microphones, speakers, optical microscanners, ultrasonic transducers, and energy harvesters, have been actively studied for future practical use. Although lead zirconate titanate  and aluminium nitride films are already widely used in MEMS devices, there is high demand to further improve piezoelectric properties to enhance device performance. Specifically, lead-free piezoelectrics are currently in demand from the viewpoint of environmental protection.Piezoelectric films have attracted much attention as sensing and actuating components in microelectromechanical systems (MEMSs)3) films5. The ef31, of BiFeO3 films is approximately −3.5 C/m2, which is lower than that of PZT films. Nonetheless, BiFeO3 films have a sufficient figure of merit (FOM) for energy harvesting applications, in which the FOM is given by 0 and εr are the permittivity of vacuum and the relative permittivity, respectively)7 because of their low permittivity (~100)9. In fact, we have demonstrated that harvesters using BiFeO3 films show output power comparable to that obtained using PZT films10. Similarly, it can be expected that BiFeO3 films will be suitable for sensing applications because of their large piezoelectric voltage constant 3 films, the direct piezoelectric response is more important than the converse piezoelectric response; however, most studies on BiFeO3 films have only characterised the latter15. We have investigated the relationship between the ef31, coefficient (determined by the direct piezoelectric response) and the crystal and domain structures using epitaxial and oriented BiFeO3 films20. It was found that films with a higher domain wall density have a larger direct piezoelectric response. For further improvement of the ef31, coefficient of BiFeO3 films, an understanding of the mechanisms is important.We developed piezoelectric MEMS vibration energy harvesters using PZT and bismuth ferrite  values of PZT films determined via direct and converse piezoelectric effects do not coincide. This discrepancy was not observed for polar wurtzite films22. These results suggest that the domain wall contribution to the piezoelectric properties of PZT films is not negligible. Although the domain wall contribution to the converse piezoelectric response has been investigated in various ways, its contribution to the direct piezoelectric response has not. As mentioned above, the direct piezoelectric response is important for sensing and energy harvesting applications. However, there is no method to characterize it on the nanoscale.Recently, Tsujiura 3 films and quantitatively measure the longitudinal piezoelectric stress coefficient  at high spatial resolution through direct piezoelectric response microscopy (DPRM), which was developed in this study. In DPRM, a modulated mechanical force is applied to the nano-region of the film surface by a conductive AFM probe. The current induced by the direct piezoelectric response is detected by a current-to-voltage (I/V) converter, which is a major difference from previously reported piezoelectric force microscopy (PFM), atomic force acoustic microscopy (AFAM)23, ultrasonic force microscopy (UFM)25 and other methods. The details of DPRM and comparison with conventional PFM are described in the Methods section. Although Gomez and co-workers measured the direct piezoelectric response using scanning probe microscopy (SPM), the spatial resolution and sensitivity are insufficient for this purpose26. By combining the DPRM results with analysis by conventional PFM, we can discuss the enhancement of the direct piezoelectric response of BiFeO3 films at domain walls.In this article, we observe the ferroelectric domain structure of epitaxially grown BiFeOF) was applied from the AFM tip to the piezoelectric film. Figure F of 1 μN; tip diameter (D) of 20 nm; and thickness (h), Young’s modulus (E), and d33 of the piezoelectric film of 200 nm, 73 GPa, and 245 pm/V, respectively. The stress was distributed in the film, and the surface around the tip was deformed. Figure Q) via the direct piezoelectric response and F under the same conditions as in Fig. Q increased linearly with F and that the proportional relationship was simply Q = d33F despite the application of uneven stress. Moreover, this relationship was almost unaffected under other conditions, as illustrated in Fig. D and h were varied from 20 to 100 nm and from 20 to 500 nm, respectively. The charges were almost independent of D and h, which suggests that DPRM can achieve quantitative analysis regardless of the measurement conditions and sample structure. Moreover, the direct piezoelectric response was hardly influenced by other contributions, such as the electrorestrictive and charging effects, because no electric field was applied in the measurement27. Therefore, DPRM should have the ability to measure the pure piezoelectric response of ultrathin piezoelectric films, which is an advantage over conventional PFM.The output signal of DPRM was analysed by the finite element method . In the simulations, a force  SrRuO3/(100) SrTiO3 and (111) SrRuO3/(111) SrTiO3 substrates, respectively, using pulsed laser deposition. The details of the samples are described elsewhere15. The thickness of these films was 500 nm. X-ray reciprocal space mapping indicated that both films had rhombohedral structures. The macroscopic remnant polarisation of the (111) and (100) films was 95 and 56 μC/cm2, respectively, and their ef31, value was −1.3 and −3.1 C/m2, respectively15.The samples used were (100) and (111) BiFeO3 film. A compressive force modulation of 1300 nN was applied. The topographic DPRM image was almost identical to that of conventional SPM, which indicates that the sinusoidally applied force did not influence the topology observation and that constant contact of the AFM probe with the sample was maintained. Figure θ) of 90° and −90° correspond to the upward and downward domains, respectively. The domain pattern was clearly observed by DPRM. The obtained pattern is almost identical to the PFM phase image shown in Fig. et al.28 reported that polarisation switching was induced by the application of the strain gradient generated by the SPM tip. Although the measurement method of Lu et al. was similar to that of DPRM, such phenomena were not observed herein, as shown in Fig. I/V converter.Figure 3 films. The relationship between ef31, (determined from the direct piezoelectric response) and the effective longitudinal piezoelectric coefficient, d33(AFM), determined from the converse piezoelectric response using SPM, is summarised in Fig. Figure 3 films was investigated by combining the PFM and DPRM measurements. Figure Ipsinθ signal of the (111) and (100) films are presented in Fig. Ip. To investigate the origin of this distribution in detail, ef33, was calculated usingk is the spring constant of the AFM probe and f and x are the operating frequency and displacement of the actuator, respectively. The BiFeO3 films were assumed to have E = 170 GPa. The cross sections of ef33, distribution for the (111) and (100) films are shown in Fig. ef33, is calculated to be 2.7 C/m2, which corresponds to the intrinsic piezoelectric response of the (111) film because this film has a single domain structure (denoted domain A). In contrast, the (100) film has different ef33, values depending on the domain, as shown in Fig. ef33, of 5.7 C/m2. The domain with 71° domain walls (domain C) has an average ef33, of 7.3 C/m2, which is 30% larger than that of domain B. The difference between the ef33, values of domains A and B must originate from the domains’ engineered configuration because all of the BiFeO3 films used in this study have rhombohedral structures. The increase in ef33, in domain C of 1.6 C/m2 compared with that in domain B indicates the contribution of the 71° domain walls to the direct piezoelectric response. This enhancement is not caused by the difference in the spontaneous polarisation directions because domain D, which has the same spontaneous polarisation direction as that of domain C and no 71° domain walls, has a smaller ef33, than domain C. Moreover, the 71° domain walls are tilted with respect to the film surface. It appears that this tilt contributed to the increase in ef33, in the peripheral area of the 71° domain walls. The maximum ef33, was observed in the region with two 71° domain walls, as shown in Fig. 29. The enhancement of the direct piezoelectric response at the 71° domain walls was observed with good reproducibility in regions other than domain C, as shown in Fig. 30.The contribution of the domain walls to the direct piezoelectric response of the BiFeOef31, is discussed. For the (111) film, which has only an intrinsic contribution, the ratio of ef31, (=−1.3 C/m2) to ef33, (=2.7 C/m2) is −2.1. As 31, the obtained ratio is reasonable, which suggests quantitative analysis with DPRM. By applying this ratio to ef33, of domain B (5.7 C/m2), the estimated intrinsic contribution of ef31, in the (100) film is −2.7 C/m2. The difference between the measured ef31, (=−3.5 C/m2), and the intrinsic contribution of ef31, is −0.8 C/m2, which corresponds to the contribution of the 71° domain walls. This value is consistent with that estimated from the enhancement of ef33, caused by the contribution of the 71° domain walls, which is 1.6 C/m2. Because the 71° domain walls are mainly formed between the domains of spontaneous polarisation in the same out-of-plane direction, these walls will remain after the poling treatment and contribute to the enhancement of the direct piezoelectric response. The domain wall contribution to the direct piezoelectric response of the BiFeO3 films is only 30% of the intrinsic contribution. This is important for energy conversion applications such as energy harvesting because the electromechanical coupling factor (k2) is proportional to the square of the piezoelectric coefficient. It is estimated that almost half of k2 originates from the domain wall contribution.Finally, the contribution of the 71° domain walls to the macroscopic direct piezoelectric response characterised by This work presented a technique to observe the domain structure of ferroelectric films through the direct piezoelectric effect and quantitatively characterise the effective longitudinal piezoelectric coefficient. Using the developed DPRM technique, a marked increase in the direct piezoelectric response in the domains with 71° domain walls was identified. This work should stimulate attempts to enhance the piezoelectric properties of ferroelectric films through the modification of domain structures, including the introduction of domain walls.An SPM  was modified to observe the direct piezoelectric response of a nanoscopic region. A schematic illustration of DPRM is shown in Fig. I/V converter to prevent the formation of parasitic capacitance between the conductive cantilever beam and the bottom electrode of the sample  is proportional to the frequency of the applied force because the charge induced via the direct piezoelectric effect is proportional to the force and the output current is given by the charge differentiation. Given that the cutoff frequency of the I/V converter was 10 kHz, all the measurements were carried out at 7.3 kHz.The piezoelectric response was detected by the output current using a lock-in-amplifier and ple Fig. . IdeallyIn the measurements, a commercially available conductive Pt/Cr-coated AFM probe (Budget Sensors: ElectriTap190) with a radius of 25 nm and a spring constant of 48 N/m was attached to a film sample. The sample was vibrated along the longitudinal axis by a laminated piezoelectric actuator that was operated by an nf-function generator (WF1965). A periodic compressive strain was applied to the film at a frequency of 7.3 kHz. The charge induced via the direct piezoelectric response was measured using a lock-in amplifier (NF LI5640). Ferroelectric domain images were obtained by measuring the direct piezoelectric response while scanning a specific area under constant force modulation.IP sin(ωt + θ), where IP is the amplitude of the induced current, ω is angular velocity, t is time and θ is phase. Therefore, the electric displacement component (D3) can be derived fromA is the contact area of the tip. Using the piezoelectric constitutive equationsSij is the strain, Tj is the stress, and Ek is electric field, e33, f can be written asThe current induced via the direct piezoelectric response is described as The strain applied to the film was calculated bye33, f  was obt, f  epitaxial film were visualised by rotating the sample in −55° increments around the (010) plane and combining the out-of-plane and in-plane converse piezoelectric responses. The details are described in Fig. S332.The mapping measurement of PFM was carried out by applying an AC voltage of 1.0 VSupplementary material


Covalent organic frameworks (COFs) have emerged as a promising light-harvesting module for artificial photosynthesis and photovoltaics. For efficient generation of free charge carriers, the donor–acceptor (D-A) conjugation has been adopted for two-dimensional (2D) COFs recently. In the 2D D-A COFs, photoexcitation would generate a polaron pair, which is a precursor to free charge carriers and has lower binding energy than an exciton. Although the character of the primary excitation species is a key factor in determining optoelectronic properties of a material, excited-state dynamics leading to the creation of a polaron pair have not been investigated yet. Here, we investigate the dynamics of photogenerated charge carriers in 2D D-A COFs by combining femtosecond optical spectroscopy and non-adiabatic molecular dynamics simulation. From this investigation, we elucidate that the polaron pair is formed through ultrafast intra-layer hole transfer coupled with coherent vibrations of the 2D lattice, suggesting a mechanism of phonon-assisted charge transfer. The donor–acceptor (D-A) conjugation has been adopted for two-dimensional (2D) covalent organic frameworks (COFs) for efficient generation of free charge carriers. Here, the authors investigate the dynamics of photogenerated charge carriers in 2D D-A COFs by combining femtosecond optical spectroscopy and non-adiabatic molecular dynamics simulation. Specifically, 2D COFs consist of π-conjugated aromatic molecules forming a 2D layer and the 2D layers stack up on top of each other to form a three-dimensional structure. Electronic interaction among the building blocks of a 2D COF and inter-layer π–π interaction among the stacked layers of COFs provide efficient pathways for photogenerated charge carriers. Due to their robust structure and high charge mobility through the framework, the COFs have the potential to serve as a primary light-harvesting module in photovoltaic and optoelectronic devices10.Covalent organic frameworks (COFs) with periodically ordered architecture have attracted much interest as a promising functional material for artificial photosynthesis and photovoltaic devices. In COFs, organic molecules are covalently bonded to each other to form two-dimensional (2D) or three-dimensional porous crystalline structures17. First, photons are absorbed by chromophores to generate charge carriers bound by Coulombic interaction . Then, the excitons diffuse toward a heterojunction, e.g., the polymer/fullerene interface, where an exciton is separated into free charge carriers . This process of charge separation in artificial light-harvesting systems occurs on ultrafast time scales ranging from tens of femtoseconds (fs) to several picoseconds16, as commonly observed in natural photosynthesis20.In artificial photosynthetic systems such as organic polymer solar cells, photons are converted into electrical energy via a series of steps22, borrowing the concept of low-bandgap polymers employing a donor–acceptor (D-A) conjugation scheme14. In those D-A COFs, the repeat units with low and high electron affinities can serve as an electron donor and an electron acceptor, respectively. As a result of the D-A conjugation, photoexcitation of 2D D-A COFs would lead to the formation of a polaron pair, which is a precursor to free charge carriers and has lower binding energy than an exciton, as in the low-bandgap D-A copolymers. Although the character of the primary excitation species is crucial for determining optoelectronic properties of a material, excited-state dynamics leading to the creation of a polaron pair in the 2D D-A COFs have not been investigated yet. According to previous studies of photoinduced charge carrier dynamics in D-A COFs22, it was suggested that light absorption induces charge transfer between each donor and acceptor units within 1.8 ps and the charges stay separated for up to 10 μs due to charge delocalization in two separate donor and acceptor arrays aligned perpendicular to stacked 2D layers. This result shows a unique molecular configuration of D-A COFs where the D-A interfaces serve as a well-ordered columnar heterojunction throughout the three-dimensional framework.For excitons to be separated into free charge carriers, the exciton-binding energy should be overcome. In this regard, for high-performance solar cells, it is desirable to have a light-harvesting system where excitations of low exciton-binding energy are generated. As a means of lowering the exciton-binding energy, researchers have recently developed 2D COFs consisting of two types of repeat units with different electron affinitiesIn this work, by applying both time-resolved spectroscopic experiment and theoretical simulation, we investigate the earliest stage  of charge carrier dynamics in 2D D-A COFs consisting of 3,4,9,10-perylenetetracarboxylic acid diimide (PDI) and free-base porphyrin. As the two constituent units have different electron affinities from each other, the 2D D-A COFs contain the D-A conjugation through π-electronic interactions between the building blocks. To elucidate the dynamics of photogenerated charge carriers in the 2D D-A COFs, fs-transient absorption (fs-TA) spectroscopy and non-adiabatic molecular dynamics (NA-MD) simulation are employed in this work. From the fs-TA measurement, we find that excited charge carriers are transferred between PDI and free-base porphyrin on ultrafast time scale with 124 fs time constant, leading to the formation of polaron pairs in the 2D lattice of COFs. From the systematic theoretical simulation based on time-dependent density functional theory (DFT), the ultrafast charge transfer can be specifically assigned to the migration of holes from PDI to free-base porphyrin unit. In addition, we demonstrate that this intra-layer charge transfer is coupled with lattice vibrations manifested as coherent oscillations of TA spectra, which is a compelling evidence for a mechanism of phonon-assisted charge transfer in the periodically ordered structure of COFs.c-axis with slipping along a-axis and/or b-axis. Moreover, the PXRD pattern is also more consistent with the staggered structure as shown in Fig. N,N-dimethylformamide (DMF) has absorption features in the entire visible region, including vibronic progression, as shown in Fig. 0 → S1 transition of PDI (peaked at 481 and 516 nm) and the Qy and Qx transitions of free-base porphyrin . The electronic interaction between PDI and free-base porphyrin units is manifested as spectral red shift and broadening of the absorption spectrum of COFs compared with the absorption spectrum of free-base porphyrin, as shown in Fig. Figure T/T, are shown in Fig. 25, the broad ESA band of free-base porphyrin overlaps with the SE of PDI, making it difficult to resolve the spectral features of porphyrin and PDI in the one-dimensional pump-probe spectrum. To overcome the spectral congestion, we performed global kinetic analysis of the TA spectra based on singular value decomposition (SVD). The details of the global kinetic analysis are described in the Supplementary Note 28. Thus, the A state is likely to be associated with PDI units of 2D COFs. The second SADS compared with the first SADS shows that the transition from A to B accompanies quenching of SE and/or spectral broadening of ESA. The SE at 490–625 nm is quenched further with the transition from B to C as can be seen in the spectral and temporal changes from the second SADS to third SADS shown in Fig. To investigate the dynamics of excited charge carriers of COFs dispersed in DMF, we performed fs-TA measurement. For excitation of COFs, we used the 40 fs pulses of 523 nm center wavelength with the bandwidth of 20 nm full-width-at-half-maximum (FWHM). This photoexcitation condition is predominantly resonant with the electronic transition of PDI units, as supported by the theoretical absorption spectrum calculated by time-dependent DFT calculation that will be discussed later. TA spectra, −∆y transition of free-base porphyrin, of which the absorption spectrum is shown in Supplementary Fig. x state of free-base porphyrin, which can be reached by Qy-to-Qx internal conversion and vibrational relaxation, as reported previously30. We note that the spectral feature of the valley shape around 645 nm in the second SADS of free-base porphyrin  and the Qy transition of free-base porphyrin (peaked at 513 nm). Although the relaxation of excited charge carriers generated from direct excitation of free-base porphyrin unit can occur, it cannot account for the large change of TA spectra induced by the A → B transition shown in Fig. 31, the relaxation of excited population in the S1 state does not accompany any significant change of TA spectra in the time window up to 2 ps. By considering our fs-TA experiment on free-base porphyrin and the previous spectroscopic study of perylene bisimide31, we can confirm that the spectral change accompanying the A → B transition is associated with charge transfer between the building units of COFs rather than relaxation of charge carriers at local chromophores.Based on the results obtained from the TA measurements on COFs and free-base porphyrin, in the TA spectra of COFs, the quenching of SE at 490–625 nm arising from PDI units concurs with the growth of SE at 645 nm arising from free-base porphyrin units. Thus, the A → B transition of COFs with the time constant of 124 fs can be attributed to the ultrafast transfer of excited charge carriers from PDI to free-base porphyrin. Meanwhile, the excitation at 523 nm can be resonant with both the SThe TA spectra of COFs shown in Fig. −1 over a broad range of probe wavelengths in the FTPS of both COFs and DMF, and these features are reproduced by the theoretical spectrum reconstructed from four Gaussian functions peaked at 364, 405, 659, and 861 cm−1. The frequencies of these peaks observed in the FTPS of DMF are very similar to the vibrational frequencies of 354, 405, 667, and 866 cm−1 observed in the Raman spectrum of DMF and thus those peaks can be assigned to the vibrations of C–N out-of-plane bending, CH3–N–CH3 in-plane bending, O=C–N in-plane bending, and N–CH3 symmetrical stretching of DMF, respectively32. In addition, the excitation pulse at 523 nm can excite the Qy transition of free-base porphyrin moiety. As shown in Supplementary Fig. −1 are distinctly different from the frequencies of 354, 405, 667, and 866 cm−1 observed in the neat DMF. Based on the previously reported resonance Raman and theoretical studies34, the two peaks at 85 and 321 cm−1 can be assigned to the torsional motion of side phenyl rings and the in-plane stretching of porphyrin, respectively. From the comparison of FTPS of COFs, neat solvent, and free-base porphyrin in the low-frequency region of <300 cm−1, we found that the COFs exhibit two prominent peaks at 76 and 285 cm−1, which are absent in the FTPS of neat DMF and free-base porphyrin monomer.Although the excitation pulse is resonant only with the electronic transition of COFs, the solvent still gives non-resonant, impulsive stimulated Raman response and thus also contributes to the FTPS. To distinguish the response of neat solvent from that of COFs dispersed in the solvent, we performed a separate fs-TA measurement on neat DMF. As shown in Fig. 37. As the long-lived oscillations of 76 and 285 cm−1 frequencies show nearly common phase over the entire range of probe spectrum, without any distinct nodal point  around the probe wavelength where the maximum of steady-state absorption spectrum of COFs is located, we can rule out the possibility that any of the oscillations arise from the ground-state vibrational coherence41. In Fig. −1 has a similar spectral shape as the SE signal of the first SADS, whereas the FTPS slice at 285 cm−1 has non-zero amplitude in the entire spectral range of the second SADS. We note that the first SADS in the 490–625 nm region is dominated by the SE of PDI units. Based on this result, we infer that the long-lived oscillations of 76 and 285 cm−1 frequencies can be assigned to molecular vibrations of excited-state COFs, specifically the former is related with the PDI units.In the 2D plot of temporal oscillations shown in Supplementary Fig. 43 was performed to investigate the ultrafast dynamics of charge carriers observed in the fs-TA measurement. The structure of COFs was modeled by using a periodic unit cell of monolayer +m notation, which denote the occupied and virtual KS states, respectively, of the simulated COF system, where the larger n (or m) means the state located farther away from the Fermi level. Due to the quasi fourfold rotational symmetry with respect to the center of free-base porphyrin unit in the 2D COFs, the nearly degenerate KS states were allowed and distinguished using the prime notation; e.g., HOMO−n′ or LUMO+m′. The low-energy singly excited states, which were chosen as the active space of our surface-hopping (SH) simulations, are labeled as Sa to Sx (note that the excited states are labeled in terms of LUMO energy in ascending order); the locations of an electron and a hole in each excited state are summarized in Supplementary Table NA-MD simulations based on time-dependent Kohn–Sham DFT (KS-DFT)0-to-Sb transition involving the KS orbitals mainly located along the vertical axis of COFs, and (2) an electronic transition from HOMO-3′ to LUMO′, which corresponds to the S0-to-Si transition involving the KS orbitals mainly located along the horizontal axis of COFs. For clarity, the directions of vertical and horizontal axes in the structure of COFs are indicated in Fig. b state decays with a time constant of 179 fs  and the electron located at PDI . Therefore, the transition from Sb to Sd or Sj involves hole transfer from PDI to porphyrin. The Si excited state, which consists of KS orbitals mainly located along the horizontal axis, also relaxes to Sd and Sj with the time constant of 173 fs, which is a time scale comparable with that of relaxation dynamics of Sb state (179 fs time constant) located along the vertical axis, as shown in Supplementary Fig. From the NA-MD simulation, we found that the photoexcitation at 523 nm employed in the fs-TA measurement induces the following: (1) an electronic transition from HOMO-3 to LUMO, which corresponds to the Sd state was also investigated. The Sd state decays slowly with a time constant of 1.55 ps  and the electron (LUMO and LUMO′) located at the PDI moieties, indicating the back transfer of holes from porphyrin (HOMO-2) to PDI (HOMO-3 and HOMO-3′) by the transition from Sd to Sb, Sc, and Si. The Se and Sk states have the hole at porphyrin (HOMO-1) and the electron at PDI (LUMO and LUMO′), indicating the relaxation of holes from HOMO-2 to HOMO-1 (at porphyrin) by the transition from Sd to Se and Sk. In the case of the Sj state, the electron is located at the horizontal PDI moieties and the hole is at the porphyrin units, indicating the transfer of electrons from vertical PDI (LUMO) to horizontal PDI (LUMO′) by the transition from Sd to Sj.From the additional set of NA-MD simulations, the relaxation dynamics of the S ps Fig. . Concomi−1 are summarized in Supplementary Table −1 is shown in Supplementary Fig. In addition to the charge carrier dynamics of COFs, vibrational normal modes of the COF monolayer were investigated in order to examine the structural motions associated with the charge carrier dynamics. For readers with interest, all calculated vibrational motions are provided using the animated GIFs in the Supplementary Data b state, which corresponds to the transition of HOMO-3 → LUMO, leads to concurrent growth of the populations of Sd and Sj states with the time constant of ~180 fs, matching the time scale of the ultrafast quenching of SE observed in the fs-TA data. As Sd and Sj states have holes at the porphyrin units and electrons at the PDI units as shown above, the transition from Sb to Sd or Sj induces hole transfer from PDI to porphyrin, resulting in the formation of a polaron pair in the 2D lattice of D-A COFs. Thus, the NA-MD simulation not only confirms the assignment of A → B transition but also reveals that it is the hole carrier that is transferred from PDI to porphyrin.The combined results of the fs-TA measurement and the NA-MD simulation provide comprehensive information on the charge carrier dynamics of COFs with the D-A conjugation. In the fs-TA measurement, it was observed that SE is quenched with 124 fs time constant by the A → B transition, which is assigned to charge transfer from PDI to porphyrin. In order to examine the excitation condition of fs-TA experiment, the theoretical linear absorption spectrum of COFs was calculated from the KS-DFT levels in conjunction with the ground-state MD simulation. As shown in Supplementary Fig. b and Sd/Sj states, respectively, and are located along the vertical axis of COFs. The similar energies of the two orbitals are expected to increase the rate of hole transfer in the vertical direction. Such quasi-degeneracy is also observed between HOMO-3′ and HOMO-2 orbitals located along the horizontal axis of COFs, suggesting ultrafast hole transfer (occurring with the transition from Si to Sd and Sj) in the horizontal direction as well.The ultrafast charge transfer identified in the present work may originate from the quasi-degeneracy of HOMO-3 and HOMO-2 orbitals, which constitute Sd occurs with 1.55 ps time constant, which matches the time scale of the B → C transition. Therefore, this kinetic component can be assigned to the relaxation of the polaron pair states, i.e., (1) porphyrin-to-PDI back transfer of hole carriers (to Sb/Sc/Si), (2) hole relaxation at porphyrin (to Se/Sk), and (3) electron transfer from vertical to horizontal PDI (to Sj)22. We note that a the current NA-MD simulation of COFs was implemented for single-layered COFs, only intra-layer charge transfer, not inter-layer charge transport, was considered. Meanwhile, in a previous TA study on D-A COFs composed of zinc phthalocyanine and naphthalene diimide 22, the earliest kinetic component with 1.8 ps time constant was observed and assigned to photoinduced electron transfer from ZnPc to NDI unit and subsequent inter-layer charge delocalization along separate donor and acceptor arrays perpendicular to stacked 2D layers. Therefore, considering the similarity of the time scales of the kinetic components obtained from the two TA studies (1.25 ps vs. 1.8 ps), the 1.25 ps component may be attributed to inter-layer charge transport.In addition to the ultrafast dynamics of hole transfer, the kinetic component of 1.25 ps time constant is identified in the TA spectra and assigned to the B → C transition. According to the NA-MD simulation, the relaxation of Sc-axis with slipping along a-axis and/or b-axis, as shown in Supplementary Fig. Although the pore size of COFs determined from the nitrogen isotherm shown in Supplementary Fig. −1 and 285 cm−1 frequencies observed in the fs-TA data reflect lattice vibrations coupled with the electronic transition of COFs. The detailed motions of those lattice vibrations can be examined with vibrational normal mode analysis of COFs. As the calculated lattice vibrations in the reticular structure of COFs show complex patterns of motions, we describe those motions in terms of vibrations of constituent units of COFs. The vibrational modes of 74.02 cm−1 and 77.12 cm−1 have the closest frequencies to the 76 cm−1 oscillation identified in the fs-TA measurement and these modes correspond to collective motions including (i) saddling motion of PDI moiety, (ii) in-plane and out-of-plane motions of free-base porphyrin moiety, and (iii) torsional motion of benzene ring that covalently links PDI and free-base porphyrin. The vibrational modes of 282.08 cm−1 and 286.26 cm−1 frequencies have the closest frequencies to the 285 cm−1 oscillation identified in the fs-TA data, and they contain (i) in-plane distortion of PDI moiety without any rotating motion of benzene linker and (ii) in-plane and out-of-plane motions of free-base porphyrin moiety. According to the vibrational normal mode analysis, the distinct difference between the 76 cm−1 mode and the 285 cm−1 mode is the change of the dihedral angle between the benzene ring and the layer plane of PDI and porphyrin; this angle changes in the 76 cm−1 mode, whereas it is almost kept constant in the 285 cm−1 mode. The torsional motion of the benzene linker in the 76 cm−1 mode can enhance its π-interaction with the PDI and porphyrin by planarization of PDI–benzene–porphyrin units and resultantly facilitates charge transfer between PDI and porphyrin. Therefore, the coherent oscillation of 76 cm−1 frequency observed in the fs-TA data is evidence of the intra-layer charge transfer mediated by the torsional motions of intervening π-bridge units. Such an ultrafast intramolecular charge transfer driven by the torsional motion of π-bridge has been also observed in various donor-bridge-acceptor copolymers45. Based on the population dynamics and the vibrational normal modes of COFs obtained from the quantum mechanical simulation, we conclude that the ultrafast dynamics observed in the fs-TA signal of 2D COFs originate from the ultrafast formation of polaron pair through hole migration between the constituent donor and acceptor units of COFs and such hole transfer is mediated by motions of lattice phonons. We summarize the results of the fs-TA measurement and the NA-MD simulations in Fig. The coherent oscillations of 76 cm21. Such hybrid COF architecture will be able to achieve efficient charge separation and charge transport of hole carriers, which are desirable for highly efficient photovoltaic devices.In this work, by combining fs-TA spectroscopy and NA-MD simulation, we directly observed ultrafast dynamics of phonon-assisted, PDI-to-porphyrin hole transfer in 2D COFs consisting of covalently linked electron donor and acceptor units. The key findings of this work can be utilized for the design of COF-based light-harvesting module applicable to high-performance optoelectronic devices. For example, the 2D D-A COFs can be combined with COFs consisting of only free-base porphyrins. The free-base porphyrin COFs in the form of well-stacked 2D layers exhibit high hole mobility through a periodically ordered macrocycle channelH,23H-porphyrin were prepared separately and then linked together by the polymerization process. In a 500 ml two-neck round-bottle flask, perylene-3,4,9,10-tetracarboxylic dianhydride  was dissolved in anhydrous pyridine (90 mL). To this solution, a solution of 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphyrin  in pyridine (40 mL) was added drop wise. After overnight stirring at 130 °C in argon atmosphere, the reaction mixture was cooled down to room temperature. The reddish-purple precipitate was filtered and washed with copious amount of distilled water, methanol, and chloroform. After drying in the oven at 110 °C, pale purple solid product was obtained. The details for the synthesis and the characterization of COFs are described in the Supplementary Note For the synthesis of COFs, perylene-3,4,9,10-tetracarboxylic dianhydride and 5,10,15,20-tetrakis(4-aminophenyl)-21c-cut sapphire window of 3 mm thickness and converted into white light continuum spanning from visible to near-infrared wavelengths by self-phase modulation. The visible portion (460–720 nm) of the white light continuum was used as broadband probe pulses without further compensation of the dispersion. The probe pulses were time-delayed with respect to the pump pulses using a motorized translation stage . By recording “pump-on” and “pump-off” probe spectra, the differential transmission (∆T/T) spectrum was obtained as a function of time. The spectra of transient signal and the reference were detected by a spectrometer  equipped with a Si CCD . In all the measurements, the polarization of the pump pulses was set to be at a magic angle (54.7°) relative to the probe polarization in order to prevent polarization-dependent signals. For the TA measurement, 2D COFs were suspended in DMF, which causes the exfoliation of the 2D COF layers from the bulk sample through sonication process. This exfoliation procedure improves solubility of COFs that is well suited for the TA spectroscopic measurement46. The pulse energy of the pump pulses was varied from 63 to 125 nJ to check the excitation energy dependence of TA signal. As the decay profiles of TA data were not dependent on the fluence of pump pulse shown in Supplementary Fig. 4), the instrumental response function of the TA measurement was determined to be 68 fs from the FWHM of cross-correlation function of the pump and the probe pulses measured at the sample position. The solvent signals were measured with neat DMF in the identical quartz cell as used in the measurement of the COFs sample dissolved in DMF.TA spectra were measured with fs laser pulses using a visible pump–broadband probe scheme. The output pulses at 800 nm wavelength from a Ti:sapphire amplified laser (Coherent Legend Elite) were split into pump and probe beams. On the pump arm, the 800 nm laser pulses were converted into the pump pulses of 523 nm wavelength and 20 nm bandwidth using a home-built, all-reflective-optic noncollinear optical parametric amplifier. The pump pulses were sent through a pair of Brewster-cut fused-silica prisms to pre-compensate for the dispersion obtained from transmissive optics and compressed to near-transform-limited pulses at the sample position. On the probe arm, the 800 nm laser pulses were sent into a 43. This method employs the classical path approximation (CPA)48—assuming the evolution of nuclear dynamics being independent of the electrons, which allows the practical investigation of the real-time relaxation dynamics of the photoexcited systems with periodic boundary conditions by means of the SH with KS-DFT states. We note that the validity of CPA has been tested against a number of systems, in which electronic excitation barely affects the nuclear dynamics according to previous studies52. In this regard, the charge-transfer dynamics, accompanied by the structural motion, can be properly explained by the NA-MD simulation based on the PYXAID code.We performed NA-MD simulations as implemented in the PYXAID code53, we first optimized a unit cell of COFs  functional54 with a plane-wave cutoff energy of 35 Ry and only the gamma point is sampled in the reciprocal space. After applying scissor operations to the virtual states to reproduce the experimental absorption energy  as follows:55 should be corrected to obtain a reasonable time constant (Supplementary Fig. 56 scheme. For the calculations of PXRD patterns and vibrational modes, we used Vienna ab initio simulation package58 with the choice of same PBE xc-functional and the plane-wave cutoff energy of 450 eV.To obtain statistically meaningful population dynamics of excited states, five 400 fs NA-MD trajectories were obtained and averaged. The time constant, Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14


Two deformation transitions are found experimentally: from coactivated twinning/detwinning (λ < 5 nm) to secondary twinning (5 nm < λ < 129 nm), and then to the dislocation glide (λ > 129 nm). The simulation results are highly consistent with the observed strong λ-effect, and reveal the intrinsic transition mechanisms induced by partial dislocation slip.Understanding the deformation behavior of metallic materials containing nanotwins (NTs), which can enhance both strength and ductility, is useful for tailoring microstructures at the micro- and nano- scale to enhance mechanical properties. Here, we construct a clear deformation pattern of NTs in austenitic stainless steel by combining in situ tensile tests with a dislocation-based theoretical model and molecular dynamics simulations. Deformation NTs are observed in situ using a transmission electron microscope in different sample regions containing NTs with twin-lamella-spacing ( The deformation mechanisms of twin boundaries in nanotwinned metallic materials are still unclear. Here the authors combine in situ transmission electron microscopy tensile tests and molecular dynamics simulations with a dislocation-based theoretical model to reveal the deformation mechanism of nanotwins. However, the strength-ductility synergy of NTed materials is controlled by the deformation mechanisms, involving dislocation pile-up at the twin boundary (TB), partial dislocation slip along the TB, and TB migration, or detwinning10. Many researchers have explored the coupling mechanisms among these deformation modes16. For example, Li et al. reported that the plastic deformation transited from inclined dislocation glide to detwinning in NTed Cu when the twin-lamella-spacing (λ) decreased from 15 to 4 nm17, and this transition might result in maximum strength in NTed Cu, as observed in experimental tests18. Other molecular dynamics (MD) simulations suggested that the movement of partial dislocations, involving 60° dislocation and screw dislocation systems, transited from cutting across TB to dislocation-induced TB migration at a critical λ of 2.4 nm21. Yuan et al.22 predicted that the Hall-Petch strengthening effect of the secondary twinning deformation was more effective than that of the primary twinning and that there is a critical λ of secondary twinning to achieve maximum strength. However, the activation of secondary twinning is still not well understood. It is challenging to clarify the coupling relationship of λ-dependent deformation behaviors in face-centered cubic (FCC) metals with NTed structure.The nanotwin (NT) is strongly thought to be a desirable structure for overcoming the dilemma between strength and ductility28. Different from the growth NTs in Cu, Ag, and Ni metals and their alloys30, the NTs in the structural steels, commonly produced by severe plastic deformation (SPD), are deformation NTs that exhibit a high density of defective structures34. However, the effects of defective TBs on the deformation behaviors are less understood in nanotwin-strengthened materials. Furthermore, martensitic transformation is the most likely deformation mode in NTed austenitic steels because of the metastable austenite phase39. Hence, it is difficult to observe or predict the deformation evolution due to indistinguishable structures in postmortem observations or the multiscale diversities in simulations. No direct evidence elucidates how to activate these deformations and what kinetics sequences occur during deformation. Thus, a full-scale deformation map of NTs should be established to provide a clear strengthening mechanism for microstructure manipulation.As the second generation of advanced high strength steels, metastable austenitic steels such as austenitic stainless steel (SS) and high manganese steel are widely used in the chemical, transportation, and aerospace industries. These steels with NTed structure also exhibit ultrahigh strength, good ductility, and high work hardeningλ-dependent effect on the deformation transitions of NTed steels. Austenitic SS is selected as a template from a structural materials perspective. We performed in situ transmission electron microscopy (TEM) tensile tests to obtain direct evidence of the deformation characteristics, applied a dislocation-based theoretical model and conducted MD simulations to reveal the deformation transition mechanisms of NTs. Both the in situ tests and the simulation results show that there are two critical λs to transit the deformation mode from twinning/detwinning to secondary twinning, and then to dislocation accumulation. We generated a deformation map of NTs as a function of λ, which may aid in the design of advanced steels with superior strength-ductility synergy.In this work, we investigate the 1 exhibits a 70° angle to the loading direction and TB2 is approximately parallel to the loading direction. Supplementary Figure 2 direction and NT 4 along the TB1 direction exhibit partial twinning, as shown in Fig. 2 direction, and NTs 5  in type 304 SS. The detailed microstructures and in situ tensile method are given in s 5 Fig. , 6 and 7s 5 Fig.  along th40, which induces an earlier formation of martensitic transformation than the other coarse grains (CG). The above observations clearly indicate the detwinning of NTs (λ < 5 nm) preferentially occurs before the martensitic transformation. Thus, the deformation sequence of the NTs (λ < 5 nm) is detwinning and martensitic transformation when the TB is closely parallel to the loading direction.When the TB is closely parallel to the tensile direction, detwinning of the primary NTs is dominant at first and then martensitic transformation is promoted , where the λ of NTs 1–5 are 129, 42, 15, 6 and 5 nm  exhibited the coactivation of twinning and detwinning under shear stress, including intersected and primary NTs. 100% of NTs with λ < 5 nm (two observed zones) exhibited detwinning and the subsequent martensitic transformation when the TBs are closely parallel to the tensile direction. Secondary twinning occurred in primary NTs (λ = 6–129 nm) with a frequency of 80% among the five observed zones. Only dislocation motion was observed in the left two zones, and the λ of these twins are at the submicrometer scale. The statistical diagram is given in Supplementary Figure λ is smaller than 5 nm, the twinning and detwinning are jointly activated under shear stress and detwinning and martensitic transformation occur successively when the TBs are closely parallel to the loading direction. When λ is 6 to 129 nm, secondary twinning is induced inside the primary twin system, and the twinning and detwinning are coactivated only in the secondary twin system. As λ increases to the submicrometer scale, dislocation motion is the featured deformation behavior, as observed in TEM tensile tests  and the trailing stress (lower bound), thus, the twinning deformation can be initiated. The two critical values, transition points I and II, are very close to the in situ TEM observation results. Furthermore, the theoretical results also predict that the confined range is strongly related to the orientation angle of the TB with the tensile stress and the types of partial dislocations. For example, the λ is 4.8–186 nm for the 60° system at 85.2°, and 6.7–237 nm for the screw system at 26.5°  was carried out to investigate the deformation transition mechanisms   Fig. . Secondaide Fig. . In the ide Fig. , leadingide Fig. . Thereaf2nd Fig. . The amp33, but they are the two classic types of dislocation systems to activate the twinning/detwinning deformation16. Furthermore, the activation of twinning/detwinning depends on the magnitude of twinning, trailing, detwinning and local stress and is influenced by the orientation angle of the TBs with loading direction. Figure λ = 2.5 nm was selected as a typical scale and the zone of unit length (L) was 100–500 nm according to the visual sight of the TEM observations. When the twinning stress is smaller than both the trailing stress and the local stress, twinning is initiated. Similarly, when the detwinning stress is less than the local stress, detwinning occurs. In Fig. L < 160 nm and larger than the trailing stress in most of the observed area (L > 160 nm), suggesting that twinning of the 60° partial system occurs with a low probability. However, the detwinning stress is lower than the local stress in the observed area (L > 40 nm), thus, detwinning is dominant. In contrast to the 60° partial system, the trailing stress of the screw system is negative at L < 370 nm and larger than the twinning stress at L > 370 nm, therefore, twinning occurs in the entire observed area . However, detwinning of the existing NTs is triggered in the entire observed area (L > 60 nm). Thus, for the screw system at a 9° angle of the TB to the loading direction, detwinning is the dominant deformation behavior in the observed area, and twinning is also activated at a low frequency. Finally, the NTs disappear due to the overwhelming majority of detwinning compared to twinning.In the case of a 9° angle, twinning is very hard to initiate for the 60° system due to a larger twinning stress than the trailing stress Fig. , and detλ from several nanometers to hundreds of nanometers using in situ TEM observations combined with a theoretical model and MD simulations. For NTs with λ < 5 nm, twinning and detwinning are concurrently activated under shear stress. The partial dislocation of the 60° system is primarily responsible for the detwinning, whereas the screw system exhibits twinning deformation. Detwinning is dominant when the TB orientation is closely parallel to the tensile direction. The formation of secondary twinning has a critical range of λ = 6–129 nm, which is highly consistent with the results of the dislocation-based theoretical model and MD simulations. Our methods are not specific to particular metals, so the conclusion should be generally applicable to other NTed FCC metals.To conclude, we established a deformation map of NTs with a 33. The specimens for in situ tests were taken 150 μm from the surface and prepared by mechanical polishing to 30 μm thickness. The 5 mm × 2 mm × 30 μm foils were thinned by twin-jet electropolishing, as illustrated in Supplementary Figure F, was uniaxial along the horizontal direction. Four samples with 14 microzones were observed through in situ TEM experiments, where seven observed zones had NTs with λ < 5 nm, five observed zones had NTs with 5 nm < λ < 129 nm, and two observed zones contained twins with λ at the submicrometer scale. Details of the sample preparation, dimension and movies are given in Supplementary Note The NTed 304 SS was produced by SMAT with the GCr15 balls randomly impacted onto the specimen surface with an impacting frequency of 20 kHz. The detailed processing parameters and microstructure features are given in ref. 41. An embedded atom method potential for the Fe–Ni–Cr system was employed to describe the atomic interaction in the samples43. The MD simulation results exhibited in this work were colored complying with the following standard: dark blue, light blue, and red represent FCC, hexagonal close packed and disordered atoms, respectively. The models were prepared to be an FCC structure with a lattice constant of 3.562 Å. The relaxed samples were subjected to uniaxial tensile simulation along the y-direction with 0° and 70° angles between the TB orientation and tensile direction.A dislocation-based theoretical model (see Supplementary Note Supplementary InformationPeer review fileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


Bacteriophage endolysins, enzymes that degrade the bacterial peptidoglycan (PG), have gained an increasing interest as alternative antimicrobial agents, due to their ability to kill antibiotic resistant pathogens efficiently when applied externally as purified proteins. Typical endolysins derived from bacteriophage that infect Gram-positive hosts consist of an N-terminal enzymatically-active domain (EAD) that cleaves covalent bonds in the PG, and a C-terminal cell-binding domain (CBD) that recognizes specific ligands on the surface of the PG. Although CBDs are usually essential for the EADs to access the PG substrate, some EADs possess activity in the absence of CBDs, and a few even display better activity profiles or an extended host spectrum than the full-length endolysin. A current hypothesis suggests a net positive charge on the EAD enables it to reach the negatively charged bacterial surface via ionic interactions in the absence of a CBD. Here, we used the PlyC CHAP domain as a model EAD to further test the hypothesis. We mutated negatively charged surface amino acids of the CHAP domain that are not involved in structured regions to neutral or positively charged amino acids in order to increase the net charge from -3 to a range from +1 to +7. The seven mutant candidates were successfully expressed and purified as soluble proteins. Contrary to the current hypothesis, none of the mutants were more active than wild-type CHAP. Analysis of electrostatic surface potential implies that the surface charge distribution may affect the activity of a positively charged EAD. Thus, we suggest that while charge should continue to be considered for future engineering efforts, it should not be the sole focus of such engineering efforts. Bacteriophage (phage) endolysins are peptidoglycan (PG) hydrolases produced by phage at the end of a lytic cycle . In the Endolysins derived from phage that infect Gram-positive hosts have very similar modular structures with one or more N-terminal enzymatically-active domains (EADs) and a C-terminal cell wall binding domain (CBD) . The EADBacillus anthracis phage endolysin, PlyL [Clostridium difficile phage endolysin, CD27L [Whereas many EADs require the presence of the CBD for binding and subsequent activity, some EADs can bind the bacterial surface independently of the CBD, and a few even have increased enzymatic activity compared to the full-length endolysin. One example is the staphylococcal phage endolysin, LysK. The LysK EAD, a cysteine-histidine amidohydrolase/endopeptidase (CHAP), alone displays higher lytic activity against staphylococci than the full-length LysK . Similarin, PlyL  and the n, CD27L .Bacillus subtilis prophage endolysin, XlyA, had a net charge (Z) of -3 at neutral pH and displayed no lytic activity against B. subtilis cells in the absence of its CBD. Site-directed mutagenesis of five non-cationic residues to lysine (K) produced a shift in net charge from Z = −3 to Z = +3, and the mutated XlyA EAD alone was able to lyse B. subtilis cells at a rate nearly identical to that of full-length XlyA. In a separate study, the addition of a positively-charged peptide enhanced the lytic activity of the λSa2lys endolysin [The reason(s) as to why some EADs can target and lyse the PG in the absence of a CBD, whereas the presence of a CBD is an absolute requirement for activity in other EADs, is unknown. However, a thought-provoking study by Low et al.  suggestendolysin , suggestIn the present work, we sought to validate Low’s hypothesis. The model EAD for this study is the CHAP domain from the PlyC endolysin . This EAThe PlyC CHAP domain  was isolated from the PlyC holoenzyme crystal structure and edited in PyMOL (atomic coordinates were only available for amino acid 310-464). Five surface and unstructured residues, Asp-311, Asp-355, Asp-363, Asp-429, and Asp-450, were selected for mutagenesis . ThroughFoldX = ΔGmut – ΔGWT). A negative ΔΔGFoldX (ΔΔGFoldX < 0) suggests that the mutation is more stable than the WT protein and should fold properly. However, 79% of the mutants were predicted to have a positive ΔΔGFoldX, meaning these mutations had destabilizing effects  for better activities and/or an altered or expanded host range. This engineering approach has been exploited by nature itself through horizontal gene transfer, such as the pneumococcal endolysin Pal whose EAD is most similar to the endolysin of dolysins . A well-dolysins ,28. Thisdolysins , Cpl-711dolysins , Csl2 [3dolysins , and PL3dolysins  result idolysins ,34.in silico and calculate the effect these mutations have on protein stability through the approximation of Gibbs free energy (ΔG). The top resulting mutants can then be made and characterized experimentally. Such an approach was recently done to introduce a thermostabilizing mutation, T406R, to the EAD of PlyC. This point mutant introduced a critical hydrogen bond between two domains and resulted in a 16 fold increase of half-life at 45 °C [Structure-guided mutagenesis is expected to expand bioengineering efforts toward modulating endolysin properties as more high resolution X-ray crystallography and NMR structures are solved. The atomic coordinates can be used for computational modeling to predict mutations that can affect enzymatic activity, substrate binding affinity, and interactions with the solvent interface or intra- or inter-domain interactions that may affect stability of the enzyme. Notably, several algorithms, such as Rosetta  and Foldat 45 °C . B. subtilis in the absence of its CBD, the mutant EAD, XlyA+5K, possessed WT XlyA activity independent of the CBD. The authors postulated that the net positive charge allowed for interaction with the “continuum of negative charge” on the secondary cell wall polymers on the bacterial surface, thus obviating the need for a CBD. To contrast this gain-of-function experiment, the authors also tested a loss-of-function engineering approach using the PlyBa04 EAD, which carries a net charge of Z = +1. Unlike the native XlyA EAD, the PlyBa04 EAD is able to lyse its target, B. cereus, as efficiently as the full-length endolysin. However, after site-directed mutagenesis to change the net charge from Z = +1 to Z = −3, the resulting PlyBa04 EAD mutant showed negligible lytic activity towards B. cereus. The authors concluded that engineering a reversal of net charge on an EAD could be used to either create or eliminate CBD dependence, which could be used in future bioengineering studies to fine-tune endolysin activity. Two years later, a separate study by Diez-Martinez et al. [Low et al.  used simz et al.  with theThe ultimate goal of our study was to create an engineered endolysin that is simple  and works on a very broad host range . Toward this end, we sought to engineer the PlyC CHAP domain to be such an enzyme using engineering principles guided by the findings of Low et al. . Toward Our results indicated that none of the positively charged CHAP mutants displayed higher lytic activity than WT CHAP. Thus, at least for the PlyC CHAP, the hypothesis developed by Low et al.  is not sStreptococcus pyogenes D471 was cultured from a −80 °C frozen stock and grown in Todd Hewitt broth supplemented with 1% yeast extract (THY) without shaking at 37 °C. E. coli strains DH5α and BL21 (DE3) were grown in Luria-Bertani (LB) broth. When needed, kanamycin (50 μg/mL) was added to the media. All bacterial cultures were grown at 37 °C in a shaking incubator unless otherwise stated. FoldX). The desirable mutants possessed ΔΔGFoldX < 0 kcal/mol (ΔΔGFoldX = ΔGmut – ΔGWT) and the mutants with the largest negative ΔΔGFoldX were then picked for experimental study. The electrostatic surface potential was imaged using CCP4MG [The crystal structure coordinates were obtained from the Protein Data Bank for PlyC (4F88), XlyA (3RDR), and XlyA+5K (3HMB). The strategy used to change the net charge (Z) of the CHAP domain was to substitute negatively charged amino acids, aspartic acid (D) and glutamic acid (E), that were surface exposed and not involved in structured regions  to neutral ) or positively charged (lysine (K)) amino acids to increase the Z score from −3 to +1 through +7 at pH 7.4. The net charges were calculated from the online Protein Calculator Version 3.4 . PyMOL  before being transformed into E. coli BL21 (DE3) for protein expression.The primers used in this study are listed in plyC  and clonE. coli BL21 (DE3) harboring the WT PlyCA CHAP domain or mutants were sub-cultured 1:100 into 1.5 L LB supplemented with kanamycin in a 4 L baffled Erlenmeyer flask at 37 °C in a shaking incubator. The culture was induced at mid-log phase (about 4 h) with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 18 °C overnight. The next morning, E. coli BL21 (DE3) bacterial cells were harvested at 5,000 rpm, resuspended in PBS, pH 7.4, sonicated, and clarified via centrifugation at 12,000 rpm at 4 °C. The soluble portion of the cell lysate was applied to a Ni-NTA resin column . The 6X His-tagged protein was washed and eluted using a gradient of imidazole from 20 mM to 500 mM in PBS buffer, pH 7.4. The protein purity was assessed on a 7.5% SDS-PAGE gel before dialysis to remove the imidazole. The 6x His-tag was removed using the Thrombin Cleavage Capture Kit  according to the protocol provided by the manufacturer.The overnight cultures of S. pyogenes D471 was harvested at 4500 rpm for 10 min, washed twice and resuspended in PBS, pH 7.4 buffer to reach an OD600 = 2.0. In a flat-bottom 96-well plate (Thermo Fisher Scientific), bacterial cells were mixed 1:1 with equimolar amounts of the PlyC CHAP domain or its mutants and the OD600 was monitored every 15 sec for 1 hour at 37 °C using a SpectraMax 190 spectrophotometer . The OD600 decrease was calculated after 1 hour treatment by calculating the difference in OD600 readings between the PBS treated bacteria and the enzyme treated bacteria. Each assay was conducted in triplicate.The activities of the PlyC CHAP domain and its mutants were evaluated via a spectrophotometric-based turbidity reduction assay as described previously . An overBacteriophage-encoded endolysins offer an emerging alternative to conventional antibiotics. These enzymes function to degrade the bacterial peptidoglycan, producing fatal osmotic lysis in susceptible Gram-positive bacteria. A cell wall binding domain (CBD) is often, but not always, required for activity. In some endolysins, the catalytic domain alone is sufficient for lysis. In these examples, it has been noted that the catalytic domains have a net positive charge, suggesting they can interact with the negatively charged bacterial surface in the absence of a CBD. Protein engineering studies support this hypothesis by showing that a change in net charge of a catalytic domain, from negative to positive, correlated with CBD independence. However, similar experiments on the PlyC endolysin catalytic domain reported here do not support the hypothesis. While not discounting the charge hypothesis, other factors, such as distribution of surface charges, may also affect the CBD dependence of an endolysin and should be considered for future protein engineering efforts.


Mycobacterium avium subsp. paratuberculosis (MAP), is the ability to track disease incidence over space and time. JD surveillance in the U.S. dairy cattle is challenging due to lack of regulatory requirements, imperfect diagnostic tests, and associated expenses, including time and labor. An alternative approach is to use voluntary testing programs. Here, data from a voluntary JD testing program, conducted by the Minnesota Dairy Herd Improvement Association, were used to: a) explore whether such a program provides representative information on JD-prevalence in Minnesota dairy herds, b) estimate JD distribution, and, c) identify herd and environmental factors associated with finding JD-positive cows. Milk samples  collected from 54,652 unique cows from 600 Minnesota dairy herds between November 2014 and April 2017 were tested using a MAP antibody ELISA. Participant representativeness was assessed by comparing the number of JD-tested herds with the number of herds required to estimate the true disease prevalence per county based on official statistics from the National Agricultural Statistical Services. Multivariable logistic regression models, with and without spatial dependence between observations, were then used to investigate the association between herd status to JD (positive/negative), as indicated by milk ELISA results, and available covariates at the herd level.One of the key steps in the management of chronic diseases in animals including Johne’s disease (JD), caused by Within the study population, at least one test-positive cow was found in 414 of 600 (69%) herds. Results indicated that large herds that test frequently and herds located in loamy or silt soils are more likely to have at least one MAP test-positive cow. After adjusting for herd size, testing frequency, and soil type, there was no spatial dependence in JD risk between neighboring dairies within 5 to 20 km. Furthermore, the importance of collecting data on herd management, feed, and biosecurity for insightful interpretations was recognized. The study suggested that, although limited, the voluntary testing database may support monitoring JD status.Results presented here help elucidate the spatial characteristics of JD in Minnesota and the study may ultimately contribute to the design and implementation of surveillance programs for the disease. Mycobacterium avium subsp. paratuberculosis (MAP) [Johne’s disease (JD) is a chronic, debilitating enteritis that affects ruminants and is caused by infection with is (MAP) . Financiis (MAP) . Reducedis (MAP) –6. In adis (MAP) . A recenis (MAP) .The management and control of a chronic disease such as JD in a proactive and organized manner is challenging in the U.S. due to lack of regulatory requirements for testing , imperfeJD is widespread in the U. S, and herd prevalence has been estimated as 60.7% in Midwestern U.S. dairies  and 91.1http://www.dhia.org/members.asp). However, it is unknown if data collected by the MNDHIA is useful as a passive surveillance tool to monitor JD in Minnesota.Due to the lack of official disease monitoring, a common alternative for evaluating the epidemiological status of JD in a region is the use of data from voluntary testing programs, such as those collected by the Minnesota Dairy Herd Improvement Association (MNDHIA). Minnesota, a Midwestern state of the U.S., has nearly 460,000 dairy cattle and is among the top ten dairy states, 6th in terms of milk cow numbers and 8th in dairy herd numbers, per 2016 statistics available from the National Agricultural Statistics Services (NASS) , 18. A pThe objectives of the study here were to a) test whether the voluntary JD testing program conducted by the MNDHIA can provide representative information on the prevalence of JD in dairy herds in Minnesota, b) estimate the JD distribution in Minnesota using data from the MNDHIA voluntary JD testing program, and c) identify possible herd and environmental variables associated with increased risk of having JD milk ELISA test-positive cows, using the available data. We hypothesized that results from the voluntary JD program might be used to evaluate JD status in an area and inform management decisions made by the testing agencies, veterinarians, and dairy producers. In evaluating the use of the MNDHIA database as a passive surveillance tool, our overarching objective was to generate evidence that could influence management decisions by recognizing modifiable factors to reduce the JD risk at the individual, herd, and regional levels. Results could therefore be useful in the design and implementation of surveillance programs for the U.S. dairy industry.During the 2.5-year study period, 600/4746 13%) dairy herds in Minnesota tested at least once for JD at MNDHIA laboratories, representing 18.7% (600/3210) of the licensed dairy herds in Minnesota with permits to ship milk for human consumption . Figure 3% dairy n = 332) and large  dairies. Figure The apparent herd-level prevalence of MAP, based on having 1+ cows with a positive milk ELISA, was 69% (414/600). The MNDHIA herds in this study included both small . Similarly, results of the fitted CAR model suggested that the spatial dependence for dairy herds located between 5 and 20 km was not significant after adjusting for the covariates, and its AIC value was higher than that of the model without the spatial component  were retained in the final multivariable model Table . Herd tuThis study suggests the use of an existing voluntary testing program as a passive surveillance system to track JD in Minnesota. We demonstrated that the MNDHIA voluntary testing program may be a useful source to investigate the JD status of Minnesota dairies, with program participants in 2014–2017 representing 13% of the dairy herds across Minnesota and coming from areas with both high and low density of dairy farms. Even though county-level JD herd prevalence could not be reliably estimated because the required sample sizes were not achieved in most counties, we were able to estimate the herd-level JD status for the study area as 69%, as 414 of the 600 herds had at least one cow tested-positive for JD milk ELISA. As per the epidemiological factor analysis, the most important epidemiological factors contributing to the JD status of a herd were herd size, testing frequency, and soil type, i.e. texture. We did not observe spatial dependence of the residuals of the regression model indicating that the observation of similar characteristics in JD status in the participant dairy herds were explained by the three covariates, namely, herd size, testing frequency, and soil type/texture. These results will be used to inform the potential use of the database as a surveillance tool and to suggest improvements in JD testing program conducted by MNDHIA.JD positive herds were distributed throughout Minnesota, although herds in the southern region were more likely to be JD milk ELISA test-positive compared to herds in the north-central region Fig. : Panel aThe herds located on silt, loam, or clay soils were more likely to be JD positive compared to the herds located on sandy soils. This observation contradicts early studies in the Midwest , which fSome of the limitations associated with this study include the limited generalizability of the results because MNDHIA data represents a purposive, non-random proportion of dairy herds in Minnesota. Moreover, the lack of information on herd characteristics other than herd size and testing frequency is another limitation that should be considered when interpreting the results. A more insightful interpretation requires herd management details including maternity pen management, which cows were chosen for testing for JD (high-risk cows or cows that were being sold), and the management decisions towards JD positive cows . AlthougAlthough MNDHIA does not currently offer a control program, through this study we recognized opportunities to improve the MNDHIA database to be used as a passive surveillance tool. These opportunities include: the determination of the number of herds to be sampled to establish the prevalence of JD in Minnesota, the determination of the number of animals to be sampled from each herd to ensure a reliable evaluation of its JD status (positive/negative), ensuring that farmers provide accurate farm location information, and regularly collecting herd-level information on other relevant factors such as biosecurity measures to facilitate a better assessment of JD status/risk. Moreover, having the disease and underlying factors collected frequently over the time would facilitate conducting spatiotemporal analysis and enable facilitate making temporal confidence of “disease free zones” instead of static estimates generated through a cross-sectional analysis.http://www.dhia.org/members.asp) and their network already acts as a record keeping system for dairy farms. Having a system to evaluate the JD status in a region would benefit the dairy industry in multiple ways, such as recognizing the differences between participant and non-participant dairy farms in the voluntary testing programs, understanding underlying risk factors and covariates in the neighborhoods, and eventually recognizing disease-free areas [https://queries.uscdcb.com/) together would have been a useful system to trace the movements/transfers or termination of individual cows, which could facilitate further investigations of how cattle movement play a role in the JD transmission. However, it is important to recognize that the choice to test for JD may not necessarily overlap temporally to make a causal association between cow transfers and JD.A JD surveillance program would be costly to establish and a voluntary testing database could be preferred for monitoring endemic pathogens causing chronic diseases like MAP. Thus, the strength of using MNDHIA data as a source is that there are 16National Dairy Herd Improvement (DHI) Association laboratories across the U.S. (Participation in voluntary testing and control programs varies due to multiple factors such as: a) farmers’ belief in the importance of JD , b) farmIn summary, results reported here suggest that a routinely generated database obtained from a voluntary testing program can be used as a passive surveillance tool to monitor the infection status and epidemiological determinants of JD in a region. However, because the risk of introduction may always be present, successful prevention and control of JD depends on ongoing willingness to continue funding surveillance and research on the disease by both animal health authorities and the support from the community and stakeholders of livestock industry , 33.Data from the voluntary JD testing program conducted by the MNDHIA were collected through a 2.5-year period, between November 1st, 2014 and April 30th, 2017. Although there were records from 723 JD testing herds, 123 herds were excluded from the study  collected from 54,652 unique cows from 600 Minnesota dairy herds were included in the study. Samples were analyzed using the IDEXX MAP Enzyme-Linked Immunosorbent Assay (ELISA)  for detection of antibodies against MAP in milk. Some herds  were tested only once during the 2.5-year study period. The median number of times a herd was tested was 2 (interquartile range between 1 and 11).Milk samples , which was based on remote sensing data creating the Land Cover Data Portal under the National Gap Analysis Project (GAP) [Layers of soil pH and soil type in Minnesota were obtained from the Natural Resource Conservation Services of the United States Department of Agriculture (USDA) , 50, 51.ct (GAP) . The Minn = 1; Cook county). To evaluate whether the study herds were representative of all Minnesota dairy herds, the number of study herds per county (tested for JD) was compared to the appropriate sample size, i.e. number of dairy herds to be sampled from each county to estimate the true herd prevalence of JD using an imperfect test and adjusting for a finite population  [Minnesota counties were classified into terciles based on the number of NASS dairy herds present . Counties without records for milk cow herds in the NASS 2012 statistics were excluded for this calculation (.com.au) , 55. Tot.com.au) . In addi.com.au) , a preciThe apparent JD prevalence, the spatial distribution of JD milk ELISA test-positive farms, and the presence of spatial autocorrelation in the risk of JD and in other covariates considered were visualized and, for the latter, estimated using Morans’ I and Getis Ord Gi* statistics , 20, 57.http://epitools.ausvet.com.au) [The output variable (herd JD status) was dichotomized as follows: 1) ‘Negative herds’ in which an appropriate number of unique cows were tested during the study period and were all negative (see below definition of appropriate number) and 2) ‘Positive herds’ in which at least one cow was tested positive on the milk ELISA test during the entire study period. The appropriate sample size to certify disease freedom in a herd, i.e. minimum required sample size per herd, using an imperfect test and adjusting for a finite population, was set for each herd using the AusVet EpiTool Epidemiological calculator (URL: .com.au) , 55. Ass.com.au) , and b) The outcome variable used in all models was the farm-level JD milk ELISA test-status . The herd size, which ranged between 1 and 1929 cows, was categorized using Jenks natural breaks method . Similarp-value < 0.2 and that were not significantly associated among them  were tested as candidate variables in the full multivariable logistic regression model. To prevent overfitting, the full model, including all possible 2-way interactions deemed biologically plausible, was subjected to backward stepwise regression based on the lowest Akaike Information Criterion (AIC) until the most parsimonious  model was fitted. The regression analysis was carried out using the R Statistical Software . For the descriptive analysis and logistic regression, we used R packages ‘Base’ [To avoid multicollinearity, a simple logistic regression was used to assess the marginal association between JD status and the covariates. The strength of the association between pairs of covariates was analyzed using Chi-square test followed by a Cramer’s V test. Variables with s ‘Base’  and ‘MASs ‘Base’ .Evidence of spatial autocorrelation in the residuals of the final regression model was assessed using the global Morans I and local Getis Ord Gi* tests. To account for potential spatial dependence in the outcome variable, a proper conditional autoregressive model (CAR) structure was included in the model  using thAdditional file 1: Figure S1. The study population . The 600 herds here 18.7% of the licensed dairy herds in Minnesota with permits to ship milk for human consumption [innesota  comparedAdditional file 2: Table S1. Environmental factors assessed for association with Johne’s disease status in Minnesota dairy herds participating in the voluntary testing program conducted by Minnesota Dairy Herd Improvement Association. Table S2. Akike information criterion (AIC) values for the model fits.


Zymogen PK is activated to PKa and cleaves substrates kininogen and FXII contributing to bradykinin generation.Monomeric PKa and dimeric homologue FXI utilize the N‐terminal apple domains to recruit substrates.A high‐resolution 1.3 Å structure of full‐length PKa reveals an active conformation of the protease and apple domains.The PKa protease and four‐apple domain disc organization is 180° rotated compared to FXI.PK) and factor XI (FXI) are apple domain‐containing serine proteases that when activated to PKa and FXIa cleave substrates kininogen, factor XII, and factor IX, respectively, directing plasma coagulation, bradykinin release, inflammation, and thrombosis pathways.Plasma prekallikrein  extend from the PKa protease domain to form interactions with the apple 1 and apple 3 domains, respectively. A major unexpected difference in the PKa structure compared to FXI is the 180° disc rotation of the apple domains relative to the protease domain. This results in a switched configuration of the latch loops such that LL2 interacts and buries portions of the apple 3 domain in the FXI zymogen whereas in PKa LL2 interacts with the apple 1 domain. Hydrogen‐deuterium exchange mass spectrometry on plasma purified human PK and PKa determined that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK, suggesting conformational change upon activation.A 1.3 Å structure of full‐length  The regulation of extracellular proteolysis is critical to blood hemostasis and innate immunity and proteases plasma prekallikrein (PK), and coagulation factors XII (FXII) and XI (FXI) act as master regulators of several cascades including coagulation, fibrinolysis, complement activation, and inflammation The PK‐HK complex can undergo autoactivation in the presence of zinc ions and negatively charged polymers PK and FXI are homologous and share 58% amino acid sequence identity with the unique feature of four tandem repeats of 90 amino acid apple domains at the N‐terminus Fig. A, which KLKB1) was cloned into the pMT‐puro vector using BglII and MluI restriction sites with a six‐histidine tag at the N‐terminus after the immunoglobulin binding chaperone protein secretion signal for expression using the Drosophila expression system . A series of constructs and mutants were evaluated to improve recombinant PK for crystallization purposes. The final recombinant PK variant utilized site‐directed mutagenesis (SDM)  to mutate two high‐entropy cluster residues E323A, K325A, K507A, and K509A, and three glycosylation sites N377Q, N434Q, and N475Q on the catalytic domain, together with the active site mutant S559A to prevent autoproteolysis and optimize PK for crystallization experiments .A gene encoding human PK (gene Drosophila S2 cells were grown in Dulbecco's modified eagle's medium (Gibco) supplemented with 10% fetal calf serum at 28 °C and transfection was performed using calcium phosphate. Cells were grown for an additional 48 h before selection with puromycin to establish stable cell lines. Proteins were expressed in serum‐free Express Five insect culture medium (Gibco) and the cells were harvested 6 days after induction with 0.5 mm CuSO4. Protein purification was performed using a Capto S column followed by a Ni‐sepharose affinity column and a Superdex 200 gel filtration column .−1) with β‐FXIIa at a ratio of 40 : 1 in 20 mm Tris‐Cl, 150 mm NaCl, pH 8 overnight at 20 °C. The reaction was stopped by adding benzamidine at a final concentration of 20 mm. PKa was further purified by gel filtration (Superdex 200 column) and then concentrated to 8.2 mg mL−1. Crystallization was performed at 20 °C and 10 °C using sparse matrix screens  in sitting drop plates. Crystals were observed for PKa in the Morpheus screen with a reservoir containing 0.1 m carboxylic acids, 0.1 m Hepes/MOPS pH 7.5, 12.5% MPD, 12.5% PEG1000, 12.5% PEG3350 and a drop containing 0.1 m carboxylic acids, 0.1 m Tris/BICINE pH 8.5, 20% PEG 500 MME, 10% PEG 20 000. Recombinant FXI was a gift from Novo Nordisk and crystallized as described previously The activated form PKa was prepared by incubating pure recombinant PK 1 mg mL mg mL−1 −1 of human plasma purified PK and PKa supplied by Enzyme Research Laboratories (termed HPK and HPKa) were diluted 1 : 7 (v : v) into labeling buffer  for 10 to 10 000 s at 20 °C by automated LEAP robot pipetting. The 0‐s time point was represented by the dilution into the H2O‐based labeling buffer. The HDX was quenched 1 : 1 (v : v) with precooled buffer containing 100 mm phosphate, 0.5 m TCEP, 0.8% formic acid, 2% acetonitrile, pH 2.5 for 180 s at 1 °C and digested on a Waters Enzymate BEH Pepsin Column (2.1 × 30 mm) at 20 μL min−1. Fragments were separated on a Waters Nano ACQUITY UPLC BEH C18 column  at 40 μL min−1 with a gradient of 40% to 90% acetonitrile. Mass spectrometry was performed using electrospray ionization in positive ion mode. Peptides with no exchange were sequenced via Protein Lynx Global Software V3.0.2, followed by amide deuterium uptake analysis done manually via Dynamx 3.0 software. Fragments with >0.2‐Da mass deviations were removed.Hydrogen deuterium exchange mass spectrometry (HDX‐MS) was performed largely as described To determine a full‐length PKa structure recombinant optimized PK active site mutant S559A was activated by proteolytic cleavage of the activation loop Arg371‐Ile372 using FXIIa. PKa crystallized in the presence of inhibitor benzamidine and the structure was determined to 1.3 Å resolution  binding pocket in the apple 3 domain To investigate whether large conformational differences exist between the PK zymogen and PKa in solution, HDX‐MS was performed on human plasma purified samples (termed HPK and HPKa). With this approach, the extent of deuterium exchange of backbone amide protons in HPK and HPKa was evaluated as a function of time. For HPK, 150 peptide fragments were assigned, achieving 94.9% sequence coverage and a redundancy of 4.99. For HPKa, 113 peptide fragments were assigned, achieving 94.7% sequence coverage and a redundancy of 3.14. Heat maps showing relative fractional deuterium uptake over exchange time were calculated with Dynamx Software. A residual heat map of each HPK and HPKa fragment from 0 to 10 000s is shown in rainbow scale (Fig. Solvent accessibility differences between the PK zymogen model and PKa structure were calculated using the program STRIDE We present the first example of a full‐length crystal structure for PKa at 1.3 Å resolution, which defines the detailed organization of the active protease domain and its relationship to the four ancillary apple domains. A comparison with a 2.6‐Å FXI zymogen structure revealed significant conformational differences in the apple 4 domain defining the molecular basis of the monomeric PKa structure versus the FXI dimer. A major conformational difference is also observed in the apple 3 domain with residues from the loop connecting the apple 3 to the apple 4 domain becoming exposed in PKa as opposed to being buried in the center of the apple domain disc in the zymogen form of FXI. It is tempting to speculate that PK may undergo conformational rearrangements upon activation based on the comparison of the FXI and PKa structures. This hypothesis is supported by HDX mass spectrometry measurements on plasma purified human PK and PKa determining that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK. The biological significance of the increased exposure of residues on the upper surface of the apple 3 domain is unknown for PK but has been proposed to occur in FXI to regulate substrate binding A comparison of PKa with the FXI structure revealed a large conformational 180° rotational rearrangement of the intact apple domain disc relative to the protease domain. Low‐resolution structural studies and biochemical data comparing the homologous FXI and active form FXIa have suggested that a large conformational shape change occurs upon activation Overall these new structures provide the first insight into the active conformation of the apple domain containing protease PKa and generate a scaffold for development of future medicines targeting PKa in diverse inflammatory, thrombotic, and vascular disorders CL purified, crystallized, and determined the structure of PKa. MP and ID generated PK expression constructs and contributed to the manuscript. GH crystallized and determined the structure of FXI. KMV and RL performed and analyzed HDX experiments. JE initiated and designed research, analyzed results, and wrote the paper with ID, KM, and RL.The authors declare no conflicts of interest.Fig. S1. HPK raw time‐dependent deuterium uptake chart for each peptide fragment.Click here for additional data file.Fig. S2.  HPKa raw time‐dependent deuterium uptake chart for each peptide fragment.Click here for additional data file.


Increased walnut flour replacement showed that physical properties, loaf volume and specific loaf volume, have declined. The sensory attributes between the unsupplemented and supplemented bread showed major differences. As an outcome, substituting 30% walnut flour gave the best overall quality of bread acceptability.The study was carried out to demonstrate the effect of walnut flour enhancement on the physical, nutritional, and sensory quality of bread. Walnut flour was prepared by soaking, deshelling, oven drying, and sieving whole walnuts. The wheat flour was supplemented with walnut flour by 0, 20, 30, 40, and 50% of the total amount. Standard procedures were taken to estimate the proximate composition of wheat and walnut flour and bread samples. A comparison between the control and supplemented bread was made, where the physical characteristics  and sensory quality were checked. The enhanced bread, where the percentage was between 20 and 50%, appeared to have a significant increase in protein, fat, linoleic acid, and  Relating to these components, they also develop greater nutritional benefits of products in regard to the protein content and other beneficial ingredients for health. Incidentally, the nutritional benefits were improved by adding these components. Walnuts, compared to wheat, have a definite chemical structure with greater nutritional benefits; therefore, they are highly recommended. Despite the improvement of the analytic features, composites have increased bread quality and nutritional benefits. They are related to enhanced blood lipoprotein profile, antioxidant activity, antiatherogenic impact, decrease of tumour initiation or advancement, repair of DNA damage, regulation of cell differentiation and proliferation control of immunological activity, induction of phase II metabolic enzymes, and inflammatory reaction , 2. Walnalcohols , walnutsalcohols . The helalcohols . Raw walalcohols . A studyalcohols . A decrealcohols . Walnutsalcohols . Also, oalcohols , 13. A nWheat flour (75% extraction) was obtained from grain silos and flour mills organization, Jeddah, Saudi Arabia. Walnuts, sugar, and salt were purchased from local markets in Jeddah, Saudi Arabia. All chemicals used are of analytical grade.Juglans regia) were initially washed completely to get rid of any adhering contaminants. They were then cooked for half an hour in an exceeding steel pot to get rid of the shells. With the help of a stainless-steel knife, the deshelled walnuts were reduced into smaller sizes. Then they were blanched in a warm water for five minutes before debilitating. Once blanched, the walnuts were dried in an exceedingly hot air kitchen appliance at 60°C for five hours to get rid of moisture. They were then processed and sieved to supply the walnut flour [The whole walnuts  followed the standard methods of American Association of Cereal Chemists . Farinog3/g)Physical characteristics of bread samples such as loaf weight, loaf volume, and specific loaf volume were evaluated. Loaf weight was measured using a laboratory scale  after half an hour when the loaves were removed from the oven and the readings recorded in grams. Loaf volume was measured as modified by Giami et al. 2004)  using th04  usingSensory evaluation of bread samples was performed 2 h after baking by 50 females selected among students, staff, and faculty members of King Abdelaziz University, Jeddah, Saudi Arabia. The sensory quality of bread was assessed using a 5-point hedonic scale  used for the external and internal properties  of the bread. The bread samples were coded with random numbers of three digits and served randomly to the panelists. Sensory analysis was carried out in three sessions, and the mean values of the scores of 50 evaluators were calculated and used in data analysis for each sample and session. p ≤ 0.05.Data were analysed using SPSS Inc., version 25, Chicago, IL, USA. Data were presented as means and standard deviation (SD). All data were subjected to variance analysis (ANOVA) and the results were compared using Turkey's test and the statistical significance meaning was defined asThe proximate composition of wheat flour and walnut flour and their composition are given in α-linolenic acid (omega 3 fatty acid) except for carbohydrate contents. The moisture values for the flours were 8.20%, 5.80%, 6.24%, 9.57%, and 7.15%, respectively. It seems to be within the usual moisture values of dried food . The five samples showed distinctions in the moisture content. There was a good indication that the low moisture of these samples determined a long shelf life. Materials that contain more than 12% moisture are considered to have essentially flour and starch and less storage stability than those with lower moisture content. Consequently, water content of 10% is commonly specified for flours and other related products. The sample AB3  had a significantly higher moisture content that differed from other samples. Various food materials have different moisture retention capabilities that can exist as occluded or absorbed water [p ≤ 0.05) content of linoleic acid and α-linolenic acid than control bread. The high content of omega 3 fatty acids in the diet provides many health benefits. It is essential for proper brain functions and has beneficial effects in cardiovascular diseases and in prevention of arthritis [In general, the chemical quality of the combined bread was affected by the supplementation of wheat flour with walnut flour. Raising the percentage of walnut supplementation increases the values for protein, ash, fat, fibre, linoleic acid (omega 6 fatty acid), and ed water –23. The p ≤ 0.05) increased the water absorption from 60% in control to 65% in wheat flour supplemented with 50% walnuts flour. This could be due to the increase of the protein solubility and content of the dough following the addition of walnuts whose protein is characterized by its high solubility and absorption capacity. Furthermore, added walnuts could result in a structural modification in the dough which may allow absorption of more water due to hydrogen bonding. High water absorption capacity of dough represents consistency which is one of the appealing characteristics in bread making. In addition, several research reports have demonstrated that supplementation of wheat flour with vegetable and legumes flour, dairy products, or protein isolates significantly increased the water absorption capacity [p ≤ 0.05) affected the gelatinization temperature, peak viscosity, and temperature at peak viscosity of fortified dough compared to control samples. Gelatinization temperature increased with increase the walnuts flour in the dough, while the peak viscosity and temperature at peak viscosity showed concomitant reduction as the walnuts flour increased. Our finding agreed with other reports which have indicated that addition of legumes protein isolates reduced the peak viscosity of the dough [The dough rheological properties of wheat flour and supplementation of wheat flour with walnuts flour were analysed by farinograph and amylograph. The farinograph results indicated that supplementation of wheat flour with walnuts flour significantly affected the water absorption, dough stability, and degree of softening . Increascapacity –28. Thiscapacity , Anton ecapacity , Gadallacapacity , and Pascapacity  as the phe dough , 34. In he dough –38. The 3) and specific volume (2.63±0.06 cm3/g) were observed at 50% walnuts flour supplementation. The increased weight and dense texture of the composite bread samples is developed by less retention of carbon dioxide in the mixed batter, whereas the decrease in the volume and specific volume of the composite loaf is due to the dilution effects on gluten with addition of walnuts flour to the wheat flour. When fermenting, the carbon dioxide produced by the yeast is being expanded and trapped in the dough causing elasticity by the gluten fraction. At the point when gluten coagulates and is affected by the heat during baking, it delivers as the framework of the loaf, which turns generally rigid and does not collapse. Furthermore, adding walnut flour causes the increase in fibre content of the composite flour which may have noticeable effects on dough properties yielding for higher water absorption, giving persistence and endurance, and smaller extensibility as to those taken without fibre supplementation [Outcomes of the physical attributes of composite bread samples incorporating various percentages of walnut flour supplementation in comparison to the control are given in entation . Likewisentation . Consequentation –42.Sensory assessment is a vital measure for quality assessment in a newly developed food product to attract consumers and to meet their requirements . The choThe current study was to determine the possibilities of using walnuts in the production of the wheat bread and determine the impact of walnuts on physical and sensory characteristics of wheat bread. Bread samples that were enhanced with walnut flour were found to be highly nutritional  compared to 100% wheat bread. Nevertheless, walnut supplemented wheat flour was substantially affected by rheological properties , physical properties , and sensory quality of bread. Supplementation of 30% walnut flour into wheat flour resulted in sensory acceptability that was best for the bread.