Discovery of deaminase functions by structure-based protein clustering.

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.

Genome-edited powdery mildew resistance in wheat without growth penalties.

Disruption of susceptibility (S) genes in crops is an attractive breeding strategy for conferring disease resistance1,2. However, S genes are implicated in many essential biological functions and deletion of these genes typically results in undesired pleiotropic effects1. Loss-of-function mutations in one such S gene, Mildew resistance locus O (MLO), confers durable and broad-spectrum resistance to powdery mildew in various plant species2,3. However, mlo-associated resistance is also accompanied by growth penalties and yield losses3,4, thereby limiting its widespread use in agriculture. Here we describe Tamlo-R32, a mutant with a 304-kilobase pair targeted deletion in the MLO-B1 locus of wheat that retains crop growth and yields while conferring robust powdery mildew resistance. We show that this deletion results in an altered local chromatin landscape, leading to the ectopic activation of Tonoplast monosaccharide transporter 3 (TaTMT3B), and that this activation alleviates growth and yield penalties associated with MLO disruption. Notably, the function of TMT3 is conserved in other plant species such as Arabidopsis thaliana. Moreover, precision genome editing facilitates the rapid introduction of this mlo resistance allele (Tamlo-R32) into elite wheat varieties. This work demonstrates the ability to stack genetic changes to rescue growth defects caused by recessive alleles, which is critical for developing high-yielding crop varieties with robust and durable disease resistance.

Investigation on vehicle occupant dummy applicability for under-foot impact loading conditions.

PURPOSE: Under-foot impact loadings can cause serious lower limb injuries in many activities, such as automobile collisions and underbody explosions to military vehicles. The present study aims to compare the biomechanical responses of the mainstream vehicle occupant dummies with the human body lower limb model and analyze their robustness and applicability for assessing lower limb injury risk in under-foot impact loading environments. METHODS: The Hybrid III model, the test device for human occupant restraint (THOR) model, and a hybrid human body model with the human active lower limb model were adopted for under-foot impact analysis regarding different impact velocities and initial lower limb postures. RESULTS: The results show that the 2 dummy models have larger peak tibial axial force and higher sensitivity to the impact velocities and initial postures than the human lower limb model. In particular, the Hybrid III dummy model presented extremely larger peak tibial axial forces than the human lower limb model. In the case of minimal difference in tibial axial force, Hybrid III's tibial axial force (7.5 KN) is still 312.5% that of human active lower limb's (2.4 KN). Even with closer peak tibial axial force values, the biomechanical response curve shapes of the THOR model show significant differences from the human lower limb model. CONCLUSION: Based on the present results, the Hybrid III dummy cannot be used to evaluate the lower limb injury risk in under-foot loading environments. In contrast, potential improvement in ankle biofidelity and related soft tissues of the THOR dummy can be implemented in the future for better applicability.

Postinvasive Bacterial Resistance Conferred by Open Stomata in Rice.

Stomata are leaf pores that regulate gas exchange and water transpiration in response to environmental cues. They also function in innate immunity by limiting pathogen entry through actively closing in so-called stomatal defense. However, roles of stomata in plant disease resistance are not fully elucidated, especially in monocots. Here, we report that non-race specific resistance of the rice abscisic acid-deficient mutant Osaba1 to Xanthomonas oryzae pv. oryzae is due to increased stomatal conductance. Reducing stomatal conductance in the Osaba1 mutant increases its susceptibility to X. oryzae pv. oryzae. Artificial opening of stomata in wild-type plants leads to enhanced resistance to X. oryzae pv. oryzae. The rice mutant es1-1 with constitutively higher stomatal conductance exhibits strong resistance to X. oryzae pv. oryzae. Additionally, Osaba1 and es1-1 are resistant to X. oryzae pv. oryzicola. The data support that open stomata confer postinvasive resistance against bacterial pathogens in rice, and such resistance probably results from decreased leaf water potential. Our findings reveal a novel role of stomata in plant immunity through modulation of leaf water status, which provides physiological insight into the interactions between plant, pathogen, and environment.

MYB75 Phosphorylation by MPK4 Is Required for Light-Induced Anthocyanin Accumulation in Arabidopsis.

Light is a major environmental cue affecting various physiological and metabolic processes in plants. Although plant photoreceptors are well characterized, the mechanisms by which light regulates downstream responses are less clear. In Arabidopsis thaliana, the accumulation of photoprotective anthocyanin pigments is light dependent, and the R2R3 MYB transcription factor MYB75/PAP1 regulates anthocyanin accumulation. Here, we report that MYB75 interacts with and is phosphorylated by MAP KINASE4 (MPK4). Their interaction is dependent on MPK4 kinase activity and is required for full function of MYB75. MPK4 can be activated in response to light and is involved in the light-induced accumulation of anthocyanins. We show that MPK4 phosphorylation of MYB75 increases its stability and is essential for light-induced anthocyanin accumulation. Our findings reveal an important role for a MAPK pathway in light signal transduction.

Direct and tunable modulation of protein levels in rice and wheat with a synthetic small molecule.

Direct control of protein level enables rapid and efficient analyses of gene functions in crops. Previously, we developed the RDDK-Shield1 (Shld1) system in the model plant Arabidopsis thaliana for direct modulation of protein stabilization using a synthetic small molecule. However, it was unclear whether this system is applicable to economically important crops. In this study, we show that the RDDK-Shld1 system enables rapid and tunable control of protein levels in rice and wheat. Accumulation of RDDK fusion proteins can be reversibly and spatio-temporally controlled by the synthetic small-molecule Shld1. Moreover, RDDK-Bar and RDDK-Pid3 fusions confer herbicide and rice blast resistance, respectively, in a Shld1-dependent manner. Therefore, the RDDK-Shld1 system provides a reversible and tunable technique for controlling protein functions and conditional expression of transgenes in crops.

The chloride channel family gene CLCd negatively regulates pathogen-associated molecular pattern (PAMP)-triggered immunity in Arabidopsis.

Chloride channel (CLC) family genes are ubiquitous from prokaryotes to eukaryotes and encode proteins with both channel and transporter activities. The Arabidopsis thaliana genome encodes seven CLC genes, and their products are found in a variety of cellular compartments and have various physiological functions. However, a role for AtCLCs in plant innate immunity has not previously been demonstrated. Here it is reported that AtCLCd is a negative regulator of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). T-DNA insertion mutants of AtCLCd exhibited enhanced responses to the elicitor, flg22. The PTI phenotypes of the clcd mutants were rescued by expression of AtCLCd. Overexpression of AtCLCd led to impaired flg22-induced responses. In line with a role for AtCLCd in PTI, the clcd mutants were more resistant to a virulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 when spray inoculated, while AtCLCd-overexpressing lines displayed increased susceptibility to this pathogen. Interestingly, flg22 treatment was found to repress the expression of AtCLCd. In addition, its expression was elevated in mutants of the flg22 pattern recognition receptor (PRR) FLS2 and the PRR regulatory proteins BAK1 and BKK1, and reduced in an FLS2-overexpressing line. These latter findings indicate that FLS2 complexes regulate the expression of AtCLCd, further supporting a role for AtCLCd in PTI.

Characteristics of head frequency response in blunt impacts: a biomechanical modeling study.

Existing evaluation criteria for head impact injuries are typically based on time-domain features, and less attention has been paid to head frequency responses for head impact injury assessment. The purpose of the current study is, therefore, to understand the characteristics of human body head frequency response in blunt impacts via finite element (FE) modeling and the wavelet packet analysis method. FE simulation results show that head frequency response in blunt impacts could be affected by the impact boundary condition. The head energy peak and its frequency increase with the increase in impact; a stiffer impact block is associated with a higher head energy peak, and a bigger impact block could result in a high proportion of the energy peak. Regression analysis indicates that only the head energy peak has a high correlation with exiting head injury criteria, which implies that the amplitude-frequency aggregation characteristic but not the frequency itself of the head acceleration response has predictability for head impact injury in blunt impacts. The findings of the current study may provide additional criteria for head impact injury evaluation and new ideas for head impact injury protection.

Experimental investigation of brain contusion characteristics and dynamic response in low-age children using an animal model.

INTRODUCTION: Brain contusion is a prevalent traumatic brain injury (TBI) in low-age children, bearing the potential for coma and fatality. Hence, it is imperative to undertake comprehensive research in this field. METHODS: This study employed 4-week-old piglets as surrogates for children and introduced self-designed devices for both free-fall drop impact tests and drop-hammer impact tests. The study explored the characteristics of brain contusion and dynamic responses of brain under these distinct testing conditions. RESULTS: Brain contusions induced by free-fall and drop-hammer conditions both were categorized as the coup injury, except that slight difference in the contusion location was observed, with contusion occurring mainly in the surrounding regions beneath the impact location under free-fall condition and the region just right beneath the impact location under drop-hammer condition. Analysis of impact force and intracranial pressure (ICP) curves indicated similar trends in impact forces under both conditions, yet different trends in ICPs. Further examination of the peak impact forces and ICPs elucidated that, with increasing impact energy, the former followed a combined power and first-order polynomial function, while the latter adhered to a power function. The brain contusion was induced at the height (energy) of 2 m (17.2 J), but not at the heights of 0.4, 0.7, 1, 1.35 and 1.7 m, when the vertex of the piglet head collided with a rigid plate. In the case of a cylindrical rigid hammer (cross-sectional area constituting 40 % of the parietal bone) striking the head, the brain contusion was observed under the energy of 21.9 J, but not under energies of 8.1 J, 12.7 J and 20.3 J. Notably, the incidence of brain contusion was more pronounced under the free-fall condition. CONCLUSIONS: These findings not only facilitate a comprehensive understanding of brain contusion dynamics in pediatric TBIs, but also contribute to the validation of theories and finite element models for piglet heads, which are commonly employed as surrogates for children.

Prime editing using CRISPR-Cas12a and circular RNAs in human cells.

Genome editing with prime editors based on CRISPR-Cas9 is limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE) and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and possess a potential multiplexing capacity in comparison to corresponding Cas9-based systems. The efficiencies of the nuclease-based systems are up to 10.42%, whereas niCPE and sniCPE reach editing frequencies of up to 24.89% and 40.75% without positive selection in human cells, respectively. A derivative system, called one-sniCPE, combines all three RNA editing components under a single promoter. By arraying CRISPR RNAs for different targets in one circular RNA, we also demonstrate low-efficiency editing of up to four genes simultaneously with the nickase prime editors niCPE and sniCPE.

Effects of different preservation on the mechanical properties of cortical bone under quasi-static and dynamic compression.

Introduction: Mechanical properties of biological tissue are important for numerical simulations. Preservative treatments are necessary for disinfection and long-term storage when conducting biomechanical experimentation on materials. However, few studies have been focused on the effect of preservation on the mechanical properties of bone in a wide strain rate. The purpose of this study was to evaluate the influence of formalin and dehydration on the intrinsic mechanical properties of cortical bone from quasi-static to dynamic compression. Methods: Cube specimens were prepared from pig femur and divided into three groups (fresh, formalin, and dehydration). All samples underwent static and dynamic compression at a strain rate from 10-3 s-1 to 103 s-1. The ultimate stress, ultimate strain, elastic modulus, and strain-rate sensitivity exponent were calculated. A one-way ANOVA test was performed to determine if the preservation method showed significant differences in mechanical properties under at different strain rates. The morphology of the macroscopic and microscopic structure of bones was observed. Results: The results show that ultimate stress and ultimate strain increased as the strain rate increased, while the elastic modulus decreased. Formalin fixation and dehydration did not affect elastic modulus significantly whereas significantly increased the ultimate strain and ultimate stress. The strain-rate sensitivity exponent was the highest in the fresh group, followed by the formalin group and dehydration group. Different fracture mechanisms were observed on the fractured surface, with fresh and preserved bone tending to fracture along the oblique direction, and dried bone tending to fracture along the axial direction. Discussion: In conclusion, preservation with both formalin and dehydration showed an influence on mechanical properties. The influence of the preservation method on material properties should be fully considered in developing a numerical simulation model, especially for high strain rate simulation.

Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

Leaf rolling controlled by the homeodomain leucine zipper class IV gene Roc5 in rice.

Leaf rolling is considered an important agronomic trait in rice (Oryza sativa) breeding. To understand the molecular mechanism controlling leaf rolling, we screened a rice T-DNA insertion population and isolated the outcurved leaf1 (oul1) mutant showing abaxial leaf rolling. The phenotypes were caused by knockout of Rice outermost cell-specific gene5 (Roc5), an ortholog of the Arabidopsis (Arabidopsis thaliana) homeodomain leucine zipper class IV gene GLABRA2. Interestingly, overexpression of Roc5 led to adaxially rolled leaves, whereas cosuppression of Roc5 resulted in abaxial leaf rolling. Bulliform cell number and size increased in oul1 and Roc5 cosuppression plants but were reduced in Roc5-overexpressing lines. The data indicate that Roc5 negatively regulates bulliform cell fate and development. Gene expression profiling, quantitative polymerase chain reaction, and RNA interference (RNAi) analyses revealed that Protodermal Factor Like (PFL) was probably down-regulated in oul1. The mRNA level of PFL was increased in Roc5-overexpressing lines, and PFL-RNAi transgenic plants exhibit reversely rolling leaves by reason of increases of bulliform cell number and size, indicating that Roc5 may have a conserved function. These are, to our knowledge, the first functional data for a gene encoding a homeodomain leucine zipper class IV transcriptional factor in rice that modulates leaf rolling.

[Advances on molecular mechanisms of plant-pathogen interactions].

Plants have established a complicated immune defense system during co-evolution with pathogens. The innate immune system of plants can be generally divided into two levels. One, named PAMP-triggered immunity (PTI), is based on the recognition of pathogen-associated molecular patterns by pattern-recognition receptors, which confers resistance to most pathogenic microbes. The other begins in cytoplasm and mainly relies on recognition of microbial effectors by plant resistance proteins in direct or indirect ways, which then initiates potent defense responses. This process, termed effector-triggered immunity (ETI), is necessary for defense against pathogens that can secret effectors to suppress the first level of immunity. Activation of these two layers of immunity in plant is based on distinguishing and recognition of "self" and "non-self" signals. Recognition of "non-self" signals can activate signal cascades, such as MAPK cascades, which will then induce defense gene expression and corresponding defense responses. In this review, we focused on underlying molecular mechanisms of plant-pathogen interactions and the latest advances of the PTI and ETI signaling network.

[Plant prime editing technique: a new genome editing tool for plants].

The CRISPR/Cas9 based prime editing (PE) technique enables all 12 types of base substitutions and precise small DNA deletions or insertions without generating DNA double-strand breaks. Prime editing has been successfully applied in plants and plays important roles in plant precision breeding. Although plant prime editing (PPE) can substantially expand the scope and capabilities of precise genome editing in plants, its editing efficiency still needs to be further improved. Here, we review the development of PPE technique, and introduce structural composition, advantages and limitations of PPE. Strategies to improve the PPE editing efficiency, including the Tm-directed PBS length design, the RT template length, the dual-pegRNA strategy, the PlantPegDesigner website, and the strategies for optimizing the target proteins of PPE, were highlighted. Finally, the prospects of future development and application of PPE were discussed.

Quantification and statistical analysis on the cranial vault morphology for Chinese children 3-10 years old.

BACKGROUND AND OBJECTIVE: Head injury is the leading cause of fatalities and disabilities in children. Characterizing the variation in cranial size/shape and thickness during growth is important for developing finite element models of child heads and evaluating head injury risk at different ages. However, the quantitative morphological features of the cranial vault (size/shape and non-uniform thickness distribution) have not been accounted for in children aged between 3 and 10 years old (YO). METHODS: Geometrically equivalent discrete points were identified on 42 head CT scans of 3-10 YO children by separation, curve dividing, and point fitting. Based on discrete points, the principal component analysis and regression (PCA&R) method was used to develop a statistical model of the cranial vault as a function of age and head circumference. RESULTS: The ontogeny of three-dimensional cranial morphology and non-uniform thickness from 3 to 10 years of age was quantified and cranial vault morphologies for 3-10 YO children were generated in 1 year intervals. CONCLUSIONS: The automatic method, the procedure of identifying discrete points from CT scans, and the developed quantitative cranial vault model are reliable and accurate.

Biomechanical analysis of thorax-abdomen response of vehicle occupant under seat belt load considering different frontal crash pulses.

PURPOSE: The purpose of this work was to understand the biomechanical response and injury risk of thorax and abdomen of vehicle front seat occupants caused by seat belt load under different frontal crash pulses. METHODS: A vehicle-seat-occupant subsystem finite element (FE) model was developed using the a assembly of vehicle front seat and seat belt together with the THUMS (Total Human body Model for Safety) AM50 (50th% Adult Male) occupant model. Then the typical vehicle frontal crash pulses from different impact scenarios were applied to the vehicle-seat-occupant subsystem FE model, and the predictions from the occupant model were analyzed. RESULTS: The modeling results indicate that the maximum sternal compression of the occupant caused by seat belt load is not sensitive to the peek of the crash pulse but sensitive to the energy contained by the crash pulse in the phrase before seat belt load reaching its limit. Injury risk analysis implies that seat belt load of the four crash scenarios considered in the current work could induce a high thorax AIS2+ injury risk (>80%) to the occupants older than 70 years, and a potential injury risk to the spleen. CONCLUSIONS: The findings suggest that control of the energy in the first 75 ms of the crash pulse is crucial for vehicle safety design, and thorax tolerance of the older population and spleen injury prevention are the key considerations in developing of seat belt system.

Analysis on alteration of road traffic casualties in western China from multi-department data in recent decade.

Background: Road traffic safety has considerably improved in China. However, the changes may differ in the economically backward and altitude higher western region. This study aims to investigate changes in the occurrence and severity of traffic casualties in western China and illuminate several key causal factors. Materials and methods: Traffic accident data from the Annual Traffic Accident Statistics Report combined with population and vehicle data from the China Statistics Bureau between 2009 and 2019, were retrospectively analyzed. Traffic accident numbers, fatalities, human injury (HI), case fatality rates (CFR), mortality per 100,000 population (MRP), and mortality per 10,000 vehicles (MRV) were compared between the western and eastern regions. The HI, CFR, MRV, and MRP between the four groups based on the altitude of cities, below 500 meters, 500 to 1,500 meters, 1,500 to 3,000 meters, and over 3,000 meters, were compared using one-way analysis of variance. One hundred and seventy-eight cases of extremely serious traffic accidents were further analyzed in terms of accident occurrence time, vehicle type, road grade, road shape, accident pattern, and accident reason. The differences of accident characteristics between the eastern and western regions were compared using the chi-square test. Results: The number of traffic accidents and fatalities decreased in low-altitude areas in western China. However, there was a significant increasing trend in the high altitude area. The HI, CFR, MRV, and MRP were higher in the western region than that in the eastern and national. Those accident indicators tended to increase with increasing altitude. And there were statistically significant differences (p < 0.05) among groups from different altitudes. Chi-square test results show that there are statistically significant differences (p < 0.05) in term of road grade, road shape, accident pattern between eastern and western. Low-grade roads, combined curved and sloping roads, and rollover were significant features associated with traffic accidents in the western region. Bad roads were the main cause of rollover accidents in western China, which will lead to more serious casualties. Over speeding, overloading, bad weather, vehicle failure, and driver error were the top five accident causes. Conclusion: Traffic accidents are increasing in high-altitude areas of western China, and they lead to more severe casualties. The characteristics of serious traffic accidents in this part of the country differ from those of the eastern regions. Improving road safety facilities, restrictions of speed, and improving medical treatment at accident scenes may be effective measures to reduce traffic accidents related injuries in the western region.