Searching for Novel Targets to Control Wheat Head Blight Disease—I-Protein Identification, 3D Modeling and Virtual Screening

Fusarium head blight (FHB) is a destructive disease of wheat and other cereals. FHB occurs in Europe, North America and around the world causing significant losses in production and endangers human and animal health. In this article, we provide the strategic steps for the specific target selection for the phytopathogen system wheat-Fusarium graminearum. The economic impact of FHB leads to the need for innovation. Currently used fungicides have been shown to be effective over the years, but recently cereal infecting Fusaria have developed resistance. Our work presents a new perspective on target selection to allow the development of new fungicides. We developed an innovative approach combining both genomic analysis and molecular modeling to increase the discovery for new chemical compounds with both safety and low environmental impact. Our protein targets selection revealed 13 candidates with high specificity, essentiality and potentially assayable with a favorable accessibility to drug activity. Among them, three proteins: trichodiene synthase, endoglucanase-5 and ERG6 were selected for deeper structural analyses to identify new putative fungicides. Overall, the bioinformatics filtering for novel protein targets applied for agricultural purposes is a response to the demand for chemical crop protection. The availability of the genome, secretome and PHI-base allowed the enrichment of the search that combined experimental data in planta. The homology modeling and molecular dynamics simulations allowed the acquisition of three robust and stable conformers. From this step, approximately ten thousand compounds have been virtually screened against three candidates. Forty-five top-ranked compounds were selected from docking results as presenting better interactions and energy at the binding pockets and no toxicity. These compounds may act as inhibitors and lead to the development of new fungicides.

Determination of Protein and Starch Content in Whole Maize Kernel by Near Infrared Reflectance Spectroscopy

Using 128 bulk-kernel samples of inbred lines and hybrids, a study was conducted toinvestigate the feasibility and method of measuring protein and starch contents inintact seeds of maize by near infrared reflectance spectroscopy (NIRS). The chemometricalgorithms of partial least square (PLS) regression was used. The results indicated thatthe calibration models developed by the spectral data pretreatment of firstderivative+multivariate scattering correction within the spectral region of 10000-4000cm-1, and first derivative + straight line subtraction in 9000-4000cm-1 were thebest for protein and starch, respectively. All these models yielded coefficients ofdetermination of calibration (R2cal) above 0.97, while R2cv and R2val of cross and externalvalidation ranged from 0.92 to 0.95, respectively; however, the root of mean squareerrors of calibration, cross and external validation (RMSEE, RMSECV and RMSEP) werebelow 1(ranged 0.3-0.7),respectively. This study demonstrated that it is feasible touse NIRS as a rapid, accurate, and none-destructive technique to predict protein andstarch contents of whole kernel in the maize quality improvement program.

Compound Danshen tablets downregulate amyloid protein precursor mRNA expression in a transgenic cell model of Alzheimer's disease Effects and a comparison with donepezil

After gene mutation, the pcDNA3.1/APP595/596 plasmid was transfected into HEK293 cells to establish a cell model of Alzheimer＇s disease. The cell model was treated with donepezil or compound Danshen tablets after culture for 72 hours. Reverse transcription-PCR showed that the mRNA expression of amyloid protein precursor decreased in all groups following culture for 24 hours, and that there was no significant difference in the amount of decrease between donepezil and compound Danshen tablets. Our results suggest that compound Danshen tablets can reduce expression of the mRNA for amyloid protein precursor in a transgenic cell model of Alzheimer＇s disease, with similar effects to donepezil.

Interventional effect of hirudin on the expression of microtubule-associated protein 2 in peripheral tissue of hematom of model rats with acute intracerebral hemorrhage

BACKGROUND： It is suspected that dissociation, destruction or synthetic disorder of microtubule-associated protein 2 （MAP-2） may participate in secondary injury of intracerebral hemorrhage （ICH）, and the reason may be related to thrombin in high concentration after ICH; therefore, the mechanism should be studied further. OBJECTIVE： To explore the effect of hirudin on expression of MAP-2 in peripheral tissue of hematom after ICH and changes of water content in brain tissue and analyze pathogenesis of thrombin in secondary injury after ICH. DESIGN ： Completely randomized grouping design and controlled animal study SEn-ING ： Department of Neurology, the First Affiliated Hospital of Jilin University MATERIALS ： The experiment was carried out in the Neurological Laboratory of the First Affiliated Hospital of Jilin University from April 2003 to April 2004. A number of 80 healthy Wistar rats, of both genders, aged 3-4 months, weighing 250-350 g, were randomly divided into 8 groups： normal control group, 6-hour ICH group, 1-day ICH group, 2-day ICH group, 3-day ICH group, 7-day ICH group, 3-day hirudin group and 7-day hirudin group with 10 in each group. Five rats from each group were selected to measure their water content, and the others were undertaken immunohistochemical stain. Hirudin was produced by Sigma Company, USA, and MAP-2 rabbit-rat polyclonal antibody was provided by Fuzhou Maixin Biotechnology Company Limited. METHODS： ① Model establishing and grouping intervention： Rats in simple ICH group were collected their blood from tails and then inserted with 50 μL non-anticoagulant auto-arterial blood into the cauda of the putamen in right brain within 5 minutes. Rats in hirudin groups were inserted with 10 U hirudin （which was diluted with saline to 20 μL） into local hematom regions within 5 minutes, and the needle was pulled out after 10 minutes. Rats in normal control group were untouched. ② Water content in peripheral tissue of hematom： Based on the ratio between dry weight and wet weight, brain tissue at bleeding side and in right frontal lobe was selected to measure dry and wet weights so as to calculate the water content [（wet weight - dry weight） /wet weight] × 100%.③ Positive expression of MAP-2： Based on immunohistochemical stain, positive MAP-2 cells were regarded as neurons and they were buffy morphological. Positive rate of MAP-2 was calculated, i.e., percentage of positive cells in each sight to total cells in all sights. ④ Statistical analysis： Data among groups were compared with one-way analysis of variance, averages were compared with SNK-q test by each other, and relation between water content and MAP-2 was analyzed with linear regression technique. MAIN OUTCOME MEASURES： Changes of water content and MAP-2 expression in peripheral tissue of hematorn at various time points after ICH and intervention of hirudin. RESULTS： All 80 rats were involved in the final analysis. ①Water content： Water content was increased at day 1, reached peak at day 3 and decreased at day 7. It was （72.31±0.32）%, （77.42±0.53）%, （78.44±0.28）%, （74.10±0.13）%, （74.85±0.51）% and （70.07±0.36）%, respectively in 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was higher than that in normal control group （63.85±0.41, q=-4.684 3 to -7.262 0, P〈 0.05）; that in 2-day and 3-day ICH groups was higher than that in 7-day ICH group （q=-3.053 4, -3.727 0, P 〈 0.05）; and that in 3-day and 7-day ICH groups was higher than that in hirudin groups at the same time points （q=-2.965 6, -2.726 4, P 〈 0.05）. ②Positive expression of MAP-2： Positive expression of MAP-2 was decreased at 6 hours after ICH, reached the lowest value at day 3 and increased at day 7. Positive rate was （78.60±0.42）%, （60.56±0.74）%, （44.60±0.26）%, （25.45±0.85）%, （32.55±0.64）%, （37.69＋0.76）%, （41.75±0.68）%, respectively in 6-hour, 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was lower than that in normal control group [（96.50±0.33）%, q= -3.074 5 to -8.128 5, P 〈 0.05]. In addition, positive cells of MAP-2 disappeared plentifully at 3-7 days after ICH, stain of positive cells were light, and only stain of plasma was positive. That in 3-day and 7-day hirudin groups was higher than that in ICH groups at the same time points （q= -3.391 8, -2.967 9, P 〈 0.05）. Moreover, positive cells of MAP-2 was formed slightly but deeply stained. ③ Results of linear regression： Water content was negatively related to MAP-2 changes at 7 days after ICH （r= -0.894 9, P〈 0.01）, i.e., water content was increased with decrease of MAP-2 expression. CONCLUSION ： The deterioration of MAP-2 may be involved in the pathogenesis of thrombin within the first week after ICH, and the local administration of hirudin can protect neurons.

All-Atom Direct Folding Simulation for Proteins Using the Accelerated Molecular Dynamics in Implicit Solvent Model

We report the results of protein folding （219M, C34, N36, 2KES, 2KHK） by the method of accelerated molecular dynamics （aMD） at room temperature with the implicit solvent model. Starting from the linear structures, these proteins successfully fold to the native structure in a lO0-ns aMD simulation. In contrast, they are failed under the traditional MD simulation in the same simulation time. Then we find that the lowest root mean square deviations of helix structures from the native structures are 0.36 A, 0.63 A, 0.52 A, 1.1 A and 0.78 A. What is more, native contacts, cluster and free energy analyses show that the results of the aMD method are in accordance with the experiment very well. All analyses show that the aMD can accelerate the simulation process, thus we may apply it to the field of computer aided drug designs.

Heuristic Quasi-physical Algorithm for Protein Structure Prediction

A three-dimensional off-lattice protein model with two species of monomers, hydrophobic and hydrophilic, is studied. Enligh- tened by the law of reciprocity among things in the physical world, a heuristic quasi-physical algorithm for protein structure prediction problem is put forward. First, by elaborately simulating the movement of the smooth elastic balls in the physical world, the algorithm finds low energy configurations for a given monomer chain. An ＂off-trap＂ strategy is then proposed to get out of local minima. Experimental results show promising performance. For all chains with lengths 13≤n ≤55, the proposed algorithm finds states with lower energy than the putative ground states reported in literatures. Furthermore, for chain lengths n = 21, 34, and 55, the algorithm finds new low energy configurations different from those given in literatures.

Optimization of Insulin Rapid Amyloid Fibrosis Conditions

Amyloid fibers are now considered as one of the possible therapeutic targets of neurodegenerative diseases due to their unique physicochemical properties and toxicity. To efficiently trace in real time how the drug inhibits protein amyloid fibrosis, it is necessary to study the conditions for rapid amyloid fibrosis. Insulin is selected as the model protein in vitro to explore the process of amyloid formation. The effects of the molar concentration of NaCl, pH and reaction temperature in the single-factor experiment are discussed and the response surface analysis is carried out. Amyloid fibrosis is labeled by Thioflavin-T(ThT). The optimal molar concentration of NaCl, pH and reaction temperature for insulin rapid amyloid fibrosis are 50.0 mmol/L, 2.02 and 54℃, respectively. With the addition of 0.1 mmol/L phenol, the half-time of insulin amyloid fibrosis is shortened from 5.4 h to 1.7 h. The insulin rapid amyloid fibrosis system provides a new approach for screening the protein amyloid fibrosis inhibitors.

Polymorphisms of the maternal Slug gene in fetal neural tube defects in a Chinese population

Several studies have demonstrated that Slug,which encodes a zinc finger of the Snail family of transcription factors,is a potential risk factor for neural tube defects.Neural tube defects tend to occur with a high rate in Shanxi province,China.The present case-control study investigated genotypic distributions and allele frequencies of Slug C1548A polymorphisms in DNA samples from59 women with a history of neural tube defect pregnancies and 73 controls during the same period from Shanxi Province,China.Results demonstrated that women with a history of neural tube defect pregnancies had significantly greater genotypic distributions of Slug AA genotypes and A allele frequencies compared with controls,and A allele Slug C1548A was a risk factor for neural tube defects（odds ratio = 3.444;95% confidence interval;2.021-5.868,P 〈 0.05）.Three-dimensional structure prediction revealed that Slug C1548A resulted in transition of aspartic acid into glutamate at position 119.This indicated that these mutations could lead to damaged protein structure and function.These findings suggest that Slug C1548A gene polymorphism is closely related to neural tube defects in a population of Han Chinese origin from Shanxi Province,China

Structural basis of SARS-CoV-2 3CL^pro and anti-COVID-19 drug discovery from medicinal plants

The recent pandemic of coronavirus disease 2019(COVID-19)caused by SARS-CoV-2 has raised global health concerns.The viral 3-chymotrypsin-like cysteine protease(3CL^pro)enzyme controls coronavirus replication and is essential for its life cycle.3CL^pro is a proven drug discovery target in the case of severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV).Recent studies revealed that the genome sequence of SARS-CoV-2 is very similar to that of SARS-CoV.Therefore,herein,we analysed the 3CL^pro sequence,constructed its 3D homology model,and screened it against a medicinal plant library containing 32,297 potential anti-viral phytochemicals/traditional Chinese medicinal compounds.Our analyses revealed that the top nine hits might serve as potential anti-SARS-CoV-2 lead molecules for further optimisation and drug development process to combat COVID-19.

Analysis on Stability of AC,GT and PC in Rice Varieties (Orzya sativa L.)

The investigation of rice varieties stability for amylose content (AC), gelatinization temperature (GT) and protein content (PC) was carried out using additive main effects and multiplicative interaction (AMMI) model in three locations and two years. Eighteen tested varieties were further clustered and evaluated based on the phenotypic values of three quality traits and their Di values from the AMMI analysis. The results showed that the genotype by environment interactions, the differences of phenotype and stability of AC, GT and PC among different genotypes and environments were all significant at 1% level. Also, only one variety, W002, had high stability for all the three quality traits. Additionally, in consideration with the phenotype of AC, GT and PC, and their stability in rice varieties, etc., four rice varieties, W002, Zaofeng 9, Guangling Xiangjing and Nanjing16, could be also applied as breeding parents to improve eating quality and stability of rice.

NetGO 3.0:Protein Language Model Improves Large-scale Functional Annotations

As one of the state-of-the-art automated function prediction(AFP)methods,NetGO 2.0 integrates multi-source information to improve the performance.However,it mainly utilizes the proteins with experimentally supported functional annotations without leveraging valuable information from a vast number of unannotated proteins.Recently,protein language models have been proposed to learn informative representations[e.g.,Evolutionary Scale Modeling(ESM)-1b embedding] from protein sequences based on self-supervision.Here,we represented each protein by ESM-1b and used logistic regression(LR)to train a new model,LR-ESM,for AFP.The experimental results showed that LR-ESM achieved comparable performance with the best-performing component of NetGO 2.0.Therefore,by incorporating LR-ESM into NetGO 2.0,we developed NetGO 3.0 to improve the performance of AFP extensively.

Nuclear Targeting of Methyl-Recycling Enzymes in Arabidopsis thaliana Is Mediated by Specific Protein Interactions

Numerous transmethylation reactions are required for normal plant growth and development. S-adenosylhomocysteine hydrolase （SAHH） and adenosine kinase （ADK） act coordinately to recycle the by-product of these reactions, S-adenosylhomocysteine （SAH） that would otherwise competitively inhibit methyltransferase （MT） activities. Here, we report on investigations to understand how the SAH produced in the nucleus is metabolized by SAHH and ADK. Localization analyses using green fluorescent fusion proteins demonstrated that both enzymes are capable of localizing to the cytoplasm and the nucleus, although no obvious nuclear localization signal was found in their sequences. Deletion analysis revealed that a 41-amino-acid segment of SAHH （GlylS^-Lys19~） is required for nuclear targeting of this enzyme. This segment is surface exposed, shows unique sequence conservation patterns in plant SAHHs, and possesses additional features of protein-protein interaction motifs. ADK and SAHH interact in Arabidopsb via this segment and also interact with an mRNA cap MT. We propose that the targeting of this complex is directed by the nuclear localization signal of the MT; other MTs may similarly target SAHH/ADK to other subcellular compartments to ensure uninterrupted transmethylation.

A New Hidden Markov Model for Protein Quality Assessment Using Compatibility Between Protein Sequence and Structure

Protein structure Quality Assessment（QA） is an essential component in protein structure prediction and analysis. The relationship between protein sequence and structure often serves as a basis for protein structure QA.In this work, we developed a new Hidden Markov Model（HMM） to assess the compatibility of protein sequence and structure for capturing their complex relationship. More specifically, the emission of the HMM consists of protein local structures in angular space, secondary structures, and sequence profiles. This model has two capabilities：（1） encoding local structure of each position by jointly considering sequence and structure information, and（2）assigning a global score to estimate the overall quality of a predicted structure, as well as local scores to assess the quality of specific regions of a structure, which provides useful guidance for targeted structure refinement. We compared the HMM model to state-of-art single structure quality assessment methods OPUSCA, DFIRE, GOAP,and RW in protein structure selection. Computational results showed our new score HMM.Z can achieve better overall selection performance on the benchmark datasets.

Single nucleotide polymorphisms of the maternal gene and their association with fetal neural tube defects in Han ethnic group in Shanxi Province, China

Background Neural tube defects are the most common human birth defects. The causes are multifactorial with complex genetic and environmental factors, although the exact genetic causes are unknown. This research was conducted to study the frequency of Msx2 gene polymorphisms in 59 women with a history of pregnancy with a neural tube defect and in 73 healthy controls. We aimed to determine the effect of this genetic polymorphism on the incidence of neural tube defects in the Han Chinese population.Methods We studied 59 mothers with at least one previous child with a neural tube defect （the case group） and 73case-control subjects during the same period, from Shanxi Province, China. We analyzed the genotypic distributions and allele frequencies of Msx2 C386T poiymorphisms in DNA samples from the case and control groups. A three-dimensional protein model was predicted using Swiss-Pdb Viewer software version 4.0. Disease association was analyzed using chi-square tests.Results Significant differences were observed in the genotypes and allele frequencies of the Msx2 C386T allele between the case and control groups （CT： 32% vs. 15%, P=0.0073 and TT 15% vs. 4%, P=0.013, respectively）. Logistic regression analysis showed that the C386T mutation is a potential risk factor for neural tube defects （P 〈0.05; OR： 3.466;95%CI： 1.831-6.560）. Three-dimensional structure prediction revealed that the Msx2 C386T mutation results in a threonine substitution for methionine at position 129 of exon 2, which might lead to structural mutations or dysfunctions in the protein encoded by Msx2.Conclusion Maternal Msx2 C386T gene polymorphisms were associated with fetal neural tube defects in Han Chinese women in Shanxi Province.

Local Conformational Constraint of Firefly Luciferase Can Affect the Energy of Bioluminescence and Enzyme Stability

Conformational dynamics contribute importantly to enzyme catalysis,such that targeted conformational constraint may affect catalysis.Firefly luciferases undergo extensive structural change during catalysis;key residues form a hydrophobic pocket,excluding water and enabling maximally energetic light production.Point mutants almost always luminesce at longer wavelengths(lower energy)than the wild type.Conformational constraint,using dipeptide analogue 3 at a position critical for optimized excited state structure,produced luciferase emission at a shorter wavelength by∼10 nm.Incomparison,introduction of conformationally constrained analogues 4,5,or 7 afforded luciferases emitting at longer wavelengths,while a related unconstrained luciferase(analogue 6)exhibited wild-type emission.The constrained luciferases tested were more stable than the wild type.Protein modeling demonstrated that the“inside”or“outside”orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength,respectively.More broadly,these results suggest that local conformational constraint can control specific elements of enzyme behavior,both in vitro and in vivo.This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position.The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.