CryoRes: Local Resolution Estimation of Cryo-EM Density Maps by Deep Learning

Recent progress in cryo-EM research has ignited a revolution in biological macromolecule structure determination. Resolution is an essential parameter for quality assessment of a cryo-EM density map, and it is known that resolution varies in different regions of a map. Currently available methods for local resolution estimation require manual adjustment of parameters and in some cases necessitate acquisition or de novo generation of so-called “half maps”. Here, we developed CryoRes, a deep-learning algorithm to estimate local resolution directly from a single final cryo-EM density map, specifically by learning resolution-aware patterns of density map voxels through supervised training on a large dataset comprising 1,174 experimental cryo-EM density maps. CryoRes significantly outperforms all of the state-of-the-art competing resolution estimation methods, achieving an average RMSE of 2.26 Å for local resolution estimation relative to the currently most reliable FSC-based method blocres, yet requiring only the single final map as input. Further, CryoRes is able to generate a molecular mask for each map, with accuracy 12.12% higher than the masks generated by ResMap. CryoRes is ultra-fast, fully automatic, parameter-free, applicable to cryo-EM subtomogram data, and freely available at https://cryores.zhanglab.net.     

基于响应面分析的高抗原活性猪水肿病大肠杆菌疫苗培养基的研制及效果评价

【背景】猪水肿病大肠杆菌引发的疾病造成了很大的危害,但现有培养基存在培养密度低的问题。【目的】研制出高抗原活性猪水肿病大肠杆菌疫苗培养基。【方法】以常用的市售猪水肿培养基为对照,通过单因素试验、爬坡试验(Plackett-Burman, PB)、响应面(Box-Behnken, BB)试验对猪水肿培养基进行响应面优化,得到猪水肿培养基最优配方。以响应面试验得到的培养基培养猪水肿病大肠杆菌,评价不同培养时间点菌株的抗原活性,制作灭活疫苗,进行动物免疫保护试验。【结果】对研制的培养基进行扩大培养验证,发现扩大培养得到的菌株活菌数可达5×10⁹ CFU/mL以上,约为对照组的2倍。制备的灭活疫苗效价可达1:140 000,并在9 h时抗原蛋白效价达到最高。【结论】本研究研制出的疫苗培养基显著提高了猪大肠杆菌菌体密度,并可提高菌体抗原活性,为猪水肿病灭活疫苗的制备提供了技术指引。     

外源海藻糖对高温胁迫下穗分化期水稻叶片生理特性及产量的影响

该研究以耐热型水稻品种Nagina22和热敏型水稻品种YR343为供试材料,采用盆栽试验,设置喷施清水+常温处理(NT0)、喷施清水+穗分化期高温胁迫(HT0),以及分别喷施5、10、15、20 mmol·L^-1外源海藻糖+高温胁迫(分别记为HT1、HT2、HT3、HT4)共6个处理,分析外源海藻糖对高温胁迫下穗分化期水稻叶片叶绿素含量、光合气体交换参数、抗氧化酶活性、渗透调节物质含量、活性氧含量等生理特性,以及籽粒产量及其构成因素的影响,为水稻抗热栽培和耐热品种的选育提供理论依据。结果表明:(1)在高温胁迫下水稻穗分化期,2个水稻品种叶片的叶绿素含量、光合气体交换参数及渗透调节物质含量均降低,叶片MDA和H₂O₂含量以及■的产生速率均上升,叶片抗氧化酶活性呈先增后降的趋势,最终显示水稻籽粒产量及其构成因素显著下降。(2)喷施外源海藻糖能显著增加高温胁迫下穗分化期水稻的每穗粒数、千粒重和结实率,从而提高籽粒产量,其中弱势粒千粒重和结实率的增幅高于强势粒,外源海藻糖最适喷施浓度为15 mmol·L^-1...     

Preparation and Characterization of a Silk Fibroin/Polyurethane Fiber Blend Membrane Containing Actinomycin X2 with Excellent Mechanical Properties and Enhanced Antibacterial Activities

Development of suitable antimicrobial biomaterials for hygienic wound dressing and healing is an important requirement for medical application. Durable mechanical properties increase the application range of biomaterial in different environmental and biological conditions. Due to the inherent brittleness of silk fibroin (SF), polyurethane fiber (PUF) was used to modify SF containing actinomycin X2 (Ac.X2) to prepare silk fibroin@actinomycin X₂/polyurethane fiber (ASF/PUF) blend membranes. The ASF/PUF blend membrane was developed by solution casting method. Incorporation of PUF improved the flexibility of material and introduction of Ac.X2 has increased antibacterial activity of materials. Excellent mechanical properties (tensile strength up to 25.7 MPa and elongation at break up to 946.5 %) of 50 % SF+50 % PUF blend membrane were proved by tensile testing machine. FT-IR spectra, TGA, contact angle and DMA were tested to prove the blend membrane's physico-chemical characteristics. ASF/PUF blend membrane displayed satisfactory antibacterial activity against S. aureus, and the cytotoxicity tests showed that the blend membrane has better biosafety compared to directly applied Ac.X2 in soluble form. These results suggest that the modification of SF through PUF for development of flexible antibacterial membranes has great potential application value in the field of silk-like material fabrication.     

A new strategy for cellulases application in high temperature industrial scenarios with syringic acid assisting

In the process of bioethanol production, more stable and active cellulase in high temperature condition is required. In this study, syringic acid was applied in cellulase hydrolysis system. At 70°C, TvEG3 activity increased 201.36%, CtBglA activity decreased 72.79% by syringic acid. With syringic acid assisting, TvEG3 thermostability was improved, CtBglA thermostability was reduced. Syringic acid scarcely affected CtCBH. In hydrolysis system with the cellulases containing TvEG3, CtCBH, and CtBglA, the reducing sugar yield improved by 28.37% with syringic acid assisting. With the molecular dynamic simulation in syringic acid system, the backbone root-mean-square deviation (RMSD) and the residue root-mean-square fluctuation (RMSF) of TvEG3, CtCBH reduced, while the RMSD and RMSF of CtBglA increased. The reduction in the number of secondary structures, especially α-helix, caused the structure of CtBglA in the presence of syringic acid to collapse at high temperature. More secondary structures in TvEG3 and more α-helix in CtCBH in the presence of syringic acid make them more stable at high temperatures. These means syringic acid can stabilize TvEG3 and CtCBH structure, destabilize CtBglA structure at high temperature. In summary, this study not only provides insight into cellulase hydrolysis at high temperature with syringic acid assisting but also demonstrates the promoting mechanism of syringic acid.     

Secretion systems and membrane-associated structures in rust fungi after high pressure freezing and freeze-fracturing

Excellent preservation and new structural details can be demonstrated in rust-infected leaf tissue after high pressure freezing and freeze-fracturing. A tubular-vesicular complex was the most remarkable cytoplasmic structure observed in cells of the bean rust fungus Uromyces appendiculatus var. appendiculatus during its establishement in its host Phaseolus vulgaris. In fungal cells undergoing intensive synthesis of wall material, this membranous system extended throughout the cytoplasm; in addition, vesicles were accumulated adjacent to the plasma membrane. Here, membrane-associated configurations were observed which seem to be involved in exo- and/or endocytotic processes. It is assumed that the tubular-vesicular complex belongs to the endomembraneous system of the bean rust fungus and that it is involved in the synthesis and secretion of wall material.     

Tachykinin NK-1 receptor probed with constrained analogues of substance P

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. -tetrahydroisoquinoleic acid (Tic), -fluorenylglycine (Flg), -diphenylalanine (Dip), the diastereoisomers of -1-indanylglycine (Ing) and -benz[ƒ]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (Δ^ZPhe, Δ^EPhe, Δ^ZNal, Δ^ENal) and (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S₇ and S₈ subsites, corresponding to residues Phe⁷ and Phe⁸ of substance P. According to the binding potencies of these substituted-SP analogues, the S₇ binding subsite is smaller than the S₈ subsite: the S₇ subsite accepts only one aromatic nucleus, while the S₈ can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S₈ binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu⁶, Pro⁹]SP(6–11), discrepancies are observed between affinity (K_i) and activity (EC₅₀) values for IPs production. While a weak correlation between K_i and EC₅₀ values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K_i) and their potencies (EC₅₀) for cAMP production (r = 0.97). The high potency (EC₅₀) observed for ‘septide-like’ molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC₅₀ values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).

According to the binding potencies of constrained analogues of phenylalanine, the S₇ binding subsite of human NK-1 receptor is small, whereas the S₈, which can accommodate three coplanar nuclei, might probably reside in the extracellular loop. The discrepancies observed between affinity (K_i) and activity (EC₅₀) values for IPs production are not an artefact of CHO cells since a good correlation was found between EC₅₀ for PI hydrolysis and those measured in guinea pig ileum bioassay.     

An Arrayed Bacteriophage P1 Genomic Library of Pneumocystis carinii

We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of ∽4800 independent clones with an average insert size of ∽55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4–5 positive clones for each, consistent with a several-fold coverage of the ∽10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library off. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen.     

Cysteine Racemization in Peptide Synthesis: A New and Easy Detection Method

A new method has been developed for the rapid determination of D-cysteine contents in synthetic peptides. It is based on the reduction of cystine residues, when present, with tris- alkylphosphines, selective derivatization of the cysteine residues with 4-vinylpyridine, followed by acid hydrolysis of the (4-pyridylethyl)cysteine –peptides. Baseline enantiomeric resolution of theD ,L -S-β-(4-pyridylethyl)cysteine, and thus quantification ofD - enantiomer contents at levels ≤1%, is easily achieved by capillary zone electrophoresis exploiting the host–guest complexation principle with crown ethers or by gas chromatography on chiral glass capillary columns upon conventional derivatization of the hydrolysate. The acid-stability of the (4-pyridylethyl)cysteine derivative prevents racemization via thiazoline intermediates and allows for standardization of the acid hydrolysis-dependent racemization.