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Abstract Background Although contrast extravasation on follow-up head computed tomography (CT) is frequently visualized after endovascular treatment, this phenomenon is rare after intravenous thrombolytic treatment in patients with acute ischemic stroke (AIS). Here, we report a case of contrast extravasation mimicking intracerebral hemorrhage (ICH) with intraventricular extension after intravenous thrombolytic treatment and computed tomography angiography (CTA). Case presentation A 52-year-old man presented with right-sided hemiparesis and hypoesthesia. Initial non-contrast head CT was negative for intracranial hemorrhage and acute ischemic changes. He received intravenous treatment with tenecteplase 3.8 h after the onset of stroke. CTA of the head and neck was performed at 4.3 h after stroke onset. It showed no stenosis or occlusion of the carotid and major intracranial arteries. At about 1.5 h after CTA, the right-sided hemiparesis deteriorated, accompanied by drowsiness, aphasia, and urinary incontinence. Immediate head CT showed hyperdense lesions with mild space-occupying effect in the left basal ganglia and both lateral ventricles. The hyperdense lesions were reduced in size on follow-up CT after 5 h. Two days later, CT showed that the hyperdense lesions in the lateral ventricles almost completely disappeared and only a small amount remained in the infarcted area. Conclusions Contrast extravasation into the brain tissue and lateral ventricles, mimicking ICH with intraventricular extension, could occur after intravenous thrombolytic treatment and CTA in a patient with AIS, which might lead to misdiagnosis and wrong treatment of the patient. The rapid resolution of intracranial hyperdense lesions is key to differentiate contrast extravasation from ICH on serial non-enhanced CT.

Abstract Background Bone morphogenetic proteins (BMPs) are part of the transforming growth factor beta (TGF-β) superfamily and play crucial roles in bone development, as well as in the formation and maintenance of various organs. Triplophysa dalaica, a small loach fish that primarily inhabits relatively high elevations and cooler water bodies, was the focus of this study. Understanding the function of BMP genes during the morphogenesis of T. dalaica helps to clarify the mechanisms of its evolution and serves as a reference for the study of BMP genes in other bony fishes. The data for the T. dalaica transcriptome and genome used in this investigation were derived from the outcomes of our laboratory sequencing. Results This study identified a total of 26 BMP genes, all of which, except for BMP1, possess similar TGF-β structural domains. We conducted an analysis of these 26 BMP genes, examining their physicochemical properties, subcellular localization, phylogenetic relationships, covariance within and among species, chromosomal localization, gene structure, conserved motifs, conserved structural domains, and expression patterns. Our findings indicated that three BMP genes were associated with unstable proteins, while 11 BMP genes were located within the extracellular matrix. Furthermore, some BMP genes were duplicated, with the majority being enriched in the GO:0008083 pathway, which is related to growth factor activity. It was hypothesized that genes within the BMP1/3/11/15 subgroup (Group I) play a significant role in the growth and development of T. dalaica. By analyzing the expression patterns of proteins in nine tissues (gonad, kidney, gill, spleen, brain, liver, fin, heart, and muscle), we found that BMP genes play diverse regulatory roles during different stages of growth and development and exhibit characteristics of division of labor. Conclusions This study contributes to a deeper understanding of BMP gene family member expression patterns in high-altitude, high-salinity environments and provides valuable insights for future research on the BMP gene family in bony fishes.

Spirulina is widely known as the “Super Nutritious Food of the Future” due to its exceptional nutritional properties. In this article, we investigated the effects of fermentation using a combination of Lactobacillus acidophilus KDB-03 and Kluyveromyces marxianus GDMCC 2.119 on Spirulina. Fermentation significantly increased the total flavonoid and phenol levels in Spirulina, and enhanced the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging capacity and ferric-reducing antioxidant power. Untargeted ultra-performance liquid chromatography-electrospray tandem mass spectrometry-based metabolomics and gas chromatography-ion mobility spectrometry analysis revealed alterations in the metabolites and flavor components of fermented Spirulina (FS). Furthermore, a mice experiment demonstrated that FS could effectively alleviate metabolism-associated fatty liver disease (MAFLD) by regulating liver metabolism, intestinal flora, and facilitating communication between the liver and gut. These findings suggest that fermentation could be a innovative approach to enhance the diversity of Spirulina products, and potentially be used as a strategy for the prevention and treatment of MAFLD.

In order to improve the utilization value of N. oceanica, the effects of enzyme-bacteria synergy treatment on its nutritional and functional components as well as its biological activities were investigated. Papain was used to hydrolyze N. oceanica, and the enzymolysis process was optimized by single factor and response surface test. After fermented by lactic acid bacteria, the contents of total phenols, peptides and flavonoids as well as the antioxidant and hypolipidemic activities in vitro before and after fermentation were measured. Sensory evaluation was also performed. The results showed that the optimum enzymolysis process of N. oceanica was as follows: Enzyme amount 5.0%, pH8.0, enzymolysis time 4.0 h, enzymolysis temperature 76 ℃. Under this condition, the content of polypeptide significantly increased by nearly 1.3 times and the total phenolic content increased by 5.1%, while the total flavonoid content decreased by 32.5%. The antioxidant activity and pancreatic lipase inhibitory activity of the fermented products were enhanced, among which the DPPH free radical scavenging rate in mixed bacteria fermented group increased by 1.2 times, and the pancreatic lipase scavenging rate in mixed bacteria fermented group increased by 59.4%. Moreover, the sensory score of the sample after fermentation was significantly improved. In conclusion, the synergistic fermentation of enzyme-bacteria can significantly improve the polypeptide content, antioxidant activity, hypolipidemia activity and sensory properties of N. oceanica, thus provides reference and technical support for the development of functional fermentation products.

Introduction Myocardial injury is a relatively common complication of traumatic brain injury (TBI). However, the incidence and clinical impact of myocardial injury characterised by elevated cardiac troponin (cTn) levels after TBI are still poorly known. The objective of our study is to assess the global incidence of myocardial injury characterised by elevated cTn in adult patients with TBI and its association with in-hospital mortality.Methods and analysis The protocol of our systematic review and meta-analysis is performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols guidelines. We will search the Medline, Embase, Cochrane Library, Scopus and Web of Science databases from inception to 1 January 2024, for observational studies in any language that reported the incidence of elevated cTn and/or in-hospital mortality associated with elevated cTn among adult patients with TBI. Two reviewers will independently assess study eligibility, extract the data and assess the risk of bias. ORs and 95% CIs will be used with a random-effects or fixed-effects model according to the estimated heterogeneity among studies assessed by the I2 index. We will perform a quantitative synthesis for the incidence of elevated cTn and in-hospital mortality data. If sufficient data are available, we will perform subgroup analysis and meta-regression to address the heterogeneity. In addition, we will perform a narrative analysis if quantitative synthesis is not appropriate.Ethics and dissemination Ethics approval was not required for this study. We intend to publish our findings in a high-quality, peer-reviewed journal.PROSPERO registration number CRD42023454686.

Acheilognathus barbatulus is distributed in Yangtze River, Yellow River and Pearl River systems in China. Genome data can help to understand the phylogenetic relationships of A. barbatulus, but its complete mitochondrial genome has not been published. We determined the complete mitochondrial genome structure and characteristics of this species and constructed a comprehensive phylogenetic tree, based on mitochondrial genome data of several species of Acheilognathus, Rhodeus and Pseudorasbora parva. The complete length of the mitochondrial genome of A. barbatulus is 16726 bp. The genome is a covalently closed double-stranded circular molecule containing 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, a D-loop and a light strand replication initiation region. The base composition of the complete mitochondrial genome is A (29.33%) > T (27.6%) > C (26.12%) > G (16.95%), showing a strong AT preference and anti-G bias. All 13 PCGs have different degrees of codon preference, except for cytochrome c oxidase 1, which uses GTG as the start codon. All the PCGs use ATG as the start codon and the stop codon is dominated by TAG. The encoded amino acids Leu and Ser exist in two types, whereas the rest are all present as one type, except for tRNASer (GCT), which lacks the D-arm and has an incomplete secondary structure, all other tRNAs can be folded to form a typical cloverleaf secondary structure. Based on the 13 PCG tandems, the Maximum Likelihood and Bayesian trees were constructed, based on the concatenated sequence of 13 PCGs for the genera Acheilognathus and Rhodeus, with Pseudorasbora parva as the outgroup. Acheilognathus barbatulus, Acheilognathus tonkinensis and Acheilognathus cf. macropterus were clustered together and the most closely related. The results of this study enrich the mitochondrial genomic data of Acheilognathus and provide molecular and genetic base information for species conservation, molecular identification and species evolution of Acheilognathinae.

In flowering Chinese cabbage, early booting is one of the most important characteristics that is linked with quality and production. Through fixed light intensity (280 μmol·m−2·s−1) and fixed intermittent lighting in flowering Chinese cabbage, there was early bolting, bud emergence, and flowering. Moreover, the aboveground fresh weight, blade area, dry weight of blade, and quantification of the leaves in flowering Chinese cabbage were significantly reduced, while the thickness of tillers, tillers height, dry weight of tillers, and tillers weight were significantly increased. The chlorophyll contents and soil–plant analysis and development (SPAD) value decreased in the early stage and increased in the later stage. The nitrate content decreased, while the photosynthetic rate, vitamin C content, soluble sugar content, soluble protein content, phenolic content, and flavonoid content increased, and mineral elements also accumulated. In order to explore the mechanism of intermittent light promoting the early bolting and flowering of ‘49d’ flowering Chinese cabbage, this study analyzed the transcriptional regulation from a global perspective using RNA sequencing. A total of 17,086 differentially expressed genes (DEGs) were obtained and 396 DEGs were selected that were closely related to early bolting. These DEGs were mainly involved in pollen wall assembly and plant circadian rhythm pathways, light action (34 DEGs), hormone biosynthesis and regulation (26 DEGs), development (21 DEGs), and carbohydrate synthesis and transport (6 DEGs). Three hub genes with the highest connectivity were identified through weighted gene co-expression network analysis (WGCNA): BrRVE, BrLHY, and BrRVE1. It is speculated that they may be involved in the intermittent light regulation of early bolting in flowering Chinese cabbage. In conclusion, intermittent light can be used as a useful tool to regulate plant growth structure, increase planting density, enhance photosynthesis, increase mineral accumulation, accelerate growth, and shorten the breeding cycle.

Objective Previous study have shown that lipid accumulation product (LAP), visceral adiposity index (VAI), triglyceride/high-density lipoprotein cholesterol ratio (TG/HDL-C) and triglycerides/glucose index (TyG index) could be simple clinical indicators of insulin resistance (IR) based on anthropometric and/or biochemical parameters. However, the rational and preferred surrogate marker of IR in different population has yet to be validated. The aim of this study was evaluating the practicability of the LAP, VAI, TG/HDL-C, and TyG in predicting IR in middle-aged Chinese population. Methods A cross-sectional study was conducted in 569 Chinese participants (mean age was 48.5; man 67.7%), and each participant completed a questionnaire survey, anthropometric measurement, and biochemical testing. One-way ANOVAs, Chi-squared test, Pearson’s correlation, and multiple logistic regression were used to evaluate the association between VAI, LAP, TG/HDL-C, and TyG with IR. To correctly discriminate individuals with insulin resistance, a receiver operating characteristic (ROC) analysis was conducted for each evaluated variable and the overall diagnostic accuracy was quantified using the area under the ROC curve (AUC). The AUC of evaluated variables were compared using a nonparametric approach. The optimal cut-off points were determined by the Youden’s index, and the corresponding sensitivity and specificity were provided. Results Significant positive correlation was identified between HOMA-IR with TG/HDL-C (r = 0.306), VAI (r = 0.217), LAP (r = 0.381), and TyG (r = 0.371), respectively (all p

BackgroundMembrane palmitoylated proteins (MPPs) are engaged in various biological processes, such as cell adhesion and cell polarity. Dysregulated MPP members have different effects on hepatocellular carcinoma (HCC) development. However, the role of MPP6 in HCC has been unknown.MethodHCC transcriptome and clinical data from different public databases were downloaded and analyzed, and the results were further validated by qRT−PCR, Western blotting and immunohistochemistry (IHC) using HCC cell lines and tissues. The association between MPP6 and prognosis, potential pathogenic mechanisms, angiogenesis, immune evasion, tumor mutation burden (TMB) and treatment response in HCC patients was analyzed by bioinformatics and IHC staining.ResultsMPP6 was significantly overexpressed in HCC, and its expression was related to T stage, pathologic stage, histologic grade and adverse prognosis in HCC patients. Gene set enrichment analysis revealed that differentially expressed genes were mainly enriched in the synthesis of genetic materials and the WNT signaling pathway. GEPIA database analysis and IHC staining suggested that MPP6 expression had a positive correlation with angiogenesis. Single-cell dataset analysis indicated that MPP6 was associated with features of the tumor microenvironment. Additional analyses discovered that MPP6 expression was inversely related to immune cell infiltration and was involved in tumor immune evasion. MPP6 expression was positively associated with TMB, and patients with high TMB had an adverse prognosis. Immunotherapy was more effective in HCC patients with low MPP6 expression, whereas those with high MPP6 expression responded better to sorafenib, gemcitabine, 5-FU, and doxorubicin.ConclusionsElevated MPP6 expression is associated with an unfavorable prognosis, angiogenesis and immune evasion in HCC. Moreover, MPP6 has the potential to be used to assess TMB and treatment response. Therefore, MPP6 might serve as a novel prognostic biomarker and therapeutic target for HCC.

Abstract Background Prenatal exposure to heavy metals is implicated in the etiology of birth defects. We investigated whether concentrations of cadmium (Cd) and lead (Pb) in umbilical cord tissue are associated with risk for neural tube defects (NTDs) and whether selected genetic variants of the fetus modify their associations. Methods This study included 166 cases of NTD fetuses/newborns and 166 newborns without congenital malformations. Umbilical cord tissue was collected at birth or elective pregnancy termination. Cd and Pb concentrations were assessed by inductively coupled plasma-mass spectrometry, and 20 single-nucleotide polymorphisms (SNPs) in 9 genes were genotyped. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to estimate the risk for NTDs in association with metal concentrations or genotype using logistic regression. Multiplicative-scale interactions between the metals and genotypes on NTD risk were assessed with logistic regression, and additive-scale interactions were estimated with a non-linear mixed effects model. Results Higher concentrations of Cd were observed in the NTD group than in the control group, but no difference was found for Pb. Concentrations of Cd above the median level showed a risk effect, while the association between Pb and NTD risk was not significant in univariate analyses. The association of Cd was attenuated after adjusting for periconceptional folic acid supplementation. Fetuses with the AG and GG genotypes of rs4880 in SOD2 (superoxide dismutase 2) tended to have a lower risk, but fetuses with the CT and TT genotypes of rs1801133 in MTHFR (5,10-methylenetetrahydrofolatereductase) have a higher risk for NTDs when compared to their respective wild-type. rs4880 and Cd exhibited a multiplicative-scale interaction on NTD risk: the association between higher Cd and the risk for NTDs was increased by over fourfold in fetuses carrying the G allele [OR 4.43 (1.30–15.07)] compared to fetuses with the wild-type genotype. rs1801133 and Cd exposure showed an additive interaction, with a significant relative excess risk of interaction [RERI 0.64 (0.02–1.25)]. Conclusions Prenatal exposure to Cd may be a risk factor for NTDs, and the risk effect may be enhanced in fetuses who carry the G allele of rs4880 in SOD2 and T allele of rs1801133 in MTHFR.

Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo β-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of β-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in β-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, β-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.

Computational image aesthetic evaluation is a computable human aesthetic perception and judgment realized by machines, which has a significant impact on a variety of applications such as image advanced search and promotional exhibition of painting arts. Various approaches have been proposed in copious literature trying to solve this challenging problem. However, there have been few attempts in reviewing works from different types of visual arts, due to their significant differences in visual features and aesthetic principles. In this survey, we present a comprehensive listing of the reviewed works on aesthetic assessment of photographs and paintings, mainly highlighting the contributions and innovations of the existing approaches. We firstly introduce aesthetic assessment benchmark datasets in different categories. Then, conventional aesthetic evaluation approaches based on handcrafted features are reviewed. Besides, we systematically evaluate recent deep learning techniques that are useful for developing robust models for aesthetic prediction tasks in scoring, distribution, attribute, and description. Moreover, the possibility of aesthetic-aware color enhancement, recomposition of photo images, and automatic generation of aesthetic-guided art paintings through computational approaches are summarized. Finally, challenges and potential future directions for this field are discussed. We hope that our survey could serve as a comprehensive reference source for future research on computational aesthetics in visual media.

For device printing, it is important for uniform films to be printed because the morphology of the film affects the performance of the device in inkjet printing. In this study, to prepare stable and well-dispersed ink, polyacrylate and silicone surfactant BD-3033H were added to ink. Then a uniform quantum dot (QD) film was obtained by optimizing the polyacrylate and silicone surfactant contents of the ink as well as the substrate temperature. The change of the polyacrylate structure from a curly chain structure to a three-dimensional network structure blocked the outward flow of QDs. Silicone surfactant BD-3033H caused Marangoni flow and made the QDs flow inward. When the substrate temperature was increased, the solvent evaporation rate was accelerated, the contact line was pinned, and the outward flow was enhanced, which changed the film structure from convex to flat. Finally, when the polyacrylate content was 12 wt%, the silicone surfactant BD-3033H content was 0.10 wt%, the substrate temperature was 40°C, and the coffee ring effect was eliminated. A uniform QD film was printed, providing a technical guarantee for the fabrication of QD devices in the future.

Assessing the aesthetic appeal of artworks has become an active research direction recently. However, previous works mainly focus on photographs and oil paintings, there have been few attempts in predicting aesthetics of Chinese ink paintings, due to their significant differences in visual features, semantic features, and aesthetic principles. Aiming at this problem, we propose a comprehensive framework, named Inkthetics, to quantify aesthetics of Chinese ink paintings based on deep learning. Firstly, an aesthetic assessment dataset is built for Chinese ink painting images. Secondly, a deep multi-view parallel convolutional neural network (DMVCNN) is designed by extracting global attribute images and multi-patches as inputs to jointly learn aesthetic features. Finally, we build a comprehensive aesthetic evaluation model by fusing the deeply-learned features with handcrafted features that rely on art expert knowledge. Experimental results show that our proposed deep network significantly outperforms existing methods on the dataset, and our proposed model can predict human aesthetic judgment with Pearson highly significant correlation of 0.843, which indicates an improvement up to 5.7% than the DMVCNN model when the handcrafted features are fused with activation from DMVCNN. Our work not only provides a deep-learning-based reference framework for computational aesthetic evaluation of Chinese paintings, but also explores to what extent can handcrafted features aid learning-based features in predicting human aesthetic perceptions.

Abstract Computational aesthetics, which bridges science and art, is emerging as a new interdisciplinary field. This paper concentrates on two main aspects of computational aesthetics: aesthetic measurement and quantification, generative art, and then proposes a design generation framework. On aesthetic measurement and quantification, we review different types of features used in measurement, the currently used evaluation methods, and their applications. On generative art, we focus on both fractal art and abstract paintings modeled on well-known artists’ styles. In general, computational aesthetics exploits computational methods for aesthetic expressions. In other words, it enables computer to appraise beauty and ugliness and also automatically generate aesthetic images. Computational aesthetics has been widely applied to many areas, such as photography, fine art, Chinese hand-writing, web design, graphic design, and industrial design. We finally propose a design generation methodology, utilizing techniques from both aesthetic measurements and generative art.

Based on a three-year field experiment under controlled condition in Ji’nan, China, the effects of peanut growth on the variation in the abundance and community structure of ammonia oxidizing bacteria (AOB) and Archaea (AOA) before and after peanut growth were investigated through quantitative PCR and cluster analysis of terminal-restriction fragment length polymorphism. Our results show that the community composition of AOA and AOB was greatly affected by the peanut growth leading to the decreased abundance of AOA and increased abundance of AOB. Furthermore, AOA and AOB community structures varied before and after peanut growth. Phylogenetic analysis indicated that all AOA and AOB community sequences were clustered into the uncultured group. Altogether, the results suggested that the abundance of AOA and AOB in soil and their community compositions can be greatly affected by the peanut growth.

Drought and heat stress are major causes of lost plant crop yield. In the future, high levels of CO2, in combination of other abiotic stress factors, will become a novel source of stress. Little is known of the mechanisms involved in the acclimation responses of plants to this combination of abiotic stress factors, though it has been demonstrated that heat shock transcription factors (Hsfs) are involved in plant response to various abiotic stresses. In this study, we performed a genome-wide identification and a systematic analysis of genes in the Hsf gene family in Brassica napus. A total of 64 genes encoding Hsf proteins were identified and classified into 3 major classes: A, B and C. We found that, unlike in other eudicots, the A9 subclass is absent in rapeseed. Further gene structure analysis revealed a loss of the only intron in the DBD domain for BnaHsf63 and -64 within class C, which is evolutionarily conserved in all Hsf genes. Transcription profile results demonstrated that most BnaHsf family genes are upregulated by both drought and heat conditions, while some are responded to a high CO2 treatment. According to the combined RNA-seq and qRT-PCR analysis, the A1E/A4A/A7 subclasses were upregulated by both drought and heat treatments. Members in class C seemed to be predominantly induced only by drought. Among BnaHsf genes, the A2/A3/B2 subclasses were regulated by all three abiotic stresses. Members in A2/B2 subclasses were upregulated by drought and heat treatments, but were downregulated under high CO2 conditions. While the A3 subclass was upregulated by all the three abiotic stresses. Various stress-related cis-acting elements, enriched in promoter regions, were correlated with the transcriptional response of BnaHsfs to these abiotic stresses. Further study of these novel groups of multifunctional BnaHsf genes will improve our understanding of plant acclimation response to abiotic stresses, and may be useful for improving the abiotic stress resistance of crop varieties.

Palmitoleic acid (C16:1) and stearic acid (C18:0) are precursors of polyunsaturated fatty acids, which are the focus of intensive global research due to their nutritional value, medicinal applications, and potential use as biofuel. Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that determine the types and amounts of fatty acids produced in plants and release fatty acids into the cytosol to be incorporated into glycerolipids. Based on amino acid sequence identity and substrate specificity, these enzymes are classified into two families, FatA and FatB. In this study, we cloned FatA and FatB thioesterases from Arachis hypogaea L. seeds and functionally expressed these genes, both individually and in tandem, in a blue-green alga Synechocystis sp. PCC6803. The heterologous expression of these genes in Synechocystis altered the fatty acid composition of lipids, resulting in a 29.5–31.6% increase in palmitoleic acid production and a 22.5–35.5% increase in stearic acid production. Moreover, the transgenic Synechocystis cells also showed significant increases in levels of oleic acid (C18:1, OA), linoleic acid (C18:2, LA), and α-linolenic acid (C18:3n3, ALA). These results suggest that the fatty acid profile of algae can be significantly improved by the heterologous expression of exogenous genes. This study not only provides insight into fatty acid biosynthesis, but also lays the foundation for manipulating the fatty acid content of cyanobacteria.

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (kcat/Km), half-lives (t1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.

Sterol 14α-demethylases from Cytochrome P450 family (CYP51s) are essential enzymes in sterol biosynthesis and well-known as the target of antifungal drugs. The 3D structure of CYP51A from Penicillium italicum (PiCYP51A) was constructed through homology modeling based on the crystal structure of human CYP51A (PDB: 3LD6). Molecular dynamics (MD) simulation was operated to relax the initial model and followed by quality assessment using PROCHECK program. On the basis of the docking information on the currently available CYP51s with the patent demethylase inhibitors (DMIs), pharmacophore-based virtual screening combined with docking analysis was performed to pick out twelve new compounds from ZINC database. Six hits revealed in the ligand database suggested potential ability to inhibit PiCYP51A. Compared to patent fungicide triazolone, the top three lead compounds had similar or higher affinity with the target enzyme, and accordingly, exhibited comparable or lower EC50 values to P. italicum isolates. The results could provide references for de novo antifungal drug design.