Omics Analysis of Blood-Responsive Regulon in Bordetella pertussis Identifies a Novel Essential T3SS Substrate

Bacterial pathogens sense specific cues associated with different host niches and integrate these signals to appropriately adjust the global gene expression. Bordetella pertussis is a Gram-negative, strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Though B. pertussis does not cause invasive infections, previous results indicated that this reemerging pathogen responds to blood exposure. Here, omics RNA-seq and LC–MS/MS techniques were applied to determine the blood-responsive regulon of B. pertussis. These analyses revealed that direct contact with blood rewired global gene expression profiles in B. pertussis as the expression of almost 20% of all genes was significantly modulated. However, upon loss of contact with blood, the majority of blood-specific effects vanished, with the exception of several genes encoding the T3SS-secreted substrates. For the first time, the T3SS regulator BtrA was identified in culture supernatants of B. pertussis. Furthermore, proteomic analysis identified BP2259 protein as a novel secreted T3SS substrate, which is required for T3SS functionality. Collectively, presented data indicate that contact with blood represents an important cue for B. pertussis cells.     

A New Optimized Wrapper Gene Selection Method for Breast Cancer Prediction

Machine-learning algorithms have been widely used in breast cancer diagnosis to help pathologists and physicians in the decision-making process. However, the high dimensionality of genetic data makes the classification process a challenging task. In this paper, we propose a new optimized wrapper gene selection method that is based on a nature-inspired algorithm (simulated annealing (SA)), which will help select the most informative genes for breast cancer prediction. These optimal genes will then be used to train the classifier to improve its accuracy and efficiency. Three supervised machine-learning algorithms, namely, the support vector machine, the decision tree, and the random forest were used to create the classifier models that will help to predict breast cancer. Two different experiments were conducted using three datasets: Gene expression (GE), deoxyribonucleic acid (DNA) methylation, and a combination of the two. Six measures were used to evaluate the performance of the proposed algorithm, which include the following: Accuracy, precision, recall, specificity, area under the curve (AUC), and execution time. The effectiveness of the proposed classifiers was evaluated through comprehensive experiments. The results demonstrated that our approach outperformed the conventional classifiers as expected in terms of accuracy and execution time. High accuracy values of 99.77%, 99.45%, and 99.45% have been achieved by SA-SVM for GE, DNA methylation, and the combined datasets, respectively. The execution time of the proposed approach was significantly reduced, in comparison to that of the traditional classifiers and the best execution time has been reached by SA-SVM, which was 0.02, 0.03, and 0.02 on GE, DNA methylation, and the combined datasets respectively. In regard to precision and specificity, SA-RF obtained the best result of 100 on GE dataset. While SA-SVM attained the best recall result of 100 on GE dataset.     

东营凹陷深部储层流度属性提取及应用

东营凹陷深部储层埋深大、储层薄,为干燥环境下沉积的红层,采用常规储层描述与预测方法难以取得好的效果。在流度属性表征方法计算公式详细推导和流度属性本质含义解剖的基础上,根据研究区岩石物理参数和流体置换的特征,建立了含油层和干层的理论模型,发现将储层主频作为优势频率提取的流度属性,具有很好的有利储层识别度,含油砂体的流度属性能量团比干层及其它地层的能量要强。对东营凹陷深部实际三维工区数据进行目标处理,过井剖面的有利储层得到很好展示;沿层流度属性切片,其储层厚度、储层流体类型及其分布范围与井点图吻合度很高,进一步验证了方法的有效性。研究成果拓展了流度属性的应用范围(以往仅局限于低频),也为深部储层预测开辟了一条新的途径,具有重要的理论意义和较好的推广价值。     

Discovery and characterization of tyrosinases from sea anemone pedal disc

Abstract

The formation of underwater adhesion is a complicated physiological process and many different types of enzymes are found to be essential apart from structural proteins. Previous studies have shown that various tyrosinases were present in marine adhesives, but little information is available about the over-expression and enzymatic characterization of these enzymes. Specifically, this study first identified four significantly up-regulated tyrosinases in the pedal disc of Haliplanella luciae by means of multi-omics technology, and made preliminary bioinformatics predictions. Sequence alignment showed that the Tyr1_Hl contained six conserved His residues that bind to copper ions, of which a tyrosinase with diphenolase activity named as Tyr1_Hl^Δ, was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Enzymatic characterization showed that the activity of Tyr1_Hl^Δ was Cu²⁺ dependent and maximum catalytic activities were in 20?mM Tris–HCl (pH 8.0) at 37?°C. In summary, we identified novel tyrosinases in the pedal disks of sea anemone for the first time and the Tyr1_Hl^Δ was successfully recombinant expressed. Our study will provide basis for future exploration of bio-adhesion mechanism and design of bio-adhesives derived from sea anemones.     

重组人淋巴细胞活化基因-3蛋白胞外段的真核表达及鉴定

目的真核表达重组人淋巴细胞活化基因-3(lymphocyte activation gene-3,LAG-3)蛋白胞外段,并进行鉴定。方法用植物血球凝集素(phytohaemagg lutinin,PHA)刺激Jurkat细胞,流式细胞术检测Jurkat细胞中LAG-3蛋白的表达;提取Jurkat细胞总mRNA,RT-PCR法扩增人LAG-3蛋白胞外段基因片段,同时在蛋白C-末端引入His标签,将其克隆入载体pcDNA3. 1+,构建重组质粒,转染Expi293F真核细胞,当细胞活率低于50%时收获细胞上清,经镍柱亲合层析纯化。纯化产物进行4%~20%SDS-PAGE、HPLC及Western blot分析,BCA法测定浓度。结果经菌液PCR、双酶切及测序鉴定,表明质粒构建正确。重组表达蛋白的相对分子质量约60 000,纯化后纯度达95%以上,与鼠抗LAG-3单克隆抗体可发生特异性结合,浓度为2. 4 mg/mL。结论成功构建了重组真核表达质粒LAG-3/pcDNA3. 1+,并于Expi293F细胞中表达,纯化获得了纯度较高的LAG-3蛋白,为后期LAG-3蛋白的相关研究及其单抗的制备奠定了基础。     

Comparison between discrete and continuous analysis of facial expressions,elicited by bitter-tasting beverages in overweight and healthy-weight individuals

The aim of the present study was to compare the information provided by a discrete and continuous data analysis of two emotions (disgust and joy), elicited by bitter-tasting beverages (coffee, yerba mate infusion and grapefruit juice) in two groups with different body mass index (BMI): overweight group (25 < BMI < 30), and healthy-weight control group (18.5 < BMI ≤ 25). Participants (n = 66; 34 females, 32 males) evaluated a total of three consecutive sips of the same beverage (taking one sip every 20 s and registering a continuous video for 60 s). The Wilcoxon test (continuous analysis) showed some changes generated as the drinks were being consumed. The biggest difference was the expression of disgust for coffee in the high BMI group, at the first sip (0–10 s). It represented 27% compared to 2% in normal BMI. The continuous analysis allowed to observe the periods where the differences were greater (0–10 and 40–50 s).     

The Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Has Digestive Capacity to Degrade Complex Substrates: Functional Characterization and Heterologous Expression of an α-Amylase

Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonus rhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.