Molecular characterization of the sans fille gene in Antheraea pernyi: A highly conserved gene during the evolution of animals

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and plays an important role in sex determination in Drosophila melanogaster. In this study, the snf gene from Antheraea pernyi (Lepidoptera: Saturniidae), an economically important insect, was isolated and characterized. The obtained 925 bp cDNA sequence contains an open reading frame of 669 bp encoding a polypeptide of 222 amino acids, showing 78% sequence identity to that from D. melanogaster. A database search revealed that SNF protein homologs are present in many animals, including invertebrates and vertebrates, with more than 70% amino acid sequence identities, suggesting that they were highly conserved during the evolution of animals. Phylogenetic analysis revealed that A. pernyi SNF was closely related to Bombyx mori SNF. Quantitative real-time PCR (qRT-PCR) analysis showed that the A. pernyi snf gene was transcribed during five larval developmental stages, and in six tested tissues (ovaries, testes, silk glands, fat body, integument, and hemolymph), with the most abundance determined in the gonads (ovaries or testes). Investigation of expression changes throughout embryonic development indicated that A. pernyi snf mRNA was expressed at a low level from days 0 to 4, and reached a maximum level at day 10, but decreased to a low level before hatching. These results suggest that the product of the snf gene may play important roles in the development of A. pernyi.     

Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.     

Efficient secretion of lipase r27RCL in Pichia pastoris by enhancing the disulfide bond formation pathway in the endoplasmic reticulum

The lipase r27RCL from Rhizopus chinensis CCTCC M201021 was heterologously expressed in Pichia pastoris GS115 by simultaneous co-expression with two secretion factors ERO1p and PDI involved in the endoplasmic reticulum (ER). Compared to the expression of the lipase alone (12,500 U/ml), co-expression with these two proteins resulted in the production of larger total quantities of enzymes. The largest increase was seen when the combined ERO1p/PDI system was co-expressed, resulting in approximately 30 % higher enzyme yields (16,200 U/ml) than in the absence of co-expressed secretion factors. The extracellular protein concentration of the recombinant strain Co XY RCL-5 reached 9.39 g/l in the 7-l fermentor. Simultaneously, the fermentation time was also shortened by about 8 h compared to that of the control. The substrate-specific consumption rate (Qs) and the product-specific production rate (Qp) were both investigated in this research. In conclusion, the space–time yield was improved by co-expression with ERO1p and PDI. This is a potential strategy for high level expression of other heterologous proteins in P. pastoris.     

Caloric restriction augments radiation efficacy in breast cancer

Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.     

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

Background

Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism.

Methodology/Principal Findings

Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats.

Conclusions/Significance

In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.     

Disrupted Topological Organization in Whole-Brain Functional Networks of Heroin-Dependent Individuals: A Resting-State fMRI Study

Neuroimaging studies have shown that heroin addiction is related to abnormalities in widespread local regions and in the functional connectivity of the brain. However, little is known about whether heroin addiction changes the topological organization of whole-brain functional networks. Seventeen heroin-dependent individuals (HDIs) and 15 age-, gender-matched normal controls (NCs) were enrolled, and the resting-state functional magnetic resonance images (RS-fMRI) were acquired from these subjects. We constructed the brain functional networks of HDIs and NCs, and compared the between-group differences in network topological properties using graph theory method. We found that the HDIs showed decreases in the normalized clustering coefficient and in small-worldness compared to the NCs. Furthermore, the HDIs exhibited significantly decreased nodal centralities primarily in regions of cognitive control network, including the bilateral middle cingulate gyrus, left middle frontal gyrus, and right precuneus, but significantly increased nodal centralities primarily in the left hippocampus. The between-group differences in nodal centralities were not corrected by multiple comparisons suggesting these should be considered as an exploratory analysis. Moreover, nodal centralities in the left hippocampus were positively correlated with the duration of heroin addiction. Overall, our results indicated that disruptions occur in the whole-brain functional networks of HDIs, findings which may be helpful in further understanding the mechanisms underlying heroin addiction.     

Detection of Airway Anomalies in?Pediatric?Patients with Cardiovascular Anomalies with Low Dose Prospective ECG-Gated Dual-Source CT

Objectives

To assess the feasibility of low-dose prospective ECG-gated dual-source CT (DSCT) in detecting airway anomalies in pediatric patients with cardiovascular anomalies compared with flexible tracheobronchoscopy (FTB).

Methods

33 pediatrics with respiratory symptoms who had been revealed cardiovascular anomalies by transthoracic echocardiography underwent FTB and contrast material–enhanced prospective ECG-triggering CT were enrolled. The study was approved by our institution review board and written informed consent was obtained from all patients’ guardian. DSCT examinations were performed to detect cardiovascular abnormalities using weight-adjusted low–dose protocol. Two radiologists independently performed CT image analysis. The FTB reports were reviewed by an experienced pulmonologist. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of DSCT in the detection of airway anomalies were assessed. The tracheobronchial stenoses revealed on FTB were graded. Effective radiation dose was calculated.

Results

Thirty cases were diagnosed with tracheobronchial narrowing and/or abnormality in 33 patients by FTB, while 3 patients had normal FTB findings. Twenty-eight cases were diagnosed with airway anomalies by CT, of which 27 were correct positive. 3 patients with normal findings at CT had findings of tracheobronchial narrowing due to tracheobronchomalacia at inspiration at FTB. Sensitivity and specificity of CT were 90.0% (95% CI: 72.3%, 97.4%) and 66.7% (95% CI: 12.5 %, 98.2 %), respectively. PPV and NPV were 96.4% (95% CI: 79.8 %, 99.8%) and 40.0% (95% CI: 7.3%, 83.0%), respectively. Overall accuracy of DSCT in detecting airway anomalies in pediatrics with cardiovascular anomalies was 87.9% (95% CI: 74.5%, 97.6%). In grading of tracheobronchial stenosis, images from CT correlated closely (r = 0.89) with those of FTB. Mean effective dose was 0.60±0.20 mSv.

Conclusion

In pediatric patients, ECG-triggered CT to evaluate congenital cardiovascular anomalies can also be used to diagnose and characterize fixed airway involvement in relation to the vascular structures.     

GenomeFingerprinter: The Genome Fingerprint and the Universal Genome Fingerprint Analysis for Systematic Comparative Genomics

Background

No attention has been paid on comparing a set of genome sequences crossing genetic components and biological categories with far divergence over large size range. We define it as the systematic comparative genomics and aim to develop the methodology.

Results

First, we create a method, GenomeFingerprinter, to unambiguously produce a set of three-dimensional coordinates from a sequence, followed by one three-dimensional plot and six two-dimensional trajectory projections, to illustrate the genome fingerprint of a given genome sequence. Second, we develop a set of concepts and tools, and thereby establish a method called the universal genome fingerprint analysis (UGFA). Particularly, we define the total genetic component configuration (TGCC) (including chromosome, plasmid, and phage) for describing a strain as a systematic unit, the universal genome fingerprint map (UGFM) of TGCC for differentiating strains as a universal system, and the systematic comparative genomics (SCG) for comparing a set of genomes crossing genetic components and biological categories. Third, we construct a method of quantitative analysis to compare two genomes by using the outcome dataset of genome fingerprint analysis. Specifically, we define the geometric center and its geometric mean for a given genome fingerprint map, followed by the Euclidean distance, the differentiate rate, and the weighted differentiate rate to quantitatively describe the difference between two genomes of comparison. Moreover, we demonstrate the applications through case studies on various genome sequences, giving tremendous insights into the critical issues in microbial genomics and taxonomy.

Conclusions

We have created a method, GenomeFingerprinter, for rapidly computing, geometrically visualizing, intuitively comparing a set of genomes at genome fingerprint level, and hence established a method called the universal genome fingerprint analysis, as well as developed a method of quantitative analysis of the outcome dataset. These have set up the methodology of systematic comparative genomics based on the genome fingerprint analysis.     

Overexpression of an Acidic Endo-β-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed

Background

Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed.

Methodology/Principal Findings

Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0–8.0, which is in agreement with the normal environment of digestive tract.

Conclusion/Significance

Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.