Primer-directed mutagenesis of linearized plasmids

Two novel mutagenesis techniques to specifically alter the sequence of plasmid DNA have been developed. In contrast to other primer-directed mutagenesis methods which require a single-stranded, closed-circular template, a linearized single strand was used as the mutagenesis template. The template is prepared by restriction enzyme digestion of covalently-closed-circular plasmid DNA. These methods are simple, require small amounts of plasmid DNA, and can result in a relatively high frequency of mutagenesis.     

Genome‐wide association study identifies loci associated with liability to alcohol and drug dependence that is associated with variability in reward‐related ventral striatum activity in African‐ and European‐Americans

Genetic influences on alcohol and drug dependence partially overlap, however, specific loci underlying this overlap remain unclear. We conducted a genome‐wide association study (GWAS) of a phenotype representing alcohol or illicit drug dependence (ANYDEP) among 7291 European‐Americans (EA; 2927 cases) and 3132 African‐Americans (AA: 1315 cases) participating in the family‐based Collaborative Study on the Genetics of Alcoholism. ANYDEP was heritable (h ² in EA = 0.60, AA = 0.37). The AA GWAS identified three regions with genome‐wide significant (GWS; P < 5E‐08) single nucleotide polymorphisms (SNPs) on chromosomes 3 (rs34066662, rs58801820) and 13 (rs75168521, rs78886294), and an insertion‐deletion on chromosome 5 (chr5:141988181). No polymorphisms reached GWS in the EA. One GWS region (chromosome 1: rs1890881) emerged from a trans‐ancestral meta‐analysis (EA + AA) of ANYDEP, and was attributable to alcohol dependence in both samples. Four genes (AA: CRKL, DZIP3, SBK3; EA: P2RX6) and four sets of genes were significantly enriched within biological pathways for hemostasis and signal transduction. GWS signals did not replicate in two independent samples but there was weak evidence for association between rs1890881 and alcohol intake in the UK Biobank. Among 118 AA and 481 EA individuals from the Duke Neurogenetics Study, rs75168521 and rs1890881 genotypes were associated with variability in reward‐related ventral striatum activation. This study identified novel loci for substance dependence and provides preliminary evidence that these variants are also associated with individual differences in neural reward reactivity. Gene discovery efforts in non‐European samples with distinct patterns of substance use may lead to the identification of novel ancestry‐specific genetic markers of risk.     

New 4,5‐seco‐20(10→5)‐abeo‐Abietane Diterpenoids with Anti‐Inflammatory Activity from Isodon lophanthoides var. graciliflorus (Benth.) H.Hara

Three new 4,5‐seco‐20(10→5)‐abeo‐abietane diterpenoids, 16‐hydroxysalvilenone ( 1 ), 15‐hydroxysalprionin ( 2 ), and 11β,15‐dihydroxysalprionin‐12‐one ( 3 ), and nine known abietane diterpenoids, 4 – 12 , along with one known sempervirane diterpenoid, hispidanol A ( 13 ), were isolated from the aerial parts of Isodon lophanthoides var. graciliflorus. The structures of compounds 1 – 3 were determined on the basis of spectroscopic methods including extensive analysis of NMR and mass spectroscopic data. All diterpenoids were tested for their TNF‐α inhibitory effects on LPS‐induced RAW264.7 cells. Compound 9 (16‐acetoxyhorminone) was the most potent with an IC₅₀ value of 3.97±0.70 μm .     

Extraction and Antioxidant Activities of Magnolia kwangsiensis Figlar & Noot. Leaf Polyphenols

Microwave‐assisted extraction was employed to extract polyphenols from the leaf of Magnolia kwangsiensis Figlar & Noot . The yield of polyphenols was 2.44±0.02 % under the optimal conditions of RSM: acetone concentration of 70 %, ratio of solvent to material of 21 mL?g^?1 and extraction time of 16 min. The antioxidant activities were evaluated in terms of total antioxidant ability, reducing power, DPPH ^? and ^? OH scavenging activity. Results showed the polyphenols presented potential antioxidant activities, especially the stronger scavenging activity on ^? OH. In term of ^? OH scavenging activity, the IC₅₀ value of NKA‐9 purification was 0.335 mg mL^?1, equivalent to 35.23 % of V_C. The IC₅₀ values of crude extract and ethyl acetate extract were 0.580 and 0.828 mg mL^?1, equivalent to 60.99 % and 87.07 % of V_C. Results indicated that M. kwangsiensis leaf polyphenols present potential antioxidant activities that make it beneficial for human health by preventing or reducing oxidative damage.     

A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize

Plant RNA virus-based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter-driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus-induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)-based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated targeted mutagenesis in wheat and maize. BSMV-based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV-mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops.     

A distance-type measure approach to the analysis of copy number variation in DNA sequencing data

Background

The next generation sequencing technology allows us to obtain a large amount of short DNA sequence (DNA-seq) reads at a genome-wide level. DNA-seq data have been increasingly collected during the recent years. Count-type data analysis is a widely used approach for DNA-seq data. However, the related data pre-processing is based on the moving window method, in which a window size need to be defined in order to obtain count-type data. Furthermore, useful information can be reduced after data pre-processing for count-type data.

Results

In this study, we propose to analyze DNA-seq data based on the related distance-type measure. Distances are measured in base pairs (bps) between two adjacent alignments of short reads mapped to a reference genome. Our experimental data based simulation study confirms the advantages of distance-type measure approach in both detection power and detection accuracy. Furthermore, we propose artificial censoring for the distance data so that distances larger than a given value are considered potential outliers. Our purpose is to simplify the pre-processing of DNA-seq data. Statistically, we consider a mixture of right censored geometric distributions to model the distance data. Additionally, to reduce the GC-content bias, we extend the mixture model to a mixture of generalized linear models (GLMs). The estimation of model can be achieved by the Newton-Raphson algorithm as well as the Expectation-Maximization (E-M) algorithm. We have conducted simulations to evaluate the performance of our approach. Based on the rank based inverse normal transformation of distance data, we can obtain the related z-values for a follow-up analysis. For an illustration, an application to the DNA-seq data from a pair of normal and tumor cell lines is presented with a change-point analysis of z-values to detect DNA copy number alterations.

Conclusion

Our distance-type measure approach is novel. It does not require either a fixed or a sliding window procedure for generating count-type data. Its advantages have been demonstrated by our simulation studies and its practical usefulness has been illustrated by an experimental data application.

     

Confined Fe2VO4⊂Nitrogen‐Doped Carbon Nanowires with Internal Void Space for High‐Rate and Ultrastable Potassium‐Ion Storage

Developing low‐cost, high‐capacity, high‐rate, and robust earth‐abundant electrode materials for energy storage is critical for the practical and scalable application of advanced battery technologies. Herein, the first example of synthesizing 1D peapod‐like bimetallic Fe₂VO₄ nanorods confined in N‐doped carbon porous nanowires with internal void space (Fe₂VO₄?NC nanopeapods) as a high‐capacity and stable anode material for potassium‐ion batteries (KIBs) is reported. The peapod‐like Fe₂VO₄?NC nanopeapod heterostructures with interior void space and external carbon shell efficiently prevent the aggregation of the active materials, facilitate fast transportation of electrons and ions, and accommodate volume variation during the cycling process, which substantially boosts the rate and cycling performance of Fe₂VO₄. The Fe₂VO₄?NC electrode exhibits high reversible specific depotassiation capacity of 380 mAh g^?1 at 100 mA g^?1 after 60 cycles and remarkable rate capability as well as long cycling stability with a high capacity of 196 mAh g^?1 at 4 A g^?1 after 2300 cycles. The first‐principles calculations reveal that Fe₂VO₄?NC nanopeapods have high ionic/electronic conductivity characteristics and low diffusion barriers for K⁺‐intercalation. This study opens up new way for investigating high‐capacity metal oxide as high‐rate and robust electrode materials for KIBs.     

Ultrathin 2D TiS2 Nanosheets for High Capacity and Long‐Life Sodium Ion Batteries

Sodium ion batteries are now attracting great attention, mainly because of the abundance of sodium resources and their cheap raw materials. 2D materials possess a unique structure for sodium storage. Among them, transition metal chalcogenides exhibit significant potential for rechargeable battery devices due to their tunable composition, remarkable structural stability, fast ion transport, and robust kinetics. Herein, ultrathin TiS₂ nanosheets are synthesized by a shear‐mixing method and exhibit outstanding cycling performance (386 mAh g^?1 after 200 cycles at 0.2 A g^?1). To clarify the variations of galvanostatic curves and superior cycling performance, the mechanism and morphology changes are systematically investigated. This facile synthesis method is expected to shed light on the preparation of ultrathin 2D materials, whose unique morphologies could easily enable their application in rechargeable batteries.     

Preliminary analysis of geographical distribution based on cold hardiness for Evergestis extimalis (Scopoli) (Lepidoptera: Pyralidae) on Qinghai–Tibet Plateau

Evergestis extimalis  (Scopoli) is a pest insect present in spring rape fields of the Qinghai–Tibet plateau. A survey of its distribution and analysis of its physiological and biochemical variances of its overwintering larvae were conducted in this study. Prior to 2006, Evergestis extimalis Scopli appeared only sporadically at the east agricultural district of Qinghai Province at 2,100 m elevation; after 2006, there have been frequent outbreaks at 2,200 m or so height. The insect's distribution has extended continuously toward higher altitudes yearly, and the scope of its damage reached 2,800 m height in 2010. These changes indicate that the cold hardiness of E. extimalis is on the rise. Physiological and biochemical analyses were performed for the insect's overwintering larvae from November 2011 to March 2012. The supercooling point (SCP) and freezing point (FP) ranged from ?6.85°C to ?12.49°C and from ?6.23°C to ?8.17°C, respectively, and both were at their respective lowest points in January 2012; the lowest points of water and fat contents (which did not vary to any extreme degree throughout the test period) were also observed in January 2012. Glycogen content varied from 2.42 mg/g to 4.56 mg/g. Protein content increased gradually at the first two months and reached its peak in January 2012 before dropping slightly. The activity of protective enzymes POD, CAT, and SOD varied with changes in environmental temperature, and each was at its lowest point in January 2012. With the exception of protein and glycerol content, other physiological and biochemical variances were generally parallel with environmental temperature, strongly indicating that E. extimalis has indeed developed cold hardiness.     

Probiotic Clostridium butyricum Improves the Growth Performance,Immune Function,and Gut Microbiota of Weaning Rex Rabbits

Probiotics could promote animal growth and enhance immune function. This study investigated the effects of Clostridium butyricum (CB) on the growth performance, intestinal immune, and gut microbiota of weaning rex rabbits. A total of 60 healthy female rabbits (5-month-old) were divided equally into four groups and mated on the same day: control group (CTRL, fed with basal feed), low-dose group (LDG, fed with basal feed + 1.0 × 10³ CFU/g CB), middle-dose group (MDG, fed with basal feed + 1.0 × 10⁴ CFU/g CB), and high-dose group (HDG, fed with basal feed + 1.0 × 10⁵ CFU/g CB). Then, 30 weaning rex rabbits (35-day-old) were collected from each group for this experiment, and they were offered the same feeds as their mother. The results demonstrated that high-dose CB treatment significantly increased average daily weight gain of weaning rex rabbits. Further studies suggested that CB enhanced small intestinal digestive enzyme activity and improved mucosal morphology and antioxidant status. Supplemented with CB, small intestinal barrier function was maintained with the upregulation of mRNA levels of ZO-1, claudin, and occludin as well as the increase of sIgA production. Moreover, the relative expressions of MyD88, TLR2, and TLR4 were elevated in HDG; simultaneously, pro-inflammatory cytokines including IL-6, INF-γ, and TNF-α were decreased after CB administration. In addition, CB showed beneficial effects in improving weaning rex rabbit intestinal microflora via increasing the abundance of beneficial bacteria. Therefore, our results indicated CB can promote rex rabbit growth, which is likely to the enhancement of immune function and the improvement of intestinal microbiota.