Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

Efficient reductive amination process for enantioselective synthesis of L-phosphinothricin applying engineered glutamate dehydrogenase

Applied Microbiology and Biotechnology - The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of...     

Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection.     

Multipotent Cancer Stem Cells Derived from Human Malignant Peritoneal Mesothelioma Promote Tumorigenesis

During the progression of malignant peritoneal mesothelioma (MPeM), tumor nodules propagate diffusely within the abdomen and tumors are characterized by distinct phenotypic sub-types. Recent studies in solid organ cancers have shown that cancer stem cells (CSCs) play a pivotal role in the initiation and progression of tumors. However, it is not known whether tumorigenic stem cells exist and whether they promote tumor growth in MPeM. In this study, we developed and characterized a CSC model for MPeM using stably expandable tumorigenic stem cells derived from patient tumors. We found morphologically distinct populations of CSCs that divide asymmetrically or symmetrically in MPeM in vitro cell culture. The MPeM stem cells (MPeMSCs) express stem cell markers c-MYC, NES and VEGFR2 and in the presence of matrix components cells form colony spheres. MPeMSCs are multipotent, differentiate into neuronal, vascular and adipose progeny upon defined induction and the differentiating cells express lineage-specific markers such as TUBB3, an early neuronal marker; vWF, VEGFA, VEGFC and IL-8, endothelial markers; and PPARγ and FABP4, adipose markers. Xenotransplantation experiments using MPeMSCs demonstrated early tumor growth compared with parental cells. Limiting dilution experiments using MPeMSCs and endothelial lineage-induced cells derived from a single MPeMSC resulted in early tumor growth in the latter group indicating that endothelial differentiation of MPeMSCs is important for MPeM tumor initiation. Our observation that the MPeM tumors contain stem cells with tumorigenic potential has important implications for understanding the cells of origin and tumor progression in MPeM and hence targeting CSCs may be a useful strategy to inhibit malignant progression.     

RNA‐seq analysis reveals new candidate genes for drip loss in a Pietrain × Duroc × Landrace × Yorkshire population

Drip loss, one of the most important meat quality traits, is characterized by low heritability. To date, the genetic factors affecting the drip loss trait have not been clearly elucidated. The objective of this study was to identify critical candidate genes affecting drip loss. First, we generated a Pietrain × Duroc × Landrace × Yorkshire commercial pig population and obtained phenotypic values for the drip loss trait. Furthermore, we constructed two RNA libraries from pooled samples of longissimus dorsi muscles with the highest (H group) and lowest (L group) drip loss and identified the differentially expressed genes (DEGs) between these extreme phenotypes using RNA‐seq technology. In total, 25 883 genes were detected in the H and L group libraries, and none was specifically expressed in only one library. Comparative analysis of gene expression levels found that 150 genes were differentially expressed, of which 127 were upregulated and 23 were downregulated in the H group relative to the L group. In addition, 68 drip loss quantitative trait loci (QTL) overlapping with 63 DEGs were identified, and these QTL were distributed mainly on chromosomes 1, 2, 5 and 6. Interestingly, the triadin (TRDN) gene, which is involved in muscle contraction and fat deposition, and the myostatin (MSTN) gene, which has a role in muscle growth, were localized to more than two drip loss QTL, suggesting that both are critical candidate genes responsible for drip loss.     

A Microarray-Based Analysis Reveals that a Short Photoperiod Promotes Hair Growth in the Arbas Cashmere Goat

Many animals exhibit different behaviors in different seasons. The photoperiod can have effects on migration, breeding, fur growth, and other processes. The cyclic growth of the fur and feathers of some species of mammals and birds, respectively, is stimulated by the photoperiod as a result of hormone-dependent regulation of the nervous system. To further examine this phenomenon, we evaluated the Arbas Cashmere goat (Capra hircus), a species that is often used in this type of research. The goats were exposed to an experimentally controlled short photoperiod to study the regulation of cyclic cashmere growth. Exposure to a short photoperiod extended the anagen phase of the Cashmere goat hair follicle to increase cashmere production. Assessments of tissue sections indicated that the short photoperiod significantly induced cashmere growth. This conclusion was supported by a comparison of the differences in gene expression between the short photoperiod and natural conditions using gene chip technology. Using the gene chip data, we identified genes that showed altered expression under the short photoperiod compared to natural conditions, and these genes were found to be involved in the biological processes of hair follicle growth, structural composition of the hair follicle, and the morphogenesis of the surrounding skin appendages. Knowledge about differences in the expression of these genes as well as their functions and periodic regulation patterns increases our understanding of Cashmere goat hair follicle growth. This study also provides preliminary data that may be useful for the development of an artificial method to improve cashmere production by controlling the light cycle, which has practical significance for livestock breeding.     

Mass Collaboration in Earthquake Risk Management

The mechanism of mass collaboration in risk management was studied during the Sichuan earthquake under a Web-based “PeopleFinder” project, where information is contributed and shared among mass contributors. The case study is provided by a great earthquake that happened in Wenchuan County, Sichuan Province, of southwestern China at 2:28 p.m. on May 12, 2008. We witnessed and experienced the rescue and relief efforts for the great earthquake. In this article, two fundamental frameworks are developed to study the mechanism of mass collaboration. Mass collaboration is proven to be effective in a big public crisis such as the Sichuan earthquake.