piRNA-Triggered MIWI Ubiquitination and Removal by APC/C in Late Spermatogenesis

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Highlights? MIWI is a substrate of APC/C, and piRNA loading is essential for MIWI ubiquitination ? piRNA loading promotes MIWI binding to the APC/C substrate-binding subunit ? MIWI and piRNAs are coordinately eliminated in late spermatids ? Inhibition of MIWI destruction in late spermatids prevents sperm maturation     

Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.     

Effects of N application on N accumulation of two Erigeron breviscapus populations from different altitudes under enhanced ultraviolet-B radiation

To study intraspecific differences in N utilization in response to enhanced UV-B radiation, field experiments were conducted on two Erigeron breviscapus populations (Huguo and Cangshan), which were respectively obtained from low altitude (UV-B sensitive) and high altitude (UBV-B resistant).The effects of soil nitrogen (N) application (0, 15, 30, 45 g m²) on free amino acid content, the activities of nitrate reductase (NR) and glutamine synthetase (GS), total nitrogen content and N mass in leaves were determined under enhanced UV-B radiation (5 kJ m²) for both populations. The results showed that under enhanced UV-B radiation: (1) increases in total N contents in leaves of the Huguo and Cangshan populations correlated with the amount of N applied. Additionally, leaf biomass of Huguo treated with 15 g m^?2 N application and Cangshan with 30 g m^?2 N application were higher than that of other treatments. Leaf N masses were highest in both E. breviscapus populations treated with 30 g m^?2 N; (2) increases in contents of free amino acids in leaves of both E. breviscapus populations correlated with the amount of applied nitrogen; (3) increases of NR activity in leaves correlated with the amount of applied nitrogen; (4) GS activity in leaves of the Huguo and Cangshan E. breviscapus populations were highest with respective N applications of 15 g m^?2 N and 30 g m^?2 N. In general, under enhanced UV-B radiation, N application might affect NR and GS and change free amino acid content, resulting in changes in total nitrogen content, biomass and N mass. The optimal amount of supplemental N for N accumulation in E. breviscapus was 30 g m^?2 N under enhanced UV-B radiation.     

A role for Ethylene-Insensitive3 in the regulation of hydrogen peroxide production during seed germination under high salinity in Arabidopsis

The Arabidopsis Ethylene-Insensitive3 (EIN3) has received attention recently and has been shown to be involved in the regulation of multitude of responses ranging from biotic stress defense and development to hormone interaction. To better understand the roles of EIN3 in plants response to salinity stress during germination and post-germination development, seeds of two EIN3 deficient mutant and a EIN3 overexpression mutant of Arabidopsis were analyzed under salinity and compared with Col-0 as control. The results showed that the ein3-1eil1-1 double mutant (lacking EIN3 and EIN3-Like1) and ein3-1 (lacking EIN3) were hypersensitive to high salinity (>150 mM NaCl), while EIN3 overexpression mutant (EIN3ox) displayed enhanced tolerance, indicating that EIN3 plays important roles during seed germination under salinity. In addition, we also found that the two EIN3 deficient mutant seedlings accumulate high levels of hydrogen peroxide (H₂O₂), which was thought to be an inhibitor of germination under salinity before, suggesting that EIN3 may function as a negative regulator of reactive oxygen species metabolism in germinating seeds under salinity. Taken together, our studies provide insights that EIN3 promotes seed germination under salinity, at least in part, through modulating concentration of H₂O₂ in germinating seeds.     

Rubia austrozhejiangensis sp. nov. (Rubiaceae) from Zhejiang,China

Rubia austrozhejiangensis Z. P. Lei, Y. Y. Zhou & R. W. Wang, a new species of Rubiaceae from China, is described and illustrated. The new species is similar to R. ovatifolia Z. Ying Zhang and R. argyi (H. Lév. & Vaniot) H. Hara ex Lauener, but differs from the former in having stems and branches cylindrical, not quadrate‐angled, long‐ovate to ovate‐lanceolate leaf blades, many‐flowered inflorescence, and smaller mericarps, 3–4 mm in diameter. In R. ovatifolia, stems and branches are quadrate‐angled, leaf blades ovate, ovate‐cordate to rounded cordate, and the inflorescences are sparsely flowered. Compared to R. argyi, the new species has cylindrical, not quadrate‐angled stems and branches, leaf blades that are long‐ovate to ovate‐lanceolate, 3–5‐veined, and slightly reflexed corolla lobes. In R. argyi, stems and branches are quadrate‐angled or winged, the corolla lobes are spreading, and the mericarps are 5–7 mm in diameter.     

An Ensemble Method for Predicting Subnuclear Localizations from Primary Protein Structures

Background

Predicting protein subnuclear localization is a challenging problem. Some previous works based on non-sequence information including Gene Ontology annotations and kernel fusion have respective limitations. The aim of this work is twofold: one is to propose a novel individual feature extraction method; another is to develop an ensemble method to improve prediction performance using comprehensive information represented in the form of high dimensional feature vector obtained by 11 feature extraction methods.

Methodology/Principal Findings

A novel two-stage multiclass support vector machine is proposed to predict protein subnuclear localizations. It only considers those feature extraction methods based on amino acid classifications and physicochemical properties. In order to speed up our system, an automatic search method for the kernel parameter is used. The prediction performance of our method is evaluated on four datasets: Lei dataset, multi-localization dataset, SNL9 dataset and a new independent dataset. The overall accuracy of prediction for 6 localizations on Lei dataset is 75.2% and that for 9 localizations on SNL9 dataset is 72.1% in the leave-one-out cross validation, 71.7% for the multi-localization dataset and 69.8% for the new independent dataset, respectively. Comparisons with those existing methods show that our method performs better for both single-localization and multi-localization proteins and achieves more balanced sensitivities and specificities on large-size and small-size subcellular localizations. The overall accuracy improvements are 4.0% and 4.7% for single-localization proteins and 6.5% for multi-localization proteins. The reliability and stability of our classification model are further confirmed by permutation analysis.

Conclusions

It can be concluded that our method is effective and valuable for predicting protein subnuclear localizations. A web server has been designed to implement the proposed method. It is freely available at http://bioinformatics.awowshop.com/snlpred_page.php.     

Raman spectroscopy detects phenotypic differences among Escherichia coli enriched for 1‐butanol tolerance using a metagenomic DNA library

Advances in Raman spectroscopy are enabling more comprehensive measurement of microbial cell chemical composition. Advantages include results returned in near real‐time and minimal sample preparation. In this research, Raman spectroscopy is used to analyze E. coli with engineered solvent tolerance, which is a multi‐genic trait associated with complex and uncharacterized phenotypes that are of value to industrial microbiology. To generate solvent tolerant phenotypes, E. coli transformed with DNA libraries are serially enriched in the presence of 0.9% (v/v) and 1.1% (v/v) 1‐butanol. DNA libraries are created using degenerate oligonucleotide primed PCR (DOP‐PCR) from the genomic DNA of E. coli, Clostridium acetobutylicum ATCC 824, and the metagenome of a stream bank soil sample, which contained DNA from 72 different phyla. DOP‐PCR enabled high efficiency library cloning (with no DNA shearing or end‐polishing) and the inclusion un‐culturable organisms. Nine strains with improved tolerance are analyzed by Raman spectroscopy and vastly different solvent‐tolerant phenotypes are characterized. Common among these are improved membrane rigidity from increasing the fraction of unsaturated fatty acids at the expense of cyclopropane fatty acids. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real‐time and is adaptable to high‐throughput screening, making it relevant to metabolic engineering.     

ssDNA Aptamer Specifically Targets and Selectively Delivers Cytotoxic Drug Doxorubicin to HepG2 Cells

Hepatocellular carcinoma (HCC) is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3) by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX) and exploited its role as a targeting ligand to deliver doxorubicin (Dox) to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5′-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol) were fully intercalated in each conjugate aptamer-Dox (ApDC) molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells.     

The Potential Biomarkers to Identify the Development of Steatosis in Hyperuricemia

Hyperuricemia (HU) often progresses to combine with non-alcoholic fatty liver disease (NAFLD) in the clinical scenario, which further exacerbates metabolic disorders; early detection of biomarkers, if obtained during the HU progression, may be beneficial for preventing its combination with NAFLD. This study aimed to decipher the biomarkers and mechanisms of the development of steatosis in HU. Four groups of subjects undergoing health screening, including healthy subjects, subjects with HU, subjects with HU combined with NAFLD (HU+NAFLD) and subjects with HU initially and then with HU+NAFLD one year later (HU→HU+NAFLD), were recruited in this study. The metabolic profiles of all subjects’ serum were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry. The metabolomic data from subjects with HU and HU+NAFLD were compared, and the biomarkers for the progression from HU to HU+NAFLD were predicted. The metabolomic data from HU→HU+NAFLD subjects were collected for further verification. The results showed that the progression was associated with disturbances of phospholipase metabolism, purine nucleotide degradation and Liver X receptor/retinoic X receptor activation as characterized by up-regulated phosphatidic acid, cholesterol ester (18:0) and down-regulated inosine. These metabolic alterations may be at least partially responsible for the development of steatosis in HU. This study provides a new paradigm for better understanding and further prevention of disease progression.     

MOLECULAR CHARACTERIZATION OF AUTOPHAGY‐RELATED GENE 5 FROM Spodoptera exigua AND EXPRESSION ANALYSIS UNDER VARIOUS STRESS CONDITIONS

Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry‐SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl‐HP cells and partially co‐localized with BmATG6‐GFP, but it almost has no significant co‐localization with GFP‐HaATG8. Then, the Western blot analysis demonstrated that GFP‐SeATG5 conjugated with ATG12. Moreover, real‐time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions.