Therapeutic peptide production in Drosophila

Bioactive peptides are important therapeutic drugs, yet conventional methods of peptide synthesis are challenged to meet increasing demand. We developed a novel and efficient means of metabolic engineering: therapeutic peptide production in Drosophila and as a proof of concept, we demonstrate production of fully matured human insulin. This in vivo system offers an innovative means to produce valuable bioactive peptides for therapies, its inherent flexibility facilitates drug development, and its ease of producing fully processed peptides simplifies metabolic engineering of new peptide products.     

Protein identification using top-down

In the last two years, because of advances in protein separation and mass spectrometry, top-down mass spectrometry moved from analyzing single proteins to analyzing complex samples and identifying hundreds and even thousands of proteins. However, computational tools for database search of top-down spectra against protein databases are still in their infancy. We describe MS-Align+, a fast algorithm for top-down protein identification based on spectral alignment that enables searches for unexpected post-translational modifications. We also propose a method for evaluating statistical significance of top-down protein identifications and further benchmark various software tools on two top-down data sets from Saccharomyces cerevisiae and Salmonella typhimurium. We demonstrate that MS-Align+ significantly increases the number of identified spectra as compared with MASCOT and OMSSA on both data sets. Although MS-Align+ and ProSightPC have similar performance on the Salmonella typhimurium data set, MS-Align+ outperforms ProSightPC on the (more complex) Saccharomyces cerevisiae data set.     

Identification and profiling of microRNAs from skeletal muscle of the common carp

The common carp is one of the most important cultivated species in the world of freshwater aquaculture. The cultivation of this species is particularly productive due to its high skeletal muscle mass; however, the molecular mechanisms of skeletal muscle development in the common carp remain unknown. It has been shown that a class of non-coding ～22 nucleotide RNAs called microRNAs (miRNAs) play important roles in vertebrate development. They regulate gene expression through sequence-specific interactions with the 3' untranslated regions (UTRs) of target mRNAs and thereby cause translational repression or mRNA destabilization. Intriguingly, the role of miRNAs in the skeletal muscle development of the common carp remains unknown. In this study, a small-RNA cDNA library was constructed from the skeletal muscle of the common carp, and Solexa sequencing technology was used to perform high throughput sequencing of the library. Subsequent bioinformatics analysis identified 188 conserved miRNAs and 7 novel miRNAs in the carp skeletal muscle. The miRNA expression profiling showed that, miR-1, miR-133a-3p, and miR-206 were specifically expressed in muscle-containing organs, and that miR-1, miR-21, miR-26a, miR-27a, miR-133a-3p, miR-206, miR-214 and miR-222 were differentially expressed in the process of skeletal muscle development of the common carp. This study provides a first identification and profiling of miRNAs related to the muscle biology of the common carp. Their identification could provide clues leading towards a better understanding of the molecular mechanisms of carp skeletal muscle development.     

Taking down the FLAG! How insect cell expression challenges an established tag-system

In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.     

Promotion of Spinal Cord Regeneration by Neural Stem Cell-Secreted Trimerized Cell Adhesion Molecule L1

The L1 cell adhesion molecule promotes neurite outgrowth and neuronal survival in homophilic and heterophilic interactions and enhances neurite outgrowth and neuronal survival homophilically, i.e. by self binding. We investigated whether exploitation of homophilic and possibly also heterophilic mechanisms of neural stem cells overexpressing the full-length transmembrane L1 and a secreted trimer engineered to express its extracellular domain would be more beneficial for functional recovery of the compression injured spinal cord of adult mice than stem cells overexpressing only full-length L1 or the parental, non-engineered cells. Here we report that stem cells expressing trimeric and full-length L1 are indeed more efficient in promoting locomotor recovery when compared to stem cells overexpressing only full-length L1 or the parental stem cells. The trimer expressing stem cells were also more efficient in reducing glial scar volume and expression of chondroitin sulfates and the chondroitin sulfate proteoglycan NG2. They were also more efficient in enhancing regrowth/sprouting and/or preservation of serotonergic axons, and remyelination and/or myelin sparing. Moreover, degeneration/dying back of corticospinal cord axons was prevented more by the trimer expressing stem cells. These results encourage the view that stem cells engineered to drive the beneficial functions of L1 via homophilic and heterophilic interactions are functionally optimized and may thus be of therapeutic value.     

Identification of Novel Piperazinylquinoxaline Derivatives as Potent Phosphoinositide 3-Kinase (PI3K) Inhibitors

Background

Development of small-molecule inhibitors targeting phosphoinositide 3-kinase (PI3K) has been an appealing strategy for the treatment of various types of cancers.

Methodology/Principal Finding

Our approach was to perform structural modification and optimization based on previously identified morpholinoquinoxaline derivative WR1 and piperidinylquinoxaline derivative WR23 with a total of forty-five novel piperazinylquinoxaline derivatives synthesized. Most target compounds showed low micromolar to nanomolar antiproliferative potency against five human cancer cell lines using MTT method. Selected compounds showed potent PI3Kα inhibitory activity in a competitive fluorescent polarization assay, such as compound 22 (IC₅₀ 40 nM) and 41 (IC₅₀: 24 nM), which induced apoptosis in PC3 cells. Molecular docking analysis was performed to explore possible binding modes between target compounds and PI3K.

Conclusions/Significance

The identified novel piperazinylquinoxaline derivatives that showed potent PI3Kα inhibitory activity and cellular antiproliferative potency may be promising agents for potential applications in cancer treatment.     

Polymorphisms on 8q24 are associated with lung cancer risk and survival in han chinese

Chromosome 8q24 is commonly amplified in many types of cancer, particularly lung cancer. Polymorphisms in this region are associated with risk of different cancers. To investigate the relationship between three single nucleotide polymorphisms (SNPs) (rs1447295, rs16901979 and rs6983267) on 8q24 and lung cancer risk, we conducted an association study in two Han Chinese populations: one population was from Zhejiang Province (576 case patients and 576 control subjects), whereas the other was from Fujian Province (576 case patients and 576 control subjects). We found that rs6983267 was significantly associated with an increased risk of lung cancer in both populations. Compared with the TT genotype, the GG genotype was associated with a significant 1.555-fold increased risk of lung cancer [95% confidence interval (CI) 1.218–1.986, P = 4.0×10⁻⁴]. This effect was more pronounced in never-smokers [odds ratio (OR) = 2.366, 95% CI 1.605–3.488, P = 1.4×10⁻⁵]. Analyses stratified by histology revealed that rs6983267 GG genotype was most associated with patients with other histological types (OR = 3.012, 95% CI 1.675–5.417, P = 2.3×10⁻⁴). The AA genotype of rs1447295 was associated with increased risk for adenocarcinoma compared with the CC genotype (OR = 2.260, 95% CI 1.174–4.353, P = 0.015). Furthermore, the GG genotype of rs6983267 was associated with worse survival in the Zhejiang population (hazard ratio (HR) = 1.646, 95% CI 1.099–2.464, P = 0.016). No association was observed for rs16901979. These results suggest that genetic variations on 8q24 may play significant roles in the development and progression of lung cancer.