Harnessing type I and type III CRISPR-Cas systems for genome editing

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR array and a donor DNA containing a non-target sequence. Transformation of a pGE plasmid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromosomal DNA degradation, leading to cell death, whereas those carrying the mutant gene would survive the cell killing and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided the protospacer adjacent motif (PAM) of an uncharacterized PAM-dependent CRISPR-Cas system can be predicted by bioinformatic analysis

Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening

Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions

Identification of the C-Reactive Protein Interaction Network Using a Bioinformatics Approach Provides Insights into the Molecular Pathogenesis of Hepatocellular Carcinoma

Background/Aims: C reactive protein (CRP) levels are elevated in many diseases, including malignant tumors and cardiovascular disorders. In this study, the protein interaction network for CRP was evaluated to determine the importance of CRP and its interacting proteins in the molecular pathogenesis of hepatocellular carcinoma (HCC). Methods: Isobaric tags for relative and absolute quantitation (iTRAQ) and mass spectrometry were used to identify CRP interacting proteins in SMMC7721 cells. Moreover, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to evaluate enriched genes and pathways for differentially expressed genes using DAVID and WebGestalt. Co-immunoprecipitation and western blot analyses were employed to assess interactions between CRP and KRT8, ANXA2, ENO2, and HSP90B1. Results: In total, 52 proteins that interact with CRP were identified. A GO analysis suggested that most of the interacting proteins were involved in CRP complexes and regulated metabolic processes. A KEGG pathway analysis suggested that most CRP-interacting proteins contribute to the TRAIL signaling pathway, Class I PI3K/Akt signaling pathway, plasma membrane estrogen receptor signaling, Nectin adhesion pathway, and S1P1 pathway. Immunoprecipitation and western blot analyses revealed interactions between CRP and KRT8, ANXA2, ENO2, and HSP90B1. Conclusions: iTRAQ based proteomic profiling revealed the network of CRP interacting proteins. This network may activate the PI3K/Akt signaling pathway, thereby contributing to the pathogenesis of HCC

Complete Genomic Sequence of Bacteriophage Felix O1†

Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage φEa21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent

A Regulatory Module Controlling Homeostasis of a Plant Immune Kinase

Plant pattern recognition receptors (PRRs) perceive microbial and endogenous molecular patterns to activate immune signaling. The cytoplasmic kinase BIK1 acts downstream of multiple PRRs as a rate-limiting component, whose phosphorylation and accumulation are central to immune signal propagation. Previous work identified the calcium-dependent protein kinase CPK28 and heterotrimeric G proteins as negative and positive regulators of BIK1 accumulation, respectively. However, mechanisms underlying this regulation remain unknown. Here we show that the plant U-box proteins PUB25 and PUB26 are homologous E3 ligases that mark BIK1 for degradation to negatively regulate immunity. We demonstrate that the heterotrimeric G proteins inhibit PUB25/26 activity to stabilize BIK1, whereas CPK28 specifically phosphorylates conserved residues in PUB25/26 to enhance their activity and promote BIK1 degradation. Interestingly, PUB25/26 specifically target non-activated BIK1, suggesting that activated BIK1 is maintained for immune signaling. Our findings reveal a multi-protein regulatory module that enables robust yet tightly regulated immune responses

Fabrication and Intermolecular Interactions of Silk Fibroin/Hydroxybutyl Chitosan Blended Nanofibers

The native extracellular matrix (ECM) is composed of a cross-linked porous network of multifibril collagens and glycosaminoglycans. Nanofibrous scaffolds of silk fibroin (SF) and hydroxybutyl chitosan (HBC) blends were fabricated using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and trifluoroacetic acid (TFA) as solvents to biomimic the native ECM via electrospinning. Scanning electronic microscope (SEM) showed that relatively uniform nanofibers could be obtained when 12% SF was blended with 6% HBC at the weight ratio of 50:50. Meanwhile, the average nanofibrous diameter increased when the content of HBC in SF/HBC blends was raised from 20% to 100%. Fourier transform infrared spectra (FTIR) and 13C nuclear magnetic resonance (NMR) showed SF and HBC molecules existed in hydrogen bonding interactions but HBC did not induce conformation of SF transforming from random coil form to β-sheet structure. X-ray diffraction (XRD) confirmed the different structure of SF/HBC blended nanofibers from both SF and HBC. Thermogravimetry-Differential thermogravimetry (TG-DTG) results demonstrated that the thermal stability of SF/HBC blend nanofibrous scaffolds was improved. The results indicated that the rearrangement of HBC and SF molecular chain formed a new structure due to stronger hydrogen bonding between SF and HBC. These electrospun SF/HBC blended nanofibers may provide an ideal tissue engineering scaffold and wound dressing

JNK signalling in cancer: In need of new, smarter therapeutic targets

Copyright © 2013 The British Pharmacological Society. This is the accepted version of the following article: Bubici, C. and Papa, S. (2014), JNK signalling in cancer: in need of new, smarter therapeutic targets. British Journal of Pharmacology, 171: 24–37, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/bph.12432/abstract.The JNKs are master protein kinases that regulate many physiological processes, including inflammatory responses, morphogenesis, cell proliferation, differentiation, survival and death. It is increasingly apparent that persistent activation of JNKs is involved in cancer development and progression. Therefore, JNKs represent attractive targets for therapeutic intervention with small molecule kinase inhibitors. However, evidence supportive of a tumour suppressor role for the JNK proteins has also been documented. Recent studies showed that the two major JNK proteins, JNK1 and JNK2, have distinct or even opposing functions in different types of cancer. As such, close consideration of which JNK proteins are beneficial targets and, more importantly, what effect small molecule inhibitors of JNKs have on physiological processes, are essential. A number of ATP-competitive and ATP-non-competitive JNK inhibitors have been developed, but have several limitations such as a lack of specificity and cellular toxicity. In this review, we summarize the accumulating evidence supporting a role for the JNK proteins in the pathogenesis of different solid and haematological malignancies, and discuss many challenges and scientific opportunities in the targeting of JNKs in cancer.Kay Kendall Leukemia Fund, Italian Association for Cancer Research and Foundation for Liver Research

A Marine Anthraquinone SZ-685C Overrides Adriamycin-Resistance in Breast Cancer Cells through Suppressing Akt Signaling

Breast cancer remains a major health problem worldwide. While chemotherapy represents an important therapeutic modality against breast cancer, limitations in the clinical use of chemotherapy remain formidable because of chemoresistance. The HER2/PI-3K/Akt pathway has been demonstrated to play a causal role in conferring a broad chemoresistance in breast cancer cells and thus justified to be a target for enhancing the effects of anti-breast cancer chemotherapies, such as adriamycin (ADR). Agents that can either enhance the effects of chemotherapeutics or overcome chemoresistance are urgently needed for the treatment of breast cancer. In this context, SZ-685C, an agent that has been previously shown, as such, to suppress Akt signaling, is expected to increase the efficacy of chemotherapy. Our current study investigated whether SZ-685C can override chemoresistance through inhibiting Akt signaling in human breast cancer cells. ADR-resistant cells derived from human breast cancer cell lines MCF-7, MCF-7/ADR and MCF-7/Akt, were used as models to test the effects of SZ-685C. We found that SZ-685C suppressed the Akt pathway and induced apoptosis in MCF-7/ADR and MCF-7/Akt cells that are resistant to ADR treatment, leading to antitumor effects both in vitro and in vivo. Our data suggest that use of SZ-685C might represent a potentially promising approach to the treatment of ADR-resistant breast cancer

FaGAPC2/FaPKc2.2 and FaPEPCK reveal differential citric acid metabolism regulation in late development of strawberry fruit

Citric acid is the primary organic acid that affects the taste of strawberry fruit. Glycolysis supplies key substrates for the tricarboxylic acid cycle (TCA cycle). However, little is known about the regulatory mechanisms of glycolytic genes on citric acid metabolism in strawberry fruits. In this study, the citric acid content of strawberry fruit displayed a trend of rising and decreasing from the initial red stage to the full red stage and then dark red stage. Thus, a difference in citric acid metabolic regulation was suspected during strawberry fruit development. In addition, overexpression of either cytoplasm glyceraldehyde-3-phosphate dehydrogenase (FxaC_14g13400, namely FaGAPC2) or pyruvate kinase (FxaC_15g00080, namely FaPKc2.2) inhibited strawberry fruit ripening and the accumulation of citric acid, leading to a range of maturity stages from partial red to full red stage. The combined transcriptome and metabolome analysis revealed that overexpression of FaGAPC2 and FaPKc2.2 significantly suppressed the expression of phosphoenolpyruvate carboxykinase (FxaC_1g21491, namely FaPEPCK) but enhanced the content of glutamine and aspartic acid. Meanwhile, the activities of PEPCK and glutamate decarboxylase (GAD) were inhibited, but the activities of glutamine synthase (GS) were increased in FaGAPC2/FaPKc2.2-overexpressed fruit. Further, functional verification demonstrated that overexpression of FaPEPCK can promote strawberry fruit ripening, resulting in a range of maturity stage from full red to dark red stage, while the citric acid synthase (CS) activities and citric acid content were significantly decreased. Overall, this study revealed that FaGAPC2/FaPKc2.2 and FaPEPCK perform an important role in reducing citric acid content in strawberry fruit, and FaGAPC2/FaPKc2.2 mainly by promoting the GS degradation pathway and FaPEPCK mainly by inhibiting the CS synthesis pathway