Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Global Analysis of Protein Phosphorylation in Yeast

The ability to rapidly restore the integrity of a broken skin barrier is critical and is the ultimate goal of therapies for hard-to-heal-ulcers. Unfortunately effective treatments to enhance healing and reduce scarring are still lacking. A deeper understanding of the physiology of normal repair and of the pathology of delayed healing is a prerequisite for the development of more effective therapeutic interventions. Transition from the inflammatory to the proliferative phase is a key step during healing and accumulating evidence associates a compromised transition with wound healing disorders. Thus, targeting factors that impact this phase transition may offer a rationale for therapeutic development. This review summarizes mechanisms regulating the inflammation-proliferation transition at cellular and molecular levels. We propose that identification of such mechanisms will reveal promising targets for development of more effective therapies.

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Transition from inflammation to proliferation: a critical step during wound healing

MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

LB-194 Lung cancer is the most prevalent form of cancer worldwide and accounts for the most cancer deaths. MicroRNAs (miRNAs) are small, non-protein coding RNAs that have recently emerged as important regulators of gene expression and direct proper cellular growth, differentiation and cell death - all mechanisms that go awry in cancer. The let-7 miRNA is postulated to function as a tumor suppressor gene in a variety of human tissues, particularly in the lung, by negatively regulating the post-transcriptional expression of multiple oncogenes including RAS, MYC, and HMGA2, as well as other cell cycle progression genes. Here we have used both in vitro and in vivo approaches to show that let-7 directly represses cancer growth in  the lung. We show that let-7 inhibits the growth of multiple human lung cancer cell lines in culture, as well as the growth of lung cancer cell xenografts in immunodeficient mice. Using the established Lox-Stop-Lox K-ras mouse lung cancer model, we find that intranasal let-7 administration can reduce tumor formation in vivo in the lungs of animals expressing a G12D activating mutation for the K-ras oncogene. These findings support the notion that let-7 functions as a tumor suppressor in the lung and indicates that this miRNA could be used as a therapeutic agent to treat lung cancer.

The let-7 microrna reduces tumor growth in mouse models of lung cancer

Aggressive fungal pathogens such as Botrytis and Verticillium spp. cause severe crop losses worldwide. We recently discovered that Botrytis cinerea delivers small RNAs (Bc-sRNAs) into plant cells to silence host immunity genes. Such sRNA effectors are mostly produced by Botrytis cinerea Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2. Here we show that expressing sRNAs that target Bc-DCL1 and Bc-DCL2 in Arabidopsis and tomato silences Bc-DCL genes and attenuates fungal pathogenicity and growth, exemplifying bidirectional cross-kingdom RNAi and sRNA trafficking between plants and fungi. This strategy can be adapted to simultaneously control multiple fungal diseases. We also show that Botrytis can take up external sRNAs and double-stranded RNAs (dsRNAs). Applying sRNAs or dsRNAs that target Botrytis DCL1 and DCL2 genes on the surface of fruits, vegetables and flowers significantly inhibits grey mould disease. Such pathogen gene-targeting RNAs represent a new generation of environmentally friendly fungicides.

/pdf/bidirectional-cross-kingdom-rnai-and-fungal-uptake-of-559u6ml7l7.pdf

Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection

Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy.

Toll-like receptors (TLRs), a critical family of pattern recognition receptors (PRRs), are responsible for the innate immune responses via signalling pathways to provide effective host defence against pathogen infections. However, TLR-signalling pathways are also likely to stringently regulate tissue maintenance and homeostasis by elaborate modulatory mechanisms. MicroRNAs (miRNAs) have emerged as key regulators and as an essential part of the networks involved in regulating TLR-signalling pathways. In this review, we highlight our understanding of the regulation of miRNA expression profiles by TLR-signalling pathways and the regulation of TLR-signalling pathways by miRNAs. We focus on the roles of miRNAs in regulating TLR-signalling pathways by targeting multiple molecules, including TLRs themselves, their associated signalling proteins and regulatory molecules, and transcription factors and functional cytokines induced by them, at multiple levels.

/pdf/micrornas-new-regulators-of-toll-like-receptor-signalling-ty4pxrk3m2.pdf

MicroRNAs: New Regulators of Toll-Like Receptor Signalling Pathways

Chronic hepatitis B virus (HBV) infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Current treatment strategies of HBV infection including the use of interferon (IFN)-α and nucleotide analogues such as lamivudine and adefovir have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. RNA interference (RNAi), by which a small interfering RNA (siRNA) induces the gene silence at a post-transcriptional level, has the potential of treating HBV infection. The successful use of chemically synthesized siRNA, endogenous expression of small hairpin RNA (shRNA) or microRNA (miRNA) to silence the target gene make this technology towards a potentially rational therapeutics for HBV infection. However, several challenges including poor siRNA stability, inefficient cellular uptake, widespread biodistribution and non-specific effects need to be overcome. In this review, we discuss several strategies for improving the anti-HBV therapeutic efficacy of siRNAs, while avoiding their off-target effects and immunostimulation. There is an in-depth discussion on the (1) mechanisms of RNAi, (2) methods for siRNA/shRNA production, (3) barriers to RNAi-based therapies, and (4) delivery strategies of siRNA for treating HBV infection.

/pdf/rnai-for-treating-hepatitis-b-viral-infection-44b3bjffst.pdf

RNAi for Treating Hepatitis B Viral Infection

Secreted extracellular vesicles play an important role in pathogen-host interactions. Increased knowledge of schistosome extracellular vesicles could provide insights into schistosome-host interactions and enable the development of novel intervention strategies to inhibit parasitic processes and lessen disease transmission. Here, we describe biochemical characterization of Schistosoma japonicum exosome-like vesicles (S. japonicum EVs). A total of 403 proteins were identified in S. japonicum EVs, and bioinformatics analyses indicated that these proteins were mainly involved in binding, catalytic activity, and translation regulatory activity. Next, we characterized the population of small RNAs associated with S. japonicum EVs. Further studies demonstrated that mammalian cells could internalize S. japonicum EVs and transfer their cargo miRNAs to recipient cells. Additionally, we found that a specific miRNA, likely originating from a final host, ocu-miR-191–5p, is also associated with S. japonicum EVs. Overall, our findings demonstrate that S. japonicum EVs could be implicated in the pathogenesis of schistosomiasis via a mechanism involving the transfer of their cargo miRNAs to hosts. Our findings provide novel insights into the mechanisms of schistosome-host interactions.

/pdf/molecular-characterization-of-s-japonicum-exosome-like-47jebcbvcv.pdf

Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions

Circulating microRNAs (miRNAs) have received considerable attention as a novel class of biomarkers for the diagnosis of cancer and as signalling molecules in mediating intercellular communication. Schistosomes, the causative agents of schistosomiasis, live in the blood vessels of a mammalian host in the adult stage. In the present study, we characterized schistosome-specific small RNA populations in the plasma of rabbits infected with Schistosoma japonicum (S. japonicum) using a deep sequencing method and then identified five schistosome-specific miRNAs, including four known miRNAs (Bantam, miR-3479, miR-10 and miR-3096), and one novel miRNA (miR-0001, miRBase ID: sja-miR-8185). Four of the five schistosome-specific miRNAs were also detected by real-time RT-PCR in the plasma of S. japonicum-infected mice. In addition, our study indicated that schistosome Argonaute 2/3 may be an excretory-secretory (ES) protein. In summary, our findings are expected to provide useful information for further development of novel biomarkers for the diagnosis of schistosomiasis and also for deeper understanding of the mechanism of host-parasite interaction.

Guofeng Cheng

Papers

Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy.

MicroRNAs: New Regulators of Toll-Like Receptor Signalling Pathways

RNAi for Treating Hepatitis B Viral Infection

Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions

Deep sequencing-based identification of pathogen-specific microRNAs in the plasma of rabbits infected with Schistosoma japonicum.