Unrelenting environmental challenges to the gut epithelium place particular demands on the local immune system. In this context, intestinal intraepithelial lymphocytes (IEL) compose a large, highly conserved T cell compartment, hypothesized to provide a first line of defence via cytolysis of dysregulated intestinal epithelial cells (IEC) and cytokine-mediated re-growth of healthy IEC. Here we show that one of the most conspicuous impacts of activated IEL on IEC is the functional upregulation of antiviral interferon (IFN)-responsive genes, mediated by the collective actions of IFNs with other cytokines. Indeed, IEL activation in vivo rapidly provoked type I/III IFN receptor-dependent upregulation of IFN-responsive genes in the villus epithelium. Consistent with this, activated IEL mediators protected cells against virus infection in vitro, and pre-activation of IEL in vivo profoundly limited norovirus infection. Hence, intraepithelial T cell activation offers an overt means to promote the innate antiviral potential of the intestinal epithelium.

The protein phosphatase inhibitor RK-682 is one of a number of potentially valuable tetronate polyketide natural products. Understanding how the tetronate ring is formed has been frustrated by the inaccessibility of the putative substrates. We report the heterologous expression of rk genes in Saccharopolyspora erythraea and reconstitution of the RK-682 pathway using recombinant enzymes, and show that RkD is the enzyme required for RK-682 formation from acyl carrier protein-bound substrates.

The RNA-binding protein LARP1 has generated interest in recent years for its role in the mTOR signalling cascade and its regulation of terminal oligopyrimidine (TOP) mRNA translation. Paradoxically, some scientists have shown that LARP1 represses TOP translation while others that LARP1 activates it. Here, we present opinions from four leading scientists in the field to discuss these and other contradictory findings.

Subclinical infection of murine norovirus (MNV) was detected in a mixed breeding group of WT and Stat1−/− mice with no outward evidence of morbidity or mortality. Investigations revealed the presence of an attenuated MNV variant that did not cause cytopathic effects in RAW264.7 cells or death in Stat1−/− mice. Histopathological analysis of tissues from WT, heterozygous and Stat1−/− mice revealed a surprising spectrum of lesions. An infectious molecular clone was derived directly from faeces (MNV-O7) and the sequence analysis confirmed it was a member of norovirus genogroup V. Experimental infection with MNV-O7 induced a subclinical infection with no weight loss in Stat1−/− or WT mice, and recapitulated the clinical and pathological picture of the naturally infected colony. Unexpectedly, by day 54 post-infection, 50 % of Stat1−/− mice had cleared MNV-O7. In contrast, all WT mice remained infected persistently. Most significantly, this was associated with liver lesions in all the subclinically infected WT mice. These data confirmed that long-term persistence in WT mice is established with specific variants of MNV and that despite a subclinical presentation, active foci of acute inflammation persist within the liver. The data also showed that STAT1-dependent responses are not required to protect mice from lethal infection with all strains of MNV.

Purpose: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a pre-activated nucleoside released. 3'-deoxyadenosine (3'-dA) is a naturally-occurring adenosine analogue with established anti-cancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. Experimental Design:We describe the synthesis of NUC-7738. We determine the IC 50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in cancer patients. Results:We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide binding protein 1. PK and tumour samples obtained from the ongoing first-in-human Phase 1 clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective pro-apoptotic agent in cancer cells with effects on the NF-B pathway.Conclusions: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and support its further clinical evaluation as a novel cancer treatment within the growing pantheon of anti-cancer ProTides. Statement of translational relevanceProTide modification of nucleoside analogues is designed to overcome the cellular resistance mechanisms that limit their efficacy. This consists of the chemical addition of a protective phosphoramidate moiety onto the parent nucleoside, in this case the natural nucleoside analogue 3'-deoxyadenosine (3'-dA) to form the ProTide NUC-7738. The mode of action of 3'-dA has been the subject of speculation as its major limitation is its short plasma half-life due to rapid enzymatic deamination by adenosine deaminase (ADA). We demonstrate that NUC-7738 is resistant to deamination by ADA and cleaved by the intracellular phosphoramidase HINT1 into 3'-dAMP for conversion to the active metabolites 3'-dADP and 3'-dATP. We show that NUC-7738 promotes proapoptotic pathways and attenuates NF-B. These findings are validated in tumour samples from patients in an ongoing first-in-human trial of NUC-7738 from which we have identified biomarkers to enrich our understanding of NUC-7738 and patients most likely benefiting from it.