Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pylS and pylT are found in a Gram-positive bacterium. Charging a tRNA(CUA) with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.

Inflammation has classically been defined histopathologically, especially by the presence of immune cell infiltrates. However, more recent studies suggest a role for "low-grade" inflammation in a variety of disorders ranging from metabolic syndrome to cancer, which is defined by modest elevations in pro-inflammatory gene expression. Consequently, there is a need for cost-effective, non-invasive biomarkers that, ideally, would have the sensitivity to detect low-grade inflammation and have a dynamic range broad enough to reflect classic robust intestinal inflammation. Herein, we report that, for assessment of intestinal inflammation, fecal lipocalin 2 (Lcn-2), measured by ELISA, serves this purpose. Specifically, using a well-characterized mouse model of DSS colitis, we observed that fecal Lcn-2 and intestinal expression of pro-inflammatory cytokines (IL-1β, CXCL1, TNFα) are modestly but significantly induced by very low concentrations of DSS (0.25 and 0.5%), and become markedly elevated at higher concentrations of DSS (1.0 and 4.0%). As expected, careful histopathologic analysis noted only modest immune infiltrates at low DSS concentration and robust colitis at higher DSS concentrations. In accordance, increased levels of the neutrophil product myeloperoxidase (MPO) was only detected in mice given 1.0 and 4.0% DSS. In addition, fecal Lcn-2 marks the severity of spontaneous colitis development in IL-10 deficient mice. Unlike histopathology, MPO, and q-RT-PCR, the assay of fecal Lcn-2 requires only a stool sample, permits measurement over time, and can detect inflammation as early as 1 day following DSS administration. Thus, assay of fecal Lcn-2 by ELISA can function as a non-invasive, sensitive, dynamic, stable and cost-effective means to monitor intestinal inflammation in mice.

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.

Various states of inflammation, including sepsis, are associated with hypoferremia, which limits iron availability to pathogens and reduces iron-mediated oxidative stress. Lipocalin 2 (siderocalin, 24p3) plays a central role in iron transport. Accordingly, Lipocalin 2-deficient mice (Lcn2KO) exhibit elevated intracellular labile iron. Here we report that LPS-induced systemic Lcn2 by 150 fold in WT mice at 24h. Relative to WT littermates, Lcn2KO mice were markedly more sensitive to endotoxemia, exhibiting elevated indices of organ damage (transaminasaemia, LDH) and increased mortality. Such exacerbated endotoxemia was associated with substantially increased caspase 3 cleavage and concomitantly elevated immune cell apoptosis. Further, cells from Lcn2KO mice were hyperresponsive to LPS ex vivo exhibiting elevated cytokine secretion. In addition, Lcn2KO mice exhibited delayed LPS-induced hypoferremia despite normal hepatic hepcidin expression and display decreased levels of the tissue redox state indicators cysteine and glutathione in liver and plasma. Desferroxamine, an iron chelator, significantly protects Lcn2KO mice from LPS-induced toxicity, including mortality, suggesting that Lcn2 may act as an antioxidant in vivo by regulating iron homeostasis. Thus, Lcn2-mediated regulation of labile iron protects the host against sepsis. Its small size and simple structure may make Lcn2 as a deployable treatment for sepsis.

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Objectives Lipocalin-2 (Lcn2) is an innate immune protein expressed by a variety of cells and is highly upregulated during several pathological conditions including immune-complex (IC) mediated inflammatory/autoimmune disorders. However, the function of Lcn2 during IC-mediated inflammation is largely unknown. Therefore our objective was to investigate the role of Lcn2 in IC-mediated diseases. Methods The upregulation of Lcn2 was determined by ELISA in three different mouse models of IC-mediated autoimmune disease: systemic lupus erythematosus, collagen-induced arthritis and serum-induced arthritis. The in vivo role of Lcn2 during IC-mediated inflammation was investigated using Lcn2 knockout (Lcn2KO) mice and their wild type (WT) littermates. Results Lcn2 levels were significantly elevated in all the three autoimmune disease models. Further, in an acute skin inflammation model, Lcn2KO mice demonstrated a 50% reduction in inflammation with histopathological analysis revealing strikingly reduced immune cell infiltration compared to WT mice. Administration of recombinant Lcn2 to Lcn2KO mice restored inflammation to levels observed in WT mice. Neutralization of Lcn2 using a monoclonal antibody significantly reduced inflammation in WT mice. In contrast, Lcn2KO mice developed more severe serum-induced arthritis compared to WT mice. Histological analysis revealed extensive tissue and bone destruction with significantly reduced neutrophil infiltration but considerably more macrophage migration in Lcn2KO mice when compared to WT. Conclusion These results demonstrate that Lcn2 may regulate immune cell recruitment to the site of inflammation, a process essential for the controlled initiation, perpetuation and resolution of inflammatory processes. Thus, Lcn2 may present a promising target in the treatment of IC-mediated inflammatory/autoimmune diseases.