Nucleic acid catalysts (ribozymes, DNA- and XNAzymes) cleave target (m)RNAs with high specificity but have shown limited efficacy in clinical application. Here we report on the in vitro evolution and engineering of a highly specific modular RNA endonuclease XNAzyme, FR6_1, composed of 2’-deoxy-2’-fluoro-β-D-arabino nucleic acid (FANA). FR6_1 overcomes activity limitations of previous DNA- and XNAzymes and can be retargeted to cleave highly structured full-length (>5 kb) BRAF and KRAS mRNAs at physiological Mg 2+ concentrations with allelic selectivity for tumour-associated ( BRAF V600E and KRAS G12D) mutations. Phosphorothioate-FANA modification enhances FR6_1 biostability and enables rapid KRAS mRNA knockdown in cultured human adenocarcinoma cells with a G12D-allele-specific component provided by in vivo XNAzyme cleavage activity. These results provide a starting point for the development of improved gene silencing agents based on FANA or other XNA chemistries.

The emergence of an RNA molecule capable of replicating itself and other RNA sequences is a central pillar of hypotheses regarding the origin of life. In vitro evolution has yielded polymerase ribozymes (PR) that can copy a range of RNA templates using nucleotide or trinucleotide triphosphates (triplets) as substrates and may give rise to a replicase activity. However, our understanding of PR function is encumbered by a lack of structural information beyond the progenitor class I ligase (cIL) ribozyme. Here, we report the structure of the complete 5TU+t1 triplet polymerase ribozyme (TPR) apoenzyme and map its structure / function landscape. The TPR is an RNA heterodimer, comprising a catalytic (5TU) and a catalytically inactive (t1) subunit held together by two kissing loop interactions and its overall structure resembles a left hand with thumb and fingers at a 70° angle. While the 5TU subunit shows partial structural homology to the cIL, the t1 accessory subunit - despite sharing the same progenitor - exhibits a dramatically reorganized secondary and tertiary structure. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme and provide a foundation for a better understanding of RNA's potential for self-replication.

Saltwater intrusion has been recognized as a major driver of ecological regime shifts in coastal wetlands, but few studies have incorporated long‐term salinity modelling data into the understanding of vegetation dynamics. This study uniquely combined artificial neural network (ANN) modelling and time series of aerial photographs to elucidate saltwater intrusion and mangrove encroachment into the freshwater floodplain of the Loxahatchee River over a period of six decades. Three ANN models were developed to simulate river salinity on a daily time step using freshwater inflow, rainfall and tide as inputs. With 8 years of measured data for training and testing, the ANN models demonstrated comparable or superior model performance for salinity simulation to their hydrodynamic counterparts. Modelling of historical salinity (1948–2011) using the ANNs indicated that the intensity of saltwater intrusion clearly correlated with watershed hydrology, which, in turn, was linked to historical watershed alterations and regional rainfall variability. Interpretation of the 1940, 1953, 1964, 1979, 1985 and 1995 aerial photographs revealed the progressive encroachment of mangroves and displacement of bald cypress and freshwater communities in the floodplain. The spatial extent of mangrove encroachment was related to the increasing mortality of bald cypress under the chronic effect of cumulative salinity exposure. The ecohydrological controls over vegetation changes in the Loxahatchee River support the ongoing ecosystem restoration programme, which aims to achieve incremental freshwater flow targets to protect and restore the floodplain ecosystem. Copyright © 2014 John Wiley & Sons, Ltd.

Dynamic myoplasty is a relatively new use for muscle flaps and has led us to revisit the mechanisms of vascular delay as a means of optimizing blood supply to muscle flaps. Despite the well-documented effectiveness of vascular delay in skin flaps, vascular delay in muscle flaps has not been widely reported. Regardless of the many mechanisms postulated in the literature as contributors to the delay effect in skin, the one element common to all these hypotheses is the importance placed on changes in the microcirculation. Based on this factor, in the present study we developed and validated an animal model in which delay-induced microvascular changes could be measured in skeletal muscle flaps. We used the hairless mouse latissimus dorsi muscle flap because its vascular distribution is similar to that of humans and its thin structure will enable us in future studies to directly view and measure its microvasculature using videomicroscopy. In 12 animals, we found that delay significantly (p < 0.01) reduced necrosis of the distal part of the muscle from 57 +/- 9 percent in nondelayed flaps (n = 7) to 22 +/- 3 percent in delayed (n = 5) flaps. In these studies, we also determined that the hairless mouse latissimus dorsi muscle flap will serve as an excellent model for defining microvascular changes throughout delay.