Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors.

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration.

Endothelins control the timing of Schwann cell generation in vitro and in vivo.

Schwann cell precursors, derivatives of the neural crest, generate Schwann cells in a process that is tightly timed, well characterized, and directly controlled by axonal signals, in particular beta-neuregulins. Here we provide evidence that endothelins (ETs) are also important for survival and lineage progression in this system. We show that ETs promote rat Schwann cell precursor survival in vitro without stimulation of DNA synthesis. Using ET receptor agonists and antagonists, we demonstrate that this action of ET is mediated by the ET(B) receptor. RT-PCR reveals the presence of ET and ET receptor mRNA in the developing rat PNS. We showed previously that in vitro beta-neuregulins promote the generation of Schwann cells from precursors on schedule and that this process can be accelerated by fibroblast growth factor 2. Here we show that although ETs promote long-term precursor survival the transition of precursors to Schwann cells is delayed. Moreover, ETs block the maturation effects of beta-neuregulins. In spotting lethal rats, in which functional ET(B) receptors are absent, we find accelerated expression of the Schwann cell marker S100 in developing nerves. These observations indicate that complex growth factor interactions control the timing of Schwann cell development in embryonic nerves and that ETs act as negative regulators of Schwann cell generation.

Real-time, sequence-specific detection of nucleic acids during strand displacement amplification.

Strand displacement amplification (SDA) is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase. Here we describe fluorogenic reporter probes that permit real-time, sequence-specific detection of targets amplified during SDA. The new probes possess the single-strand half of a BsoBI recognition sequence flanked on opposite sides by a fluorophore and a quencher. The probes also contain target-binding sequences located 3' to the BsoBI site. Fluorophore and quencher are maintained in sufficiently close proximity that fluorescence is quenched in the intact single-stranded probe. If target is present during SDA, the probe is converted into a fully double-stranded form and is cleaved by the restriction enzyme BsoBI, which also serves as the nicking agent for SDA. Fluorophore and quencher diffuse apart upon probe cleavage, causing increased fluorescence. Target replication may thus be followed in real time during the SDA reaction. Probe performance may be enhanced by embedding the fluorogenic BsoBI site within the loop of a folded hairpin structure. The new probe designs permit detection of as few as 10 target copies within 30 min in a closed-tube, real-time format, eliminating the possibility of carry-over contamination. The probes may be used to detect RNA targets in SDA mixtures containing reverse transcriptase. Furthermore, a two-color competitive SDA format permits accurate quantification of target levels from the real-time fluorescence data.

Quantitative reverse transcription strand displacement amplification: quantitation of nucleic acids using an isothermal amplification technique.

Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting.

Interactions and survival of enteric viruses in soil materials.

There were marked differences in the abilities of eight different soil materials to remove and retain viruses from settled sewage, but for each soil material the behavior of two different viruses, poliovirus type 1 and reovirus type 3, was often similar. Virus adsorption to soil materials was rapid, the majority occurring within 15 min. Clayey materials efficiently adsorbed both viruses from wastewater over a range of pH and total dissolved solids levels. Sands and organic soil materials were comparatively poor adsorbents, but in some cases their ability to adsorb viruses increased at low pH and with the addition of total dissolved solids or divalent cations. Viruses in suspensions of soil material in settled sewage survived for considerable time periods, despite microbial activity. In some cases virus survival was prolonged in suspensions of soil materials compared to soil-free controls. Although sandy and organic soil materials were poor virus adsorbents when suspended in wastewater, they gave >/=95% virus removal from intermittently applied wastewater as unsaturated, 10-cm-deep columns. However, considerable quantities of the retained viruses were washed from the columns by simulated rainfall. Under the same conditions, clayey soil material removed >/=99.9995% of the viruses from applied wastewater, and none were washed from the columns by simulated rainfall.