Nephronophthisis-Associated CEP164 Regulates Cell Cycle Progression,Apoptosis and Epithelial-to-Mesenchymal Transition

We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys.     

Complete genome sequence of Staphylothermus hellenicus P8

Staphylothermus hellenicus belongs to the order Desulfurococcales within the archaeal phylum Crenarchaeota. Strain P8(T) is the type strain of the species and was isolated from a shallow hydrothermal vent system at Palaeochori Bay, Milos, Greece. It is a hyperthermophilic, anaerobic heterotroph. Here we describe the features of this organism together with the complete genome sequence and annotation. The 1,580,347 bp genome with its 1,668 protein-coding and 48 RNA genes was sequenced as part of a DOE Joint Genome Institute (JGI) Laboratory Sequencing Program (LSP) project.     

Testing target‐specific bait delivery for controlling feral pigs in a tropical rainforest

Mitigation of feral pig (Sus scrofa) impacts in Australia’s Wet Tropics World Heritage Area has been impeded by the lack of a target‐specific method for delivering toxic baits in the region. This study evaluated methods to reduce bait‐take by susceptible nontarget species without inhibiting bait‐take by pigs, to enable more effective pig management. We predicted that dingoes would not select an unprocessed corn bait and that other potential nontarget bait consumers would be unable to access the same bait presented under a lightweight cover. Neither of these methods was expected to reduce bait selection or access by pigs. We tested these predictions by monitoring animal interactions with covered and uncovered corn baits, and covered corn and manufactured baits. Use of corn as a bait substrate effectively prevented bait‐take by dingoes. Covering baits substantially reduced bait‐take by other nontarget species and completely prevented nontarget bait‐take when uncovered feed was provided simultaneously. The corn bait preparation was highly acceptable and accessible to feral pigs. We conclude that the methods evaluated here could enable the consideration of poison baiting as a viable method for controlling feral pigs in the World Heritage Area, where it has previously been unavailable.     

A Panel of 4 microRNAs Facilitates the Prediction of Left Ventricular Contractility after Acute Myocardial Infarction

Background

Prediction of clinical outcome after acute myocardial infarction (AMI) is challenging and would benefit from new biomarkers. We investigated the prognostic value of 4 circulating microRNAs (miRNAs) after AMI.

Methods

We enrolled 150 patients after AMI. Blood samples were obtained at discharge for determination of N-terminal pro-brain natriuretic peptide (Nt-proBNP) and levels of miR-16, miR-27a, miR-101 and miR-150. Patients were assessed by echocardiography at 6 months follow-up and the wall motion index score (WMIS) was used as an indicator of left ventricular (LV) contractility. We assessed the added predictive value of miRNAs against a multi-parameter clinical model including Nt-proBNP.

Results

Patients with anterior AMI and elevated Nt-proBNP levels at discharge from the hospital were at high risk of subsequent impaired LV contractility (follow-up WMIS>1.2, n = 71). A combination of the 4 miRNAs (miR-16/27a/101/150) improved the prediction of LV contractility based on clinical variables (P = 0.005). Patients with low levels of miR-150 (odds ratio [95% confidence interval] 0.08 [0.01–0.48]) or miR-101 (0.19 [0.04–0.97]) and elevated levels of miR-16 (15.9 [2.63–95.91]) or miR-27a (4.18 [1.36–12.83]) were at high risk of impaired LV contractility. The 4 miRNA panel reclassified a significant proportion of patients with a net reclassification improvement of 66% (P = 0.00005) and an integrated discrimination improvement of 0.08 (P = 0.001).

Conclusion

Our results indicate that panels of miRNAs may aid in prognostication of outcome after AMI.     

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of 3×10⁻⁵ mutants/locus and 125,000–300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose–response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

Estimating age of immobilized elephants from teeth impressions using dental silicon

High precision condensation dental silicon, Zetalabor^TM, was used to create moulds of the lower jaw molars from 22 immobilized African elephants (Loxodonta africana Blumenback) during radio collaring operations. These moulds were used to determine the elephant's age using Laws and Jachmann's molar aging criteria. The technique proved easy and fast and produced useful imprints in 90% of the cases. We found our age estimates, based on physical appearance, made prior to immobilizations were relatively accurate, with 75% within ±3 years and 95% within ±5 years from the age indicated from molar evaluation. When re‐collaring the same individuals in 2–3 years, new moulds will be made to compare a known time period with the degree of tooth wear. This will provide verification of Laws age estimates from free‐ranging elephants.