Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93–99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.     

Sequence analysis of three pigmentation genes in the Newfoundland population of Canis latrans links the Golden Retriever Mc1r variant to white coat color in coyotes

Three genes, Mc1r, Agouti, and CBD103, interact in a type-switching process that controls much of the pigmentation variation observed in mammals. A deletion in the CBD103 gene is responsible for dominant black color in dogs, while the white-phased black bear (“spirit bear”) of British Columbia, Canada, is the lightest documented color variant caused by a mutation in Mc1r. Rare all-white animals have recently been discovered in a new northeastern population of the coyote in insular Newfoundland and Labrador, Canada. To investigate the causative gene and mutation of white coat in coyotes, we sequenced the three type-switching genes in white and dark-phased animals from Newfoundland. The only sequence variants unambiguously associated with white color were in Mc1r, and one of these variants causes the amino acid variant R306Ter, a premature stop codon also linked to coat color in Golden Retrievers and other dogs with yellow/red coats. The allele carrying R306Ter in coyotes matches that in the Golden Retriever at other variable amino acid sites and hence may have originated in these dogs. Coyotes experienced introgression with wolves and dogs as they colonized northeastern North America, and coyote/Golden Retriever interactions have been observed in Newfoundland. We speculate that natural selection, with or without a founder effect, may contribute to the observed frequency of white coyotes in Newfoundland, as it has contributed to the high frequency of white bears, and of a domestic dog-derived CBD allele in gray wolves.     

Geographic range and structure of cryptic genetic diversity among Pacific North American populations of the non-native amphipod Grandidierella japonica

Reconstructing the invasion history of aquatic invasive species can enhance understanding of invasion risks by recognizing areas most susceptible to invasion and forecasting future spread based on past patterns of population expansion. Here we reconstruct the invasion history of the Japanese amphipod Grandidierella japonica Stephensen 1938 combining information from historical collection data with molecular genetic data to better understand post-invasion range expansion and anthropogenic connectivity across the Pacific coast of North America. Compilation of collection data from bays and estuaries of the Pacific North American coast show many new localities have been colonized in the last two decades, moving outward from harbors and bays with high commercial traffic into smaller coastal locations dominated by local recreational traffic. DNA barcode sequence data for G. japonica reveals two distinct clades: one found in San Francisco Bay and sites to the north, and one also found in San Francisco Bay and sites to the south. The two clades differ by an average 7.28 % genetic distance, large enough to consider these invasive amphipods two separate species. Both northern and southern clades exhibit low levels of genetic diversity, suggesting a single introduction event for each. The presence of cryptic diversity within this invasive amphipod highlights the need for more extensive study of the invasive and native populations of aquatic invasive invertebrates to address questions of taxonomy, diversity, and invasion history.     

Additive Effect of Anemia and Renal Impairment on Long-Term Outcome after Percutaneous Coronary Intervention

Introduction

Anemia and renal impairment are important co-morbidities among patients with coronary artery disease undergoing Percutaneous Coronary Intervention (PCI). Disease progression to eventual death can be understood as the combined effect of baseline characteristics and intermediate outcomes.

Methods

Using data from a prospective cohort study, we investigated clinical pathways reflecting the transitions from PCI through intermediate ischemic or hemorrhagic events to all-cause mortality in a multi-state analysis as a function of anemia (hemoglobin concentration <120 g/l and <130 g/l, for women and men, respectively) and renal impairment (creatinine clearance <60 ml/min) at baseline.

Results

Among 6029 patients undergoing PCI, anemia and renal impairment were observed isolated or in combination in 990 (16.4%), 384 (6.4%), and 309 (5.1%) patients, respectively. The most frequent transition was from PCI to death (6.7%, 95% CI 6.1–7.3), followed by ischemic events (4.8%, 95 CI 4.3–5.4) and bleeding (3.4%, 95% CI 3.0–3.9). Among patients with both anemia and renal impairment, the risk of death was increased 4-fold as compared to the reference group (HR 3.9, 95% CI 2.9–5.4) and roughly doubled as compared to patients with either anemia (HR 1.7, 95% CI 1.3–2.2) or renal impairment (HR 2.1, 95% CI 1.5–2.9) alone. Hazard ratios indicated an increased risk of bleeding in all three groups compared to patients with neither anemia nor renal impairment.

Conclusions

Applying a multi-state model we found evidence for a gradient of risk for the composite of bleeding, ischemic events, or death as a function of hemoglobin value and estimated glomerular filtration rate at baseline.     

The Impact of Renal Impairment on Long-Term Safety and Effectiveness of Drug-Eluting Stents

Background

Renal impairment (RI) is associated with impaired prognosis in patients with coronary artery disease. Clinical and angiographic outcomes of patients undergoing percutaneous coronary intervention (PCI) with the use of drug-eluting stents (DES) in this patient population are not well established.

Methods

We pooled individual data for 5,011 patients from 3 trials with the exclusive and unrestricted use of DES (SIRTAX - N = 1,012, LEADERS - N = 1,707, RESOLUTE AC - N = 2,292). Angiographic follow-up was available for 1,544 lesions. Outcomes through 2 years were stratified according to glomerular filtration rate (normal renal function: GFR≥90 ml/min; mild RI: 90<GFR≥60 ml/min; moderate/severe RI GFR<60 ml/min).

Results

Patients with moderate/severe RI had an increased risk of cardiac death or myocardial infarction ([MI], OR 2.14, 95%CI 1.36–3.36), cardiac death (OR 2.21, 95%CI 1.10–4.46), and MI (OR 2.02, 95%CI 1.19–3.43) compared with patients with normal renal function at 2 years follow-up. There was no difference in cardiac death or MI between patients with mild RI compared to those with normal renal function (OR 1.10, 95%CI 0.75–1.61). The risk of target-lesion revascularization was similar for patients with moderate/severe RI (OR 1.17, 95%CI 0.70–1.95) and mild RI (OR 1.16, 95%CI 0.81–1.64) compared with patients with normal renal function. In-stent late loss and in-segment restenosis were not different for patients with moderate/severe RI, mild RI, and normal renal function.

Conclusions

Renal function does not affect clinical and angiographic effectiveness of DES. However, prognosis remains impaired among patients with moderate/severe RI.     

An organ culture system to model early degenerative changes of the intervertebral disc

Introduction  

Back pain, a significant source of morbidity in our society, is related to the degenerative changes of the intervertebral disc. At present, the treatment of disc disease consists of therapies that are aimed at symptomatic relief. This shortcoming stems in large part from our lack of understanding of the biochemical and molecular events that drive the disease process. The goal of this study is to develop a model of early disc degeneration using an organ culture. This approach is based on our previous studies that indicate that organ culture closely models molecular events that occur in vivo in an ex vivo setting.     

Clinical outcome of high-risk patients with severe aortic stenosis and reduced left ventricular ejection fraction undergoing medical treatment or TAVI

Introduction

Reduced left ventricular function in patients with severe symptomatic valvular aortic stenosis is associated with impaired clinical outcome in patients undergoing surgical aortic valve replacement (SAVR). Transcatheter Aortic Valve Implantation (TAVI) has been shown non-inferior to SAVR in high-risk patients with respect to mortality and may result in faster left ventricular recovery.

Methods

We investigated clinical outcomes of high-risk patients with severe aortic stenosis undergoing medical treatment (n = 71) or TAVI (n = 256) stratified by left ventricular ejection fraction (LVEF) in a prospective single center registry.

Results

Twenty-five patients (35%) among the medical cohort were found to have an LVEF≤30% (mean 26.7±4.1%) and 37 patients (14%) among the TAVI patients (mean 25.2±4.4%). Estimated peri-interventional risk as assessed by logistic EuroSCORE was significantly higher in patients with severely impaired LVEF as compared to patients with LVEF>30% (medical/TAVI 38.5±13.8%/40.6±16.4% versus medical/TAVI 22.5±10.8%/22.1±12.8%, p <0.001). In patients undergoing TAVI, there was no significant difference in the combined endpoint of death, myocardial infarction, major stroke, life-threatening bleeding, major access-site complications, valvular re-intervention, or renal failure at 30 days between the two groups (21.0% versus 27.0%, p = 0.40). After TAVI, patients with LVEF≤30% experienced a rapid improvement in LVEF (from 25±4% to 34±10% at discharge, p = 0.002) associated with improved NYHA functional class at 30 days (decrease ≥1 NYHA class in 95%). During long-term follow-up no difference in survival was observed in patients undergoing TAVI irrespective of baseline LVEF (p = 0.29), whereas there was a significantly higher mortality in medically treated patients with severely reduced LVEF (log rank p = 0.001).

Conclusion

TAVI in patients with severely reduced left ventricular function may be performed safely and is associated with rapid recovery of systolic left ventricular function and heart failure symptoms.     

Lack of Developmental Redundancy between Unc45 Proteins in Zebrafish Muscle Development

Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p) protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b^−/−; unc45a^−/− mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b^−/− and unc45b^−/−; unc45a^−/− embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.