The relationship between eDNA particle concentration and organism abundance in nature is strengthened by allometric scaling.

Organism abundance is a critical parameter in ecology, but its estimation is often challenging. Approaches utilizing eDNA to indirectly estimate abundance have recently generated substantial interest. However, preliminary correlations observed between eDNA concentration and abundance in nature are typically moderate in strength with significant unexplained variation. Here, we apply a novel approach to integrate allometric scaling coefficients into models of eDNA concentration and organism abundance. We hypothesize that eDNA particle production scales nonlinearly with mass, with scaling coefficients < 1. Wild populations often exhibit substantial variation in individual body size distributions; we therefore predict that the distribution of mass across individuals within a population will influence population-level eDNA production rates. To test our hypothesis, we collected standardized body size distribution and mark-recapture abundance data using whole-lake experiments involving nine populations of brook trout. We correlated eDNA concentration with three metrics of abundance: density (individuals/ha), biomass (kg/ha) and allometrically scaled mass (ASM) (∑(individual mass0.73 )/ha). Density and biomass were both significantly positively correlated with eDNA concentration (adj. r2  = 0.59 and 0.63, respectively), but ASM exhibited improved model fit (adj. r2  = 0.78). We also demonstrate how estimates of ASM derived from eDNA samples in "unknown" systems can be converted to biomass or density estimates with additional size-structure data. Future experiments should empirically validate allometric scaling coefficients for eDNA production, particularly where substantial intraspecific size distribution variation exists. Incorporating allometric scaling may improve predictive models to the extent that eDNA concentration may become a reliable indicator of abundance in nature.

Small population size and low genomic diversity have no effect on fitness in experimental translocations of a wild fish.

Little empirical work in nature has quantified how wild populations with varying effective population sizes and genetic diversity perform when exposed to a gradient of ecologically important environmental conditions. To achieve this, juvenile brook trout from 12 isolated populations or closed metapopulations that differ substantially in population size and genetic diversity were transplanted to previously fishless ponds spanning a wide gradient of ecologically important variables. We evaluated the effect of genome-wide variation, effective population size (Ne), pond habitat, and initial body size on two fitness correlates (survival and growth). Genetic variables had no effect on either fitness correlate, which was determined primarily by habitat (pond temperature, depth, and pH) and initial body size. These results suggest that some vertebrate populations with low genomic diversity, low Ne, and long-term isolation can represent important sources of variation and are capable of maintaining fitness in, and ultimately persisting and adapting to, changing environments. Our results also reinforce the paramount importance of improving available habitat and slowing habitat degradation for species conservation.

Characterisation of the HLA-DRB1*07:01 biomarker for lapatinib-induced liver toxicity during treatment of early-stage breast cancer patients with lapatinib in combination with trastuzumab and/or taxanes.

HLA-DRB1*07:01 allele carriage was characterised as a risk biomarker for lapatinib-induced liver injury in a large global study evaluating lapatinib, alone and in combination with trastuzumab and taxanes, as adjuvant therapy for advanced breast cancer (adjuvant lapatinib and/or trastuzumab treatment optimisation). HLA-DRB1*07:01 carriage was associated with serum alanine aminotransferase (ALT) elevations in lapatinib-treated patients (odds ratio 6.5, P=3 × 10-26, n=4482) and the risk and severity of ALT elevation for lapatinib-treated patients was higher in homozygous than heterozygous HLA-DRB1*07:01 genotype carriers. A higher ALT case incidence plus weaker HLA association observed during concurrent administration of lapatinib and taxane suggested a subset of liver injury in this combination group that was HLA-DRB1*07:01 independent. Furthermore, the incidence of ALT elevation demonstrated an expected correlation with geographic HLA-DRB1*07:01 carriage frequency. Robust ALT elevation risk estimates for HLA-DRB1*07:01 may support causality discrimination and safety risk management during the use of lapatinib combination therapy for the treatment of metastatic breast cancer.

Current practice in total-body irradiation: results of a Canada-wide survey.

BACKGROUND: Total-body irradiation (tbi) is used to condition patients before bone marrow transplant. A variety of tbi treatment strategies have been described and implemented, but no consensus on best practice has been reached. We report on the results of a survey created to assess the current state of tbi delivery in Canada. RESULTS: A 19-question survey was distributed to 49 radiation oncology programs in Canada. Responses were received from 20 centres, including 12 centres that perform tbi. A variety of tbi dose prescriptions was reported, although 12 Gy in 6 fractions was used in 11 of the 12 centres performing tbi. Half of the centres also reported using a dose prescription unique to their facility. Most centres use an extended-distance parallel-opposed-pair technique, with the patient standing or lying on a stretcher against a wall. Others translate the patient under the beam, sweep the beam over the patient, or use a more complicated multi-field technique. All but 1 centre indicated that they attenuate the lung dose; only 3 centres indicated attenuating the dose for other organs at risk. The survey also highlighted the considerable resources used for tbi, including extra staff, prolonged planning and treatment times, and use of locally developed hardware or software. CONCLUSIONS: At transplant centres, tbi is commonly used, but there is no commonly accepted approach to planning and treatment delivery. The important discrepancies in practice between centres in Canada creates an opportunity to prompt more discussion and collaboration between centres, improving consistency and uniformity of practice.

Evolutionary hypotheses for a constraint to life-history resilience in depleted Salmo salar populations.

Phenotypic plasticity underlies much of the variation in life-history expression in fishes. An understanding of potential constraints on life-history plasticity thus may be critical for assessing the resiliency of populations or species to environmental change. Here, several evolutionary hypotheses are formulated for why a depleted lineage of Canadian Atlantic salmon Salmo salar populations continues to express an apparently maladaptive life history in the face of severe marine mortality. These hypotheses include: (1) reduced genetic variability for expressing plasticity, (2) constraints from genetic architecture, (3) constraints from gene flow, (4) phylogenetic constraints or irreversible evolutionary transitions, (5) environmental constraints to plasticity and (6) a restriction to population rescue from evolutionary-demographic feedbacks. This S. salar lineage is intriguing to consider for understanding resilience or the lack thereof, because it has life-history attributes that should favour resilience (e.g. a high degree of iteroparity, variable age at maturity and the presence of both long- and short-distance migration ecotypes). In particular, the discussion centres on the question of why S. salar females, in contrast to males, do not adopt a non-anadromous life history and mature in fresh water, given extremely high marine mortality among anadromous individuals of both sexes. A salient implication, with possibly significant conservation ramifications, is that fishes may exhibit substantial plasticity and potential for adapting to environmental change, but still be incapable of responding to certain environmental changes due to sex-specific constraints to life-history plasticity.

Puny males punch above their weight to preserve genetic diversity in a declining Atlantic salmon population.

Many salmonid fish populations have anadromous (i.e. migratory) and nonanadromous individuals co-existing in sympatry. The nonanadromous individuals, frequently males, mature at a much smaller size in freshwater without undergoing marine migrations and often successfully fertilize many eggs laid by anadromous females. Because these small males do not recruit to fisheries, they are often not regarded in high esteem by fishers. In this issue of Molecular Ecology, Johnstone et al. (2013) demonstrate that by substantially contributing to reproduction, such males help maintain genetic diversity in a declining population of Atlantic salmon (Salmo salar). Their results show that estimates of effective population size (Ne), obtained by counting the number of anadromous adults returning from sea and correcting for unequal sex ratios, are lower than estimates generated from genetic markers. Many mechanisms are expected to reduce Ne below the adult census population size (N); the opposite pattern of Ne > N observed by Johnstone et al. (2013) is difficult to explain unless the reproductive effort of nonanadromous males is accounted for. The results have important implications for the conservation of small populations and highlight the challenges of relating Ne to N in organisms with complex life histories.

Multigenerational hybridisation and its consequences for maternal effects in Atlantic salmon.

Outbreeding between segregating populations can be important from an evolutionary, conservation and economical-agricultural perspective. Whether and how outbreeding influences maternal effects in wild populations has rarely been studied, despite both the prominent maternal influence on early offspring survival and the known presence of fitness effects resulting from outbreeding in many taxa. We studied several traits during the yolk-feeding stage in multigenerational crosses between a wild and a domesticated Atlantic salmon (Salmo salar) population up to their third-generation hybrid in a common laboratory environment. Using cross-means analysis, we inferred that maternal additive outbreeding effects underlie most offspring traits but that yolk mass also underlies maternal dominant effects. As a consequence of the interplay between additive and dominant maternally controlled traits, offspring from first-generation hybrid mothers expressed an excessive proportion of residual yolk mass, relative to total mass, at the time of first feeding. Their residual yolk mass was 23-97% greater than those of other crosses and 31% more than that predicted by a purely additive model. Offspring additive, epistatic and epistatic offspring-by-maternal outbreeding effects appeared to further modify this largely maternally controlled cross-means pattern, resulting in an increase in offspring size with the percentage of domesticated alleles. Fitness implications remain elusive because of unknown phenotype-by-environment interactions. However, these results suggest how mechanistically co-adapted genetic maternal control on early offspring development can be disrupted by the effects of combining alleles from divergent populations. Complex outbreeding effects at both the maternal and offspring levels make the prediction of hybrid phenotypes difficult.

Kinship analysis of brook trout Salvelinus fontinalis during their breeding migration.

Microsatellite markers were used to test whether groups of pre-spawning adult brook trout Salvelinus fontinalis from the same population and captured at the same location during their breeding migration comprised kin. Only weak evidence for kin associations was found at the onset of breeding: the proportion of kin captured at the same location was low and similar to the proportion found across all locations and the average relatedness of S. fontinalis captured at the same location was low. A dilution of kin associations from the feeding to breeding phase is hypothesized to stem from mainly natural mortality that reduces family size by the adult stage. The results illustrate the dynamic nature of kin associations between consecutive life stages, even within the same fish population.

Extent and scale of local adaptation in salmonid fishes: review and meta-analysis.

What is the extent and scale of local adaptation (LA)? How quickly does LA arise? And what is its underlying molecular basis? Our review and meta-analysis on salmonid fishes estimates the frequency of LA to be ∼55-70%, with local populations having a 1.2 times average fitness advantage relative to foreign populations or to their performance in new environments. Salmonid LA is evident at a variety of spatial scales (for example, few km to>1000 km) and can manifest itself quickly (6-30 generations). As the geographic scale between populations increases, LA is generally more frequent and stronger. Yet the extent of LA in salmonids does not appear to differ from that in other assessed taxa. Moreover, the frequency with which foreign salmonid populations outperform local populations (∼23-35%) suggests that drift, gene flow and plasticity often limit or mediate LA. The relatively few studies based on candidate gene and genomewide analyses have identified footprints of selection at both small and large geographical scales, likely reflecting the specific functional properties of loci and the associated selection regimes (for example, local niche partitioning, pathogens, parasites, photoperiodicity and seasonal timing). The molecular basis of LA in salmonids is still largely unknown, but differential expression at the same few genes is implicated in the convergent evolution of certain phenotypes. Collectively, future research will benefit from an integration of classical and molecular approaches to understand: (i) species differences and how they originate, (ii) variation in adaptation across scales, life stages, population sizes and environmental gradients, and (iii) evolutionary responses to human activities.

Y-box protein-1 controls transforming growth factor-beta1 translation in proximal tubular cells.

Transforming growth factor-beta1 (TGF-beta1) mRNA has low basal translational efficiency in proximal tubule cells; however, its translation is stimulated by profibrotic cytokines. We studied the role of the multifunctional Y-box protein-1 (YB-1) in regulating proximal tubule cell TGF-beta1 translation. Using RNA-electrophoretic mobility shift assays and ultraviolet crosslinking, we found two protein complexes of 50 and 100 kDa, which bound to the TGF-beta1 mRNA 5'-untranslated region. Supershift studies using antibodies to YB-1 showed that both sites contained YB-1 as did studies with recombinant YB-1, which demonstrated that it was sufficient to form both complexes. RNA competition experiments confirmed YB-1 binding to the two predicted binding sites; one with high affinity and the other with lower affinity. Strong basal YB-1 association with TGF-beta1 mRNA was found in proximal tubule cells, which decreased when platelet-derived growth factor was used to activate TGF-beta1 translation. In contrast, knockdown of proximal tubule cell YB-1 expression abrogated TGF-beta1 synthesis. Our results suggest that TGF-beta1 translation in proximal tubule cells requires YB-1 binding to a high-affinity site in the 5'-untranslated region of its mRNA; however, binding to a low-affinity site inhibits basal translation.

Ocular inflammation and hemorrhage as initial manifestations of uveal malignant melanoma. Incidence and prognosis.

A consecutive series of 450 eyes enucleated because of a malignant melanoma of the choroid or ciliary body was reviewed in an effort to establish the incidence of ocular inflammation or intraocular hemorrhage as the initial clinical manifestation. Thirty-five patients had these clinical signs initially. Twenty-two (4.9%) initially had ocular inflammations. Eight of these had an episcleritis, and the remaining 14 had some form of uveitis, endophthalmitis, or panophthalmitis. Five of those with episcleritis had tumors in the ciliary body, and all six patients who developed panophthalmitis had necrotic choroidal melanomas. Thirteen patients (2.9%) were initially observed with some form of intraocular hemorrhage. Follow-up information was available for 26 of the 35 patients selected for detailed study. Fourteen patients died of metastatic disease. We believe the relatively poor prognosis of these tumors depends on three factors: cell type, maximal diameter of the tumor, and extraocular extension.

Allopatric origins of sympatric brook charr populations: colonization history and admixture.

Natural selection is presumed to be the driving force behind the occurrence of phenotypically and genetically divergent populations in sympatry within many north temperate freshwater fishes. If, however, these populations have different ancestral origins, history could also contribute to their divergence. We previously found evidence for the role of selection in the evolution of divergent outflow and inflow breeding populations of migratory brook charr (Salvelinus fontinalis) inhabiting postglacial Mistassini Lake (Quebec, Canada). Here, we show that these populations do not have a common origin, through the use of admixture and spatial analyses with seven microsatellite loci. Divergent populations clustered into two different population groups when compared to samples from surrounding drainages, although inflow populations appeared to be more admixed between the two population groups than the outflow population. These results are noteworthy since outflow and inflow populations were monomorphic at mitochondrial DNA (338-bp sequence of the control region) and are only moderately differentiated (mean F(ST) = 0.10). Colonization by two ancestral populations was also consistent with known outflow direction changes throughout lake formation. In addition to providing insight into how phenotypic divergence in sympatry may have been affected by the nature (i.e. timing and direction) of colonization of ancestral populations, our results also suggest that ancestral populations may have differed in their ability to colonize certain lake habitats.

Adaptive evolutionary conservation: towards a unified concept for defining conservation units.

Recent years have seen a debate over various methods that could objectively prioritize conservation value below the species level. Most prominent among these has been the evolutionarily significant unit (ESU). We reviewed ESU concepts with the aim of proposing a more unified concept that would reconcile opposing views. Like species concepts, conflicting ESU concepts are all essentially aiming to define the same thing: segments of species whose divergence can be measured or evaluated by putting differential emphasis on the role of evolutionary forces at varied temporal scales. Thus, differences between ESU concepts lie more in the criteria used to define the ESUs themselves rather than in their fundamental essence. We provide a context-based framework for delineating ESUs which circumvents much of this situation. Rather than embroil in a befuddled debate over an optimal criterion, the key to a solution is accepting that differing criteria will work more dynamically than others and can be used alone or in combination depending on the situation. These assertions constitute the impetus behind adaptive evolutionary conservation.

Pseudomonas aeruginosa in swimming pools related to the incidence of otitis externa infection.

A study is reported which demonstrates that four out of twenty-four people developed otitis externa as a result of swimming in a pool with a high incidence of Pseudomonas aeruginosa. Serotyping and phage typing were used to establish that P. aeruginosa was the etiolgoical event responsible for the outbreaks of the infection.

Persistence of Pseudomonas aeruginosa in chlorinated swimming pools.

Various types of swimming pools were investigated for the quantitative isolation of Pseudomonas aeruginosa. Incidence of the organism increased when the free chlorine residual dropped below 0.4 mg/L in pool water which ad a pH of 6.9-8.9. As the water pH became more alkaline the efficiency of disinfection decreased. Excessive slime production caused certain strains to become more resistent to chlorine treatment. Immunotyping and phage typing, used to study the dynamics of P. aeruginosa populations in swimming pool waters, demonstrated that high densities of the organism consisted mainly of single predominant strains.

Topoisomerase II alpha promoter trans-activation early in monocytic differentiation of HL-60 human leukemia cells.

The cytotoxic efficacy of antitumor drugs targeted at DNA topoisomerase II (topo II) in many cases varies in direct proportion to cellular topo II content. To investigate the transcriptional control of the predominant alpha form of topo II, the 5' flanking region of the human topo II alpha gene (positions -562 to +90) was subcloned into a firefly luciferase reporter plasmid and transiently transfected into HL-60 human leukemia cells, a line capable of monocytic differentiation after treatment with various agents. Early in phorbol-12-myristate-13-acetate (30 nM)-induced differentiation (18-24 hr after treatment), an unexpected 3-5-fold activation of topo II alpha gene promoter activity was observed. Activation was observed in HL-60 cells and U-937 cells, but not in HeLa human cervical carcinoma cells. Sodium butyrate (NaB) (0.4 mM) also led to activation (4-17-fold) of the topo II alpha promoter in HL-60 and U-937 cells. Promoter sequences between position -90 and position +90 mediated the inducing effects of NaB. This NaB-dependent promoter-reporter induction was partly mirrored by a transient approximately 2-fold increase in endogenous topo II alpha enzyme. The stimulus for promoter activation could be partly attributed to a 2-fold increase in DNA synthesis at 16 hr for NaB, but not phorbol-12-myristate-13-acetate. Regardless of the primary stimulus for topo II alpha promoter trans-activation, it could be bypassed by treatment of HL-60 cells with NaB for 48 hr before transfection, revealing the expected 60-70% suppression of topo II alpha promoter activity. Further study of topo II alpha promoter down-regulation later in monocytic differentiation may serve as a model for elucidating the transcriptional mechanisms that may also be exploited by tumor cells expressing intrinsic or acquired resistance to topo II-directed drugs.

Co-insertional replication is responsible for tandem multimer formation during plasmid integration into the Dictyostelium genome.

We investigated the establishment of integrating transformation vectors in the genome of Dictyostelium discoideum to gain insight into the formation of the plasmid insertions and to investigate the conditions that determine the number of plasmid copies present in such insertions. Transformation vectors conferring resistance to neomycin and/or blasticidin were introduced into the cell as a calcium phosphate coprecipitate or by electroporation. The integration of the plasmid DNA was based on either recombinational integration of plasmids or restriction enzyme-mediated integration. The genomic DNA of the resulting transformants was examined by Southern blot analysis of pulsed-field gels and by the recently published method of direct electroporation into Escherichia coli. The number of insertion sites was found to be dependent on the transformation method used, and the minimum number of plasmid copies per insertion site required for resistance depended on the type and the concentration of the selective drug. Cotransformation studies revealed a strictly homogeneous composition of vector multimers from any given insertion site. This suggests that multimers arise by co-insertional replication of a single plasmid monomer, rather than by subsequent additional insertion events involving homologous recombination. The multimerization of the integrated vector only occurred when the insertion was established by homologous recombination. Moreover, the number of plasmid copies appeared to be random, was established at the time of the transformation, and did not change with subsequent alterations to the selection regime.

A rapid, small scale method for characterization of plasmid insertions in the Dictyostelium genome.

A rapid, simple method for characterization of plasmid insertions in the Dictyostelium discoideum genome was developed. It is based on the capability of linear plasmid multimers in the insertions to recircularize efficiently in Escherichia coli cells. This recombinational recircularization of plasmid multimers provides a highly sensitive and reliable tool for determining whether individual Dictyostelium transformants resulted from restriction enzyme-mediated integration (REMI) or from recombinational integration of plasmid (RIP). The method also reveals any rearrangements in RIP insertions and provides an estimate of the vector copy number in any particular transformant.

Efficient circularization in Escherichia coli of linear plasmid multimers from Dictyostelium discoideum genomic DNA.

Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid multimers cyclize in E. coli by homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of the recA gene product but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. discoideum genome ensured that none of the E. coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of D. discoideum.