Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random ...integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.
This study advances understanding of the flow dependency of invertebrate drift in rivers and its relevance to drift-feeding fish. Background drift concentration varied spatially and with flow over ...natural flow recession (lower mid-range to low flow) in a reach of a New Zealand river, largely consistent with passive entrainment. Seven taxonomic groups (dominated by Leptophlebiidae and Chironomidae) exhibited positive drift concentration–flow relationships, and one (sandy/stony-cased caddisflies (Conoesucidae)) exhibited negative relationships. A mechanistic drift transport model accurately predicted the slope, but not y intercept, of the drift concentration–flow relationship for the total drift community that positively responded to flow but performed more poorly at the taxon or size-class level. Partitioning the relative influence of drift entry and dilution revealed that positive drift concentration–flow relationships arose from entry overwhelming dilution with increasing flow. Drift transport models have potential for predicting relative (%) effects of flow change on concentration and rate of drift-prone invertebrates. This paves the way for drift transport models to inform inputs to net rate of energy intake models for drift-feeding fish.
Proteins can form reversible mixed disulfides with glutathione (GSH). It has been hypothesized that protein glutathionylation may represent a mechanism of redox regulation, in a fashion similar to ...that mediated by protein phosphorylation. We investigated whether GSH has a signaling role in the response of HL60 cells to hydrogen peroxide ( H2O2), in addition to its obvious antioxidant role. We identified early changes in gene expression induced at different times by H2O2treatment, under conditions that increase protein glutathionylation and minimal toxicity. We then investigated the effect of prior GSH depletion by buthionine sulfoximine and diethylmaleate on this response. The analysis revealed 2,016 genes regulated by H2O2. Of these, 215 genes showed GSH-dependent expression changes, classifiable into four clusters displaying down- or up-regulation by H2O2, either potentiated or inhibited by GSH depletion. The modulation of 20 selected genes was validated by real-time RT-PCR. The biological process categories overrepresented in the largest cluster (genes whose up-regulation was inhibited by GSH depletion) were NF-κB activation, transcription, and DNA methylation. This cluster also included several cytokine and chemokine ligands and receptors, the redox regulator thioredoxin interacting protein, and the histone deacetylase sirtuin. The cluster of genes whose up-regulation was potentiated by GSH depletion included two HSPs (HSP40 and HSP70) and the AP-1 transcription factor components Fos and FosB. This work demonstrates that GSH, in addition to its antioxidant and protective function against oxidative stress, has a specific signaling role in redox regulation.
Understanding what drives variation in fish abundance at reference sites provides perspective for assessing the effects of human alterations to river flow and land use. We examined temporal variation ...in fish community abundance in a headwater tributary of a large river in the upper South Island (New Zealand) over 10 years. We were interested in the influence of natural flow variation and temperature on native fish abundance within the context of potential competition/predation pressure from juvenile introduced trout. Results from biannual sampling highlighted the dominating influence of floods on fish population dynamics, overriding biotic effects. We found no evidence for adverse effects of trout on native fish, and flow-related habitat performed more poorly in explaining variation in fish abundance than low- and median-flow statistics. Differences in temporal variation in abundance between species were largely consistent with life histories that provide resistance and (or) resilience to flood flows and relative insensitivity to low flows. Long-term data (≥10 years) are needed for detecting meaningful trends and quantifying the effects of human activities on fish community abundance.
Understanding of colony specific properties of cyanobacteria in the natural environment has been challenging because sampling methods disaggregate colonies and there are often delays before they can ...be isolated and preserved.
is a ubiquitous cyanobacteria that forms large colonies in situ and often produces microcystins, a potent hepatotoxin. In the present study a new cryo-sampling technique was used to collect intact
colonies in situ by embedding them in a sheet of ice. Thirty-two of these
colonies were investigated with image analysis, liquid chromatography-mass spectrometry, quantitative polymerase chain reaction and high-throughput sequencing to assess their volume, microcystin quota and internal transcribed spacer (ITS) genotype diversity. Microcystin quotas were positively correlated to colony volume (
= 0.32;
= 0.004). Individual colonies had low
ITS genotype diversity and one ITS operational taxonomic unit predominated in all samples. This study demonstrates the utility of the cryo-sampling method to enhance the understanding of colony-specific properties of cyanobacteria with higher precision than previously possible.
Clonal hematopoiesis (CH) is a common aging-associated condition with increased risk of hematologic malignancy. Knowledge of the mechanisms driving evolution from CH to overt malignancy has been ...hampered by a lack of in vivo models that orthogonally activate mutant alleles. Here, we develop independently regulatable mutations in DNA methyltransferase 3A (Dnmt3a) and nucleophosmin 1 (Npm1), observed in human CH and AML, respectively. We find Dnmt3a mutation expands hematopoietic stem and multipotent progenitor cells (HSC/MPPs), modeling CH. Induction of mutant Npm1 after development of Dnmt3a-mutant CH causes progression to myeloproliferative disorder (MPD), and more aggressive MPD is observed with longer latency between mutations. MPDs uniformly progress to acute myeloid leukemia (AML) following transplant, accompanied by a decrease in HSC/MPPs and an increase in myeloid-restricted progenitors, the latter of which propagate AML in tertiary recipient mice. At a molecular level, progression of CH to MPD is accompanied by selection for mutations activating Ras/Raf/MAPK signaling. Progression to AML is characterized by additional oncogenic signaling mutations (Ptpn11, Pik3r1, Flt3) and/or mutations in epigenetic regulators (Hdac1, Idh1, Arid1a). Together, our study demonstrates that Npm1 mutation drives evolution of Dnmt3a-mutant CH to AML and rate of disease progression is accelerated with longer latency of CH.
Reference benchmarks are needed to assess the contemporary status of rivers and to establish restoration targets. We developed predictive models to estimate site-specific reference values for a ...macroinvertebrate community index (MCI), which is used to indicate a range of human impacts on wadeable streams. We compared three statistical modelling approaches - general linear, boosted regression tree (BRT) and random forest (RF) - and tested the effect of spatial scale on predictive accuracy by developing national and regional BRT models. Using fitted flexible models (BRT, RF) and resetting predictors to reflect natural state provided the most accurate predictions of reference condition. Variation in reference MCI predictions from national and regional models was within the range observed from methodological and temporal variability. The proportion of native vegetation in upstream catchments was the primary predictor of MCI scores in all models, while secondary predictors varied regionally.
Setting numeric in-stream objectives (limits, criteria) to inform limits on catchment loads for major land-use stressors is a promising policy instrument to prevent ecosystem degradation. Management ...objectives can be informed by thresholds identified from stressor–response shapes of ecological indicators based on field survey data. Use of multiple structural and functional indicators and different organism groups provides multiple lines of evidence to make objectives more robust. We measured a suite of ecological indicators during a regional field survey in New Zealand. We built flexible boosted regression tree (BRT) models with a predictor set consisting of nutrient, sediment, and environmental variables and investigated the fitted functions for different types of thresholds across each stressor gradient. Congruence of impact initiation (II) thresholds for N among macroinvertebrate metrics and 2 periphyton indicators provided multiple lines of evidence for ecosystem change with small increases in N concentrations above background levels. Impact cessation (IC) on macroinvertebrate metrics at total N = ∼0.5 mg/L (below N concentrations that saturate important ecosystem processes) highlighted sensitivity of macroinvertebrate communities to eutrophication. We found few stressor–response relationships for sediment. We suggest use of sediment-specific macroinvertebrate metrics and a reliable measure of deposited fine sediment in the future. Few indicators responded to phosphorus (P) concentration. Limited information for setting P objectives highlights the need to develop alternative indicators of P loading. Statistical analysis based on single-stressor inferential threshold models suggested that these models carry high risk of identifying spurious thresholds and are less suitable for setting management objectives. II and IC thresholds of multiple ecological indicators can be used to set robust objectives aimed at different levels of protection of ecosystem health.
Summary
1. Modification of natural landscapes and land‐use intensification are global phenomena that can result in a range of differing pressures on lotic ecosystems. We analysed national‐scale ...databases to quantify the relationship between three land uses (indigenous vegetation, urbanisation and agriculture) and indicators of stream ecological integrity. Boosted regression tree modelling was used to test the response of 14 indicators belonging to four groups – water quality (at 578 sites), benthic invertebrates (at 2666 sites), fish (at 6858 sites) and ecosystem processes (at 156 sites). Our aims were to characterise the ecological response curves of selected functional and structural metrics in relation to three land uses, examine the environmental moderators of these relationships and quantify the relative utility of metrics as indicators of stream ecological integrity.
2. The strongest indicators of land‐use effects were nitrate + nitrite, delta‐15 nitrogen value (δ15N) of primary consumers and the Macroinvertebrate Community Index (a biotic index of organic pollution), while the weakest overall indicators were gross primary productivity, benthic invertebrate richness and fish richness. All indicators declined in response to removal of indigenous vegetation and urbanisation, while variable responses to agricultural intensity were observed for some indicators.
3. The response curves for several indicators suggested distinct thresholds in response to urbanisation and agriculture, specifically at 10% impervious cover and at 0.1 g m−3 nitrogen concentration, respectively.
4. Water quality and ecosystem process indicators were influenced by a combination of temperature, slope and flow variables, whereas for macroinvertebrate indicators, catchment rainfall, segment slope and temperature were significant environmental predictor variables. Downstream variables (e.g. distance to the coast) were significant in explaining residual variation in fish indicators, not surprisingly given the preponderance of diadromous fish species in New Zealand waterways. The inclusion of continuous environmental variables used to develop a stream typology improved model performance more than the inclusion of stream type alone.
5. Our results reaffirm the importance of accounting for underlying spatial variation in the environment when quantifying relationships between land use and the ecological integrity of streams. Of distinctive interest, however, were the contrasting and complementary responses of different indicators of stream integrity to land use, suggesting that multiple indicators are required to identify land‐use impact thresholds, develop environmental standards and assign ecological scores for reporting purposes.
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