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Prostate cancer (PrCa) is highly heritable, and although rare variants contribute significantly to PrCa risk, few have been identified to date. Herein, whole-genome sequencing was performed in a large PrCa family featuring multiple affected relatives spanning several generations. A rare, predicted splice site EZH2 variant, rs78589034 (G > A), was identified as segregating with disease in all but two individuals in the family, one of whom was affected with lymphoma and bowel cancer and a female relative. This variant was significantly associated with disease risk in combined familial and sporadic PrCa datasets (n = 1551; odds ratio [OR] = 3.55, P = 1.20 × 10−5 ). Transcriptome analysis was performed on prostate tumour needle biopsies available for two rare variant carriers and two wild-type cases. Although no allele-dependent differences were detected in EZH2 transcripts, a distinct differential gene expression signature was observed when comparing prostate tissue from the rare variant carriers with the wild-type samples. The gene expression signature comprised known downstream targets of EZH2 and included the top-ranked genes, DUSP1, FOS, JUNB and EGR1, which were subsequently validated by qPCR. These data provide evidence that rs78589034 is associated with increased PrCa risk in Tasmanian men and further, that this variant may be associated with perturbed EZH2 function in prostate tissue. Disrupted EZH2 function is a driver of tumourigenesis in several cancers, including prostate, and is of significant interest as a therapeutic target.

Migratory species are particularly vulnerable to climate change because habitat throughout their entire migration cycle must be suitable for the species to persist. For migratory species in rivers, predicting climate change impacts is especially difficult because there is a lack of spatially continuous and seasonally varying stream temperature data, habitat conditions can vary for an individual throughout its life cycle, and vulnerability can vary by life stage and season. To predict thermal impacts on migratory riverine populations, we first expanded a spatial stream network model to predict mean monthly temperature for 465,775 river km in the western U.S., and then applied simple yet plausible future stream temperature change scenarios. We then joined stream temperature predictions to 44,396 spatial observations and life-stage-specific phenology (timing) for 26 ecotypes (i.e., geographically distinct population groups expressing one of the four distinct seasonal migration patterns) of Chinook salmon (Oncorhynchus tshawytscha), a phenotypically diverse anadromous salmonid that is ecologically and economically important but declining throughout its range. Thermal stress, assessed for each life stage and ecotype based on federal criteria, was influenced by migration timing rather than latitude, elevation, or migration distance such that sympatric ecotypes often showed differential thermal exposure. Early-migration phenotypes were especially vulnerable due to prolonged residency in inland streams during the summer. We evaluated the thermal suitability of 31,699 stream km which are currently blocked by dams to explore reintroduction above dams as an option to mitigate the negative effects of our warmer stream temperature scenarios. Our results showed that negative impacts of stream temperature warming can be offset for almost all ecotypes if formerly occupied habitat above dams is made available. Our approach of combining spatial distribution and phenology data with spatially explicit and temporally explicit temperature predictions enables researchers to examine thermal exposure of migrating populations that use seasonally varying habitats.

Social hierarchies within groups define the distribution of resources and provide benefits that support the collective group or favor dominant members. The progression of individuals through social hierarchies is a valuable characteristic for quantifying population dynamics. On coral reefs, a number of small site-attached fish maintain size-based hierarchical communities where individuals queue through social ranks. The cost of waiting in a lower-ranked position is outweighed by the reduced risk of eviction and mortality. Clownfish exist in stable social groups with subordinate individuals queuing to be part of the dominant breeding pair. Site attachment to their host anemone, complex social interactions, and relatively low predation rates make them ideal model organisms to assess changes in group dynamics through time in their natural environment. Here, we investigate the rank changes, and isometric growth rates of individual orange clownfish, Amphiprion percula, from 247 naturally occurring social groups in Kimbe Island, Papua New Guinea (5°12’13.54” S, 150°22’32.69” E). We use DNA profiling to assign and track individuals over an eight-year time period in 2011 and 2019. Over half of the individuals survived alongside two or three members of their original social group, with twelve breeding pairs persisting over the study period. Half of the surviving individuals increased in rank and experienced double the growth rate of those that maintained their rank. Examining rank change over a long-term period in a wild fish population gives new insights and highlights the complexity and importance of rank and social hierarchy in communal site-attached reef fish.Subject Area: behavior, ecology, evolution

The Escherichia coli SOS response to DNA damage, discovered and conceptualized by Evelyn Witkin and Miroslav Radman, is the prototypic DNA-damage stress response that upregulates proteins of DNA protection and repair, a radical idea when formulated in the late 1960s and early 1970s. SOS-like responses are now described across the tree of life, and similar mechanisms of DNA-damage tolerance and repair underlie the genome instability that drives human cancer and aging. The DNA damage that precedes damage responses constitutes upstream threats to genome integrity and arises mostly from endogenous biology. Radman’s vision and work on SOS, mismatch repair, and their regulation of genome and species evolution, were extrapolated directly from bacteria to humans, at a conceptual level, by Radman, then many others. We follow his lead in exploring bacterial molecular genomic mechanisms to illuminate universal biology, including in human disease, and focus here on some events upstream of SOS: the origins of DNA damage, specifically at chromosome fragile sites, and the engineered proteins that allow us to identify mechanisms. Two fragility mechanisms dominate: one at replication barriers and another associated with the decatenation of sister chromosomes following replication. DNA structures in E. coli, additionally, suggest new interpretations of pathways in cancer evolution, and that Holliday junctions may be universal molecular markers of chromosome fragility.

Caught in the act, DNA reaction intermediates in bacterial genomes may illuminate disease-driving regions in human chromosomes., Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

The persistence of a parasite species in an ecological community is determined both by its environmental tolerance and host breadth. The relative contribution of these niche characteristics to parasite community structure is challenging to parse because host persistence is also a consequence of extrinsic environmental factors. We investigated haemosporidian parasites (genera Plasmodium, Leucocytozoon, and Haemoproteus) in a clade of avian hosts in eastern North America. Species in this clade of Catharus thrushes occupy specific elevational bands in a non‐phylogenetically determined manner. This allowed us to tease apart the effects of host relatedness and elevation on parasite community structure, diversity, and infection prevalence. We screened blood and tissue samples from 414 adult birds from four mountain ranges in the Appalachian Highlands for blood parasites using a cytochrome‐b‐nested PCR protocol and identified parasite lineages by sequencing. We found an overall infection prevalence of 88.4% and identified a total of 38 parasite lineages including six novel lineages. Parasite community patterns varied by genus. Host relatedness rather than elevational zone predicted the beta turnover and phylobeta turnover of Leucocytozoon parasites, indicating that closely related rather than geographically proximate host species had more similar parasites. This pattern was not seen in Plasmodium parasites because the diversity of this parasite genus varied considerably in the high elevational zones among mountain ranges, that is, a sky‐island effect. Haemoproteus parasites were rare in this study system. Our study suggests that the mechanisms that underlie community structuring vary between haemosporidian genera due to differences in the degree of host sharing among lineages.

To assess the toxicity and accumulation (total and subcellular partitioning) of cadmium (Cd) and mercury (Hg), juvenile eastern oysters, Crassostrea virginica, were exposed for 4 weeks to a range of concentrations (Control, Low (1×), and High (4×)). Despite the 4-fold increase in metal concentrations, oysters from the High-Cd treatment (2.4 μM Cd) attained a body burden that was only 2.4-fold greater than that of the Low-Cd treatment (0.6 μM Cd), while oysters from the High-Hg treatment (0.056 μM Hg) accumulated 8.9-fold more Hg than those from the Low-Hg treatment (0.014 μM Hg). This fold difference in total Cd burdens was, in general, mirrored at the subcellular level, though binding to heat-denatured proteins in the High-Cd treatment was depressed (only 1.6-fold higher than the Low-Cd treatment). Mercury did not appear to appreciably partition to the subcellular fractions examined in this study, with the fold difference in accumulation between the Low- and High-Hg treatments ranging from 1.5-fold (heat-stable proteins) to 4.6-fold (organelles). Differences in toxicological impairments (reductions in condition index, protein content, and ETS activity) exhibited by oysters from the High-Cd treatment may be partially due to the nature of how different metals partition to subcellular components in the oysters, though exact mechanisms will require further examination.

Genome-wide association studies (GWAS) have identified multiple associations with emphysema apicobasal distribution (EABD), but the biological functions of these variants are unknown. To characterize the functions of EABD-associated variants, we integrated GWAS results with 1) expression quantitative trait loci (eQTL) from the Genotype Tissue Expression (GTEx) project and subjects in the COPDGene (Genetic Epidemiology of COPD) study and 2) cell type epigenomic marks from the Roadmap Epigenomics project. On the basis of these analyses, we selected a variant near ACVR1B (activin A receptor type 1B) for functional validation. SNPs from 168 loci with P values less than 5 × 10(−5) in the largest GWAS meta-analysis of EABD were analyzed. Eighty-four loci overlapped eQTL, with 12 of these loci showing greater than 80% likelihood of harboring a single, shared GWAS and eQTL causal variant. Seventeen cell types were enriched for overlap between EABD loci and Roadmap Epigenomics marks (permutation P

Background Previous genome-wide association studies (GWAS) have identified 42 loci (P < 5 × 10−8) associated with risk of colorectal cancer (CRC). Expanded consortium efforts facilitating the discovery of additional susceptibility loci may capture unexplained familial risk. Methods We conducted a GWAS in European descent CRC cases and control subjects using a discovery–replication design, followed by examination of novel findings in a multiethnic sample (cumulative n = 163 315). In the discovery stage (36 948 case subjects/30 864 control subjects), we identified genetic variants with a minor allele frequency of 1% or greater associated with risk of CRC using logistic regression followed by a fixed-effects inverse variance weighted meta-analysis. All novel independent variants reaching genome-wide statistical significance (two-sided P < 5 × 10−8) were tested for replication in separate European ancestry samples (12 952 case subjects/48 383 control subjects). Next, we examined the generalizability of discovered variants in East Asians, African Americans, and Hispanics (12 085 case subjects/22 083 control subjects). Finally, we examined the contributions of novel risk variants to familial relative risk and examined the prediction capabilities of a polygenic risk score. All statistical tests were two-sided. Results The discovery GWAS identified 11 variants associated with CRC at P < 5 × 10−8, of which nine (at 4q22.2/5p15.33/5p13.1/6p21.31/6p12.1/10q11.23/12q24.21/16q24.1/20q13.13) independently replicated at a P value of less than .05. Multiethnic follow-up supported the generalizability of discovery findings. These results demonstrated a 14.7% increase in familial relative risk explained by common risk alleles from 10.3% (95% confidence interval [CI] = 7.9% to 13.7%; known variants) to 11.9% (95% CI = 9.2% to 15.5%; known and novel variants). A polygenic risk score identified 4.3% of the population at an odds ratio for developing CRC of at least 2.0. Conclusions This study provides insight into the architecture of common genetic variation contributing to CRC etiology and improves risk prediction for individualized screening.

Background: Supraphysiological insulin and incretin responses to a cereal-based diet have been described in horses and ponies with insulin dysregulation (ID). However, the hormonal responses to grazing have not yet been described. Objectives: To determine if there is a difference in the insulin and incretin responses to grazing pasture between insulin-dysregulated and healthy ponies. Animals: A cohort of 16 ponies comprising 5 with normal insulin regulation (NIR), 6 with moderate ID (MID), and 5 with severe ID (SID). Methods: In this case-control study, an oral glucose test (OGT) was used to determine the insulin responsiveness of each pony to PO carbohydrate before grazing pasture (4 hours) for 3 consecutive days. Serial blood samples collected during grazing were analyzed for glucose, insulin, glucose-dependent insulinotropic peptide (GIP) and active glucagon-like peptide-1 (aGLP-1), and compared among pony groups and day of pasture access. Results: The area under the insulin curve when grazing increased with ID severity (P

To further dissect the genetic architecture of colorectal cancer (CRC), we performed whole-genome sequencing of 1,439 cases and 720 controls, imputed discovered sequence variants and Haplotype Reference Consortium panel variants into genome-wide association study data, and tested for association in 34,869 cases and 29,051 controls. Findings were followed up in an additional 23,262 cases and 38,296 controls. We discovered a strongly protective 0.3% frequency variant signal at CHD1. In a combined meta-analysis of 125,478 individuals, we identified 40 new independent signals at P, Reporting Summary. Further information on experimental design is available in the Life Sciences Reporting Summary linked to this article.

Aim: The role of migratory birds in the spread of parasites is poorly known, in part because migratory strategies and behaviours potentially affecting transmission are not easy to study. We investigated the dynamics of infection by blood parasites through the annual cycle of a long-distance Nearctic–Neotropical migratory songbird to examine the role of this species in dispersing parasites between continents. Location: The Americas. Taxon: Grey-cheeked Thrush (Catharus minimus, Aves, Passeriformes, Turdidae), Birds. Methods: We used molecular and microscopy screening of haemosporidian parasites (Plasmodium, Haemoproteus, and Leucocytozoon) to examine the prevalence, distribution, and diversity of lineages through the annual cycle (breeding, migration, and wintering) of the grey-cheeked thrush in North and Central America, Santa Marta mountains, the Andes, and the Amazon. We aimed to identify transmission areas, to examine the degree of sharing of haemosporidian lineages with resident birds in various areas and to assess the potential role of immunologically naive juvenile individuals in parasite transmission. Results: Prevalence and lineage diversity of haemosporidians varied significantly over time, being higher during breeding and fall and spring migration, and declining during wintering. Grey-cheeked thrush shared few parasite lineages with tropical resident birds and slightly more lineages with other migratory and resident boreal species. We detected gametocytes in blood during spring migration stopover, but these were of lineages not found in resident tropical birds, indicating relapses of parasites transmitted elsewhere. Transmission likely occurs mostly on the breeding grounds, where juveniles and adults carried lineages restricted to closely related species of thrushes and other species of boreal birds. Main conclusions: Long-distance migratory songbirds are likely not important dispersers of blood parasites because there are ecological and evolutionary barriers to the interchange of parasites across vastly separated areas. Further work with thorough spatial and temporal sampling across other species, and considering the role of vectors, is necessary to understand the ecological and evolutionary factors explaining the distribution of parasites over broad scales.

Intravenous Immunoglobulin’s (IVIG’s) are prepared from thousands of donor plasmas and as a result comprise an extreme broad mix and depth of Antibody (Ab) specificities. IVIG formulations available in Australia are from both local and overseas donor sources and extracted using a variety of purification methods and immunoglobulin purities, with the Australia-derived and prepared IVIG listed at >98% and the overseas-derived preparations at ≥ 95%. Because of these differences it was predicted that the formulations might individually vary in composition and that together with obvious genetic and geographic antigenic (Ag) environment differences may result in notable variability between formulations. Hence a focussed comparative proteomic profiling of IVIG formulations was undertaken to identify notable similarities and differences across products. Comparisons between formulations did reveal marked qualitative differences in 2D-gel Antibody profiling that included parameters of isoelectric charge (pI), as well as immunoglobulin (Ig) monomer to dimer ratio variability between products, including high molecular weight (MW) immunoglobulin multimers for some. These notable differences were in part quite likely a product of the respective purification methods used, and capacity to select (or de-select) for antibodies of such different properties. Furthermore, for identification of non-Ig proteins carried over from plasma through purifications Mass spectrometry was performed. This identified a few such ancillary proteins, and their identities, in general, differed between formulations. Proteins detected included the most abundant protein of plasma, albumin, as well as other mostly large and abundant proteins; RAG1 - V(D)J recombination activating protein1, gelsolin, complement Factor-B, serotransferrin, tetranectin, NADH ubiquinone oxidoreductase, caspase 3 and VEGFR1. An alternate strategy used commercial Multiplex xMAP assay to detect cytokines, which are small and present in plasma at trace but highly active quantities. This revealed various different cytokine profiles across the formulations studied. The identification of additional proteins, and especially cytokines in IVIGs, is particularly notable, and the positive, negative or null biological relevance for clinical use, needs resolution. Collectively these findings reveal marked differences between Australian and overseas-derived (non-Australian) IVIGs in immunoglobulin composition and biochemical characteristics, and presence of additional carry-over proteins from plasma. These findings prompt the need for further evaluation of the micro-compositions of individual formulations. Perhaps detailed mining and improved comparative understanding of each IVIG formulation, may enable highly tailored and strategic clinical use of certain formulations that are personalised best-fit treatments for particular conditions. Such as in treatment of a neuropathy, as compared to another formulation, more suited for treating a particular infectious disease. The most salient and overarching study conclusion is need for caution in attributing equivalence across IVIGs.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients’ demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Diverse DNA structures occur as reaction intermediates in various DNA-damage and -repair mechanisms, most of which results from replication stress. We harness the power of proteins evolutionarily optimized to bind and "trap" specific DNA reaction-intermediate structures, to quantify the structures, and discern the mechanisms of their occurrence in cells. The engineered proteins also allow genomic mapping of sites at which specific DNA structures occur preferentially, using a structure-trapping protein and ChIP-seq- or Cut-and-Tag-like methods. Genome-wide identification of sites with recurrent DNA-damage intermediates has illuminated mechanisms implicated in genome instability, replication stress, and chromosome fragility. Here, we describe X-seq, for identifying sites of recurrent four-way DNA junctions or Holliday-junctions (HJs). X-seq uses an engineered, catalysis-defective mutant of Escherichia coli RuvC HJ-specific endonuclease, RuvCDefGFP. X-seq signal indicates sites of recombinational DNA repair or replication-fork stalling and reversal. We also describe methods for genomic mapping of 3'-single-stranded DNA ends with SsEND-seq, in E. coli. Both methods allow genomic profiling of DNA-damage and -repair intermediates, which can precede genome instability, and are expected to have many additional applications including in other cells and organisms.

Characterising and monitoring biological diversity to foster sustainable ecosystems is highly recommended as urban centres rapidly expand. However, much of New York City’s biodiversity remains undescribed, including in the historically degraded, but recovering Bronx River Estuary. In a pilot study to identify organisms and characterise biodiversity patterns there, 18S rRNA gene amplicons (V1–V3 region), obtained from river sediments and surface waters of Hunts Point Riverside and Soundview Parks, were sequenced. Across 48 environmental samples collected over three seasons in 2015 and 2016, following quality control and contaminant removal, 2,763 Amplicon Sequence Variants (ASVs) were identified from 1,918,463 sequences. Rarefaction analysis showed sufficient sampling depth, and community composition varied over time and by substrate at the study sites over the sampling period. Protists, plants, fungi and animals, including organisms of management concern, such as Eastern oysters (Crassostrea virginica), wildlife pathogens and groups related to Harmful Algal Blooms, were detected. The most common taxa identified in river sediments were annelid worms, nematodes and diatoms. In the water column, the most commonly observed organisms were diatoms, algae of the phylum Cryptophyceae, ciliates and dinoflagellates. The presented dataset demonstrates the reach of 18S rRNA metabarcoding for characterising biodiversity in an urban estuary.

Biodiversity monitoring is an essential component of restoration efforts. We sequenced 16S rRNA gene amplicons from sediments and waters of Hunts Point Riverside Park and Soundview Park, located in a historically degraded but recovering urban estuary in New York. In total, 16,165 unique amplicon sequence variants were recovered, and Proteobacteria was the dominant phylum.

The equine microbiome can change in response to dietary alteration and might play a role in insulin dysregulation. The aim of this study was to determine the effect of adding pasture to a hay diet on the fecal bacterial microbiome of both healthy and insulin-dysregulated ponies. Fecal samples were collected from sixteen ponies before and after dietary change to enable bacterial 16S rRNA sequencing of the V3-V4 region. The dominant phyla in all samples were the Firmicutes and Bacteroidetes. The evenness of the bacterial populations decreased after grazing pasture, and when a pony was moderately insulin-dysregulated (P=0.001). Evenness scores negatively correlated with post-prandial glucagon-like peptide-1 concentrations after a hay-only diet (r2;=-0.7, P=0.001). A change in diet explained 3% of fecal microbiome variability. We conclude that metabolically healthy ponies have greater microbial stability when challenged with a subtle dietary change, compared to moderately insulin-dysregulated ponies.

Perfluoroalkyl substances (PFAS) are ubiquitous and persistent environmental contaminants, yet knowledge of their biological effects and mechanisms of action is limited. The highest aqueous PFAS concentrations are found in areas where bacteria are relied upon for functions such as nutrient cycling and contaminant degradation, including fire-training areas, wastewater treatment plants, and landfill leachates. This research sought to elucidate one of the mechanisms of action of PFAS by studying their uptake by bacteria and partitioning into model phospholipid bilayer membranes. PFAS partitioned into bacteria as well as model membranes (phospholipid liposomes and bilayers). The extent of incorporation into model membranes and bacteria was positively correlated to the number of fluorinated carbons. Furthermore, incorporation was greater for perfluorinated sulfonates than for perfluorinated carboxylates. Changes in zeta potential were observed in liposomes but not bacteria, consistent with PFAS being incorporated into the phospholipid bilayer membrane. Complementary to these results, PFAS were also found to alter the gel-to-fluid phase transition temperature of phospholipid bilayers, demonstrating that PFAS affected lateral phospholipid interactions. This investigation compliments other studies showing that sulfonated PFAS and PFAS with more than seven fluorinated carbons have a higher potential to accumulate within biota than carboxylated and shorter-chain PFAS.

We examined species limits, admixture, and genetic structure among populations in the Bicknell’s Thrush (Catharus bicknelli)–Gray-cheeked Thrush (C. minimus) species complex to establish the geographic and temporal context of speciation in this group, which is a model system in ecology and a high conservation priority. We obtained mitochondrial ND2 sequences from 186 Bicknell’s Thrushes, 77 Gray-cheeked Thrushes, and 55 individuals of their closest relative, the Veery (C. fuscescens), and genotyped a subset of individuals (n = 72) at 5,633 anonymous single nucleotide polymorphic (SNP) loci. Between-species sequence divergence was an order of magnitude greater than divergence within each species, divergence was dated to the late Pleistocene (420 kbp) based on Bayesian coalescence estimation, and a coalescent model (IMa) revealed almost no gene flow between species based on ND2. SNP data were consistent with mitochondrial results and revealed low levels of admixture among species (3 of 37 Bicknell’s Thrushes, no Gray-cheeked Thrushes, and no Veeries were >2% admixed). Species distribution models projected to the Last Glacial Maximum suggest that Bicknell’s Thrush and Gray-cheeked Thrush resided in primarily allopatric refugia in the late Pleistocene, consistent with the genetic data that support reproductive isolation over an extended period of time. Our genetic data suggest that both species underwent demographic expansions, possibly as they expanded out of Pleistocene refugia into their current ranges. We conclude that Bicknell’s Thrush and Gray-cheeked Thrush are 2 distinct species-level lineages despite low levels of Gray-cheeked Thrush introgression in Bicknell’s Thrushes, and divergence has been maintained by a long history of allopatry in subtly different habitats. Their unique phylogeography among boreal forest birds indicates that either cryptic species breaks in eastern North America are still undiscovered, or another factor, such as divergent natural selection, high migratory connectivity, or interspecific competition, played a role in their divergence.

Prognostic genomic biomarkers that can be measured at diagnosis to aid choice of treatment options are unavailable for most common cancers. This is due in part to the poor quality and quantity of available diagnostic specimens for discovery research and to limitations in genomic technologies. Recent technical advances now enable high-density molecular analyses using suboptimal biological specimens. Here we describe the optimization of a transcriptome-specific protocol for use with formalin-fixed, paraffin-embedded (FFPE) diagnostic prostate cancer (PrCa) specimens. We applied the Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq Kit) to RNA samples extracted from 36 tumor-enriched and 16 adjacent normal tissues (ADJNT) from 37 FFPE PrCa specimens over a series of eight pilot studies, incorporating protocol modifications from Pilots 2 to 5. Data quality were measured by (1) the total number of mapped reads; (2) the percentage of reads that mapped to AmpliSeq target regions (OnTarget%); (3) the percentage of genes on the AmpliSeq panel with a read count ≥10 (TargetsDetected%); and (4) comparing the gene read-count distribution of the prostate tissue samples with the median gene read-count distribution of cell line-derived RNA samples. Modifications incorporated into Pilot study 5 provided gene expression data equivalent to cell line-derived RNA samples. These modifications included the use of freshly cut slides for macrodissection; increased tissue section thickness (8 µm); RNA extraction using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher); 18 target amplification cycles; and processing six samples per Ion PI chip. This protocol will facilitate the discovery of prognostic biomarkers for cancer by allowing researchers to exploit previously underutilized diagnostic FFPE specimens.

Sister species that diverged in allopatry in similar environments are expected to exhibit niche conservatism. Using ecological niche modeling and a multivariate analysis of climate and habitat data, I test the hypothesis that the Bicknell's Thrush (Catharus bicknelli) and Gray-cheeked Thrush (C. mimimus), sister species that breed in the North American boreal forest, show niche conservatism. Three tree species that are important components of breeding territories of both thrush species were combined with climatic variables to create niche models consisting of abiotic and biotic components. Abiotic-only, abiotic+biotic, and biotic-only models were evaluated using the area under the curve (AUC) criterion. Abiotic+biotic models had higher AUC scores and did not over-project thrush distributions compared to abiotic-only or biotic-only models. From the abiotic+biotic models, I tested for niche conservatism or divergence by accounting for the differences in the availability of niche components by calculating (1) niche overlap from ecological niche models and (2) mean niche differences of environmental values at occurrence points. Niche background similarity tests revealed significant niche divergence in 10 of 12 comparisons, and multivariate tests revealed niche divergence along 2 of 3 niche axes. The Bicknell's Thrush breeds in warmer and wetter regions with a high abundance of balsam fir (Abies balsamea), whereas Gray-cheeked Thrush often co-occurs with black spruce (Picea mariana). Niche divergence, rather than conservatism, was the predominant pattern for these species, suggesting that ecological divergence has played a role in the speciation of the Bicknell's Thrush and Gray-cheeked Thrush. Furthermore, because niche models were improved by the incorporation of biotic variables, this study validates the inclusion of relevant biotic factors in ecological niche modeling to increase model accuracy.

Genomic instability underlies many cancers and generates genetic variation that drives cancer initiation, progression, and therapy resistance. In contrast with classical assumptions that mutations occur purely stochastically at constant, gradual rates, microbes, plants, flies, and human cancer cells possess mechanisms of mutagenesis that are upregulated by stress responses. These generate transient, genetic-diversity bursts that can propel evolution, specifically when cells are poorly adapted to their environments—that is, when stressed. We review molecular mechanisms of stress-response-dependent (stress-induced) mutagenesis that occur from bacteria to cancer, and are activated by starvation, drugs, hypoxia, and other stressors. We discuss mutagenic DNA break repair in Escherichia coli as a model for mechanisms in cancers. The temporal regulation of mutagenesis by stress responses and spatial restriction in genomes are common themes across the tree of life. Both can accelerate evolution, including the evolution of cancers. We discuss possible anti-evolvability drugs, aimed at targeting mutagenesis and other variation generators, that could be used to delay the evolution of cancer progression and therapy resistance.

BACKGROUND Global measures of peripheral blood DNA methylation have been associated with risk of some malignancies, including breast, bladder, and gastric cancer. Here, we examined genome-wide measures of peripheral blood DNA methylation in prostate cancer and its non-aggressive and aggressive disease forms. METHODS We used a matched, case-control study of 687 incident prostate cancer samples, nested within a larger prospective cohort study. DNA methylation was measured in pre-diagnostic, peripheral blood samples using the Illumina Infinium HM450K BeadChip. Genome-wide measures of DNA methylation were computed as the median M-value of all CpG sites and according to CpG site location and regulatory function. We used conditional logistic regression to test for associations between genome-wide measures of DNA methylation and risk of prostate cancer and its subtypes, and by time between blood draw and diagnosis. RESULTS We observed no associations between the genome-wide measure of DNA methylation based on all CpG sites and risk of prostate cancer or aggressive disease. Risk of non-aggressive disease was associated with higher methylation of CpG islands (OR = 0.80; 95%CI = 0.68–0.94), promoter regions (OR = 0.79; 95%CI = 0.66–0.93), and high density CpG regions (OR = 0.80; 95%CI = 0.68–0.94). Additionally, higher methylation of all CpGs (OR = 0.66; 95%CI = 0.48–0.89), CpG shores (OR = 0.62; 95%CI = 0.45–0.84), and regulatory regions (OR = 0.68; 95% CI = 0.51–0.91) was associated with a reduced risk of overall prostate cancer within 5 years of blood draw but not thereafter. CONCLUSIONS A reduced risk of overall prostate cancer within 5 years of blood draw and non-aggressive prostate cancer was associated with higher genome-wide methylation of peripheral blood DNA. While these data have no immediate clinical utility, with further work they may provide insight into the early events of prostate carcinogenesis. Prostate 77:471–478, 2017. © 2017 Wiley Periodicals, Inc.

Microbes and human cells possess mechanisms of mutagenesis activated by stress responses. Stress-inducible mutagenesis mechanisms may provide important models for mutagenesis that drives host-pathogen interactions, antibiotic resistance, and possibly much of evolution generally. In Escherichia coli , repair of DNA double-strand breaks is switched to a mutagenic mode, using error-prone DNA polymerases, via the SOS DNA damage and general (σ S ) stress responses. We investigated small RNA (sRNA) clients of Hfq, an RNA chaperone that promotes mutagenic break repair (MBR), and found that GcvB promotes MBR by allowing a robust σ S response, achieved via opposing the membrane stress (σ E ) response. Cells that lack gcvB were MBR deficient and displayed reduced σ S -dependent transcription but not reduced σ S protein levels. The defects in MBR and σ S -dependent transcription in Δ gcvB cells were alleviated by artificially increasing σ S levels, implying that GcvB promotes mutagenesis by allowing a normal σ S response. Δ gcvB cells were highly induced for the σ E response, and blocking σ E response induction restored both mutagenesis and σ S -promoted transcription. We suggest that GcvB may promote the σ S response and mutagenesis indirectly, by promoting membrane integrity, which keeps σ E levels lower. At high levels, σ E might outcompete σ S for binding RNA polymerase and so reduce the σ S response and mutagenesis. The data show the delicate balance of stress response modulation of mutagenesis. IMPORTANCE Mutagenesis mechanisms upregulated by stress responses promote de novo antibiotic resistance and cross-resistance in bacteria, antifungal drug resistance in yeasts, and genome instability in cancer cells under hypoxic stress. This paper describes the role of a small RNA (sRNA) in promoting a stress-inducible-mutagenesis mechanism, mutagenic DNA break repair in Escherichia coli . The roles of many sRNAs in E. coli remain unknown. This study shows that Δ gcvB cells, which lack the GcvB sRNA, display a hyperactivated membrane stress response and reduced general stress response, possibly because of sigma factor competition for RNA polymerase. This results in a mutagenic break repair defect. The data illuminate a function of GcvB sRNA in opposing the membrane stress response, and thus indirectly upregulating mutagenesis.

Bacterial populations vary in their stress tolerance and population structure depending upon whether growth occurs in well-mixed or structured environments. We hypothesized that evolution in biofilms would generate greater genetic diversity than well-mixed environments and lead to different pathways of antibiotic resistance. We used experimental evolution and whole genome sequencing to test how the biofilm lifestyle influenced the rate, genetic mechanisms, and pleiotropic effects of resistance to ciprofloxacin in

Mutations drive evolution and were assumed to occur by chance: constantly, gradually, roughly uniformly in genomes, and without regard to environmental inputs, but this view is being revised by discoveries of molecular mechanisms of mutation in bacteria, now translated across the tree of life. These mechanisms reveal a picture of highly regulated mutagenesis, up-regulated temporally by stress responses and activated when cells/organisms are maladapted to their environments—when stressed—potentially accelerating adaptation. Mutation is also nonrandom in genomic space, with multiple simultaneous mutations falling in local clusters, which may allow concerted evolution—the multiple changes needed to adapt protein functions and protein machines encoded by linked genes. Molecular mechanisms of stress-inducible mutation change ideas about evolution and suggest different ways to model and address cancer development, infectious disease, and evolution generally.

Genome-wide association studies (GWAS) and fine-mapping efforts to date have identified more than 100 prostate cancer (PrCa)-susceptibility loci. We meta-analyzed genotype data from a custom high-density array of 46,939 PrCa cases and 27,910 controls of European ancestry with previously genotyped data of 32,255 PrCa cases and 33,202 controls of European ancestry. Our analysis identified 62 novel loci associated (PC, p.Pro1054Arg) in ATM and rs2066827 (OR = 1.06; P = 2.3 × 10(−9); T>G, p.Val109Gly) in CDKN1B. The combination of all loci captured 28.4% of the PrCa familial relative risk, and a polygenic risk score conferred an elevated PrCa risk for men in the ninetieth to ninety-ninth percentiles (relative risk = 2.69; 95% confidence interval (CI): 2.55–2.82) and first percentile (relative risk = 5.71; 95% CI: 5.04–6.48) risk stratum compared with the population average. These findings improve risk prediction, enhance fine-mapping, and provide insight into the underlying biology of PrCa(1).

2019 Klein et al., Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies., HLK was supported by NIH grant R01-CA146940. AA was supported by the European research Council (ERC2014- ADG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2016-75058-P), and the Junta de Andalucía (BIO1238). JLA was supported by NIH grant R35G-GM119788. CHF was supported by NIH grants P01- GM105473 and R01-GM122880. SMG was supported by the Swiss national Science Foundation and the Novartis Research Foundation. DAG was supported by National Institutes of Health (Intramural Research Program Project Z1AES103266. JEH was supported by NIH grants R35- GM127029 and P01-GM105473. GI was supported by NIH grants R01-GM125650 and R01-GM080600. SJ-R was supported by NIH grant R35-GM118077. MCK was supported by National Science Foundation Graduate Research Fellowship DGE-1122492, NIH training grant T32-GM007223 and NIH grant DP20D008429. RDK and CDP were supported by NIH grants R01-GM26017 and R01-GM50006. AK was supported by NIH grant R01-GM073115. SAEL was supported by grants from Agence Nationale de la Recherche ANR-14- CE10-0010-01 and the Foundation pour la Recherche Médicale Equipe FRM DEQ20160334889. SEL was supported by NIH grant R01-GM071011. SMM was supported by NIH grants R01-GM60987 and P01-GM105473. TDP was supported by NIH grant R35-GM118020. SMR was supported by a gift from WM Keck Foundation, NIH Director’s Pioneer Award DP1-CA174424 and NIH grant R35-GM122598. DMF was supported by the Cancer Prevention and Research Institute of Texas, Baylor College of Medicine Comprehensive Cancer Training Program Postdoctoral Fellowship RP160283. RR was supported by NIH grant R35-GM118180 and JM-H was supported by a Marie Curie International Outgoing Fellowship and the ANR-12-PDOC-0035-01. LSS was supported by NIH grant R35-GM126997. PZ was supported by National Science Centre Poland 2015/19/103859 and FNP First TEAM/2016-1/9. NK was supported by NIH grant R01-GM116007 and the Welch Foundation (AU1875). ML was supported by grants from the Danish Agency for Science, Technology and Innovation, the Villum Foundation and the Danish National Research Foundation (DNRF115). AM was supported by NIH grants R35-GM127006 and R03- ES029306.

We tested the in vivo potential of a MHC class I-restricted blocking peptide to sufficiently lower an anti-viral CTL response for preventing virus-induced CTL-mediated autoimmune diabetes (insulin-dependent diabetes mellitus (IDDM)) in vivo without affecting systemic viral clearance. By designing and screening several peptides with high binding affinities to MHC class I H-2Db for best efficiency in blocking killing of target cells by lymphocytic choriomeningitis virus (LCMV) and other viral CTL, we identified the peptide for this study. In vitro, it selectively lowered CTL killing restricted to the Db allele, which correlated directly with the affinity of the respective epitopes. Expression of the blocking peptide in the target cell lowered recognition of all Db-restricted LCMV epitopes. In addition, in vitro expansion of LCMV memory CTL was prevented, resulting in decreased IFN-γ secretion. In vivo, a 2-wk treatment with this peptide lowered the LCMV Db-restricted CTL response by over threefold without affecting viral clearance. However, the CTL reduction by the peptide treatment was sufficient to prevent LCMV-induced IDDM in rat insulin promoter-LCMV-glycoprotein transgenic mice. Following LCMV infection, these mice develop IDDM, which depends on Db-restricted anti-self (viral) CTL. Precursor numbers of splenic LCMV-CTL in peptide-treated mice were reduced, but their cytokine profile was not altered, indicating that the peptide did not induce regulatory cells. Further, non-LCMV-CTL recognizing the blocking peptide secreted IFN-γ and did not protect from IDDM. This study demonstrates that in vivo treatment with a MHC class I blocking peptide can prevent autoimmune disease by directly affecting expansion of autoreactive CTL.

Background: Global DNA methylation has been reported to be associated with urothelial cell carcinoma (UCC) by studies using blood samples collected at diagnosis. Using the Illumina HumanMethylation450 assay, we derived genome-wide measures of blood DNA methylation and assessed them for their prospective association with UCC risk. Methods: We used 439 case–control pairs from the Melbourne Collaborative Cohort Study matched on age, sex, country of birth, DNA sample type, and collection period. Conditional logistic regression was used to compute odds ratios (OR) of UCC risk per s.d. of each genome-wide measure of DNA methylation and 95% confidence intervals (CIs), adjusted for potential confounders. We also investigated associations by disease subtype, sex, smoking, and time since blood collection. Results: The risk of superficial UCC was decreased for individuals with higher levels of our genome-wide DNA methylation measure (OR=0.71, 95% CI: 0.54–0.94; P=0.02). This association was particularly strong for current smokers at sample collection (OR=0.47, 95% CI: 0.27–0.83). Intermediate levels of our genome-wide measure were associated with decreased risk of invasive UCC. Some variation was observed between UCC subtypes and the location and regulatory function of the CpGs included in the genome-wide measures of methylation. Conclusions: Higher levels of our genome-wide DNA methylation measure were associated with decreased risk of superficial UCC and intermediate levels were associated with reduced risk of invasive disease. These findings require replication by other prospective studies.

Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other’s antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM.

A study was conducted to determine the volatile organic compounds (VOCs) associated with rice grain aroma in 37 commonly grown lines within Uganda, as well as elites. The aim of the study was to identify potential volatile biochemical markers, if any, for the rice grain aroma trait. Certified rice seeds were obtained from the Uganda National Crops Resources Research Institute germplasm collection. The seeds were sown into experimental plots, under field conditions and the mature paddy harvested. Polished rice grains were heated to 80 oC and the liberated VOCs subjected to untargeted metabolite analysis using gas chromatography-time-of-flight mass spectrometry. In total, nine functional groups were present; hydrocarbons, alcohols, ketones, aldehydes, N-containing compounds, S-containing compounds, esters, oxygen heterocycles and carboxylic acids. More specifically, 148 VOCs were identified across the 37 rice lines, of which 48 (32.4%) including 2-acetyl-1-pyrroline (2-AP) appeared to elucidate the difference between non-aromatic and aromatic rice. Furthermore, 41 (27.7%) VOCs were found to be significantly correlated with 2-AP abundance, the principle rice aroma compound. Amongst the 41 VOCs, only ten compounds were found to contribute highly towards variation in 2-AP abundance, indicative of their possible modulation roles in regard to rice aroma. Within the ten influential volatiles, three aroma active compounds; toluene, 1-hexanol, 2-ethyl and heptane, 2,2,4,6,6-pentamethyl- were established as the most reliable biochemical surrogates to the rice aroma trait. Thus, the aforementioned compounds may be used in rice breeding programme for enhancing development of the grain aroma trait.

Accelerating sea-level rise (SLR) threatens salt marshes globally, and thus understanding how these ecosystems respond to this stress is vital to increasing their resiliency. In order to maintain their surface elevation as sea level rises, salt marshes must add below ground biomass and accrete sediment. In many cases, biota can significantly affect marsh accretionary and erosional processes by stabilizing or helping to mobilize sediment, which in turn can profoundly affect the morphological evolution of the marsh. However, these effects are poorly understood. Bioturbation by dense populations of the marsh crab Sesarma reticulatum has been found to facilitate expansion of tidal creeks and creek bank erosion in multiple areas. The influence of S. reticulatum bioturbation on sediment erodibility has not yet been quantified, due to difficulties involved in measuring the processes in a field setting. In this study, we used a laboratory flume to examine the effects of burrowing by common marsh crab species S. reticulatum and Uca pugnax on sediment surface roughness and erodibility. Measurements and observations of surface elevation, flow velocities, and sediment movement indicate that burrowing and feeding by S. reticulatum, and to a lesser extent, Uca pugnax, increase surface roughness and decrease the threshold velocities and shear stresses required for sediment erosion. Erosion associated with observed burrowing is of a similar magnitude to mean annual marsh surface accumulation, and, consequently, this mechanism for sediment loss in heavily burrowed areas has the potential to produce large-scale morphological changes. Taxonomy Ecological engineering, Coastal setting, Salt marsh, Geomorphological aspects, Soil erosion. Regional index terms USA, Texas, Galveston.

FitzGerald, D.; Ryerson, O.; Hughes, Z.J.; Black, S.; Georgiou, I.; Hein, C., and Novak, A., 2020. Long-term variability in inorganic sediment contribution to the Great Marsh, Massachusetts. In: Malvarez, G. and Navas, F. (eds.), Global Coastal Issues of 2020. Journal of Coastal Research, Special Issue No. 95, pp. 490–494. Coconut Creek (Florida), ISSN 0749-0208.In New England, the marsh platform sits above mean high water and is flooded about 8 times a month. This high marsh, dominated by Spartina patens, has a vertical accretion rate of ∼2.5 mm/yr, significantly lower than the 6-7 mm/yr accretion rate of low marsh, dominated by Spartina alterniflora. Accelerating sea-level rise will eventually cause the high marsh to transition to low marsh; the rate of this change is controlled, in part, by the contribution of inorganic sediment. Temporal and spatial variability of inorganic marsh sedimentation during the past ∼ 2.5 ka was examined at the Great Marsh, Massachusetts, using twenty cores, each ∼180-cm long, along five transects. Transects were aligned perpendicular to bays and major channels at different compass quadrants to assess influences of wind and tidal flow during storms. Cores were sampled every 20 cm to determine percent organic matter (loss on ignition) and grain size distribution (using a laser particle size analyzer). Data show a weak correlation for grain size versus distance from the nearest channel or bay. Additionally, no consistent vertical trends in grain size were observed, either within individual sampling sites nor among coring transects. Instead, we find coarsening upwards trends at some sites, likely due to increased channelization and tidal velocity as the marsh matured. Elsewhere, sediments fine upwards in response to deepening bays and channels. Finally, transects exhibiting variable grain size trends are likely influenced by ice rafted sediment. These results demonstrate the complexity of sedimentation on the marsh platform and the challenge in accurately predicting patterns of sediment transport using a single suspended grain size in hydrodynamic models in these systems.

Mitochondrial uncoupling was one of the earliest pharmacologic approaches to the treatment of obesity-related diseases. However, low mitochondrial specificity and negative off-target effects limited the viability of such agents. BAM15 is a small molecule recently identified for its potent ability to uncouple mitochondria. Thus, the purpose of this investigation was to explore the physiological effects of BAM15 on skeletal muscle mitochondrial function and glucose metabolism. C2C12 myotubes were incubated with either vehicle or BAM15 at varying concentrations for 16 hours. Cell viability was assessed by detection of dehydrogenase activity. Cytotoxicity was measured by a Caspase 3/7 activity assay. Glucose uptake was measured using [3H]-2-deoxy-D-glucose in the presence and absence of insulin. Oxygen consumption rates were measured using an XF24 extracellular flux analyzer. Cells were harvested for the assessment of plasma membrane GLUT4 and protein signaling via Western blot. Cells treated with BAM15 were viable up to 100 μM, and lacked toxicity up to 50 μM, compared to vehicle. BAM15 significantly increased glucose uptake (P Disclosure M.E. Hull: None. C.L. Axelrod: None. K.M. Fitzgerald: None. J. Sacks: None. J.P. Kirwan: None.

Recent work has revealed that large numbers of promoters in bacteria are located inside genes. In contrast, almost all studies of transcription have focused on promoters upstream of genes. Bacterial promoters are recognized by Sigma factors that associate with initiating RNA polymerase. In Escherichia coli, one Sigma factor recognizes the majority of promoters, and six “alternative” Sigma factors recognize specific subsets of promoters. One of these alternative Sigma factors, FliA (σ28), recognizes promoters upstream of many flagellar genes. We previously showed that most E. coli FliA binding sites are located inside genes. However, it was unclear whether these intragenic binding sites represent active promoters. Here, we construct and assay transcriptional promoter-lacZ fusions for all 52 putative FliA promoters previously identified by ChIP-seq. These experiments, coupled with integrative analysis of published genome-scale transcriptional datasets, reveal that most intragenic FliA binding sites are active promoters that transcribe highly unstable RNAs. Additionally, we show that widespread intragenic FliA-dependent transcription is a conserved phenomenon, but that the specific promoters are not themselves conserved. We conclude that intragenic FliA-dependent promoters and the resulting RNAs are unlikely to have important regulatory functions. Nonetheless, one intragenic FliA promoter is broadly conserved, and constrains evolution of the overlapping protein-coding gene. Thus, our data indicate that intragenic regulatory elements can influence protein evolution in bacteria, and suggest that the impact of intragenic regulatory sequences on genome evolution should be considered more broadly.AUTHOR SUMMARYRecent genome-scale studies of bacterial transcription have revealed thousands of promoters inside genes. In a few cases, these promoters have been shown to transcribe functional RNAs. However, it is unclear whether most intragenic promoters have important biological function. Similarly, there are likely to be thousands of intragenic binding sites for transcription factors, but very few have been functionally characterized. Moreover, it is unclear what impact intragenic promoters and transcription factor binding sites have on evolution of the overlapping genes. In this study, we focus on FliA, a broadly conserved Sigma factor that is responsible for initiating transcription of many flagellar genes. We previously showed that FliA directs RNA polymerase to ~50 genomic sites in Escherichia coli. In our current study, we show that while most intragenic FliA promoters are actively transcribed, very few are conserved in other species. This suggests that most FliA promoters are not functional. Nonetheless, one intragenic FliA promoter is highly conserved, and we show that this promoter constrains evolution of the overlapping protein-coding gene. Given the enormous number of regulatory DNA sites within genes, we propose that the evolution of many genes is constrained by these elements.

Genome-wide association studies (GWAS) and fine-mapping efforts to date have identified more than 100 prostate cancer (PrCa)-susceptibility loci. We meta-analyzed genotype data from a custom high-density array of 46,939 PrCa cases and 27,910 controls of European ancestry with previously genotyped data of 32,255 PrCa cases and 33,202 controls of European ancestry. Our analysis identified 62 novel loci associated (P < 5.0 × 10-8) with PrCa and one locus significantly associated with early-onset PrCa (≤55 years). Our findings include missense variants rs1800057 (odds ratio (OR) = 1.16; P = 8.2 × 10-9; G>C, p.Pro1054Arg) in ATM and rs2066827 (OR = 1.06; P = 2.3 × 10-9; T>G, p.Val109Gly) in CDKN1B. The combination of all loci captured 28.4% of the PrCa familial relative risk, and a polygenic risk score conferred an elevated PrCa risk for men in the ninetieth to ninety-ninth percentiles (relative risk = 2.69; 95% confidence interval (CI): 2.55-2.82) and first percentile (relative risk = 5.71; 95% CI: 5.04-6.48) risk stratum compared with the population average. These findings improve risk prediction, enhance fine-mapping, and provide insight into the underlying biology of PrCa 1 .

Vertebrate consumers can be important drivers of the structure and functioning of ecosystems, including the soil and litter invertebrate communities that drive many ecosystem processes. Burrowing seabirds, as prevalent vertebrate consumers, have the potential to impact consumptive effects via adding marine nutrients to soil (i.e. resource subsidies) and non-consumptive effects via soil disturbance associated with excavating burrows (i.e. ecosystem engineering). However, the exact mechanisms by which they influence invertebrates are poorly understood. We examined how soil chemistry and plant and invertebrate communities changed across a gradient of seabird burrow density on two islands in northern New Zealand. Increasing seabird burrow density was associated with increased soil nutrient availability and changes in plant community structure and the abundance of nearly all the measured invertebrate groups. Increasing seabird densities had a negative effect on invertebrates that were strongly influenced by soil-surface litter, a positive effect on fungal-feeding invertebrates, and variable effects on invertebrate groups with diverse feeding strategies. Gastropoda and Araneae species richness and composition were also influenced by seabird activity. Generalized multilevel path analysis revealed that invertebrate responses were strongly driven by seabird engineering effects, via increased soil disturbance, reduced soil-surface litter, and changes in trophic interactions. Almost no significant effects of resource subsidies were detected. Our results show that seabirds, and in particular their non-consumptive effects, were significant drivers of invertebrate food web structure. Reductions in seabird populations, due to predation and human activity, may therefore have far-reaching consequences for the functioning of these ecosystems.

Voltage-gated calcium channels (Cav) and their associated proteins are pivotal signalling complexes in excitable cell physiology. In nerves and muscle, Cav tailor calcium influx to processes including neurotransmission, muscle contraction and gene expression. Cav comprise a pore-forming α1 and modulatory β and α2δ subunits - the latter targeted by anti-epileptic and anti-nociceptive gabapentinoid drugs. However, the mechanisms of gabapentinoid action are unclear, not least because detailed structure-function mapping of the α2δ subunit remains lacking. Using molecular biology and electrophysiological approaches we have conducted the first systematic mapping of α2δ subunit structure-function. We generated a series of cDNA constructs encoding chimera, from which successive amino acids from the rat α2δ-1 subunit were incorporated into a Type 1 reporter protein - PIN-G, to produce sequential extensions from the transmembrane (TM) region towards the N-terminus. By successive insertion of a TGA stop codon, a further series of N- to C-terminal extension constructs lacking the TM region, were also generated. Using this approach we have defined the minimal region of α2δ-1 - we term the R-domain (Rd), that appears to contain all the machinery necessary to support the electrophysiological and trafficking effects of α2δ-1 on Cav. Structural algorithms predict that Rd is conserved across all four α2δ subunits, including RNA splice variants, and irrespective of phyla and taxa. We suggest, therefore, that Rd likely constitutes the major locus for physical interaction with the α1 subunit and may provide a target for novel Cav therapeutics.

Because of advances in targeted therapies, the clinical evaluation of cutaneous melanoma is increasingly based on a combination of traditional histopathology and molecular pathology. Therefore, it is necessary to expand our knowledge of the molecular events that accompany the development and progression of melanoma to optimize clinical management. The central objective of this study was to increase our knowledge of the mutational events that complement melanoma progression. High-throughput genotyping was adapted to query 159 known single nucleotide mutations in 33 cancer-related genes across two melanoma cohorts from Ireland (n = 94) and Belgium (n = 60). Results were correlated with various clinicopathological characteristics. A total of 23 mutations in 12 genes were identified, that is - BRAF, NRAS, MET, PHLPP2, PIK3R1, IDH1, KIT, STK11, CTNNB1, JAK2, ALK, and GNAS. Unexpectedly, we discovered significant differences in BRAF, MET, and PIK3R1 mutations between the cohorts. That is, cases from Ireland showed significantly lower (P

Genetic studies have identified single nucleotide polymorphisms (SNPs) associated with the risk of prostate cancer (PC). It remains unclear whether such genetic variants are associated with disease aggressiveness. The NCI-SPORE Genetics Working Group retrospectively collected clinicopathologic information and genotype data for 36 SNPs which at the time had been validated to be associated with PC risk from 25,674 cases with PC. Cases were grouped according to race, Gleason score (Gleason ≤6, 7, ≥8) and aggressiveness (non-aggressive, intermediate, and aggressive disease). Statistical analyses were used to compare the frequency of the SNPs between different disease cohorts. After adjusting for multiple testing, only PC-risk SNP rs2735839 (G) was significantly and inversely associated with aggressive (OR=0.77; 95% CI 0.69–0.87) and high-grade disease (OR=0.77; 95% CI 0.68–0.86) in European men. Similar associations with aggressive (OR=0.72; 95% CI 0.58–0.89) and high-grade disease (OR=0.69; 95% CI 0.54–0.87) were documented in African-American subjects. The G allele of rs2735839 was associated with disease aggressiveness even at low PSA levels (

Spiders in the endemic New Zealand genus Pahoroides (Araneae: Synotaxidae) are all morphologically very similar. Two species, Pahoroides whangarei and Pahoroides confusa, coexist in Pukenui Forest near Whangarei, Northland, living in vegetation close to the ground. We tested the hypothesis that P. whangarei and P. confusa occupy distinct ecological niches defined more by conditions at the microhabitat level than at the macrohabitat level. Twenty random sites within Pukenui Forest, from which a range of macrohabitat and microhabitat variables were collected, were sampled by beating vegetation for Pahoroides spiders. Both species were distributed throughout the forest, and in many cases, were found at the same sites. However, neither the presence nor abundance of one Pahoroides species was associated with that of the other. Whereas macrohabitat variables such as aspect, litter cover and overall plant community composition were not associated with either species, a significant association was found between P...

The HOXB13 G84E variant is associated with risk of prostate cancer (PCa), however the role this variant plays in PCa development is unknown. This study examined 751 cases, 450 relatives and 355 controls to determine the contribution of this variant to PCa risk in Tasmania and investigated HOXB13 gene and protein expression in tumours from nine G84E heterozygote variant and 13 wild-type carriers. Quantitative PCR and immunohistochemistry showed that HOXB13 gene and protein expression did not differ between tumour samples from variant and wild-type carriers. Allele-specific transcription revealed that two of seven G84E carriers transcribed both the variant and wild-type allele, while five carriers transcribed the wild-type allele. Methylation of surrounding CpG sites was lower in the variant compared to the wild-type allele, however overall methylation across the region was very low. Notably, tumour characteristics were less aggressive in the two variant carriers that transcribed the variant allele compared to the five that did not. This study has shown that HOXB13 expression does not differ between tumour tissue of G84E variant carriers and non-carriers. Intriguingly, the G84E variant allele was rarely transcribed in carriers, suggesting that HOXB13 expression may be driven by the wild-type allele in the majority of carriers.

Background Between 20% and 35% of prostate cancer (PCa) patients who undergo treatment with curative intent (ie, surgery or radiation therapy) for localized disease will experience biochemical recurrence (BCR). Alterations in the insulin-like growth factor (IGF) axis and PTEN expression have been implicated in the development and progression of several human tumors including PCa. We examined the expression of the insulin receptor (INSR), IGF-1 receptor (IGF-1R), PTEN, and AKT in radical prostatectomy tissue of patients who developed BCR post-surgery. Methods Tissue microarrays (TMA) of 130 patients post-radical prostatectomy (65 = BCR, 65 = non-BCR) were stained by immunohistochemistry for INSR, IGF-1R, PTEN, and AKT using optimized antibody protocols. INSR, IGF1-R, PTEN, and AKT expression between benign and cancerous tissue, and different Gleason grades was assessed. Kaplan-Meier survival curves were used to examine the relationship between proteins expression and BCR. Results INSR (P

Tumor-growth-factor-beta signaling helps cancer cells to evolve and become resistant to drugs by down-regulating accurate DNA repair.