Groundwater in the Inner Bluegrass Karst Region, Kentucky

The hydrogeology of about 12% of the 5600 km2 Inner Bluegrass Karst Region of central Kentucky was investigated by water tracing and other techniques. Using fluorescent dyes adsorbed on fabric and charcoal detectors, 96 traces (average length 2.7 km, maximum length 15 km) resulted in the identification of 38 groundwater basins (with areas up to 15 km2). Within the basins, subsurface flow is in a dendritic conduit system at depths up to 30 m below the surface, while in the interbasin areas which separate them flow is generally less than 5 m deep. Each groundwater basin discharges at a spring whose median discharge is approximately 20 l/s·km2 of basin area. The largest spring (Royal Spring) in the study area has a median discharge greater than 300 l/s (Meinzer second magnitude). The Ordovician Lexington Limestone which underlies the region is thin bedded with shale partings and argillaceous units. Within groundwater basins, sinkhole drains and other conduits have breached the interbedded shales and descend nearly vertically to a level determined by equilibrium flow in the larger conduits. The general location and flow directions in groundwater basins is probably determined by a potentiometric gradient prior to conduit development, and some basins are localized by a favorably oriented regional joint set or other structural element. Otherwise, lithologic and structural factors have little influence in the occurrence and flow of subsurface water in the region

Transport of black carbon to polar regions: Sensitivity and forcing by black carbon

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95651/1/grl29697.pd

The role of interfacial lipids in stabilizing membrane protein oligomers

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways1 but is often difficult to define2 or predict3. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT4, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors

Multi-Modality Therapeutics with Potent Anti-Tumor Effects: Photochemical Internalization Enhances Delivery of the Fusion Toxin scFvMEL/rGel

BACKGROUND: There is a need for drug delivery systems (DDS) that can enhance cytosolic delivery of anti-cancer drugs trapped in the endo-lysosomal compartments. Exposure of cells to specific photosensitizers followed by light exposure (photochemical internalization, PCI) results in transfer of agents from the endocytic compartment into the cytosol. METHODOLOGY AND PRINCIPAL FINDINGS: The recombinant single-chain fusion construct scFvMEL/rGel is composed of an antibody targeting the progenitor marker HMW-MAA/NG2/MGP/gp240 and the highly effective toxin gelonin (rGel). Here we demonstrate enhanced tumor cell selectivity, cytosolic delivery and anti-tumor activity by applying PCI of scFvMEL/rGel. PCI performed by light activation of cells co-incubated with scFvMEL/rGel and the endo-lysosomal targeting photosensitizers AlPcS(2a) or TPPS(2a) resulted in enhanced cytotoxic effects against antigen-positive cell lines, while no differences in cytotoxicity between the scFvMEL/rGel and rGel were observed in antigen-negative cells. Mice bearing well-developed melanoma (A-375) xenografts (50-100 mm(3)) were treated with PCI of scFvMEL/rGel. By 30 days after injection, approximately 100% of mice in the control groups had tumors>800 mm(3). In contrast, by day 40, 50% of mice in the PCI of scFvMEL/rGel combination group had tumors<800 mm(3) with no increase in tumor size up to 110 days. PCI of scFvMEL/rGel resulted in a synergistic effect (p<0.05) and complete regression (CR) in 33% of tumor-bearing mice (n = 12). CONCLUSIONS/SIGNIFICANCE: This is a unique demonstration that a non-invasive multi-modality approach combining a recombinant, targeted therapeutic such as scFvMEL/rGel and PCI act in concert to provide potent in vivo efficacy without sacrificing selectivity or enhancing toxicity. The present DDS warrants further evaluation of its clinical potential

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it might also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched towards the C-termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur prior to assembly. Using high-throughput imaging of protein homomers in vivo in E. coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Association of genomic domains in BRCA1 and BRCA2 with prostate cancer risk and aggressiveness

Pathogenic sequence variants (PSV) in BRCA1 or BRCA2 (BRCA1/2) are associated with increased risk and severity of prostate cancer. Weevaluated whether PSVs inBRCA1/2 were associated with risk of overall prostate cancer or high grade (Gleason 8+) prostate cancer using an international sample of 65 BRCA1 and 171 BRCA2 male PSV carriers with prostate cancer, and 3,388 BRCA1 and 2,880 BRCA2 male PSV carriers without prostate cancer. PSVs in the 30 region of BRCA2 (c.7914+) were significantly associated with elevated risk of prostate cancer compared with reference bin c.1001c.7913 [HR = 1.78; 95% confidence interval (CI), 1.25-2.52; P = 0.001], as well as elevated risk of Gleason 8+ prostate cancer (HR = 3.11; 95% CI, 1.63-5.95; P = 0.001). c.756-c.1000 was also associated with elevated prostate cancer risk (HR = 2.83; 95% CI, 1.71-4.68; P = 0.00004) and elevated risk of Gleason 8+prostate cancer (HR = 4.95; 95% CI, 2.12-11.54; P = 0.0002). No genotype-phenotype associations were detected for PSVs in BRCA1. These results demonstrate that specific BRCA2 PSVs may be associated with elevated risk of developing aggressive prostate cancer. Significance: Aggressive prostate cancer risk in BRCA2 mutation carriers may vary according to the specific BRCA2 mutation inherited by the at-risk individual.Peer reviewe

Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element

A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30-to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74-0.81, p = 3.1 x 10(-31)).Peer reviewe