The desire for variety: Italian wall lizard (Podarcis siculus) populations introduced to the United States via the pet trade are derived from multiple native-range sources

Tests of invasion success often require comparisons between introduced and native populations, but determining the native-range sources for introduced populations can be difficult. Molecular markers can help clarify the geographic extent of native-range sources, helping to identify which populations are appropriate for comparative studies. The Italian Wall Lizard (Podarcis siculus) was introduced multiple times to the United States with extant populations in California, Kansas, New Jersey, and New York. We used phylogeographic analysis of mtDNA sequences (cytb gene) for individuals sampled from these introduced populations and across the native range to identify the number of independent introductions and the location of the source populations. Haplotypes sampled from introduced populations were nested within three geographically distinct, well-supported clades that together encompassed a large portion of the native range. Combining these phylogeographic results with documentation of the introductions revealed putative sources: California individuals are derived from Sicily; Kansas and New York populations are from Tuscany near Florence; and the New Jersey population is likely from the Adriatic coastal region, but a more specific locality is not possible. The pet trade dominates the invasion pathway for P. siculus introductions to the US. The genetically and geographically diverse sampling of its native range may be driven by the desire for phenotypic variety in the pet trade, a hypothesis that needs future testing.     

Temporal dynamics of plant community regeneration sources during tallgrass prairie restoration

Ecological restoration aims to augment and steer the composition and contribution of propagules for community regeneration in degraded environments. We quantified patterns in the abundance, richness, and diversity of seed and bud banks across an 11-year chronosequence of restored prairies and in prairie remnants to elucidate the degree to which the germinable seed bank, emerged seedlings, belowground buds, and emerged ramets were related to community regeneration. There were no directional patterns in the abundance, richness, or diversity of the germinable seed bank across the chronosequence. Emerged seedling abundance of sown species decreased during restoration. Richness and diversity of all emerged seedlings and non-sown emerged seedling species decreased across the chronosequence. Conversely, abundance and richness of belowground buds increased with restoration age and belowground bud diversity of sown species increased across the chronosequence. Numbers of emerged ramets also increased across the chronosequence and was driven primarily by the number of graminoid ramets. There were no temporal changes in abundance and richness of sown and non-sown emerged ramets, but diversity of sown emerged ramets increased across the chronosequence. This study demonstrates that after initial seeding, plant community structure in restored prairies increasingly reflects the composition of the bud bank.     

Characterization of the Late Endosomal ESCRT Machinery in Trypanosoma brucei

The multivesicular body (MVB) is a specialized Rab7+ late endosome (LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well‐defined MVB, but contain orthologs of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in approximately threefold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin‐dependent lysosomal degradation of invariant surface glycoproteins (ISG65) (Leung et al., Traffic 2008;9:1698–1716). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and novel role(s) in LE function in trypanosomes .     

Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post‐symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early‐warning sentinels potentially have tremendous utility as wide‐area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis‐acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time‐course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.     

Comprehensive genotyping of the USA national maize inbred seed bank

Background

Genotyping by sequencing, a new low-cost, high-throughput sequencing technology was used to genotype 2,815 maize inbred accessions, preserved mostly at the National Plant Germplasm System in the USA. The collection includes inbred lines from breeding programs all over the world.

Results

The method produced 681,257 single-nucleotide polymorphism (SNP) markers distributed across the entire genome, with the ability to detect rare alleles at high confidence levels. More than half of the SNPs in the collection are rare. Although most rare alleles have been incorporated into public temperate breeding programs, only a modest amount of the available diversity is present in the commercial germplasm. Analysis of genetic distances shows population stratification, including a small number of large clusters centered on key lines. Nevertheless, an average fixation index of 0.06 indicates moderate differentiation between the three major maize subpopulations. Linkage disequilibrium (LD) decays very rapidly, but the extent of LD is highly dependent on the particular group of germplasm and region of the genome. The utility of these data for performing genome-wide association studies was tested with two simply inherited traits and one complex trait. We identified trait associations at SNPs very close to known candidate genes for kernel color, sweet corn, and flowering time; however, results suggest that more SNPs are needed to better explore the genetic architecture of complex traits.

Conclusions

The genotypic information described here allows this publicly available panel to be exploited by researchers facing the challenges of sustainable agriculture through better knowledge of the nature of genetic diversity.     

Caloric restriction augments radiation efficacy in breast cancer

Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.     

Involvement of miRNAs in the Differentiation of Human Glioblastoma Multiforme Stem-Like Cells

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.     

Programmatic Cost Evaluation of Nontargeted Opt-Out Rapid HIV Screening in the Emergency Department

Background

The Centers for Disease Control and Prevention recommends nontargeted opt-out HIV screening in healthcare settings. Cost effectiveness is critical when considering potential screening methods. Our goal was to compare programmatic costs of nontargeted opt-out rapid HIV screening with physician-directed diagnostic rapid HIV testing in an urban emergency department (ED) as part of the Denver ED HIV Opt-Out Trial.

Methods

This was a prospective cohort study nested in a larger quasi-experiment. Over 16 months, nontargeted rapid HIV screening (intervention) and diagnostic rapid HIV testing (control) were alternated in 4-month time blocks. During the intervention phase, patients were offered HIV testing using an opt-out approach during registration; during the control phase, physicians used a diagnostic approach to offer HIV testing to patients. Each method was fully integrated into ED operations. Direct program costs were determined using the perspective of the ED. Time-motion methodology was used to estimate personnel activity costs. Costs per patient newly-diagnosed with HIV infection by intervention phase, and incremental cost effectiveness ratios were calculated.

Results

During the intervention phase, 28,043 eligible patients were included, 6,933 (25%) completed testing, and 15 (0.2%, 95% CI: 0.1%–0.4%) were newly-diagnosed with HIV infection. During the control phase, 29,925 eligible patients were included, 243 (0.8%) completed testing, and 4 (1.7%, 95% CI: 0.4%–4.2%) were newly-diagnosed with HIV infection. Total annualized costs for nontargeted screening were $148,997, whereas total annualized costs for diagnostic HIV testing were $31,355. The average costs per HIV diagnosis were $9,932 and $7,839, respectively. Nontargeted HIV screening identified 11 more HIV infections at an incremental cost of $10,693 per additional infection.

Conclusions

Compared to diagnostic testing, nontargeted HIV screening was more costly but identified more HIV infections. More effective and less costly testing strategies may be required to improve the identification of patients with undiagnosed HIV infection in the ED.     

Mechanistic Phenotypes: An Aggregative Phenotyping Strategy to Identify Disease Mechanisms Using GWAS Data

A single mutation can alter cellular and global homeostatic mechanisms and give rise to multiple clinical diseases. We hypothesized that these disease mechanisms could be identified using low minor allele frequency (MAF<0.1) non-synonymous SNPs (nsSNPs) associated with “mechanistic phenotypes”, comprised of collections of related diagnoses. We studied two mechanistic phenotypes: (1) thrombosis, evaluated in a population of 1,655 African Americans; and (2) four groupings of cancer diagnoses, evaluated in 3,009 white European Americans. We tested associations between nsSNPs represented on GWAS platforms and mechanistic phenotypes ascertained from electronic medical records (EMRs), and sought enrichment in functional ontologies across the top-ranked associations. We used a two-step analytic approach whereby nsSNPs were first sorted by the strength of their association with a phenotype. We tested associations using two reverse genetic models and standard additive and recessive models. In the second step, we employed a hypothesis-free ontological enrichment analysis using the sorted nsSNPs to identify functional mechanisms underlying the diagnoses comprising the mechanistic phenotypes. The thrombosis phenotype was solely associated with ontologies related to blood coagulation (Fisher''s p = 0.0001, FDR p = 0.03), driven by the F5, P2RY12 and F2RL2 genes. For the cancer phenotypes, the reverse genetics models were enriched in DNA repair functions (p = 2×10−5, FDR p = 0.03) (POLG/FANCI, SLX4/FANCP, XRCC1, BRCA1, FANCA, CHD1L) while the additive model showed enrichment related to chromatid segregation (p = 4×10−6, FDR p = 0.005) (KIF25, PINX1). We were able to replicate nsSNP associations for POLG/FANCI, BRCA1, FANCA and CHD1L in independent data sets. Mechanism-oriented phenotyping using collections of EMR-derived diagnoses can elucidate fundamental disease mechanisms.