Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stem–loops; HIV-1 TAR and adenovirus VA_I-AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA–protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.     

A simulation model of the Devils Hole pupfish population using monthly length-frequency distributions

The Devils Hole pupfish, Cyprinodon diabolis, is a federally-endangered fish that is endemic to Devils Hole, a discontiguous part of Death Valley National Park in Nye County, Nevada. Due to its status, Devils Hole pupfish monitoring must be non-obtrusive and thereby exclude techniques that require handling fish. Due to a recent decline in pupfish abundance, Devils Hole pupfish managers have expressed a need for a model that describes population dynamics. This population model would be used to identify vulnerable life history stage(s) and inform management actions. We constructed a set of individual-based simulation models designed to explore effects of population processes and evaluate assumptions. We developed a baseline model, whose output best resembled both observed length-frequency data and predicted intra-annual abundance patterns. We then ran simulations with 5 % increases in egg-larval, juvenile, and adult survival rates to better understand Devils Hole pupfish life history, thereby helping identify vulnerable life history stages that should become the target of management actions. Simulation models with temporally constant adult, juvenile, and egg-larval survival rates were able to reproduce observed length-frequency distributions and predicted intra-annual population patterns. In particular, models with monthly adult and juvenile survival rates of 80 % and an egg-larval survival rate of 4.7 % replicated patterns in observed data. Population growth was most affected by 5 % increases in egg-larval survival, whereas adult and juvenile survival rates had similar but lesser effects on population growth. Outputs from the model were used to assess factors suspected of influencing Devils Hole pupfish population decline.     

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.     

Influence of Plasma Processing on Recovery and Analysis of Circulating Nucleic Acids

Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp^® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.     

A Novel Source of Cultured Podocytes

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.     

Representation of Ecosystem Services by Terrestrial Protected Areas: Chile as a Case Study

Protected areas are increasingly considered to play a key role in the global maintenance of ecosystem processes and the ecosystem services they provide. It is thus vital to assess the extent to which existing protected area systems represent those services. Here, for the first time, we document the effectiveness of the current Chilean protected area system and its planned extensions in representing both ecosystem services (plant productivity, carbon storage and agricultural production) and biodiversity. Additionally, we evaluate the effectiveness of protected areas based on their respective management objectives. Our results show that existing protected areas in Chile do not contain an unusually high proportion of carbon storage (14.9%), agricultural production (0.2%) or biodiversity (11.8%), and also represent a low level of plant productivity (Normalized Difference Vegetation Index of 0.38). Proposed additional priority sites enhance the representation of ecosystem services and biodiversity, but not sufficiently to attain levels of representation higher than would be expected for their area of coverage. Moreover, when the species groups were assessed separately, amphibians was the only one well represented. Suggested priority sites for biodiversity conservation, without formal protection yet, was the only protected area category that over-represents carbon storage, agricultural production and biodiversity. The low representation of ecosystem services and species’ distribution ranges by the current protected area system is because these protected areas are heavily biased toward southern Chile, and contain large extents of ice and bare rock. The designation and management of proposed priority sites needs to be addressed in order to increase the representation of ecosystem services within the Chilean protected area system.