Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines

Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ～0.5 and led to siRNA/polymer complexes of ～100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia.     

Brain Networks Supporting Execution of Mathematical Skills versus Acquisition of New Mathematical Competence

This fMRI study examines how students extend their mathematical competence. Students solved a set of algebra-like problems. These problems included Regular Problems that have a known solution technique and Exception Problems that but did not have a known technique. Two distinct networks of activity were uncovered. There was a Cognitive Network that was mainly active during the solution of problems and showed little difference between Regular Problems and Exception Problems. There was also a Metacognitive Network that was more engaged during a reflection period after the solution and was much more engaged for Exception Problems than Regular Problems. The Cognitive Network overlaps with prefrontal and parietal regions identified in the ACT-R theory of algebra problem solving and regions identified in the triple-code theory as involved in basic mathematical cognition. The Metacognitive Network included angular gyrus, middle temporal gyrus, and anterior prefrontal regions. This network is mainly engaged by the need to modify the solution procedure and not by the difficulty of the problem. Only the Metacognitive Network decreased with practice on the Exception Problems. Activity in the Cognitive Network during the solution of an Exception Problem predicted both success on that problem and future mastery. Activity in the angular gyrus and middle temporal gyrus during feedback on errors predicted future mastery.     

Aspen Increase Soil Moisture,Nutrients, Organic Matter and Respiration in Rocky Mountain Forest Communities

Development and change in forest communities are strongly influenced by plant-soil interactions. The primary objective of this paper was to identify how forest soil characteristics vary along gradients of forest community composition in aspen-conifer forests to better understand the relationship between forest vegetation characteristics and soil processes. The study was conducted on the Fishlake National Forest, Utah, USA. Soil measurements were collected in adjacent forest stands that were characterized as aspen dominated, mixed, conifer dominated or open meadow, which includes the range of vegetation conditions that exist in seral aspen forests. Soil chemistry, moisture content, respiration, and temperature were measured. There was a consistent trend in which aspen stands demonstrated higher mean soil nutrient concentrations than mixed and conifer dominated stands and meadows. Specifically, total N, NO₃ and NH₄ were nearly two-fold higher in soil underneath aspen dominated stands. Soil moisture was significantly higher in aspen stands and meadows in early summer but converged to similar levels as those found in mixed and conifer dominated stands in late summer. Soil respiration was significantly higher in aspen stands than conifer stands or meadows throughout the summer. These results suggest that changes in disturbance regimes or climate scenarios that favor conifer expansion or loss of aspen will decrease soil resource availability, which is likely to have important feedbacks on plant community development.     

Insights from Characterizing Extinct Human Gut Microbiomes

In an effort to better understand the ancestral state of the human distal gut microbiome, we examine feces retrieved from archaeological contexts (coprolites). To accomplish this, we pyrosequenced the 16S rDNA V3 region from duplicate coprolite samples recovered from three archaeological sites, each representing a different depositional environment: Hinds Cave (∼8000 years B.P.) in the southern United States, Caserones (1600 years B.P.) in northern Chile, and Rio Zape in northern Mexico (1400 years B.P.). Clustering algorithms grouped samples from the same site. Phyletic representation was more similar within sites than between them. A Bayesian approach to source-tracking was used to compare the coprolite data to published data from known sources that include, soil, compost, human gut from rural African children, human gut, oral and skin from US cosmopolitan adults and non-human primate gut. The data from the Hinds Cave samples largely represented unknown sources. The Caserones samples, retrieved directly from natural mummies, matched compost in high proportion. A substantial and robust proportion of Rio Zape data was predicted to match the gut microbiome found in traditional rural communities, with more minor matches to other sources. One of the Rio Zape samples had taxonomic representation consistent with a child. To provide an idealized scenario for sample preservation, we also applied source tracking to previously published data for Ötzi the Iceman and a soldier frozen for 93 years on a glacier. Overall these studies reveal that human microbiome data has been preserved in some coprolites, and these preserved human microbiomes match more closely to those from the rural communities than to those from cosmopolitan communities. These results suggest that the modern cosmopolitan lifestyle resulted in a dramatic change to the human gut microbiome.     

Performance of LBSap Vaccine after Intradermal Challenge with L. infantum and Saliva of Lu. longipalpis: Immunogenicity and Parasitological Evaluation

In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21⁺) and both CD4⁺ and CD8⁺ T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4⁺ T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-β1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.     

Host Modulators of H1N1 Cytopathogenicity

Influenza A virus infects 5-20% of the population annually, resulting in ～35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here we employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human bronchial epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to an expanding body of studies directed at decoding vulnerabilities in the command and control networks specified by influenza virulence factors.     

Severe Childhood Malaria Syndromes Defined by Plasma Proteome Profiles

Background

Cerebral malaria (CM) and severe malarial anemia (SMA) are the most serious life-threatening clinical syndromes of Plasmodium falciparum infection in childhood. Therefore it is important to understand the pathology underlying the development of CM and SMA, as opposed to uncomplicated malaria (UM). Different host responses to infection are likely to be reflected in plasma proteome-patterns that associate with clinical status and therefore provide indicators of the pathogenesis of these syndromes.

Methods and Findings

Plasma and comprehensive clinical data for discovery and validation cohorts were obtained as part of a prospective case-control study of severe childhood malaria at the main tertiary hospital of the city of Ibadan, an urban and densely populated holoendemic malaria area in Nigeria. A total of 946 children participated in this study. Plasma was subjected to high-throughput proteomic profiling. Statistical pattern-recognition methods were used to find proteome-patterns that defined disease groups. Plasma proteome-patterns accurately distinguished children with CM and with SMA from those with UM, and from healthy or severely ill malaria-negative children.

Conclusions

We report that an accurate definition of the major childhood malaria syndromes can be achieved using plasma proteome-patterns. Our proteomic data can be exploited to understand the pathogenesis of the different childhood severe malaria syndromes.     

Modeling Future Conservation of Hawaiian Honeycreepers by Mosquito Management and Translocation of Disease-Tolerant Amakihi

Avian malaria is an important cause of the decline of endemic Hawaiian honeycreepers. Because of the complexity of this disease system we used a computer model of avian malaria in forest birds to evaluate how two proposed conservation strategies: 1) reduction of habitat for mosquito larvae and 2) establishment of a low-elevation, malaria-tolerant honeycreeper (Hawaii Amakihi) to mid-elevation forests would affect native Hawaiian honeycreeper populations. We evaluated these approaches in mid-elevation forests, where malaria transmission is seasonal and control strategies are more likely to work. Our model suggests the potential benefit of larval habitat reduction depends on the level of malaria transmission, abundance of larval cavities, and the ability to substantially reduce these cavities. Permanent reduction in larval habitat of >80% may be needed to control abundance of infectious mosquitoes and benefit bird populations. Establishment of malaria-tolerant Amakihi in mid-elevation forests increases Amakihi abundance, creates a larger disease reservoir, and increases the abundance of infectious mosquitoes which may negatively impact other honeycreepers. For mid-elevation sites where bird populations are severely affected by avian malaria, malaria-tolerant Amakihi had little impact on other honeycreepers. Both management strategies may benefit native Hawaiian honeycreepers, but benefits depend on specific forest characteristics, the amount of reduction in larval habitat that can be achieved, and how malaria transmission is affected by temperature.