Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (K(i) values of 710 nM-169 μM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC(50) ～23.1 μM).     

European society of intensive care medicine study of therapeutic hypothermia (32-35°C) for intracranial pressure reduction after traumatic brain injury (the Eurotherm3235Trial)

Background

Severe malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.

Methods/Design

This prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo (both arms receiving standard anti-malarial therapy), among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.

Discussion

Noteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.

Trial Registration

ClinicalTrials.gov Identifier: NCT01255215     

Cytokine profile of a human bone marrow cell culture under the influence of UHMW-PE wear particles]

There is considerable evidence that orthopaedic wear debris plays a crucial role in the pathology of aseptic loosening of joint prostheses. The purpose of the present study was to evaluate the influence of ultra-high-molecular-weight polyethylene (UHMW-PE) on the cytokine response in a modified in vitro model. UHMW-PE particles (psi < 7.5 microm) were suspended in soluble collagen type I and subsequently solidified in different concentrations (105,106 and 107 particles per well) on the bottom of the wells. Human bone marrow cells in a concentration of 3 x 106 cells per well were seeded on the collagen-particle substrata and maintained for up to 12 days. The cytokine response (IL-1_, IL-6 and TNF-_) of the cells to the particles were examined by ELISA compared to cells on control collagen surfaces without any particles. Assays for viability using LDH activity were done immediately. Light and scanning microscopic evaluation revealed that the UHMWPE particles, which have built large conglomerates (psi7.5_m), were mainly surrounded by the cells and less phagocytosed. The results of the cytokine release revealed significant differences in interleukin (IL)6, tumor necrosis factor (TNF)- _ and IL-1beta. The cell viability was not affected by the UHMW-PE particles. The results demonstrate that the particle induced cytokine response by UHMW-PE is mainly by the release of Interleukin 6 and TNF- _. Moreover the results confirm that the present method is useful to evaluate the in vitro effects of UHMW-PE wear particles with direct particle cell contact.     

Delineation of the cell-extrinsic apoptosis pathway in the zebrafish

The mammalian extrinsic apoptosis pathway is triggered by Fas ligand (FasL) and Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL). Ligand binding to cognate receptors activates initiator caspases directly in a death-inducing signaling complex. In Drosophila, TNF ligand binding activates initiator caspases indirectly, through JNK. We characterized the extrinsic pathway in zebrafish to determine how it operates in a nonmammalian vertebrate. We identified homologs of FasL and Apo2L/TRAIL, their receptors, and other components of the cell death machinery. Studies with three Apo2L/TRAIL homologs demonstrated that they bind the receptors zHDR (previously linked to hematopoiesis) and ovarian TNFR (zOTR). Ectopic expression of these ligands during embryogenesis induced apoptosis in erythroblasts and notochord cells. Inhibition of zHDR, zOTR, the adaptor zFADD, or caspase-8-like proteases blocked ligand-induced apoptosis, as did antiapoptotic Bcl-2 family members. Thus, the extrinsic apoptosis pathway in zebrafish closely resembles its mammalian counterpart and cooperates with the intrinsic pathway to trigger tissue-specific apoptosis during embryogenesis in response to ectopic Apo2L/TRAIL expression.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.     

Naturally Induced Humoral Immunity to West Nile Virus Infection in Raptors

West Nile virus (WNV) infection can be fatal to many bird species, including numerous raptors, though population- and ecosystem-level impacts following introduction of the virus to North America have been difficult to document. Raptors occupy a diverse array of habitats worldwide and are important to ecosystems for their role as opportunistic predators. We documented initial (primary) WNV infection and then regularly measured WNV-specific neutralizing antibody titers in 16 resident raptors of seven species, plus one turkey vulture. Most individuals were initially infected and seroconverted between July and September of 2003, though three birds remained seronegative until summer 2006. Many of these birds became clinically ill upon primary infection, with clinical signs ranging from loss of appetite to moderate neurological disease. Naturally induced WNV neutralizing antibody titers remained essentially unchanged in some birds, while eight individuals experienced secondary rises in titer presumably due to additional exposures at 1, 2, or 3 years following primary infection. No birds experienced clinical signs surrounding or following the time of secondary exposure, and therefore antibodies were considered protective. Results of this study have implications for transmission dynamics of WNV and health of raptor populations, as well as the interpretation of serologic data from free-ranging and captive birds. Antibodies in raptors surviving WNV may persist for multiple years and protect against potential adverse effects of subsequent exposures.     

Lifestyle Factors and Inflammation: Associations by Body Mass Index

Chronic inflammation, which is associated with obesity, may play a role in the etiology of several diseases. Thus, reducing inflammation may offer a disease-prevention strategy, particularly among the obese. Several modifiable factors have been associated with inflammation, including: dietary fiber intake, saturated fat intake, physical activity, smoking, alcohol, and use of certain supplements and medications (glucosamine, chondroitin, fish oil, vitamin E, statins and aspirin). To study whether these associations differ by body mass index (BMI), we used data on 9,895 adults included in the 1999–2004 cycles of the National Health and Nutrition Examination Survey (NHANES). Survey-weighted linear regression was used to evaluate the associations between modifiable factors and serum high-sensitivity C-reactive protein (hsCRP) concentrations across the following groups: underweight/normal weight (BMI<25 kg/m²), overweight (25-<30 kg/m²) and obese (30+ kg/m²). While several factors were significantly associated with decreased hsCRP among the normal weight or overweight groups (increased fiber intake, lower saturated fat intake, physical activity, not smoking, and use of chondroitin, fish oil and statins), only increasing dietary fiber intake and moderate alcohol consumption were associated with reduced hsCRP among the obese. Effect modification by BMI was statistically significant for the saturated fat-hsCRP and smoking-hsCRP associations. These results suggest that posited anti-inflammatory drugs and behaviors may be less strongly associated with inflammation among the obese than among lower weight persons.     

Structuring features of lake districts: landscape controls on lake chemical responses to drought

1. Within a lake district of relatively homogeneous geomorphology, the responses of lakes to climate are influenced by the complexity of the hydrogeologic setting, position in the landscape, and lake‐specific biological and physical features. We examined lake chemical responses to drought in surface water‐ and groundwater‐dominated districts to address two general questions. (1) Are spatial patterns in chemical dynamics among lakes uniform and synchronous within a lake district, suggesting broad geomorphic controls; variable in a spatially explicit pattern, with synchrony related to landscape position, suggesting hydrologic flowpath controls; or spatially unstructured and asynchronous, suggesting overriding control by lake‐specific factors? (2) Are lake responses to drought a simple function of precipitation quantity or are they dictated by more complex interactions among climate, unique lake features, and hydrologic setting? 2. Annual open‐water means for epilimnetic concentrations of chloride, calcium, sulfate, ANC, DOC, total nitrogen, silica, total phosphorus, and chlorophyll a measured between 1982 and 1995 were assembled for lakes in the Red Lake and ELA districts of north‐western Ontario, the Muskoka – Dorset district in south‐central Ontario, and the Northern Highland district of Wisconsin. Within each district, we compared responses of lakes classified by landscape position into highland or lowland, depending on relative location within the local to regional hydrologic flow system. Synchrony, defined as a measure of the similarity in inter‐annual dynamics among lakes within a district, was quantified as the Pearson product‐moment correlation (r) between two lakes with observations paired by year. To determine if solute concentrations were directly related to interannual variations in precipitation quantity, we used regression analysis to fit district‐wide slopes describing the relationship between each chemical variable and annual (June to May) and October to May (Oct–May) precipitation. 3. Among lakes in each of the three Ontario districts, the pattern of chemical response to interannual shifts in precipitation was spatially uniform. In these surface water‐ dominated districts, solute concentrations were generally a simple function of precipitation. Conservative solutes, like calcium and chloride, tended to be more synchronous and were negatively related to precipitation. Solutes such as silica, total phosphorus, and chlorophyll a, which are influenced by in‐lake processes, were less synchronous and relationships with precipitation tended to be positive or absent. 4. In the groundwater‐dominated Northern Highland lakes of Wisconsin, we observed spatial structure in drought response, with lowland lakes more synchronous than highland lakes. However, there was no evidence for a direct relationship between any solute and precipitation. Instead, increases in the concentration of the conservative ion calcium during drought were not followed by a symmetrical return to pre‐drought conditions when precipitation returned to normal or above‐average values. 5. For calcium, time lags in recovery from drought appeared related to hydrologic features in a complex way. In the highland Crystal Lake, calcium concentrations tracked lake stage inversely, with a return to pre‐drought concentrations and lake stage five years after the drought. This pattern suggests strong evaporative controls. In contrast, after five years of normal precipitation, calcium in the lowland Sparkling Lake had not returned to pre‐drought conditions despite a rebound in lake stage. This result suggests that calcium concentrations in lowland lakes were controlled more by regional groundwater flowpaths, which track climatic signals more slowly. 6. Temporal dynamics driven by climate were most similar among lakes in districts that have a relatively simple hydrology, such as ELA. Where hydrologic setting was more complex, as in the groundwater‐dominated Northern Highland of Wisconsin, the expression of climate signals in lakes showed lags and spatial patterns related to landscape position. In general, we expect that landscape and lake‐specific factors become increasingly important in lake districts with more heterogeneous hydrogeology, topography or land use. These strong chemical responses to climate need to be considered when interpreting the responses of lakes to other regional disturbances.     

Midgestational maternal urine steroid markers of fetal Smith-Lemli-Opitz (SLO) syndrome (7-dehydrocholesterol 7-reductase deficiency).

Smith-Lemli-Opitz syndrome (SLOS) is a malformation syndrome associated with 7-dehydrocholesterol (7DHC) 7-reductase deficiency. Although SLOS can be detected in an affected fetus before midpregnancy by measurement of 7DHC levels in amniotic fluid or chorionic villus cells, a noninvasive, more routine method is needed. Accordingly, this study was instigated to search for specific steroids in maternal urine in an affected pregnancy that reflect the 7-reductase deficiency of the fetus, ie, steroids retaining 7,8-unsaturation. Steroids were characterized by gas chromatography/mass spectrometry after urinary extraction, conjugate separation, and derivatization. Most steroids in maternal urine from a patient carrying a SLOS fetus were identified as progesterone metabolites, and these were entirely conventional, showing no evidence of additional unsaturation. Unsaturated homologues of the cortisol metabolites were also not detected. However, unsaturated homologues of pregnane-3,16,20-triols and pregnane-3,17,20-triol were found. Most likely, these are 7,8-unsaturated homologues, but 8,9-unsaturation is also possible because of the known activity of delta7-delta8-isomerase on 7DHC, which results in 8DHC being a prominent sterol in SLOS. Among these novel human steroids, the following were provisionally characterized: 5beta-pregn-7(or 8)-ene-3alpha,17alpha,20alpha-triol, 5beta-pregn-7(or 8)-ene-3alpha,16alpha,20alpha-triol, and 5alpha-pregn-7(or 8)-ene-3,16alpha,20alpha-triol. Confirmation of the position of unsaturation will require steroid synthesis. These novel steroids are not present in normal pregnancy urine and, therefore, are valuable for prenatal diagnosis of SLOS. In addition, separate studies have shown that 5beta-pregn-7(or 8)-ene-3alpha,17alpha,20alpha-triol is present in urine of children and adults with SLOS, and so is a useful analyte for confirmation of the disorder throughout life.     

Characterization and partial purification of a keratinocyte-derived growth factor with wound-healing properties

We have previously described the mitogenic and wound-healing properties of keratinocyte-conditioned medium (KCM). In this study we investigated the effect of KCM on the activation of second messenger systems and the expression of proto-oncogene in cultured human skin fibroblasts. We also present a partial purification of the factor responsible for the mitogenic and wound-healing effects of KCM. KCM was shown to increase the expression of the proto-oncogenes c-fos, c-myc and c-jun. The effect of KCM on three second messenger systems was investigated. The extracellular release of choline metabolites was increased by 40 per cent when cells were stimulated with KCM whereas the formation of cAMP and hydrolysis of phosphatidyl inositol (PI) was unaffected. KCM was purified by ion exchange chromatography and filtration. The biologically active fraction was eluted from an SP column and retained its activity after filtration through a 3-kDa filter. The fraction was inactivated by heat and acid, indicative of a peptide origin. Furthermore, the active fraction was shown to increase the extracellular release of choline metabolites and to stimulate re-epithelialization in wounds in human skin in vitro comparable to KCM. The study indicates that human keratinocytes produce a <3 kDa peptide which may be partly responsible for the growth stimulatory and wound-healing properties of KCM. © 1997 John Wiley & Sons, Ltd.