Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies

Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.     

Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.     

Association of driver air bags with driver fatality: a matched cohort study

ObjectiveTo estimate the association of driver air bag presence with driver fatality in road traffic crashes.DesignMatched pair cohort study.SettingAll passenger vehicle crashes in the United States during 1990-2000 inclusive.Subjects51 031 driver-passenger pairs in the same vehicle.ResultsDrivers with an air bag were less likely to die than drivers without an air bag (adjusted relative risk 0.92 (95% confidence interval 0.88 to 0.96)). This estimate was nearly the same whether drivers wore a seat belt (adjusted relative risk 0.93) or not (0.91). Air bags were associated with more protection for women (0.88 (0.82 to 0.93)), than for men (0.94 (0.90 to 0.99)). Drivers wearing a seat belt were less likely to die than unbelted drivers (0.35 (0.33 to 0.36)). Belted drivers with an air bag were less likely to die than unbelted drivers without an air bag (0.32 (0.30 to 0.34)).ConclusionsIf the associations are causal the average risk of driver death was reduced 8% (95% confidence interval 4% to 12%) by an air bag. Benefit was similar for belted and unbelted drivers and was slightly greater for women. However, seat belts offered much more protection than air bags.

What is already known on this topic

Studies have estimated that driver air bags reduce the risk of death in a road vehicle crash by 10-14%These studies disagree as to whether benefit is greater for drivers wearing a seat belt or for unbelted drivers

What this study adds

Having an air bag was associated with an 8% reduction in the risk of death, whether the driver was belted or notThe reduction in risk was greater for women (12%) than for men (6%)Seat belts provided much greater protection, with seat belt use reducing the risk of death by 65% (or by 68% in combination with an air bag)     

Primary Human Immunodeficiency Virus Type 2 (HIV-2) Isolates Infect CD4-Negative Cells via CCR5 and CXCR4: Comparison with HIV-1 and Simian Immunodeficiency Virus and Relevance to Cell Tropism In Vivo

Cell surface receptors exploited by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) for infection are major determinants of tropism. HIV-1 usually requires two receptors to infect cells. Gp120 on HIV-1 virions binds CD4 on the cell surface, triggering conformational rearrangements that create or expose a binding site for a seven-transmembrane (7TM) coreceptor. Although HIV-2 and SIV strains also use CD4, several laboratory-adapted HIV-2 strains infect cells without CD4, via an interaction with the coreceptor CXCR4. Moreover, the envelope glycoproteins of SIV of macaques (SIV(MAC)) can bind to and initiate infection of CD4(-) cells via CCR5. Here, we show that most primary HIV-2 isolates can infect either CCR5(+) or CXCR4(+) cells without CD4. The efficiency of CD4-independent infection by HIV-2 was comparable to that of SIV, but markedly higher than that of HIV-1. CD4-independent HIV-2 strains that could use both CCR5 and CXCR4 to infect CD4(+) cells were only able to use one of these receptors in the absence of CD4. Our observations therefore indicate (i) that HIV-2 and SIV envelope glycoproteins form a distinct conformation that enables contact with a 7TM receptor without CD4, and (ii) the use of CD4 enables a wider range of 7TM receptors to be exploited for infection and may assist adaptation or switching to new coreceptors in vivo. Primary CD4(-) fetal astrocyte cultures expressed CXCR4 and supported replication by the T-cell-line-adapted ROD/B strain. Productive infection by primary X4 strains was only triggered upon treatment of virus with soluble CD4. Thus, many primary HIV-2 strains infect CCR5(+) or CXCR4(+) cell lines without CD4 in vitro. CD4(-) cells that express these coreceptors in vivo, however, may still resist HIV-2 entry due to insufficient coreceptor concentration on the cell surface to trigger fusion or their expression in a conformation nonfunctional as a coreceptor. Our study, however, emphasizes that primary HIV-2 strains carry the potential to infect CD4(-) cells expressing CCR5 or CXCR4 in vivo.     

Substituent control of DNA binding modes in a series of chalcogenoxanthylium photosensitizers as determined by isothermal titration calorimetry and topoisomerase I DNA unwinding assay

The DNA binding efficacy and preferred mode of binding of a series of rhodamine-related chalcogenoxanthylium dyes was investigated by isothermal titration calorimetry (ITC) using ctDNA, [poly(dCdG)](2) and [poly(dAdT)](2), and by a topoisomerase I DNA unwinding (Topo I) assay. The dyes of this study showed tight binding to ctDNA with binding constants, K(b), on the order of 10(6)-10(7)M(-1). The ITC and Topo I assay studies suggested that the 9-substituent has a strong impact on binding modes ranging from an apparent preference for intercalation with a 9-2-thienyl substituent (similar binding to [poly(dCdG)](2) and [poly(dAdT)](2), re-supercoiling of DNA in the Topo I assay at <10(-5)M dye), to mixed binding modes with 9-phenyl derivatives (2- to 3-fold preference for binding to [poly(dAdT)](2), re-supercoiling of DNA in the Topo I assay at approximately 2 x 10(-5)M dye), to minor groove binding in a 9-(2-thienyl-5-diethylcarboxamide) derivative (strong preference for binding to [poly(dAdT)](2), did not show complete re-supercoiling in the Topo I assay). No binding to ctDNA was observed in one derivative with a 9-(3-thienyl-2-diethylcarboxamide) substituent, which cannot be co-planar with the xanthylium core. In series of dyes where the chalcogen atom was varied, the selenoxanthylium derivatives had 2- to 3-fold higher values of K(b) than the corresponding xanthylium, thioxanthylium, or telluroxanthylium derivatives, which all showed comparable values of K(b). The chalcogen atom appeared to have little influence on binding mode.