Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates

Nitrogen-containing-bisphosphonates (N-BPs) are widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance

Use of Mutated Self-Cleaving 2A Peptides as a Molecular Rheostat to Direct Simultaneous Formation of Membrane and Secreted Anti-HIV Immunoglobulins

In nature, B cells produce surface immunoglobulin and secreted antibody from the same immunoglobulin gene via alternative splicing of the pre-messenger RNA. Here we present a novel system for genetically programming B cells to direct the simultaneous formation of membrane-bound and secreted immunoglobulins that we term a “Molecular Rheostat”, based on the use of mutated “self-cleaving” 2A peptides. The Molecular Rheostat is designed so that the ratio of secreted to membrane-bound immunoglobulins can be controlled by selecting appropriate mutations in the 2A peptide. Lentiviral transgenesis of Molecular Rheostat constructs into B cell lines enables the simultaneous expression of functional b12-based IgM-like BCRs that signal to the cells and mediate the secretion of b12 IgG broadly neutralizing antibodies that can bind and neutralize HIV-1 pseudovirus. We show that these b12-based Molecular Rheostat constructs promote the maturation of EU12 B cells in an in vitro model of B lymphopoiesis. The Molecular Rheostat offers a novel tool for genetically manipulating B cell specificity for B-cell based gene therapy

Relative replication capacity of phenotypic SIV variants during primary infections differs with route of inoculation

BACKGROUND: Previous studies of human and simian immunodeficiency virus (HIV and SIV) have demonstrated that adaptive mutations selected during the course of infection alter viral replicative fitness, persistence, and pathogenicity. What is unclear from those studies is the impact of transmission on the replication and pathogenicity of the founding virus population. Using the SIV-macaque model, we examined whether the route of infection would affect the establishment and replication of two SIVmne variants of distinct in vitro and in vivo biological characteristics. For these studies, we performed dual-virus inoculations of pig-tailed macaques via intrarectal or intravenous routes with SIVmneCl8, a miminally pathogenic virus, and SIVmne027, a highly pathogenic variant that replicates more robustly in CD4(+ )T cells. RESULTS: The data demonstrate that SIVmne027 is the dominant virus regardless of the route of infection, indicating that the capacity to replicate efficiently in CD4(+ )T cells is important for fitness. Interestingly, in comparison to intravenous co-infection, intrarectal inoculation enabled greater relative replication of the less pathogenic virus, SIVmneCl8. Moreover, a higher level of SIVmneCl8 replication during primary infection of the intrarectally inoculated macaques was associated with lower overall plasma viral load and slower decline in CD4(+ )T cells, even though SIVmne027 eventually became the dominant virus. CONCLUSIONS: These results suggest that the capacity to replicate in CD4(+ )T cells is a significant determinant of SIV fitness and pathogenicity. Furthermore, the data also suggest that mucosal transmission may support early replication of phenotypically diverse variants, while slowing the rate of CD4(+ )T cell decline during the initial stages of infection

Comparison of the High-Temperature Oxidation Behavior of Subsolvus and Supersolvus Treated Advanced Powder Metallurgy Disk Alloys

No abstract availabl

Observability of Earth-skimming Ultra-high Energy Neutrinos

Neutrinos with energies above 10^8 GeV are expected from cosmic ray interactions with the microwave background and are predicted in many speculative models. Such energetic neutrinos are difficult to detect, as they are shadowed by the Earth, but rarely interact in the atmosphere. Here we propose a novel detection strategy: Earth-skimming neutrinos convert to charged leptons that escape the Earth, and these leptons are detected in ground level fluorescence detectors. With the existing HiRes detector, neutrinos from some proposed sources are marginally detectable, and improvements of two orders of magnitude are possible at the proposed Telescope Array.Comment: 4 pages, 3 figure

Octopamine Neuromodulation Regulates Gr32a-Linked Aggression and Courtship Pathways in Drosophila Males

Chemosensory pheromonal information regulates aggression and reproduction in many species, but how pheromonal signals are transduced to reliably produce behavior is not well understood. Here we demonstrate that the pheromonal signals detected by Gr32a-expressing chemosensory neurons to enhance male aggression are filtered through octopamine (OA, invertebrate equivalent of norepinephrine) neurons. Using behavioral assays, we find males lacking both octopamine and Gr32a gustatory receptors exhibit parallel delays in the onset of aggression and reductions in aggression. Physiological and anatomical experiments identify Gr32a to octopamine neuron synaptic and functional connections in the suboesophageal ganglion. Refining the Gr32a-expressing population indicates that mouth Gr32a neurons promote male aggression and form synaptic contacts with OA neurons. By restricting the monoamine neuron target population, we show that three previously identified OA-FruM neurons involved in behavioral choice are among the Gr32a-OA connections. Our findings demonstrate that octopaminergic neuromodulatory neurons function as early as a second-order step in this chemosensory-driven male social behavior pathway