Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 ^-/- ;Ybx3 ^-/- double mutants using a previously reported Ybx2 ^-/- model and a newly generated global Ybx3 ^-/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets.     

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.     

Discovery of novel α7 nicotinic receptor antagonists

Two distinct families of small molecules were discovered as novel α7 nicotinic acetylcholine receptor (nAChR) antagonists by pharmacophore-based virtual screening. These novel antagonists exhibited selectivity for the neuronal α7 subtype over other nAChRs and good brain penetration. Neuroprotection was demonstrated by representative compounds 7i and 8 in a mouse seizure-like behavior model induced by the nerve agent diisopropylfluorophosphate (DFP). These novel nAChR antagonists have potential use as antidote for organophosphorus nerve agent intoxication.     

The comparative antimalarial properties of weak base and neutral synthetic ozonides

Thirty-three N-acyl 1,2,4-dispiro trioxolanes (secondary ozonides) were synthesized. For these ozonides, weak base functional groups were not required for high antimalarial potency against Plasmodium falciparum in vitro, but were necessary for high antimalarial efficacy in Plasmodium berghei-infected mice. A wide range of Log P/D_{pH 7.4} values were tolerated, although more lipophilic ozonides tended to be less metabolically stable.     

Extensive in vivo metabolite-protein interactions revealed by large-scale systematic analyses

Natural small compounds comprise most cellular molecules and bind proteins as substrates, products, cofactors, and ligands. However, a large-scale investigation of in?vivo protein-small metabolite interactions has not been performed. We developed a mass spectrometry assay for the large-scale identification of in?vivo protein-hydrophobic small metabolite interactions in yeast and analyzed compounds that bind ergosterol biosynthetic proteins and protein kinases. Many of these proteins bind small metabolites; a few interactions were previously known, but the vast majority are new. Importantly, many key regulatory proteins such as protein kinases bind metabolites. Ergosterol was found to bind many proteins and may function as a general regulator. It is required for the activity of Ypk1, a mammalian AKT/SGK kinase homolog. Our study defines potential key regulatory steps in lipid biosynthetic pathways and suggests that small metabolites may play a more general role as regulators of protein activity and function than previously appreciated.     

Densitometric, morphometric and mechanical distributions in the human proximal femur

With the prevalent use of DXA-measured BMD to assess pathologic hip fractures and its recently reported lack of reliability to predict fracture or account for efficacy of anti-resorptive therapy, it is reasonable to assess whether variations in the primary and secondary tensile and compressive trabecular microstructure can account for variations in proximal femur strength in comparison to DXA-measured BMD. To that end, microstructural and densitometric measures of trabecular bone specimens, from discrete sites within the proximal femur, were correlated with their mechanical properties. We hypothesize that accounting for regional variations in trabecular microstructure will improve predictions of proximal femur strength and stiffness compared to bone density measured by DXA. Forty-seven samples (seven donors) from seven distinct sites of human proximal femur underwent DXA and muCT imaging and mechanical testing. The results revealed significant variations in BMC, morphometric indices and mechanical properties within the proximal femur. This work has demonstrated that the mechanical performance of each sub-region is highly dependent on the corresponding trabecular microstructure. BMD measured by DXA at standard regions of interest cannot resolve the variations in trabecular density and microstructure that govern the mechanical behavior of the proximal femur. This work suggests that a quantitative Singh index that uses high resolution QCT to monitor the trabecular microstructure at specific sub-regions of the proximal femur may allow better predictions of hip fracture risk in individual patients and an improved assessment of changing bone structure in response to pharmacological interventions.     

Knockdown and mortality comparisons among spinosad-, imidacloprid-, and methomyl-containing baits against susceptible Musca domestica (Diptera: Muscidae) under laboratory conditions

The activity of spinosad, imidacloprid, and methomyl baits and technical actives were assessed against susceptible house flies, Musca domestica L. (Diptera: Muscidae). In a feeding assay, imidacloprid affected flies more rapidly than methomyl or spinosad, but spinosad was 2.7 times more potent than methomyl and 8 times more potent than imidacloprid. The profile of technical actives correlated with their respective fly bait formulations in laboratory assays. Although having the most rapid onset of activity in laboratory tests, up to 50% of flies remained alive after exposure to imidacloprid bait. In contrast, <5% of flies survived 24-h exposure to spinosad or methomyl baits. High temperature reduced the knockdown activity of imidacloprid bait and slowed the speed of kill for spinosad and methomyl baits over a 24-h exposure period. Spinosad and methomyl baits were also superior to imidacloprid when applied to the floors of environmentally controlled rooms at label recommended rates, providing good fly control for up to 21 d. The fact that a significant percentage of flies exposed to imidacloprid were rapidly knocked down but subsequently remained alive in all of the assays suggested that flies were recovering from initial exposure to this compound. Given its favorable safety profile, a high degree of initial and residual activity comparable with methomyl and lack of cross-resistance to other chemistries, spinosad bait may be a valuable component of house fly control programs to help control or delay the emergence of resistant populations.     

Positional artifacts in microarrays: experimental verification and construction of COP, an automated detection tool

Microarray technology is currently one of the most widely-used technologies in biology. Many studies focus on inferring the function of an unknown gene from its co-expressed genes. Here, we are able to show that there are two types of positional artifacts in microarray data introducing spurious correlations between genes. First, we find that genes that are close on the microarray chips tend to have higher correlations between their expression profiles. We call this the 'chip artifact'. Our calculations suggest that the carry-over during the printing process is one of the major sources of this type of artifact, which is later confirmed by our experiments. Based on our experiments, the measured intensity of a microarray spot contains 0.1% (for fully-hybridized spots) to 93% (for un-hybridized ones) of noise resulting from this artifact. Secondly, we, for the first time, show that genes that are close on the microtiter plates in microarray experiments also tend to have higher correlations. We call this the 'plate artifact'. Both types of artifacts exist with different severity in all cDNA microarray experiments that we analyzed. Therefore, we develop an automated web tool-COP (COrrelations by Positional artifacts) to detect these artifacts in microarray experiments. COP has been integrated with the microarray data normalization tool, ExpressYourself, which is available at http://bioinfo.mbb.yale.edu/ExpressYourself/. Together, the two can eliminate most of the common noises in microarray data.