Summary Mitochondria are complex organelles whose dysfunction underlies a broad spectrum of human diseases. Identifying all the proteins resident in this organelle and understanding how they integrate into pathways represent major challenges in cell biology. Toward this goal, we performed mass spectrometry, GFP tagging, and machine learning to create a mitochondrial compendium of 1098 genes and their protein expression across 14 mouse tissues. We link poorly characterized proteins in this inventory to known mitochondrial pathways by virtue of shared evolutionary history. Using this approach we predict 19 proteins to be important for the function of complex I (CI) of the electron transport chain. We validate a subset of these predictions using RNAi, including C8orf38, which we further show harbors an inherited mutation in a lethal, infantile CI deficiency. Our results have important implications for understanding CI function and pathogenesis, and more generally, illustrate how our compendium can serve as a foundation for systematic investigations of mitochondria.

Two types of radially symmetric three-dimensional nonlinear photonic crystals with the cylindrical structure and the egglike structure are proposed, from which the conical and the spherical quadratic harmonic waves can be produced, respectively, by three-dimensional quasi-phase matching. First, the cylindrical structures with periodic and aperiodic modulations of the nonlinear coefficient are both studied, showing their significant advantages compared to the corresponding two-dimensional structures. The dependencies of the transverse and the longitudinal phase-matching periods on harmonic propagating directions are also calculated and analyzed. Then, the egglike structure is designed by programming and the distribution of reciprocal vectors is presented, indicating its ability to generate the spherical harmonic as a point light source. The investigation of the intensity distribution on the spherical wavefront is also performed, showing its strong dependence on the harmonic polarization and the quadratic nonlinear coefficients.

The capital, operating, and overall costs of a dedicated continuous manufacturing process to synthesize an active pharmaceutical ingredient (API) and formulate it into tablets are estimated for a production scale of 2000 t of tablets per year, with raw material cost, production yield, and API loading varied over broad ranges. Costs are compared to batch production in a dedicated facility. Synthesis begins with a key organic intermediate three synthetic steps before the final API; results are given for key intermediate (KI) costs of $100 to $3000/kg, with drug loadings in the tablet of 10 and 50 wt %. The novel continuous process described here is being developed by an interdisciplinary team of 20 researchers. Since yields are not yet well-known, and continuous processes typically have better yields than batch ones, the overall yields of the continuous processes with recycling were set equal to that of the batch process. Without recycling, yields are 10% lower, but less equipment is required. The continuous process has not been built at large scale, so Wroth factors and other assumptions were used to estimate costs. Capital expenditures for continuous production were estimated to be 20 to 76% lower, depending on the drug loading, KI cost, and process chosen; operating expenditures were estimated to be between 40% lower and 9% higher. The novel continuous process with recycling coupled to a novel direct tablet formation process yields the best overall cost savings in each drug loading/KI price scenario: estimated savings range from 9 to 40%. Overall cost savings are also given assuming the yield in the continuous case is 10% above and 10% below that of the batch process. Even when yields in the continuous case are lower than in the batch case, savings can still be achieved because the labor, materials handling, CapEx, and other savings compensate.

A series of tubes: The continuous manufacture of a finished drug product starting from chemical intermediates is reported. The continuous pilot-scale plant used a novel route that incorporated many advantages of continuous-flow processes to produce active pharmaceutical ingredients and the drug product in one integrated system.

Abstract-Toxicity of fresh waters with high total dissolved solids has been shown to be dependent on the specific ionic composition of the water. To provide a predictive tool to assess toxicity attributable to major ions, we tested the toxicity of over 2,900 ion solutions using the daphnids, Ceriodaphnia dubia and Daphnia magna, and fathead minnows (Pimephales promelas). Multiple logistic regression was used to relate ion composition to survival for each of the three test species. In general, relative ion toxicity was K ϩ Ͼ ഠ Mg 2ϩ Ͼ Cl Ϫ Ͼ ; Na ϩ and Ca 2ϩ were not significant variables in the regressions, suggesting that the toxicityof Na ϩ and Ca 2ϩ salts was primarily attributable to the corresponding anion. For C. dubia and D. magna, toxicity of Cl Ϫ , , 2 Ϫ SO 4 and K ϩ was reduced in solutions enriched with more than one cation. Final regression models showed a good quality of fit to the data (R 2 ϭ 0.767-0.861). Preliminary applications of these models to field-collected samples indicated a high degree of accuracy for the C. dubia model, while the D. magna and fathead minnow models tended to overpredict ion toxicity.

Fatty acid uptake into 3T3 L1 adipocytes is predominantly transporter mediated. Here we show that, during 3T3 L1 adipocyte differentiation, expression of fatty acid transport proteins (FATPs) 1 and 4 is induced. Using subcellular membrane fractionation and immunofluorescence microscopy, we demonstrate that, in adipocytes, insulin induces plasma membrane translocation of FATPs from an intracellular perinuclear compartment to the plasma membrane. This translocation was observed within minutes of insulin treatment and was paralleled by an increase in long chain fatty acid (LCFA) uptake. In contrast, treatment with TNF-alpha inhibited basal and insulin-induced LCFA uptake and reduced FATP1 and -4 levels. Thus, hormonal regulation of FATP activity may play an important role in energy homeostasis and metabolic disorders such as type 2 diabetes.

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with β-actin–ECFP and L-fimbrin–EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.