Mycobacterial Acid Tolerance Enables Phagolysosomal Survival and Establishment of Tuberculous Infection In Vivo.

The blockade of phagolysosomal fusion is considered a critical mycobacterial strategy to survive in macrophages. However, viable mycobacteria have been observed in phagolysosomes during infection of cultured macrophages, and mycobacteria have the virulence determinant MarP, which confers acid resistance in vitro. Here we show in mice and zebrafish that innate macrophages overcome mycobacterial lysosomal avoidance strategies to rapidly deliver a substantial proportion of infecting bacteria to phagolysosomes. Exploiting the optical transparency of the zebrafish, we tracked the fates of individual mycobacteria delivered to phagosomes versus phagolysosomes and discovered that bacteria survive and grow in phagolysosomes, though growth is slower. MarP is required specifically for phagolysosomal survival, making it an important determinant for the establishment of mycobacterial infection in their hosts. Our work suggests that if pathogenic mycobacteria fail to prevent lysosomal trafficking, they tolerate the resulting acidic environment of the phagolysosome to establish infection.National Institutes of Health (Grant IDs: R37AI054503, R01 AI076327, 5T32HD007233, 5F30HL110455), Wellcome Trust, National Institute of Health Research Cambridge Biomedical Research CentreThis is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.chom.2016.07.00

The Science Performance of JWST as Characterized in Commissioning

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies.Comment: 5th version as accepted to PASP; 31 pages, 18 figures; https://iopscience.iop.org/article/10.1088/1538-3873/acb29

Measuring Actin Dynamics Using Correlation Analysis

Correlation analysis is a tool commonly used to obtain information about the dynamics of biofilaments such as actin and microtubules in cells. This approach is particularly useful when individual identification of filaments is difficult in vivo. The goal of this project is to develop a coarse-grained model of dynamic actin filaments using Brownian dynamics simu- lations, and to validate correlation analysis as a viable technique to measure the dynamics, and determine its range of applicability. Particular focus is on the dependence on the density of filaments, as well as polymerization and depolymerization

A Coarse-Grained Computational Model of Kinesin-Microtubule Gliding Assays on Lipid Bilayers

Cytoskeletal filaments and their associated motor proteins play key roles in many cellular processes including cell division, locomotion, and cargo transport. One type of cytoskeletal filament, the microtubule, acts as the rigid structural skeleton of eukaryotic cells and forms the spindle during mitosis. Kinesin, a motor protein associated with the microtubule, processively steps along the microtubule hand-over-hand to transport cargo and to reorganize the microtubule network structure. Much has been done in recent years to characterize the biophysical properties of these proteins, and one of the most common methods is the gliding assay, performed on glass surfaces. Motors in vivo are often embedded in the plasma membrane or lipid vesicles, however, in which they can diffuse. This leads to quantitative and qualitative differences in behavior that are not reproduced in glass gliding assays. Here, we study the diffusion of kinesin-1 motors across a lipid bilayer and their accumulation onto microtubules using coarse-grained computer simulations. By comparing the resulting behavior to gliding assay experiments performed on a lipid bilayer, we can determine the on-rate of the motors onto the microtubule under biologically relevant conditions, and also shed light into finite size effects as well as the limit of the mean-field approach. We also develop a model for relating the velocity of the microtubule to the various mechanistic properties of the simulation, which agrees with both the high- and low-density regimes

Creating Video Games for the Visually Impaired

In the 1970s when video games first emerged, it was virtually impossible to create a game that did not involve some sort of visuals. This is mainly due to a lack of technology, as almost all computers, games, and electronic speakers and displays could only output crude, pixilated visuals and rough, synthesized sound. However, technology has steadily improved to the point where games that do not need visuals are a possibility. - Games for the visually impaired are an untapped industry, especially with the technology that exists today. By conducting interviews with the visually impaired we discovered what types of games the visual impaired prefer, what they would enjoy to play, and what ideas they have for developing these games

HDL-like discs for assaying membrane proteins in drug discovery

To broaden the use of the recombinant high-density lipoprotein (rHDL) approach to the characterization of lead compounds, we investigated the pharmacology of the human beta-2-adrenoceptor in nanolipid bilayers (rHDL) with a broad set of beta-adrenoceptor antagonists. To that end, we developed a homogeneous copper-chelate scintillation proximity binding assay (SPA) in order to compare receptor-ligand binding affinities before and after reconstitution into rHDLs. Our results clearly suggest that the receptor in rHDLs display the same pharmacology than in cell membranes and that rHDLs can be used not only to measure affinities for a range of ligands but also to study binding kinetics

An alternative thiol-reactive dye to analyze ligand interactions with the chemokine receptor CXCR2 using a new thermal shift assay format

Integral membrane proteins (IMP) play an important role in many cellular events and are involved in numerous pathological processes. Therefore, understanding the structure and function of IMPs is a crucial prerequisite to enable successful targeting of these proteins with low molecular weight (LMW) ligands early on in the discovery process. To optimize IMP purification/crystallization and to identify/characterize LMW ligand/target interactions, robust, reliable, high-throughput and sensitive biophysical methods are needed. Here, we describe a differential scanning fluorimerty (DSF) screening method using the thiol-reactive BODIPY®-FL-cystine dye to monitor thermal unfolding of the G-protein coupled receptor (GPCR), CXCR2. To validate this method the seven-transmembrane protein CXCR2 was analyzed with a set of well-characterized antagonists. This study showed that the new DSF assay assessed reliably the stability of CXCR2 in 384well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY® assay format as a general tool to investigate membrane proteins and their interaction partners

PARP-inhibition reprograms macrophages toward an anti-tumor phenotype

Poly(ADP)ribosylation inhibitors (PARPis) are toxic to cancer cells with homologous recombination (HR) deficiency but not to HR-proficient cells in the tumor microenvironment (TME), including tumor-associated macrophages (TAMs). As TAMs can promote or inhibit tumor growth, we set out to examine the effects of PARP inhibition on TAMs in BRCA1-related breast cancer (BC). The PARPi olaparib causes reprogramming of TAMs toward higher cytotoxicity and phagocytosis. A PARPi-related surge in NAD+ increases glycolysis, blunts oxidative phosphorylation, and induces reverse mitochondrial electron transport (RET) with an increase in reactive oxygen species (ROS) and transcriptional reprogramming. This reprogramming occurs in the absence or presence of PARP1 or PARP2 and is partially recapitulated by addition of NAD derivative methyl-nicotinamide (MNA). In vivo and ex vivo, the effect of olaparib on TAMs contributes to the anti-tumor efficacy of the PARPi. In vivo blockade of the “don’t-eat-me signal” with CD47 antibodies in combination with olaparib improves outcomes in a BRCA1-related BC model.publishedVersio