The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

The Molecular Alarmone (p)ppGpp Mediates Stress Responses, Vancomycin Tolerance, and Virulence in Enterococcus faecalis▿

The stringent response is a global bacterial response to stress that is mediated by accumulation of the alarmone (p)ppGpp. In this study, treatment with mupirocin was shown to induce high levels of (p)ppGpp production in Enterococcus faecalis, indicating that this nosocomial pathogen can mount a classic stringent response. In addition, (p)ppGpp was found to accumulate in cells subjected to heat shock, alkaline shock, and inhibitory concentrations of vancomycin. Sequence analysis of the E. faecalis genome indicated that (p)ppGpp synthesis is catalyzed by the bifunctional synthetase/hydrolase RelA and the RelQ small synthase. The (p)ppGpp profiles of ΔrelA, ΔrelQ, and ΔrelAQ strains revealed that RelA is the major enzyme responsible for the accumulation of (p)ppGpp during antibiotic or physical stresses, while RelQ appears to be responsible for maintaining basal levels of alarmone during homeostatic growth. Compared to its parent, the ΔrelA strain was more susceptible to several stress conditions, whereas complete elimination of (p)ppGpp in a ΔrelAQ double mutant restored many of the stress-sensitive phenotypes of ΔrelA. Interestingly, growth curves and time-kill studies indicated that tolerance to vancomycin is enhanced in the ΔrelA strain but diminished in the ΔrelQ and ΔrelAQ strains. Finally, virulence of the ΔrelAQ strain but not of the ΔrelA or ΔrelQ strain was significantly attenuated in the Caenorhabditis elegans model. Taken together, these results indicate that (p)ppGpp pools modulate environmental stress responses, vancomycin tolerance, and virulence in this important nosocomial pathogen

Association of BLM and BRCA1 during Telomere Maintenance in ALT Cells

<div><p>Fifteen percent of tumors utilize recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. The mechanisms underlying ALT are unclear but involve several proteins involved in homologous recombination including the BLM helicase, mutated in Bloom's syndrome, and the BRCA1 tumor suppressor. Cells deficient in either BLM or BRCA1 have phenotypes consistent with telomere dysfunction. Although BLM associates with numerous DNA damage repair proteins including BRCA1 during DNA repair, the functional consequences of BLM-BRCA1 association in telomere maintenance are not completely understood. Our earlier work showed the involvement of BRCA1 in different mechanisms of ALT, and telomere shortening upon loss of BLM in ALT cells. In order to delineate their roles in telomere maintenance, we studied their association in telomere metabolism in cells using ALT. This work shows that BLM and BRCA1 co-localize with RAD50 at telomeres during S- and G2-phases of the cell cycle in immortalized human cells using ALT but not in cells using telomerase to maintain telomeres. Co-immunoprecipitation of BRCA1 and BLM is enhanced in ALT cells at G2. Furthermore, BRCA1 and BLM interact with RAD50 predominantly in S- and G2-phases, respectively. Biochemical assays demonstrate that full-length BRCA1 increases the unwinding rate of BLM three-fold in assays using a DNA substrate that models a forked structure composed of telomeric repeats. Our results suggest that BRCA1 participates in ALT through its interactions with RAD50 and BLM.</p></div

Role of Clp Proteins in Expression of Virulence Properties of Streptococcus mutans▿ †

Mutational analysis revealed that members of the Clp system, specifically the ClpL chaperone and the ClpXP proteolytic complex, modulate the expression of important virulence attributes of Streptococcus mutans. Compared to its parent, the ΔclpL strain displayed an enhanced capacity to form biofilms in the presence of sucrose, had reduced viability, and was more sensitive to acid killing. The ΔclpP and ΔclpX strains displayed several phenotypes in common: slow growth, tendency to aggregate in culture, reduced autolysis, and reduced ability to grow under stress, including acidic pH. Unexpectedly, the ΔclpP and ΔclpX mutants were more resistant to acid killing and demonstrated enhanced viability in long-term survival assays. Biofilm formation by the ΔclpP and ΔclpX strains was impaired when grown in glucose but enhanced in sucrose. In an animal study, the average number of S. mutans colonies recovered from the teeth of rats infected with the ΔclpP or ΔclpX strain was slightly lower than that of the parent strain. In Bacillus subtilis, the accumulation of the Spx global regulator, a substrate of ClpXP, has accounted for the ΔclpXP phenotypes. Searching the S. mutans genome, we identified two putative spx genes, designated spxA and spxB. The inactivation of either of these genes bypassed phenotypes of the clpP and clpX mutants. Western blotting demonstrated that Spx accumulates in the ΔclpP and ΔclpX strains. Our results reveal that the proteolysis of ClpL and ClpXP plays a role in the expression of key virulence traits of S. mutans and indicates that the underlying mechanisms by which ClpXP affect virulence traits are associated with the accumulation of two Spx orthologues

BRCA1 and BLM co-localize at telomeres in ALT cells.

<p>(<b>A</b>) Cells were synchronized to G2 (7h post release after double thymidine block) and stained with antibodies to BRCA1 and BLM and telomeres labeled by FISH with a PNA probe (upper panel) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103819#pone-0103819-g001" target="_blank">Figure 1</a>. Yellow arrows indicate foci with BLM-BRCA1-telomere co-localization.</p

BRCA1 and BLM co-localize at telomeres along with RAD50 at different phases of cell cycle in ALT cells.

<p>U2OS cells were synchronized and stained with antibodies to BRCA1 or BLM (grey), Rad50 (green) and telomeres were labeled by FISH with a PNA probe (Red); nuclei were stained with DAPI. Representative images are shown as MIP image. White boxes indicate co-localizations of BRCA1 (<b>panel A</b>) or BLM (<b>panel B</b>) with Rad50 and telomere FISH. Co-localizations were confirmed by 3D rendering of multiple channels (volume rendering) using Metamorph analysis software. Enlargements of rendered co-localizations are shown at the right side and indicated by arrow. Co-localized foci of BRCA1 (or BLM), Rad50 and telomere were quantified per nucleus in unsynchronized, G1, S and G2 synchronized U2OS cells and represented graphically (right side of each panel). Each open circle represents an individual cell. Red bar indicate mean values.</p

BRCA1 stimulates BLM helicase activity on telomeric repeat forks.

<p>(<b>A</b>) Fork substrates (1F, 2F and 4F) used for the study. The number of telomeric repeat units (GGGATT) are boxed in each fork substrate – 1F contains one repeat, 2F contains two repeats and 4F contains four repeats. (<b>B</b>) BLM helicase activity on fork substrates. Increasing amounts of BLM (0.6 nM, 1.2 nM and 4.2 nM) were used for each substrate, reactions were terminated after 12 minutes and analyzed by native PAGE. HD refers to heat-denatured substrate. (<b>C</b>) Effect of BRCA1 on BLM helicase activity on 2F substrate. Helicase reactions were performed for 12 minutes with 1.2 nM BLM and increasing concentrations of BRCA1 (0–4 nM). Reactions were terminated and analyzed by native PAGE. The amount of substrate unwound (%) was plotted as a function of BRCA1 (nM). A representative gel and corresponding graph are shown. (<b>D</b>) Stimulation of BLM activity by BRCA1 on telomeric and non-telomeric substrates. BLM helicase activity was tested in the presence of 3 nM BRCA1 on fork substrates that contained two telomeric repeats (panel A) or a non-telomeric sequence instead of the repeat units. Reactions were analyzed by native PAGE and quantitated on a phosphorimager. The fold stimulation of BLM activity by BRCA1 for each substrate is shown as histogram. (<b>E</b>) BRCA1 enhances the rate of unwinding of telomeric fork by BLM. Kinetics of BLM unwinding was analyzed in the presence of 1.2 nM BLM and 3 nM BRCA1. Reactions were analyzed as in C. The amount of substrate unwound (%) was plotted as a function time (minutes) and fitted to Michaelis-Menton kinetics using Kaleidagraph. A representative gel and corresponding graph are shown.</p

Co-immunoprecipitations of BRCA1, RAD50 and BLM from U2OS cells at different phases of the cell cycle.

<p>Cells were synchronized by double-thymidine block and extracts made at different cell cycle phases. Extracts were immunoprecipitated using indicated antibodies, separated by 8% SDS-PAGE and analyzed by western blotting using indicated antibodies. <b>(A and B) Immunoprecipitation of BRCA1 and BLM by RAD50.</b> Immunoprecipitation was performed using αRAD50 antibody on extracts representing unsynchronized cells or cells at G1-, S- and G2-phases of cell cycle. Controls included immunoprecipitation in the absence of αRAD50 antibody (Beads) or immunoprecipitation using IgG. Extracts in the absence of immunoprecipitation were included in the gels as input. Western blotting was performed using αBRCA1 (panel A) or αBLM antibody (panel B). Cellular extracts used for immunoprecipitation corresponding to each stage were separated on an 8% SDS-PAGE and analyzed by western blotting with αLamin B antibody to show equivalent loading (panel B lower blot). <b>(C) Immunoprecipitation of RAD50 by BLM.</b> Immunoprecipitation was performed using αBLM antibody on extracts representing unsynchronized cells or cells at G1-, S- and G2-phases of cell cycle. Controls included immunoprecipitation in the absence of αRAD50 antibody (Beads), immunoprecipitation using IgG or immunoprecipitation of extracts from a BLM-deficient cell line (GM8505). Extracts in the absence of immunoprecipitation were included in the gel as input. Western Blotting was performed using αRad50 antibody.</p

BRCA1 and BLM co-localize at telomeres only in ALT cells and physically interact predominantly during G2.

<p>(<b>A</b>) Quantitation of percent of cells with BLM-BRCA1, BRCA1-telomere, and BLM-BRCA1-telomere co-localized foci in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103819#pone-0103819-g003" target="_blank">Figure 3</a>. (<b>B</b>) Upper panel: Co-immunoprecipitation of BLM with BRCA1 from U2OS cells. Cellular extracts (20 µg each) prepared at different stages of cell cycle were immunoprecipitated with an αBRCA1 antibody, separated on an 8% SDS-PAGE and analyzed by western blotting with αBLM antibody. Lower panel: Cellular extracts (20 µg each) used in upper panel corresponding to each stage were separated on an 8% SDS-PAGE and analyzed by western blotting with αLamin B antibody to show equivalent loading. The arrow mark indicates the position of BLM or Lamin B on the blot. Extracts from BLM-deficient cell line GM8505 served as a control.</p