Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate

Ontogeny of midazolam glucuronidation in preterm infants

Purpose: In preterm infants, the biotransformation of midazolam (M) to 1-OH-midazolam (OHM) by cytochrome P450 3A4 (CYP3A4) is developmentally immature, but it is currently unknown whether the glucuronidation of OHM to 1-OH-midazolam glucuronide (OHMG) is also decreased. The aim of our study was to investigate the urinary excretion of midazolam and its metabolites OHM and OHMG in preterm neonates following the intravenous (IV) or oral (PO) administration of a single M dose. Methods: Preterm infants (post-natal age 3-13 days, gestational age 26-34 4/7 weeks) scheduled to undergo a stressful procedure received a 30-min IV infusion (n=15) or a PO bolus dose (n=7) of 0.1 mg/kg midazolam. The percentage of midazolam dose excreted in the urine as M, OHM and OHMG up to 6 h post-dose was determined. Results: The median percentage of the midazolam dose excreted as M, OHM and OHMG in the urine during the 6-h interval after the IV infusion was 0.44% (range 0.02-1.39%), 0.04% (0.01-0.13%) and 1.57% (0.36-7.7%), respectively. After administration of the PO bolus dose, the median percentage of M, OHM and OHMG excreted in the urine was 0.11% (0.02-0.59%), 0.02% (0.00-0.10%) and 1.69% (0.58-7.31%), respectively. The proportion of the IV midazolam dose excreted as OHMG increased significantly with postconceptional age (r=0.73, p <0.05). Conclusion: The glucuronidation of OHM appears immature in preterm infants less than 2 weeks of age. The observed increase in urinary excretion of OHMG with postconceptional age likely reflects the combined maturation of glucuronidation and renal function

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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Classifying the evolutionary and ecological features of neoplasms

The consensus conference was supported by Wellcome Genome Campus Advanced Courses and Scientific Conferences. C.C.M. is supported in part by US NIH grants P01 CA91955, R01 CA149566, R01 CA170595, R01 CA185138 and R01 CA140657 as well as CDMRP Breast Cancer Research Program Award BC132057. M.J. is supported by NIH grant K99CA201606. K.S.A. is supported by NCI 5R21 CA196460. K. Polyak is supported by R35 CA197623, U01 CA195469, U54 CA193461, and the Breast Cancer Research Foundation. K.J.P. is supported by NIH grants CA143803, CA163124, CA093900 and CA143055. D.P. is supported by the European Research Council (ERC-617457- PHYLOCANCER), the Spanish Ministry of Economy and Competitiveness (BFU2015-63774-P) and the Education, Culture and University Development Department of the Galician Government. K.S.A. is supported in part by the Breast Cancer Research Foundation and NCI R21CA196460. C.S. is supported by the Royal Society, Cancer Research UK (FC001169), the UK Medical Research Council (FC001169), and the Wellcome Trust (FC001169), NovoNordisk Foundation (ID 16584), the Breast Cancer Research Foundation (BCRF), the European Research Council (THESEUS) and Marie Curie Network PloidyNet. T.A.G. is a Cancer Research UK fellow and a Wellcome Trust funded Investigator. E.S.H. is supported by R01 CA185138-01 and W81XWH-14-1-0473. M.Gerlinger is supported by Cancer Research UK and The Royal Marsden/ICR National Institute of Health Research Biomedical Research Centre. M.Ge., M.Gr., Y.Y., and A.So. were also supported in part by the Wellcome Trust [105104/Z/14/Z]. J.D.S. holds the Edward B. Clark, MD Chair in Pediatric Research, and is supported by the Primary Children's Hospital (PCH) Pediatric Cancer Research Program, funded by the Intermountain Healthcare Foundation and the PCH Foundation. A.S. is supported by the Chris Rokos Fellowship in Evolution and Cancer. Y.Y. is a Cancer Research UK fellow and supported by The Royal Marsden/ICR National Institute of Health Research Biomedical Research Centre. E.S.H. was supported in part by PCORI grants 1505–30497 and 1503–29572, NIH grants R01 CA185138, T32 CA093245, and U10 CA180857, CDMRP Breast Cancer Research Program Award BC132057, a CRUK Grand Challenge grant, and the Breast Cancer Research Foundation. A.R.A.A. was funded in part by NIH grant U01CA151924. A.R.A.A., R.G. and J.S.B. were funded in part by NIH grant U54CA193489

Using a Role-Driven Race Equity Reform Approach to Mitigate the Effects of America's History of Racism on Food Insecurity

Food insecurity, or the lack of reliable access to sufficient quantities of nutritious food, affects African Americans and other minorities disproportionately. This paper examines how America’s history of racism created and sustains the Nation’s racially disparate food system. Food insecurity contributes to hunger. This paper contemplates disparities in other American systems, including education and criminal justice, as exemplars of the broader ramifications of hunger. Finally, the paper examines the potential of individual action to address problems in any system. It champions the adoption of a role-driven race equity reform strategy as a tool to confront the current food insecurity. The strategy emphasizes the capacity of individuals to use the inherent authority of roles at any level of an organization to create change. The paper contends that individual actors, both within and without the food system, can work toward achieving more equitable outcomes in the Nation’s food system

Using a Role-Driven Race Equity Reform Approach to Mitigate the Effects of America's History of Racism on Food Insecurity

Food insecurity, or the lack of reliable access to sufficient quantities of nutritious food, affects African Americans and other minorities disproportionately. This paper examines how America’s history of racism created and sustains the Nation’s racially disparate food system. Food insecurity contributes to hunger. This paper contemplates disparities in other American systems, including education and criminal justice, as exemplars of the broader ramifications of hunger. Finally, the paper examines the potential of individual action to address problems in any system. It champions the adoption of a role-driven race equity reform strategy as a tool to confront the current food insecurity. The strategy emphasizes the capacity of individuals to use the inherent authority of roles at any level of an organization to create change. The paper contends that individual actors, both within and without the food system, can work toward achieving more equitable outcomes in the Nation’s food system

Immunofluorescence microscopy analysis of TbIP₃R.

<p>(A) TbIP₃R co-localized with TbVP1 in acidocalcisomes of PCF trypanosomes (Pearson's correlation coefficient of 0.8399). <i>Yellow</i> in merge images indicates co-localization. <i>Scale bars</i> = 10 µm. (B) Western blot analysis of TbIP₃R expressed in PCF trypanosomes using polyclonal anti-TbIP₃R antibody. Lysate containing 30 µg of protein from PCF trypanosomes was subjected to SDS/PAGE on 4–15% polyacrylamide gel, and transferred to a nitrocellulose membrane. Molecular weight markers at <i>left</i> and <i>arrow</i> shows the band corresponding to TbIP₃R.</p

Immunofluorescence microscopy and western blot analysis of V-H⁺-ATPase subunit <i>a</i> in PCF trypanosomes.

<p>Epitope-tagged V-H⁺-ATPase subunit <i>a</i> co-localizes with TbVP1 to the acidocalcisomes (A), with TbGRASP to the Golgi complex (B) with TbCATL (C) and with p67 (D) and to lysosomes (Pearson's correlation coefficients of 0.631, 0.539, 0.804, and 0.754, respectively). <i>Yellow</i> in merge images indicate co-localization (also shown with <i>arrows</i> in (B–D)). <i>Scale bars</i> for (A–D) = 10 µm. (E) Confirmation of tagging by western blot analyses with monoclonal anti-HA in PCF trypanosomes. HRP-conjugated goat anti-mouse was used as a secondary antibody. Magic Mark XP (Invitrogen) was used as a molecular weight marker and <i>arrow</i> shows band corresponding to TbVA<i>a</i>. Tubulin (Tub) was used as a loading control (<i>bottom panel</i>).</p

Distribution on iodixanol gradients of organellar markers from PCF trypanosomes.

<p>(A) Photograph showing bands obtained after the second iodixanol gradient centrifugation. Fraction 5 corresponds to the purified acidocalcisomes. (B) Protein distribution. (C) TbVP1 activity (measured as the AMDP-sensitive P_i release) is concentrated in fractions 3 and 5. (D) Mitochondrial marker distribution, succinate cytochrome c reductase. (E) Glycosomal marker distribution, hexokinase. (F) Lysosomal marker distribution, α-mannosidase. In (B–F) the <i>y-</i>axis indicates relative distribution; the <i>x-</i>axis indicates fraction number; <i>bars</i> show means ± SD (as a percentage of the total recovered activity) from two or three independent experiments. (G) SDS-PAGE of Fraction 5 from a representative acidocalcisome (ACCS) fractionation stained with Coomassie brilliant blue. The relative intensities of the bands were obtained from a bitmap file of the gel image and is shown on the right. Background was subtracted. The approximate localization of the acidocalcisome proteins identified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004555#ppat-1004555-t002" target="_blank">Table 2</a> is shown. BenchMark protein markers are shown at the left.</p