The Interlaboratory Robustness Of Next-Generation Sequencing (IRON) Study Phase II: Deep-Sequencing Analyses Of Hematological Malignancies Performed In 8,867 Cases By An International Network Involving 27 Laboratories

Introduction: Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8).

Aims: In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany).

Results: We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies.

Conclusion: Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far.

Genotoxic effects in occupational exposure to formaldehyde: A study in anatomy and pathology laboratories and formaldehyde-resins production

ABSTRACT – Background: According to the Report on Carcinogens, formaldehyde ranks 25th in the overall U.S. chemical production, with more than 5 million tons produced each year. Given its economic importance and widespread use, many people are exposed to formaldehyde environmentally and/or occupationally. Presently, the International Agency for Research on Cancer classifies formaldehyde as carcinogenic to humans (Group 1), based on sufficient evidence in humans and in experimental animals. Manyfold in vitro studies clearly indicated that formaldehyde can induce genotoxic effects in proliferating cultured mammalian cells. Furthermore, some in vivo studies have found changes in epithelial cells and in peripheral blood lymphocytes related to formaldehyde exposure. Methods: A study was carried out in Portugal, using 80 workers occupationally exposed to formaldehyde vapours: 30 workers from formaldehyde and formaldehyde-based resins production factory and 50 from 10 pathology and anatomy laboratories. A control group of 85 non-exposed subjects was considered. Exposure assessment was performed by applying simultaneously two techniques of air monitoring: NIOSH Method 2541 and Photo Ionization Detection equipment with simultaneously video recording. Evaluation of genotoxic effects was performed by application of micronucleus test in exfoliated epithelial cells from buccal mucosa and peripheral blood lymphocytes. Results: Time-weighted average concentrations not exceeded the reference value (0.75 ppm) in the two occupational settings studied. Ceiling concentrations, on the other hand, were higher than reference value (0.3 ppm) in both. The frequency of micronucleus in peripheral blood lymphocytes and in epithelial cells was significantly higher in both exposed groups than in the control group (p < 0.001). Moreover, the frequency of micronucleus in peripheral blood lymphocytes was significantly higher in the laboratories group than in the factory workers (p < 0.05). A moderate positive correlation was found between duration of occupational exposure to formaldehyde (years of exposure) and micronucleus frequency in peripheral blood lymphocytes (r = 0.401; p < 0.001) and in epithelial cells (r = 0.209; p < 0.01). Conclusions: The population studied is exposed to high peak concentrations of formaldehyde with a long-term exposure. These two aspects, cumulatively, can be the cause of the observed genotoxic endpoint effects. The association of these cytogenetic effects with formaldehyde exposure gives important information to risk assessment process and may also be used to assess health risks for exposed worker

Letters from the Secretary of War to the Committee of Ways and Means, in relation to the number of Militia called into the public service in 1813, to a provision for paying the bounties and premiums to soldiers lately authorized, and to the strength of the army.--

At head of title: [78].

Orderly book, Second Regiment of U.S. Dragoons, New York, 1812-1813

The Second U.S. Regiment of Light Dragoons was formed in January 1812. In March of 1814, it merged with the First U.S. Regiment of Light Dragoons to form the Regiment of Light Dragoons.

Orderly book, Second Regiment of U.S. Dragoons, New York, 1812-1813

An orderly book of the Second Regiment of U.S. Dragoons, New York, dated August 14, 1812-July 29, 1813. The book contains orders pertaining to day-to-day military matters, such as punishments for disobedience, court-martial proceedings, camp rules and regulations, and guidelines for interacting with civilians in the vicinity of the camp. The Regiment was stationed at various locations in upstate New York and Canada, including Greenbush, Albany, Sackets Harbor, Utica, Geneva, Fort Niagara and Fort George. General Henry Dearborn originally commanded the Regiment at Greenbush. Names noted in this book include:E. Beebe, Deputy Adjt. General; William King, Capt. 15th; John Chandler, General ;W. Gamewood, Major ;James Burns, Colonel;John Woodford, Major; Andrew McDowell, Capt.; Abm. Gustis, Major; C.W. Hunter, Brigade Major; Selden, Captain; Holland, Captain; Harris, Captain; Clarkson, Lieutenant; Johnson, Lieutenant; Robert Craig, Adjt.; R.G. Hith, A.A. General. Also included with the orderly book are a monthly return form, a contract for medical services, and a bonus pay voucher for Thomas Blunt. The monthly return form is partially completed and dated January 1813 at Greenbush, New York. It is signed by Captain Jonas Holland. The contract is dated May 20, 1812, between John Dodge, physician and surgeon, and Jonas Holland. The contract describes the services required of the physician and the salary to be paid. The bonus pay voucher is dated April 25, 1813, for $8.00 paid to Thomas Blunt by Captain Jonas Holland for 'enlisting into the army of the United States for five years'.

Report of the Secretary of War, communicating; in compliance witha resolution of the Senate, a map showing the operations of the army of the United States in Texas and the adjacent Mexican Stateson the Rio Grande, accompanied by astronomical observations, and descriptive and military memoirs of the country.

UANL

Remote sensing of intertidal morphological change in Morecambe Bay, U.K., between 1991 and 2007

Tidal Flats are important examples of extensive areas of natural environment that remain relatively unaffected by man. Monitoring of tidal flats is required for a variety of purposes. Remote sensing has become an established technique for the measurement of topography over tidal flats. A further requirement is to measure topographic changes in order to measure sediment budgets. To date there have been few attempts to make quantitative estimates of morphological change over tidal flat areas. This paper illustrates the use of remote sensing to measure quantitative and qualitative changes in the tidal flats of Morecambe Bay during the relatively long period 1991–2007. An understanding of the patterns of sediment transport within the Bay is of considerable interest for coastal management and defence purposes. Tidal asymmetry is considered to be the dominant cause of morphological change in the Bay, with the higher currents associated with the flood tide being the main agency moulding the channel system. Quantitative changes were measured by comparing a Digital Elevation Model (DEM) of the intertidal zone formed using the waterline technique applied to satellite Synthetic Aperture Radar (SAR) images from 1991–1994, to a second DEM constructed from airborne laser altimetry data acquired in 2005. Qualitative changes were studied using additional SAR images acquired since 2003. A significant movement of sediment from below Mean Sea Level (MSL) to above MSL was detected by comparing the two Digital Elevation Models, though the proportion of this change that could be ascribed to seasonal effects was not clear. Between 1991 and 2004 there was a migration of the Ulverston channel of the river Leven north-east by about 5 km, followed by the development of a straighter channel to the west, leaving the previous channel decoupled from the river. This is thought to be due to independent tidal and fluvial forcing mechanisms acting on the channel. The results demonstrate the effectiveness of remote sensing for measurement of long-term morphological change in tidal flat areas. An alternative use of waterlines as partial bathymetry for assimilation into a morphodynamic model of the coastal zone is also discussed.

The uncertain consequences of transferring bacterial strains between laboratories - rpoS instability as an example

Abstract Background Microbiological studies frequently involve exchanges of strains between laboratories and/or stock centers. The integrity of exchanged strains is vital for archival reasons and to ensure reproducible experimental results. For at least 50 years, one of the most common means of shipping bacteria was by inoculating bacterial samples in agar stabs. Long-term cultures in stabs exhibit genetic instabilities and one common instability is in rpoS. The sigma factor RpoS accumulates in response to several stresses and in the stationary phase. One consequence of RpoS accumulation is the competition with the vegetative sigma factor σ70. Under nutrient limiting conditions mutations in rpoS or in genes that regulate its expression tend to accumulate. Here, we investigate whether short-term storage and mailing of cultures in stabs results in genetic heterogeneity. Results We found that samples of the E. coli K-12 strain MC4100TF exchanged on three separate occasions by mail between our laboratories became heterogeneous. Reconstruction studies indicated that LB-stabs exhibited mutations previously found in GASP studies in stationary phase LB broth. At least 40% of reconstructed stocks and an equivalent proportion of actually mailed stock contained these mutations. Mutants with low RpoS levels emerged within 7 days of incubation in the stabs. Sequence analysis of ten of these segregants revealed that they harboured each of three different rpoS mutations. These mutants displayed the classical phenotypes of bacteria lacking rpoS. The genetic stability of MC4100TF was also tested in filter disks embedded in glycerol. Under these conditions, GASP mutants emerge only after a 3-week period. We also confirm that the intrinsic high RpoS level in MC4100TF is mainly due to the presence of an IS1 insertion in rssB. Conclusions Given that many E. coli strains contain high RpoS levels similar to MC4100TF, the integrity of such strains during transfers and storage is questionable. Variations in important collections may be due to storage-transfer related issues. These results raise important questions on the integrity of bacterial archives and transferred strains, explain variation like in the ECOR collection between laboratories and indicate a need for the development of better methods of strain transfer.

Compartments of the foot: topographic anatomy

Recent publications have renewed the debate regarding the number of foot compartments. There is also no consensus regarding allocation of individual muscles and communication between compartments. The current study examines the anatomic topography of the foot compartments anew using 32 injections of epoxy-resin and subsequent sheet plastination in 12 cadaveric foot specimens. Six compartments were identified: dorsal, medial, lateral, superficial central, deep forefoot, and deep hindfoot compartments. Communication was evident between the deep hindfoot compartment and the superficial central and deep central forefoot compartments. In the hindfoot, the neurovascular bundles were located in separate tissue sheaths between the central hindfoot compartment and the medial compartment. In the forefoot, the medial and lateral bundles entered the deep central forefoot compartment. The deep central hindfoot compartment housed the quadratus plantae muscle, and after calcaneus fracture could develop an isolated compartment syndrome.

Physics and Science Policy Implications of Reduction of the U.S. Nuclear Weapons Arsenal

Maintaining the readiness U.S. nuclear weapons stockpile is a fundamental pillar of American defense policy. The biggest issue facing the national laboratories that are charged with this mission is replenishing the highly skilled and specialized manpower that is necessary for this task. This lecture presents both physics and science policy questions that must be answered. It also presents employment opportunities for young scientists and engineers.

Na 1 Nachlass Max Horkheimer, 568 - Korrespondenzen u.a. mit Margot von Mendelssohn (p. VI 28, 232-393)

133 Briefe zwischen Max Horkheimer und Margot von Mendelssohn; 2 Briefe zwischen Margot von Mendelssohn und Western Defense Command and Fourth Army (San Francisco), 1942; 1 Briefe an die Wartime Civil Control (San Francisco) von Margot von Mendelssohn, 1.12.1942; 1 Brief von Max Horkheimer an die Wartime Civil Control (San Francisco), [1942]; 3 Briefe an das Police Department (West Los Angeles) von Max Horkheimer, 1942; 1 Brief von Margot von Mendelssohn an L. M. Newberry, 03.09.1942; 1 Brief an L. M. Newberry von Max Horkheimer, 31.08.1942; 1 Brief von Max Horkheimer an U. S. Attorney (Los Angeles), 16.06.1942; 1 Brief von der Hebrew Sheltering and Immigrant Aid Society (New York) an Max Horkheimer, 29.10.1941; 1 Brief an das Department of State (Washington D. C.) von F. Pollock, 30.07.1941; 1 Brief von Leo Löwenthal an das Department of State (Washington D. C.), 28.07.1941;

The influence of seawater pH on U/ Ca ratios in the scleractinian cold-water coral Lophelia pertusa

The increasing pCO2 in seawater is a serious threat for marine calcifiers and alters the biogeochemistry of the ocean. Therefore, the reconstruction of past-seawater properties and their impact on marine ecosystems is an important way to investigate the underlying mechanisms and to better constrain the effects of possible changes in the future ocean. Cold-water coral (CWC) ecosystems are biodiversity hotspots. Living close to aragonite undersaturation, these corals serve as living laboratories as well as archives to reconstruct the boundary conditions of their calcification under the carbonate system of the ocean. We investigated the reef-building CWC Lophelia pertusa as a recorder of intermediate ocean seawater pH. This species-specific field calibration is based on a unique sample set of live in situ collected L. pertusa and corresponding seawater samples. These data demonstrate that uranium speciation and skeletal incorporation for azooxanthellate scleractinian CWCs is pH dependent and can be reconstructed with an uncertainty of ±0.15. Our Lophelia U / Ca-pH calibration appears to be controlled by the high pH values and thus highlighting the need for future coral and seawater sampling to refine this relationship. However, this study recommends L. pertusa as a new archive for the reconstruction of intermediate water mass pH and hence may help to constrain tipping points for ecosystem dynamics and evolutionary characteristics in a changing ocean.