Antibody validation of immunohistochemistry for biomarker discovery: Recommendations of a consortium of academic and pharmaceutical based histopathology researchers

As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for ‘‘research use’’ continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1–3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier

Bottom current modification of turbidite lobe complexes

Submarine lobes form at the distal end of sediment gravity flow systems and are globally important sinks for sediment, anthropogenic pollutants and organic carbon, as well as forming hydrocarbon and CO2 reservoirs. Deep-marine, near bed or bottom currents can modify gravity flow pathways and sediment distribution by directly interacting with the flow or by modifying seafloor morphology. Deciphering the nature of gravity- and bottom currents interaction, particularly in ancient systems, remains a challenge due to the lack of integrated datasets and the necessary oceanographic framework. Here we analyse high-resolution 3D seismic reflection and core data from the Upper Cretaceous interval offshore Tanzania to reveal the interaction of turbidite lobes with fine-grained sediment waves and contourite drift deposits. Contourite drift morphology governs the large-scale confinement style and shape of lobes that range from frontally confined and crescent shaped, to laterally confined and elongated, to semi-confined lobes. Core data reveals massive to cross-laminated high density turbidites in the lobe axis position that show no direct interaction between gravity flows and contour currents. Lobe off-axis and fringe deposits consist of parallel- and ripple-laminated, low density turbidites, which are inter-bedded with bioturbated, muddy siltstones that represent the toes of contourite drifts. Starved ripples, and streaks of up to fine-grained sandstone above individual turbidite beds indicate reworking by bottom currents. This facies distribution reflects the temporal interaction of quasi-steady bottom currents and turbidity currents that interact with the topography and build lobes over short periods of time. Frontally confined turbidity currents form lobes in a fill-and-spill fashion, in which the confinement of turbidity currents causes rapid deposition and obscures any bottom current signal. Lateral confinement causes increased turbidity current runout length, and promotes the development of lobe fringes with a high proportion of bottom current reworked sands. During times when sediment gravity flows are subordinate, contourites accumulate on top of the lobe, confining the next flow and thus modifying the overall stacking pattern of the lobe complex. Although sediment volumes of these bottom current modified lobe complexes are comparable to other deep-marine systems, bottom currents considerably influence facies distribution and deposit architecture

Mixed axial and transverse deep-water systems: The Cretaceous post-rift Lysing Formation, offshore Norway

Deep-water stratigraphic successions from syn- to post-rift stages are an archive of evolving physiographic configurations, and can record axial and transverse sedimentary sources. The healing of topography decreases the influence of syn-rift structures on sedimentation patterns and transport processes over time, which leads to a long-term transition from dominantly axial to transverse dispersal patterns. The Halten and Dønna terraces, offshore mid-Norway, comprise a series of rift-related sub-basins established during the Jurassic, which were infilled with sediments during the Cretaceous. The Cretaceous Lysing Formation developed as slope- and basin-floor fans within a series of weakly confined post-rift sub-basins with some shallow marine deposits interpreted on the basin margins. A deep-water setting is supported by seismic interpretation, and bed type and architectural element analysis in all cored and uncored wells in the area. We document that an axial submarine fan system was active throughout the post-rift stage due to subtle inherited topography from syn-rift structures, which interacted with locally sourced transverse sediment sources. This led to a complicated stratigraphic architecture, with lobe fringe deposits of the axial fan system juxtaposed with channel-fills and channel-lobe transition zone deposits of transverse systems. The refined palaeogeographic reconstruction of the Lysing Formation illustrates how subtle topography can impact sediment routing patterns many millions of years after the end of rifting and can be used for palaeoenvironmental interpretations in other post-rift settings

Crystal structure of Cu-Sn-In alloys around the {\eta} phase field studied by neutron diffraction

The study of the Cu-Sn-In ternary system has become of great importance in recent years, due to new environmental regulations forcing to eliminate the use of Pb in bonding technologies for electronic devices. A key relevant issue concerns the intermetallic phases which grow in the bonding zone and are determining in their quality and performance. In this work, we focus in the {\eta}-phase (Cu2In or Cu6Sn5) that exists in both end binaries and as a ternary phase. We present a neutron diffraction study of the constitution and crystallography of a series of alloys around the 60 at.% Cu composition, and with In contents ranging from 0 to 25 at.%, quenched from 300\degreeC. The alloys were characterized by scanning electron microscopy, probe microanalysis and high-resolution neutron diffraction. The Rietveld refinement of neutron diffraction data allowed to improve the currently available model for site occupancies in the hexagonal {\eta}-phase in the binary Cu-Sn as well as in ternary alloys. For the first time, structural data is reported in the ternary Cu-Sn-In {\eta}-phase as a function of composition, information that is of fundamental technological importance as well as valuable input data for ongoing modelisations of the ternary phase diagram.Comment: 8 pages, 10 figure

Real World Data in Health Technology Assessment of Complex Health Technologies

The available evidence on relative effectiveness and risks of new health technologies is often limited at the time of health technology assessment (HTA). Additionally, a wide variety in real-world data (RWD) policies exist among HTA organizations. This study assessed which challenges, related to the increasingly complex nature of new health technologies, make the acceptance of RWD most likely. A questionnaire was disseminated among 33 EUnetHTA member HTA organizations. The questions focused on accepted data sources, circumstances that allowed for RWD acceptance and barriers to acceptance. The questionnaire was validated and tested for reliability by an expert panel, and pilot-tested before dissemination via LimeSurvey. Twenty-two HTA organizations completed the questionnaire (67%). All reported accepting randomized clinical trials. The most accepted RWD source were patient registries (19/22, 86%), the least accepted were editorials and expert opinions (8/22, 36%). With orphan treatments or companion diagnostics, organizations tended to be most likely to accept RWD sources, 4.3-3.2 on a 5-point Likert scale, respectively. Additional circumstances were reported to accept RWD (e.g., a high disease burden). The two most important barriers to accepting RWD were lacking necessary RWD sources and existing policy structures. European HTA organizations seem positive toward the (wider) use of RWD in HTA of complex therapies. Expanding the use of patient registries could be potentially useful, as a large share of the organizations already accepts this source. However, many barriers still exist to the widespread use of RWD. Our results can be used to prioritize circumstances in which RWD might be accepted

The Origin and 3D Architecture of a Km-Scale Deep-Water Scour-Fill: Example From the Skoorsteenberg Fm, Karoo Basin, South Africa

Scours, and scour fields, are common features on the modern seafloor of deep-marine systems, particularly downstream of submarine channels, and in channel-lobe-transition-zones. High-resolution images of the seafloor have improved the documentation of the large scale, coalescence, and distribution of these scours in deep-marine systems. However, their scale and high aspect ratio mean they can be challenging to identify in outcrop. Here, we document a large-scale, composite erosion surface from the exhumed deep-marine stratigraphy of Unit 5 from the Permian Karoo Basin succession in South Africa, which is interpreted to be present at the end of a submarine channel. This study utilizes 24 sedimentary logs, 2 cored boreholes, and extensive palaeocurrent and thickness data across a 126 km2 study area. Sedimentary facies analysis, thickness variations and correlation panels allowed identification of a lower heterolithic-dominated part (up to 70 m thick) and an upper sandstone-dominated part (10–40 m thick) separated by an extensive erosion surface. The lower part comprises heterolithics with abundant current and sinusoidal ripples, which due to palaeocurrents, thickness trends and adjacent depositional environments is interpreted as the aggradational lobe complex fringes. The base of the upper part comprises 2-3 medium-bedded sandstone beds interpreted as precursor lobes cut by a 3–4 km wide, 1–2 km long, and up to 28 m deep, high aspect ratio (1:100) composite scour surface. The abrupt change from heterolithics to thick-bedded sandstones marks the establishment of a new sediment delivery system, which may have been triggered by an updip channel avulsion. The composite scour and subsequent sandstone fill support a change from erosion- and bypass-dominated flows to depositional flows, which might reflect increasingly sand-rich flows as a new sediment route matured. This study provides a unique outcrop example with 3D stratigraphic control of the record of a new sediment conduit, and development and fill of a large-scale composite scour surface at a channel mouth transition zone, providing a rare insight into how scours imaged on seafloor data can be filled and preserved in the rock record

Molecular markers for tracking the origin and worldwide distribution of invasive strains of <i>Puccinia striiformis</i>

Investigating the origin and dispersal pathways is instrumental to mitigate threats and economic and environmental consequences of invasive crop pathogens. In the case of Puccinia striiformis causing yellow rust on wheat, a number of economically important invasions have been reported, e.g., the spreading of two aggressive and high temperature adapted strains to three continents since 2000. The combination of sequence-characterized amplified region (SCAR) markers, which were developed from two specific AFLP fragments, differentiated the two invasive strains, PstS1 and PstS2 from all other P. striiformis strains investigated at a worldwide level. The application of the SCAR markers on 566 isolates showed that PstS1 was present in East Africa in the early 1980s and then detected in the Americas in 2000 and in Australia in 2002. PstS2 which evolved from PstS1 became widespread in the Middle East and Central Asia. In 2000, PstS2 was detected in Europe, where it never became prevalent. Additional SSR genotyping and virulence phenotyping revealed 10 and six variants, respectively, within PstS1 and PstS2, demonstrating the evolutionary potential of the pathogen. Overall, the results suggested East Africa as the most plausible origin of the two invasive strains. The SCAR markers developed in the present study provide a rapid, inexpensive, and efficient tool to track the distribution of P. striiformis invasive strains, PstS1 and PstS2

Cover to Volume 3

The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth

Scalable In Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers

Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues