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News Article | May 17, 2017
Site: marketersmedia.com

InspireMD, Inc. (NYSE MKT: NSPR) (NYSE MKT: NSPR.WS) ("InspireMD" or the "Company"), a leader in embolic prevention systems (EPS) / thrombus management technologies and neurovascular devices, today announced the publication of the Investigator Initiated IRON-GUARD Italian clinical registry in the peer reviewed journal EuroIntervention, which appeared in the May 9th issue. IRON-GUARD was an independent, multicentre, multi-disciplinary clinical study treating patients with carotid artery disease using the CGuardTM EPS (embolic prevention system) in 12 Italian centers. The IRON-GUARD registry enrolled 200 patients, and showed results of 100% technical success placing the device, and zero incidence of major adverse cardiovascular events (MACE), comprised of death, major stroke or myocardial infarction, in all patients at 30 days. Five (2.5%) minor strokes and two transient ischemic attacks (1%) were observed, which were resolved by 30 days. Dr. Francesco Spezialie, stated, "In our multi-center, multi-specialty experience, use of the CGuard EPS in routine clinical practice was associated with no major peri-procedural neurologic complications and a total elimination of post-procedural neurologic complications after 30 days. The CGuard EPS has shown itself to be a promising treatment for carotid lesions." James Barry, PhD, Chief Executive Officer of InspireMD, commented, "We were pleased to see the results of the IRON-GUARD registry accepted and published in EuroIntervention. These results further validate the positive clinical outcomes of other trials and registries utilizing CGuardTM EPS and the data is quite consistent with those other clinical trials. To our knowledge, this now appears to be the largest registry utilizing CGuard. Following on from the very successful Paradigm 101 study, IRON-GUARD is a further high quality independent registry which continues to give us confidence in CGuard becoming the standard-of-care for treatment of carotid artery disease due to the significant safety advantages of our device." EuroIntervention is a monthly peer-reviewed journal of interventional cardiovascular medicine that has become one of the benchmarks in its field. EuroIntervention is the official journal of EuroPCR and the European Association of Percutaneous Cardiovascular Interventions (EAPCI). The Journal is endorsed by the European Society of Cardiology (ESC) and has a distinguished European and International editorial board led by Prof Patrick W. Serruys from the Erasmus MC, Rotterdam. InspireMD seeks to utilize its proprietary MicroNet™ technology to make its products the industry standard for embolic protection and to provide a superior solution to the key clinical issues of current stenting in patients with a high risk of distal embolization, no reflow and major adverse cardiac events. InspireMD intends to pursue applications of this MicroNet technology in coronary, carotid (CGuard™), neurovascular, and peripheral artery procedures. InspireMD's common stock is quoted on the NYSE MKT under the ticker symbol NSPR and certain warrants are quoted on the NYSE MKT under the ticker symbol NSPR.WS. This press release contains "forward-looking statements." Such statements may be preceded by the words "intends," "may," "will," "plans," "expects," "anticipates," "projects," "predicts," "estimates," "aims," "believes," "hopes," "potential" or similar words. Forward-looking statements are not guarantees of future performance, are based on certain assumptions and are subject to various known and unknown risks and uncertainties, many of which are beyond the Company's control, and cannot be predicted or quantified and consequently, actual results may differ materially from those expressed or implied by such forward-looking statements. Such risks and uncertainties include, without limitation, risks and uncertainties associated with (i) market acceptance of our existing and new products, (ii) negative clinical trial results or lengthy product delays in key markets, (iii) an inability to secure regulatory approvals for the sale of our products, (iv) intense competition in the medical device industry from much larger, multinational companies, (v) product liability claims, (vi) product malfunctions, (vii) our limited manufacturing capabilities and reliance on subcontractors for assistance, (viii) insufficient or inadequate reimbursement by governmental and other third party payers for our products, (ix) our efforts to successfully obtain and maintain intellectual property protection covering our products, which may not be successful, (x) legislative or regulatory reform of the healthcare system in both the U.S. and foreign jurisdictions, (xi) our reliance on single suppliers for certain product components, (xii) the fact that we will need to raise additional capital to meet our business requirements in the future and that such capital raising may be costly, dilutive or difficult to obtain and (xiii) the fact that we conduct business in multiple foreign jurisdictions, exposing us to foreign currency exchange rate fluctuations, logistical and communications challenges, burdens and costs of compliance with foreign laws and political and economic instability in each jurisdiction. More detailed information about the Company and the risk factors that may affect the realization of forward looking statements is set forth in the Company's filings with the Securities and Exchange Commission (SEC), including the Company's Annual Report on Form 10-K and its Quarterly Reports on Form 10-Q. Investors and security holders are urged to read these documents free of charge on the SEC's web site at http://www.sec.gov. The Company assumes no obligation to publicly update or revise its forward-looking statements as a result of new information, future events or otherwise.


News Article | May 17, 2017
Site: www.accesswire.com

Zero incidence of major adverse cardiovascular events including no major strokes BOSTON, MA / ACCESSWIRE / May 17, 2017 / InspireMD, Inc. (NYSE MKT: NSPR) (NYSE MKT: NSPR.WS) ("InspireMD" or the "Company"), a leader in embolic prevention systems (EPS) / thrombus management technologies and neurovascular devices, today announced the publication of the Investigator Initiated IRON-GUARD Italian clinical registry in the peer reviewed journal EuroIntervention, which appeared in the May 9th issue. IRON-GUARD was an independent, multicentre, multi-disciplinary clinical study treating patients with carotid artery disease using the CGuardTM EPS (embolic prevention system) in 12 Italian centers. The IRON-GUARD registry enrolled 200 patients, and showed results of 100% technical success placing the device, and zero incidence of major adverse cardiovascular events (MACE), comprised of death, major stroke or myocardial infarction, in all patients at 30 days. Five (2.5%) minor strokes and two transient ischemic attacks (1%) were observed, which were resolved by 30 days. Dr. Francesco Spezialie, stated, "In our multi-center, multi-specialty experience, use of the CGuard EPS in routine clinical practice was associated with no major peri-procedural neurologic complications and a total elimination of post-procedural neurologic complications after 30 days. The CGuard EPS has shown itself to be a promising treatment for carotid lesions." James Barry, PhD, Chief Executive Officer of InspireMD, commented, "We were pleased to see the results of the IRON-GUARD registry accepted and published in EuroIntervention. These results further validate the positive clinical outcomes of other trials and registries utilizing CGuardTM EPS and the data is quite consistent with those other clinical trials. To our knowledge, this now appears to be the largest registry utilizing CGuard. Following on from the very successful Paradigm 101 study, IRON-GUARD is a further high quality independent registry which continues to give us confidence in CGuard becoming the standard-of-care for treatment of carotid artery disease due to the significant safety advantages of our device." EuroIntervention is a monthly peer-reviewed journal of interventional cardiovascular medicine that has become one of the benchmarks in its field. EuroIntervention is the official journal of EuroPCR and the European Association of Percutaneous Cardiovascular Interventions (EAPCI). The Journal is endorsed by the European Society of Cardiology (ESC) and has a distinguished European and International editorial board led by Prof Patrick W. Serruys from the Erasmus MC, Rotterdam. InspireMD seeks to utilize its proprietary MicroNet™ technology to make its products the industry standard for embolic protection and to provide a superior solution to the key clinical issues of current stenting in patients with a high risk of distal embolization, no reflow and major adverse cardiac events. InspireMD intends to pursue applications of this MicroNet technology in coronary, carotid (CGuard™), neurovascular, and peripheral artery procedures. InspireMD's common stock is quoted on the NYSE MKT under the ticker symbol NSPR and certain warrants are quoted on the NYSE MKT under the ticker symbol NSPR.WS. This press release contains "forward-looking statements." Such statements may be preceded by the words "intends," "may," "will," "plans," "expects," "anticipates," "projects," "predicts," "estimates," "aims," "believes," "hopes," "potential" or similar words. Forward-looking statements are not guarantees of future performance, are based on certain assumptions and are subject to various known and unknown risks and uncertainties, many of which are beyond the Company's control, and cannot be predicted or quantified and consequently, actual results may differ materially from those expressed or implied by such forward-looking statements. Such risks and uncertainties include, without limitation, risks and uncertainties associated with (i) market acceptance of our existing and new products, (ii) negative clinical trial results or lengthy product delays in key markets, (iii) an inability to secure regulatory approvals for the sale of our products, (iv) intense competition in the medical device industry from much larger, multinational companies, (v) product liability claims, (vi) product malfunctions, (vii) our limited manufacturing capabilities and reliance on subcontractors for assistance, (viii) insufficient or inadequate reimbursement by governmental and other third party payers for our products, (ix) our efforts to successfully obtain and maintain intellectual property protection covering our products, which may not be successful, (x) legislative or regulatory reform of the healthcare system in both the U.S. and foreign jurisdictions, (xi) our reliance on single suppliers for certain product components, (xii) the fact that we will need to raise additional capital to meet our business requirements in the future and that such capital raising may be costly, dilutive or difficult to obtain and (xiii) the fact that we conduct business in multiple foreign jurisdictions, exposing us to foreign currency exchange rate fluctuations, logistical and communications challenges, burdens and costs of compliance with foreign laws and political and economic instability in each jurisdiction. More detailed information about the Company and the risk factors that may affect the realization of forward looking statements is set forth in the Company's filings with the Securities and Exchange Commission (SEC), including the Company's Annual Report on Form 10-K and its Quarterly Reports on Form 10-Q. Investors and security holders are urged to read these documents free of charge on the SEC's web site at http://www.sec.gov. The Company assumes no obligation to publicly update or revise its forward-looking statements as a result of new information, future events or otherwise.


News Article | November 17, 2016
Site: www.prnewswire.co.uk

Publikation im Journal of Clinical Virology demonstriert Effektivität und einfache Handhabung für Routinetests AUSTIN, Texas, 17. November 2016 /PRNewswire/ -- Die Luminex Corporation (NASDAQ: LMNX) gab heute bekannt, dass die international renommierte Abteilung für Virologie im Erasmus Medical Center in Rotterdam, Niederlande, das ARIES® System und Flu A/B & RSV CE-IVD Assay für klinische Tests in einer kürzlich veröffentlichten Studie evaluiert hat. Das Erasmus MC ist ein Konsultationszentrum zu viralen Infektionen für die Weltgesundheitsorganisation und dient ebenfalls in den Niederlanden als nationales Referenzzentrum für Influenza und aufkommende Infektionen. In der Studie zeigten die ARIES® Plattform und das Flu A/B & RSV Assay eine hohe klinische Spezifizität und Sensitivität, die mit laborentwickelten RT-PCR Assays vergleichbar sind, sowie bessere Ergebnisse in diesem Zusammenhang als jene von etablierten Assays, wie z. B. immunchromatographische Tests und direkte Immunfluoreszenz-Tests. Die Anwenderfreundlichkeit ist laut Studie mit anderen schnellen molekularen Testplattformen gleichzusetzen. Die ARIES® Plattform ist insofern einzigartig, da man mit ihr ebenfalls intern entwickelte Assays in Echtzeit mit generischen ARIES® Kassetten durchführen kann. „Wir sind sehr stolz, dass eines der führenden medizinischen Zentren für klinische Virologie nach einer sorgfältigen Beurteilung die Effektivität und leichte Handhabung von ARIES festgestellt hat", sagte Thomas Pracht, Managing Director für EMEIA bei der Luminex Corporation. „Die Erasmus-Studie zeigt die Qualität und Leistung der ARIES® Plattform und den Nutzen des Systems für Molekulardiagnose-Labore, die MDx-Testverfahren „außerhalb des Takts" durchführen wollen – in anderen Worten, außerhalb der offiziellen Dienstzeiten für Tests oder beispielsweise Statistiktests zwischen den Testläufen während der offiziellen Dienstzeiten im Labor. Die Studie unterstreicht ebenfalls die Bedeutung der Tests mit Direktauswirkung, bei denen die Resultate zu unmittelbaren Folgen für die Patientenbehandlung und die klinische Entscheidungsfindung führen – selbst außerhalb der normalen Dienstzeiten." Lesen Sie die komplette Studie „Performance evaluation of a rapid molecular diagnostic, MultiCode based, sample-to-answer assay for the simultaneous detection of Influenza A, B and respiratory syncytial viruses", veröffentlicht im Journal of Clinical Virology, Dezember 2016, Volume 85, Seiten 65-70. Über die Luminex Corporation Luminex hat sich das Ziel gesetzt, Labore bei der Suche nach zuverlässigen, zeitnahen und umsetzbaren Antworten zu unterstützen, um letztlich zur Förderung der Gesundheit beizutragen. Wir bieten ein breites Spektrum von Lösungen, die in verschiedenen Märkten eingesetzt werden können, unter anderem in den Bereichen klinische Diagnostik, pharmazeutische Wirkstoffentwicklung, Genom- und Proteomikforschung, biologische Abwehrforschung und Lebensmittelsicherheit. Unser Unternehmen stellt Antworten schneller bereit, vereinfacht gleichzeitig die Komplexität und verschafft sichere Erkenntnisse auf der Grundlage einer nahtlosen Anwendererfahrung. Weitere Informationen über Luminex erhalten Sie, wenn Sie luminexcorp.com aufrufen. Aussagen in dieser Pressemitteilung, welche die Absichten, Pläne, Überzeugungen, Erwartungen oder Prognosen von Luminex bzw. der Geschäftsführung von Luminex zu zukünftigen Ereignissen ausdrücken, sind sogenannte vorausschauende Aussagen. Zu den vorausschauenden Aussagen in dieser Pressemitteilung gehören Aussagen über die Entwicklung und die Prüfungsfortschritte unserer Pipeline-Produkte sowie über deren regulatorische Zulassung. Begriffe wie „glauben", „erwarten", „beabsichtigen", „prognostizieren", „überzeugt", „werden", „könnten", „sollten" und ähnliche Ausdrücke weisen auf derartige vorausschauende Aussagen im Sinne des Private Securities Litigation Reform Act von 1995 hin und machen sie kenntlich. In diesem Zusammenhang wird ausdrücklich darauf hingewiesen, dass sich die tatsächlichen Ergebnisse oder Leistungen des Unternehmens wesentlich von jenen unterscheiden könnten, die in solchen vorausschauenden Aussagen prognostiziert werden. Zu den Faktoren, die dazu führen könnten, dass die tatsächlichen Ergebnisse oder Leistungen von Luminex erheblich abweichen, gehören Risiken und Unwägbarkeiten u. a. im Zusammenhang mit unserer Fähigkeit, Produkte zeitnah auf dem Markt einzuführen, mit dem zeitlichen Ablauf regulatorischer Zulassungen und dem Ergebnis klinischer Studien ebenso wie die im Abschnitt „Risikofaktoren" in den von Luminex bei der Securities and Exchange Commission auf den Formularen 10-K und 10-Q eingereichten und erörterten Risiken. Die hier enthaltenen vorausschauenden Aussagen geben die Einschätzung von Luminex zum Zeitpunkt der Veröffentlichung dieser Pressemitteilung wieder und Luminex lehnt ausdrückliche jede Absicht, Verpflichtung oder Zusicherung ab, öffentlich Aktualisierungen oder Berichtigungen an vorausschauenden Aussagen vorzunehmen, um Änderungen der Erwartungen von Luminex in diesem Zusammenhang oder Änderungen aufgrund von Ereignissen, Bedingungen oder Umständen, auf denen vorausschauenden Aussagen basieren, widerzuspiegeln.


News Article | December 9, 2016
Site: www.eurekalert.org

20 to 40 percent of the patients with multiple myeloma - a type of leukaemia - have a defect in the ribosome, the protein factory of the cell. These patients have a poorer prognosis than patients with intact ribosomes. At the same time, they respond better to a drug that already exists. These are the findings of a study by the Laboratory for Disease Mechanisms in Cancer at KU Leuven (University of Leuven), Belgium. Multiple myeloma (MM, also known as Kahler's disease) is a blood cancer whereby the plasma cells in the bone marrow start proliferating malignantly. MM cannot be cured and is most common among older people. Various treatments exist to temporarily suppress the disease, but the challenge is determining to which treatment the patient will respond best. Doctoral student Isabel Hofman (KU Leuven) discovered defects in the ribosome of MM patients. "The ribosome is the protein factory of a cell. In MM patients, one part of the ribosome is produced less in 20 to 40 percent of the patients, depending on how aggressive the cancer is. We suspect that their cells are still producing protein, but that the balance is somewhat disrupted. In any case, we found that these people have a poorer prognosis than MM patients with an intact ribosome," explains Professor Kim De Keersmaecker, head of the KU Leuven Laboratory for Disease Mechanisms in Cancer. One possible treatment for MM is the use of proteasome inhibitors. "The proteasome is the protein demolition machine in a cell. There's a type of drugs, including Bortezomib, that inhibits its functioning. How the defects in the ribosome influence the proteasome is not quite clear yet. But we discovered that patients with a defective ribosome respond better to Bortezomib. In other words, their poorer prognosis can be offset by this treatment. On the basis of these findings, we can now develop tests to identify defects in the ribosome and thus determine which therapy will have most effect in a specific patient." The notion that cancer is related to ribosome defects is a relatively new concept in science. "A few years ago, we discovered defects in the ribosome of patients with acute lymphatic leukaemia. Now we know that the same applies to MM. In all likelihood, this will also hold true for other types of cancer. Our next research goal is finding out for which cancers this is the case, how the link between ribosome and proteasome works, and what the possibilities are of drugs that target the ribosome itself." This study is a collaboration between KU Leuven, University Hospitals Leuven, and the Erasmus MC Rotterdam, among others.


INGELHEIM, Germany--(BUSINESS WIRE)--New results from a global survey of over 150 people living with idiopathic pulmonary fibrosis (IPF), a life-threatening rare lung disease, demonstrate that while many patients were involved in treatment decisions, opportunities exist to improve the level of dialogue between patients and healthcare professionals. The new findings from the survey, supported by Boehringer Ingelheim, are released to coincide with Rare Disease Day whose theme in 2017 is ‘research’. Encouragingly, the results show that 2 out of 3 patients (64%) were involved in the decision-making process for their IPF treatment.1 However, one out of every three patients (30%) said they were not involved in treatment discussions at all.1 " It is vital that IPF patients are actively involved in the treatment decisions that affect them,” said Liam Galvin, Secretary of the European Idiopathic Pulmonary Fibrosis & Related Disorders Federation (EU-IPFF). “ Open discussion about the lifestyle needs and priorities of patients is key to making the right choice of therapy that can slow the progression of IPF and other options that could assist with its symptoms and management.” The new findings expand on insights from a 2015 global survey of over 400 pulmonologists. Analysis of the results shows that whilst patients and physicians agreed on one of the key treatment priorities – to maintain lung function for as long as possible – patient and physician views on other priorities regarding IPF treatment differed.1,2 In addition to maintaining lung function, patients identified reducing the risk of the condition suddenly worsening and being on a medication with manageable side-effects among their top priorities for IPF treatment.1,2 However, physicians cited being on a treatment that allows patients to continue day-to-day activities as normally as possible and being on an effective treatment independent of the stage of disease as their view of patients’ top priorities for IPF treatment besides maintaining lung function for as long as possible.2 This variation in what is seen as most important by patients and physicians highlights the benefit of open dialogue to compare these potentially competing priorities. The new survey findings also highlight that as many as 1 in every 3 patients involved in the decision-making process (30%) said that in coming to an agreement about appropriate medical treatment for their IPF, further discussion about treatment choice was not really needed and they strictly followed their doctor or nurse’s treatment recommendation.1 Furthermore, only 40% of those involved said that they were “actively” involved in the decision making-process before agreeing to a certain treatment.1 "IPF can make a patient feel their world has turned upside-down, and they may want to leave treatment decisions to their doctor,” said Dr. Marlies Wijsenbeek, pulmonologist, Erasmus MC, The Netherlands. “It is important that patients engage in discussion. This provides their doctor with relevant information so that the right decision is made at the right time on the appropriate treatment option that best suits their needs. This applies both for medication as well as other non-medical support that may be of benefit.” The global survey of 152 patients was designed to provide insights into IPF and the realities of living with the condition. These new findings have been released to support the 2017 Rare Disease Day theme of ‘research’. As a leader in medical research, Boehringer Ingelheim is committed to transforming fibrosing lung diseases from fatal diseases to chronic, treatable ones. As part of this commitment, Boehringer Ingelheim is currently enrolling patients to participate in the SENSCIS™ study – (Safety and Efficacy of Nintedanib in Systemic SClerosIS), which is the largest trial to date in people with systemic sclerosis who have also developed interstitial lung disease (SSc-ILD). Please click on the link below for ‘Notes to Editors’ and ‘References’: This press release is issued from our Corporate Headquarters in Ingelheim, Germany and is intended to provide information about our global business.


News Article | October 12, 2016
Site: www.nature.com

No sample-size estimate was calculated before the study was executed. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Endoscopic, colorectal and duodenal biopsy samples were obtained from individuals of different ages that had been admitted for suspected inflammation. One individual (donor 1) showed no inflammation during colonoscopy, but was later diagnosed with microscopic colitis. The other individuals were found to be healthy based on standard histological examination. Endoscopic biopsies were performed at the University Medical Center Utrecht and the Wilhelmina Children’s Hospital. The patients’ informed consent was obtained and this study was approved by the ethical committee of University Medical Center Utrecht. Additionally, normal tissue was isolated from resected colon segments at >5 cm distance from a tumour in three colorectal cancer patients (donors 3, 4 and 19). The colonic tissues were obtained at The Diakonessen Hospital Utrecht with informed consent and the study was approved by the ethical committee. Liver biopsies (0.5–1 cm3) were obtained from donor livers during transplantations performed at the Erasmus Medical Center, Rotterdam. Both liver and colon biopsies were obtained from donor 18. The Medical Ethical Council of the Erasmus MC approved the use of this material for research purposes, and informed consent was provided by all donors and/or relatives. Dissociated colon and small intestinal crypts were isolated from the biopsies and cultured for 1 - 2 weeks under conditions that are optimal for stem-cell proliferation, as previously described5. Liver cells were isolated from human liver biopsies and cultured as previously described3. From these cultures, single cells were sorted by flow cytometry and clonally expanded (Extended Data Fig. 1a). Clonal ASC cultures were subsequently established by manual picking of individual organoids derived from single cells and in vitro expansion for a period of ~6 weeks. DNA libraries for Illumina sequencing were generated using standard protocols (Illumina) from 200 ng - 1 μg of genomic DNA isolated from the clonally expanded ASC cultures with genomic tips (Qiagen). The libraries were sequenced with paired-end (2 × 100 bp) runs using Illumina HiSeq 2500 sequencers to a minimal depth of 30× base coverage. Samples of donors 1, 2, 3, 4, 10, 12, 13, 15, 16, 18 and 19 were sequenced using Illumina HiSeq X Ten sequencers to equal depth. The reference samples, blood or biopsy, were sequenced similarly. Sequence reads were mapped against human reference genome GRCh37 using Burrows–Wheeler Aligner v0.5.9 mapping tool27 with settings ‘bwa mem -c 100 –M’. Sequence reads were marked for duplicates using Sambamba v0.4.7 (ref. 28) and realigned per donor using Genome Analysis Toolkit (GATK) IndelRealigner v2.7.2 and sequence read-quality scores were recalibrated with GATK BaseRecalibrator v2.7.2. Alignments from different libraries of the same ASC culture were combined into a single BAM file. Raw variants were multi-sample (per donor) called using the GATK UnifiedGenotyper v2.7.2 (ref. 29) and GATK-Queue v2.7.2 with default settings and additional option ‘EMIT_ALL_CONFIDENT_SITES’. The quality of variant and reference positions was evaluated using GATK VariantFiltration v2.7.2 with options ‘–filterExpression “MQ0 ≥4 && ((MQ0 / (1.0 * DP)) > 0.1)”–filterName “HARD_TO_VALIDATE”–filterExpression “QUAL < 30.0 “–filterName “VeryLowQual”–filterExpression “QUAL > 30.0 && QUAL < 50.0 “–filterName “LowQual”–filterExpression “QD < 1.5 “–filterName “LowQD”’. To obtain high-quality catalogues of somatic point mutations, we applied a comprehensive filtering procedure (Extended Data Fig. 1b). We considered variants that were passed by VariantFiltration and had a GATK phred-scaled quality score ≥100. Subsequently, for each ASC culture, we considered the positions with a base coverage of at least 20× in both the culture and the reference sample (blood or biopsy). Furthermore, we only regarded variants at autosomal chromosomes. We excluded variant positions that overlapped with single-nucleotide polymorphisms (SNPs) in the SNP database (dbSNP) v137.b37 (ref. 30). Furthermore, we excluded all positions that were found to be variable in at least two of three unrelated individuals (that is, donor 5, 6 and X (not in study)) to exclude recurrent sequencing artefacts. To obtain somatic point mutations, we filtered out all variants with any evidence of the alternative allele in the reference sample. We validated the clonal origin of the sequenced ASC cultures by analysing the variant allele frequencies (VAFs) of the somatic mutations. Two cultures (donor 14, cell b and donor 17, cell c) showed a shift in the peak of the somatic heterozygous mutations to the left, indicating that they did not arise from a single stem cell, and were therefore excluded from the analysis (Extended Data Fig. 2). Finally, for all cultures we excluded point mutations with a VAF < 0.3 to exclude mutations that were potentially induced in vitro after the (first) clonal step (Extended Data Fig. 1b–d). The number of mutations that passed each filtering step for the samples of donor 5 and 6 is depicted in Extended Data Fig. 1c. The overlap of the point mutations between ASCs of the same donor is depicted in Extended Data Fig. 4d. We evaluated our mutation filtering procedure by independent validations of 374 pre-selected positions that were either discarded or passed during filtering using amplicon-based next-generation sequencing. To this end, primers were designed ~250 nucleotides 5′ and 3′ from the candidate point mutations to obtain amplicons of ~500 bp (primer sequences available upon request). These regions were PCR-amplified for both the organoid cultures and reference samples of donor 5 and 6, using 5 ng genomic DNA, 1× PCR Gold Buffer (Life Technologies), 1.5 mM MgCl , 0.2 mM of each dNTP and 1 unit of AmpliTaq Gold (Life Technologies) in a final volume of 10 μl. This which was held at 94 °C for 60 s followed by 15 cycles at 92 °C for 30 s, 65 °C for 30 s (with a decrement of 0.2 °C per cycle) and 72 °C for 60 s; followed by 30 cycles of 92 °C for 30 s, 58 °C for 30 s and 72 °C for 60 s; with a final extension at 72 °C for 180 s. The PCR products were pooled and barcoded per culture. Illumina sequence libraries were generated according to the manufacturer’s protocol. Subsequently, the libraries were pooled and sequenced using the MiSeq platform (2 × 250 bp) to an average depth of ~100×. Alignment and variant-calling was performed as described above. For each ASC we evaluated those positions with at least 20× coverage for both culture and reference sample, and defined positive positions as those with a call in culture, with a VAF ≥ 0.3 and no call in the reference sample. Subsequently, we determined the number of confirmed negatives of the positions that were filtered out for each filter step (Extended Data Fig. 1d). Moreover, we determined the number of confirmed positive of the positions that passed all filters (Extended Data Fig. 1e, f). We expanded 10 initial clonal organoid cultures from small intestine and liver for a further 3–5 months (equivalent to ~20 weekly passages), upon which we isolated single cells and subjected them to clonal expansion to obtain sufficient DNA for WGS (Extended Data Fig. 6a). This approach allowed us to catalogue the mutations that accumulated in single ASCs during the culturing period between the two clonal steps. To this end, we selected the somatic point mutations that were unique to the sub-clonal cultures and not present in the corresponding original clonal cultures and therefore acquired during the in vitro expansion. We evaluated the specificity of our mutation-discovery procedure by determining the confirmation rate of the mutations identified in the original clone in the corresponding subclone. Only positions that had a coverage of ≥20× in both the original clonal and corresponding subclonal culture as well as in the reference sample were evaluated. On average, 91.1% ± 4.87 (mean ± s.d). of these point mutations were confirmed in the subclonal cultures (Extended Data Fig. 3). The surveyed area per ASC was calculated as the number of positions coverage ≥20× in both culture and the reference sample. The percentage of the whole non-N autosomal genome (GCRh37: 2,682,655,440 bp) that is surveyed in each ACS is depicted in Extended Data Table 1. For each ASC the total number of identified somatic point mutations was extrapolated to the whole non-N autosomal genome using its surveyed area. Subsequently, a linear mixed-effects regression model was fitted to estimate the effect of age on the number of somatic point mutations for each tissue using the nlme R package31, 32, in which ‘donor’ is modelled as a random effect to resolve the non-independence that results from having multiple measurements per donor. A two-tailed t-test was performed to test whether the slope is significantly different from zero (that is to say, whether the fixed age effect in the linear mixed model is statistically significant). The intercept of the regression lines with the y axis represents the somatic mutations present at birth (that have accumulated in the tissue lineage during prenatal development) plus the noise levels in the data and the mutations that have accumulated during the first week(s) of culturing proceeding the clonal step (see above). Since all cells were assessed in a similar manner, noise levels will be comparable and therefore will not bias the mutation rate (slope) estimates. The slope of the regression line was used to estimate the fixed age effect on somatic point mutation rate per tissue. To exclude the possibility that differences in surveyed areas between ASCs bias our results, we performed the age correlation and spectrum analyses on a subset of mutations that are located in genomic regions that are surveyed (≥20×) in all samples in this study. This consensus surveyed area comprises 38.2% of the autosomal non-N genome and both the mutation rate and spectra were highly similar to those in Fig. 1c (Extended Data Fig. 4a–c), indicating that the differences in surveyed areas between the clones do not bias our conclusions. To generate a conserved DNA replication timing profile for the human genome, we downloaded 16 Repli-seq data sets from the ENCODE project33 at the University of California, SantaCruz (UCSC) genome browser34 (GRCh37/hg19). The data consisted of Wavelet-smoothed values per 1-kb bin throughout the genome for 15 different cell lines (BJ, BG02ES, GM06990, GM12801, GM12812, GM12813, GM12878, HeLa-S3, HepG2, HUVEC, IMR90, K562, MCF-7, NHEK and SK-N-SH). We considered the median values of all cell lines per bin, thereby excluding cell-specific values. We arbitrarily divided the genome into early- (≥60), intermediate- (>33 & <60) and late- (≤33) replicating bins (Fig. 3b). To generate a conserved chromatin-association profile for the human genome, we downloaded data containing the H3K9me3 signal per 25-nucleotide bin throughout the genome for 22 different cell lines (A549, AG04450, DND41, GM12878, H1-hESC, HeLa-S3, HepG2, HMEC, HSMM, HSMMt, HUVEC, K562, monocytes-CD14+_RO1746, NH-A, NHDF-Ad, NHEK, NHLF, osteoblasts, MCF-7, NT2-D1, PBMC and U2OS) and the H3K27ac signal for 9 different cell lines (CD20+_RO01794, DND41, H1-hESC, HeLa-S3, HSMM, monocytes-CD14+_RO1746, NH-A, NHDF and osteoblasts). Data were downloaded from the ENCODE project33 at the UCSC browser34 (GRCh37/hg19) and the median values of all cell lines per bin were calculated. Next, we determined the distribution of the fractions of all bins (genome-wide). According to the shape of the resulting graph, we considered bins with an H3K9me3 value ≥4, or an H3K27ac value ≥2, as associated with that chromatin mark. Finally, exonic sequences were defined as all exonic regions reported in Ensembl v75 (GCRh37)35. We determined whether somatic point mutations were enriched or depleted in the genomic regions described above. To this end, we determined how many point mutations were observed in each genomic region for each donor. Next, we calculated the number of bases that were surveyed in each genomic region and calculated the expected number of point mutations by multiplying this surveyed length with the genome-wide point-mutation frequency. The log (observed/expected) of the mutations in the genomic regions was used as a measure of the effect size of the depletion or enrichment. One-tailed binomial tests were performed to calculate the statistical significance of deviations from the expected number of mutations in the genomic regions using pbinom31; P < 0.05 was considered significant. The occurrences of all 96-trinucleotide changes were counted for each ASC and averaged per donor. Three mutational signatures were extracted using NMF36. To determine the replication bias of signatures, we determined whether the point mutations were located in an intermediate, early or late replicating region (as defined above) using GenomicRanges37 and repeated the NMF on a 288 count matrix (96 trinucleotides × 3 replication timing regions). Similarly, we looked at transcriptional strand bias by performing NMF on a 192 count matrix (96 trinucleotides × 2 strands). To this end, we selected all point mutations that fall within gene bodies and checked whether the mutated C or T was located on the transcribed or non-transcribed strand. We defined the transcribed units of all protein coding genes based on Ensembl v75 (GCRh37)35 and included introns and untranslated regions. The dN/dS ratio was determined as described previously18. In brief, we used 192 rates, one for each of the possible trinucleotide changes in both strands. For each substitution type, we counted the number of potential synonymous and non-synonymous mutations in the protein-coding sequences of the human genome, using the longest DNA coding sequence as the reference sequence for each gene. Poisson regression was used to obtain maximum-likelihood estimates and confidence intervals of the normalized ratio of non-synonymous versus synonymous mutations (dN/dS ratio). The dN/dS ratio was tested against neutrality (dN/dS = 1) using a likelihood-ratio test. Intestinal ASCs were isolated from the proximal part of the small intestine of randomly chosen ~2-year-old mice (one male and one female) carrying the Lgr5-EGFP-Ires-CreERT2 allele (mice were C57BL/6 background) by sorting for GFPhigh cells. Subsequently, three Lgr5-positive cells per animal were clonally expanded as described4. All experiments were approved by the Animal Care Committee of the Royal Dutch Academy of Sciences according to the Dutch legal ethical guidelines. DNA isolated from the intestinal ASC cultures isolated from mouse 1 were sequenced with paired-end (75 and 35 bp) runs using SOLiD 5500 sequencers (Life Technologies) to an average depth of ~18× base coverage. Intestinal ASC cultures of mouse 2 were sequenced using Illumina HiSeq 2500 sequencers as described above. Sequence reads were aligned using Burrows–Wheeler Aligner to the mouse reference genome (NCBIM37) and point mutations were called using the GATK UnifiedGenotyper v2.7.2 as described above. Post-processing filters for the intestinal ASCs of mouse 1 (analysed by SOLiD sequencing) were as follows: a minimum depth of 10×, variant uniquely called in one intestinal stem cell without more than one alternative allele found at the same position in the other ASCs of the same mouse, a GATK a phred-scaled quality score ≥100, variant absent in mouse 2, variant position absent in the dbSNP (build 128) and a VAF ≥ 0.25. Post-processing filters for the intestinal ASCs of mouse 2 (analysed by Illumina sequencing) were as described above for the human mutation data. Mutations identified in the indicated genes in colorectal or liver cancers were downloaded from cBioPortal (http://www.cbioportal.org/). Only point mutations that resulted in a missense, nonsense or splice-site mutation were considered. To detect copy-number variations (CNVs), BAM files were analysed for read-depth variations by CNVnator v0.2.7 (ref. 39) with a bin size of 1 kb and Control-FREEC v6.740 with a bin size of 5 kb. Highly variable regions, defined as harbouring germline CNVs in at least three control samples, were excluded from the analysis. To obtain somatic CNVs, we excluded CNVs for which there was evidence in the reference sample (blood/biopsy) of the same individual. Resulting candidate CNV regions were assessed for additional structural variants on the paired-end and split-read level through DELLY v0.3.3 (ref. 41). Based on these results, we excluded five candidate CNV regions as mapping artefacts on the read-depth level and acquired base-pair accuracy of the involved breakpoints for the other events. This also revealed the tandem orientation of the duplication events and the complex structural variation in the colon sample. Reported gene definitions (Extended Data Table 2) are based on Ensembl v75 (GCRh37)35. Common fragile sites overlapping the events were detected using existing definitions42. LINE/SINE elements within 100 bp of the breakpoints were determined with the repeat element annotation43 from the UCSC genome browser34 GCRh37 (retrieved 26 October 2015). All code and filtered vcf files are freely available under a MIT License at https://wgs11.op.umcutrecht.nl/mutational_patterns_ASCs/ and https://github.com/CuppenResearch/MutationalPatterns/.


News Article | December 7, 2016
Site: www.prweb.com

Levels of a protein in the blood associated with heart disease are also linked to early-stage brain damage, according to a study appearing online in the journal Radiology. Heart disease and brain disease exact a major burden on society, and the incidence is expected to increase significantly due to the rapidly aging population. Damage to both organs often occurs at a subclinical stage, or before signs and symptoms of disease are evident. A substance, or marker, in the blood indicative of subclinical heart disease and brain diseases like stroke and dementia could speed the initiation of treatments and lifestyle changes, potentially slowing or even reversing the disease’s course. One promising marker is N-terminal Pro-B-type natriuretic peptide (NT-proBNP), a protein released into the blood in response to cardiac wall stress. Blood serum levels of NT-proBNP rise when heart failure worsens and drop when it gets better. While previous studies have shown a link between heart disease and brain disease, less is known about the association between NT-proBNP and the entire spectrum of imaging markers of subclinical brain damage, like brain volume and white matter integrity. Researchers from the Netherlands recently investigated this association in 2,397 community-dwelling middle-aged and elderly non-demented people without a clinical diagnosis of heart disease. The patients were drawn from the landmark Rotterdam Study, an ongoing, population-based study of more than 10,000 people from a suburb of Rotterdam, the Netherlands. When the researchers compared serum levels of NT-proBNP with MRI findings, they discovered a clear association between higher NT-proBNP levels and brain damage. “We found that higher serum levels of NT-proBNP were associated with smaller brain volumes, in particular with smaller gray matter volume, and with poorer organization of the brain’s white matter,” said Meike W. Vernooij, M.D., Ph.D., the study’s lead author and a neuroradiologist at Erasmus MC University Medical Center in Rotterdam. The findings imply a close link between the heart and brain even in presumably healthy individuals, Dr. Vernooij said. There are several hypotheses to explain the link between cardiac dysfunction and subclinical brain damage, according to Dr. Vernooij. For instance, decreases in blood flow could lead to cerebral microvascular damage or problems in the function of the blood-brain barrier, a network of blood vessels that allow essential nutrients into the brain while blocking potentially harmful substances. Inflammatory factors associated with cardiac stress could also harm the barrier, leading to increased permeability and damage to the brain. While NT-proBNP is currently used in a clinical setting to rule out heart failure, it is too early to say if it can play a similar role for subclinical brain damage, as the new study only looked at people at one point in time. “We cannot rule out that the observed subclinical brain damage led to increased levels of NT-proBNP,” Dr. Vernooij said. “However, from a biological perspective, and based on animal studies, it is more likely that cardiac dysfunction affects brain changes rather than vice versa.” Further research, including follow-up brain MRI studies and measurements of NT-proBNP, will be needed to clarify the relationship between cardiac dysfunction and subclinical brain disease, the researchers said. “N-Terminal Pro-B-Type Natriuretic Peptide and Subclinical Brain Damage in the General Population.” Collaborating with Dr. Vernooij were Hazel I. Zonneveld, M.D., M. Arfan Ikram, M.D., Ph.D., Albert Hofman, M.D., Ph.D., Wiro J. Niessen, Ph.D., Aad van der Lugt, M.D., Ph.D., Gabriel P. Krestin, M.D., Ph.D., and Oscar H. Franco, M.D., Ph.D. Radiology is edited by Herbert Y. Kressel, M.D., Harvard Medical School, Boston, Mass., and owned and published by the Radiological Society of North America, Inc. (http://radiology.rsna.org/) RSNA is an association of more than 54,000 radiologists, radiation oncologists, medical physicists and related scientists promoting excellence in patient care and health care delivery through education, research and technologic innovation. The Society is based in Oak Brook, Ill. (RSNA.org)


News Article | April 29, 2016
Site: www.biosciencetechnology.com

Researchers reporting in the Cell Press journal Current Biology on April 28 have found a gene that helps explain why some people appear more youthful than others. The gene in question, known as MC1R, is already well known for producing red hair and pale skin. Now, it appears that variation in this same gene is also related to differences in how old people look to other people--their perceived age. People carrying particular MC1R variants in their DNA look, on average, almost 2 years older than they are. "For the first time, a gene has been found that explains in part why some people look older and others younger for their age," said Manfred Kayser of Erasmus MC University Medical Center Rotterdam in the Netherlands. Earlier studies had shown that a person's perceived age is influenced by a combination of genetic and environmental factors in roughly equal parts. Interestingly, perceived age has also been shown to predict a person's health and mortality, suggesting that the age we perceive a person to be from the appearance of their face might also be related in important ways to a person's biological age and health. To further investigate in the new study, Kayser and his colleague David Gunn at the UK- and Netherlands-based company Unilever searched the genomes of more than 2,600 elderly Dutch Europeans from the Rotterdam Study for DNA variants associated with differences in perceived facial age and wrinkling as estimated from digital facial images. The strongest hits for perceived facial age were for DNA variants in the MC1R gene. This finding was confirmed in two other large European studies. As Kayser and Gunn report, individuals carrying particular MC1R variants looked almost 2 years older for their age. The association between these DNA variants and perceived age wasn't influenced by age, sex, skin color, or sun damage. In addition to its role in skin color, MC1R is also known to play a role in other biological processes, such as inflammation and DNA damage repair. The researchers say the gene's influence on these processes might be the reason it links to youthful looks. While the researchers note that this gene is just one of many factors that influence perceived age, they plan to continue exploring exactly how it influences our looks and identify other genes that influence perceived age. Ultimately, the researchers think that this line of work can offer important insights into our health and the nature of aging itself. "We believe that using the perception of age is one of the best and most exciting ways to measure how 'well' people are aging, which we hope will lead to further breakthroughs in aging and health research in the near future," David Gunn said.


The current invention pertains to a method for screening and discovery of compounds capable of inhibiting, preventing, delaying or reducing genome maintenance disorders and consequences thereof, in particular ageing related symptoms and disorders. The current invention provides a method for screening and discovery of compounds that arc capable of inhibiting, preventing, delaying or reducing genome maintenance disorders and consequences thereof. The invention exploits animal models that comprise deficiencies in their genome maintenance systems, such as DNA repair systems, and display premature, enhanced, accelerated or segmental ageing phenotypes. These animal models can be advantageously applied to screen compounds and thereby develop schemes of intervention to treat, delay, inhibit, prevent or cure ageing related symptoms. The current invention thus provides a new and powerful tool to screen and/or discover therapeutically active compounds to treat ageing related symptoms and diseases. On the same basis it permits screening and discovery of compounds that influence ischemia, reperfusion damage in organ/tissue transplantation, chemotherapy and stem cell transplantation.


The current invention pertains to a method for screening and discovery of compounds capable of inhibiting, preventing, delaying or reducing genome maintenance disorders and consequences thereof, in particular ageing related symptoms and disorders. The current invention provides a method for screening and discovery of compounds that arc capable of inhibiting, preventing, delaying or reducing genome maintenance disorders and consequences thereof. The invention exploits animal models that comprise deficiencies in their genome maintenance systems, such as DNA repair systems, and display premature, enhanced, accelerated or segmental ageing phenotypes. These animal models can be advantageously applied to screen compounds and thereby develop schemes of intervention to treat, delay, inhibit, prevent or cure ageing related symptoms. The current invention thus provides a new and powerful tool to screen and/or discover therapeutically active compounds to treat ageing related symptoms and diseases. On the same basis it permits screening and discovery of compounds that influence ischemia, reperfusion damage in organ/tissue transplantation, chemotherapy and stem cell transplantation.

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