Podcasts about Microarray

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537409v1?rss=1 Authors: Matsumoto, T., Higaki, T., Takatsuka, H., Kutsuna, N., Ogata, Y., Hasezawa, S., Umeda, M., Inada, N. Abstract: ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in the Arabidopsis thaliana genome are grouped into four subclasses, and subclass I ADFs, ADF1-4, are all expressed throughout the plant. Previously, we showed that subclass I ADFs function in the regulation of the response against powdery mildew fungus as well as in the regulation of cell size and endoreduplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through a microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in both adf4 and transgenic lines where expression of subclass I ADFs are downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0. A. thaliana possesses eight ACTIN genes, among which ACT2, -7, and -8 are expressed in vegetative organs. The chromocenter size in act7, but not in the act2/8 double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed in adf4 and ADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat (NLR) genes, which are involved in effector-triggered plant immunity, was reduced in adf4 and ADF1-4Ri. qRT-PCR confirmed the altered expressions shown with microarray analysis. Overall, these results suggest that ADF regulates various aspects of plant physiology through its role in regulation of nuclear organization and gene expression. The mechanism how ADF and ACTIN regulate nuclear organization and gene expression is discussed. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

(00:31) Could you provide a little bit about yourself and your background? (02:04) Could you provide an overview of the chromosomal microarray assay? (05:49) Why should the chromosomal microarray assay be used over other available assays, such as FISH? (08:56) Does the chromosomal microarray provide good benefits in terms of gain of 7 and loss of 10, and EGFR amplification? (12:03) What's the value of utilizing the test alongside a neuro-specific NGS panel, such as Mayo Clinic Laboratories' NONCP panel? (17:20) How are the results used in patient care? (19:41)Is there anything else you feel it's important to highlight?

This week, please join author Mads Liisberg and Guest Host Mercedes Carnethon as they discuss the article "Clinical Characteristics, Incidences, and Mortality Rates for Type A and B Aortic Dissections: A Nationwide Danish Population-Based Cohort Study from 1996 to 2016." Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. We're your co-host. I'm Dr. Carolyn Lam, Associate Editor from the National Heart Center and Duke, National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, Associate Editor, Director of the Poly Heart Center at VCU Health in Richmond, Virginia. Dr. Carolyn Lam: Greg, today's feature paper is about aortic dissections and it's the first nationwide population based study investigating the clinical characteristic, incidents, and mortality based on validated diagnosis of aortic dissection in a national patient registry. You want to hear more? Well, you have to just keep listening. Let's go on though first to discuss the other really important papers in today's issue, shall we? Dr. Greg Hundley: Absolutely. Dr. Carolyn Lam: You know what, Greg? I'm going to start while you grab a coffee. I want to talk about high sensitivity cardiac troponins and how they have allowed the use of strategies in the emergency department, for example, to rapidly rule out acute MI within one to three hours and potentially facilitate early discharge of low-risk patients. Now, the ability to rapidly rule out MI of course depends on the turnaround time of these high sensitivity cardiac troponin results from the central laboratory, which is often delayed due to specimen transport and handling and all these things. So, point-of-care assays can reduce this turnaround time by even 40 minutes, and early studies have actually used frozen plasma bio banks to assess these point-of-care assays... But no study has evaluated these point-of-care assays with fresh whole blood to safely rule out MI in the emergency department. That is until today's paper. So in today's study led by corresponding other Doctor Fred Apple from Hennepin Medical Center in Minneapolis, Minnesota and his team, they aimed to derive and validate an optimal high sensitivity cardiac troponin threshold concentration using whole blood point-of-care troponin eye assay on a single sample at presentation in the emergency department to identify patients at low risk of index MI for potential early discharge. Dr. Greg Hundley: Fascinating study Carolyn. So point-of-care testing, high sensitivity troponin from whole blood in the ED. So what did they find? Dr. Carolyn Lam: Among consecutive emergency department patients from two prospective observational studies with suspected acute coronary syndrome, a point-of-care, whole blood, high sensitivity cardiac troponin eye assay, the Atellica VTli provided a sensitivity of 98.9% and a negative predictive value of 99.5% for ruling out MI. A single measurement using a cutoff of less than four nanograms per liter for whole blood was successful in rapidly identifying patients at low risk of MI cardiac and all cause death and unplanned revascularization at 30 days. Dr. Greg Hundley: Very nice Carolyn, and could be quite practical. So Carolyn, my next paper comes to us from the world of preclinical science and it pertains to cardiac regeneration. So cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes. However, certain animals readily regenerate lost myocardium via process involving dedifferentiation, which unlocks their proliferative capacities. So inspired by this concept, these investigators led by Professor Patrick Hsieh from Academic Sinica, generated mice with inducible cardiomyocyte specific expression of the Yamanaka factors enabling adult cardiomyocyte reprogramming and dedifferentiation in vivo. Dr. Carolyn Lam: Wow. So what did they find, Greg? Dr. Greg Hundley: Right, Carolyn. So two days following induction, adult cardiomyocytes presented with a dedifferentiated phenotype, an increase proliferation in vivo. Microarray analysis revealed that the up-regulation of ketogenesis was central to this process. Now adenovirus driven HMGCS2 over-expression induced ketogenesis in adult cardiomyocytes and recapitulated cardiomyocyte dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue. And HMGCS2 knockout mice showed impaired cardiac function and response to injury, and so in summary, Carolyn, these data demonstrated the importance of HMGCS2 induced ketogenesis as a means to regulate metabolic response to cardiomyocyte injury, thus allowing cell dedifferentiation and proliferation as a regenerative response. Dr. Carolyn Lam: Wow, that's so cool. From cell regeneration to autoimmunity in this next paper. Now autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. However, the functional features of cardiac autoimmunity in humans remain undefined due to the challenge of studying immune responses in situ. Now, these authors previously described a subset of c-Met expressing memory T lymphocytes, which preferentially migrate to cardiac tissue in mice and humans. In today's study, these authors led by co-corresponding authors, Dr. Federica Marielli-Berg and Saidi Mohidden from William Harvey Research Institute, Barts and the London Faculty of Medicine and Dentistry, and Queen Mary University of London, and their colleagues performed in-depth phenotyping of peripheral blood T cells in groups of patients with inflammatory and non-inflammatory cardiomyopathies, patients with non-cardiac autoimmunity and healthy controls... And they found that c-Met positive T cells were selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met positive T-cells were distinct from c-Met negative T cells, including preferential proliferation to cardiac myosin and co-production of multiple cytokines. Further, circulating c-Met positive T cell subpopulations in different heart muscle diseases identified distinct and overlapping mechanisms of heart inflammation. Furthermore, validation studies in experimental autoimmune myocarditis showed that elevations of auto-antigens specific c-Met positive T cells in peripheral blood, marked the loss of immune tolerance to the heart. Importantly, disease development could be halted by pharmacologic c-Met inhibition indicating a positive role for these c-Met positive T cells. Dr. Greg Hundley: All right, Carolyn, as you always ask me. So what's the take home message here? Dr. Carolyn Lam: This study demonstrates that the detection of circulating c-Met positive T cells may have utility in the diagnosis and monitoring of adaptive cardiac inflammation and additionally defined new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury... And this is discussed in an editorial by doctors at Abplanalp, Merten, and Dimmeler. Dr. Greg Hundley: Very nice, Carolyn. Wow. More fantastic preclinical science. Well, in the mail of the bag today, there is a Research Letter by Professor Burr entitled “Cannabis Inhalation Acutely Reduces Muscle Sympathetic Nerve Activity in Humans.” Dr. Carolyn Lam: There's an ECG Challenge by Dr. Reddy entitled “Shortness of Breath and Near Syncope During Exertion In a Child, When Patient Worry Syndrome.” There's also a Perspective by Dr. Weitz on what is the future of Factor 11 inhibitors. Dr. Greg Hundley: Well Carolyn, I'm looking forward to learning more about aortic dissections and that large Danish population-based study. Wow. Dr. Carolyn Lam: That's great. Let's go Greg. Dr. Mercedes Carnethon: Well, welcome to this episode of Circulation on the Run. My name is Mercedes Carnethon, an Associate Editor of Circulation, and Professor and Vice Chair of Preventive Medicine at Northwestern University. I'm really excited today to be here with the senior author of a really exciting paper that we're featuring on clinical characteristics, incidences and mortality rates for aortic dissections type A and B, a nationwide Danish population-based cohort study, and we have with us today Mads Liisberg. So welcome today. Dr. Mads Liisberg: Thank you. Dr. Mercedes Carnethon: So thank you so much for joining us and really thank you for sharing your important research with Circulation. This topic is so critically important, particularly given the high mortality rates associated with aortic dissections. Can you tell us a little bit about the work that you and your co-authors did in this important space? Dr. Mads Liisberg: Yes. Well actually the work originated when I started my PhD thesis and we got a registry data dump from the Danish medical registries and we found that almost none of our patients in the registry were registered with a specific aortic dissection code. So we did a validation study on the same time period from 1999 to 2006 where we went through all these medical records to ensure that we had the right aortic dissection TC 10 codes on population. Then we went a bit further and looked at the clinical characteristics of this patient, 'cause that's one of the really major things about Danish medical registries in our country, is that we have access to not only every patient's specific in hospital contacts, but also their medicine abuse, their drug use based on a TC code. So we can go really deep into each and everyone's drug history. When we did this study, we wanted to find out if the incidence rates during this timeframe had changed, which we find that it did, but also looking at mortality rates because, as you said, it's really high risk disease to be diagnosed with. So that's more the rationale for this study. Dr. Mercedes Carnethon: Thank you so much for sharing that. Certainly we know that the mortality rates from this are very high. I note that you report some changes over time between 1996 and 2016 in the incidents of these types of aortic dissections. So what did you find about the patterns of change in type A aortic dissections? Dr. Mads Liisberg: We found that it almost doubled from the beginning of our time period to the last, and the question is why is this? 'Cause that's one of the thing that the data doesn't reveal. We are only able to see that the incidence actually rises, but is it because that they are underdiagnosed in the beginning? Or is it because that we are better diagnosing in the end of the study that really are progressed? Dr. Mercedes Carnethon: That's a good question. I noted that when you studied the correlates of aortic dissection, you identified a number of characteristics and what stood out to me was the finding about the strong association of hypertension. I'm less aware of patterns of hypertension in the Danish population. Do you think that the changes in the prevalence of hypertension in the population contributed at all to these findings? Dr. Mads Liisberg: Well, most certainly, 'cause when you look at the prevalence of hypertension throughout any person's lifespan, the older they get the more likely they are to suffer from hypertension, and the Danish population has aged quite a lot in recent years. So I think that's one of the main reasons we find this, and also that most of our arctic dissection patients are actually quite old, which would correlate with hypertension as well. Dr. Mercedes Carnethon: One thing I really like, and you pointed this out, is really the richness of the data that you have in your health system, and I wonder, just going even a little deeper on the hypertension question, given that it is the most common medical diagnosis worldwide, were you able to study characteristics of hypertension that would be more strongly associated with aortic dissection? So for example, duration of hypertension, severity, prevalence of hypertension control? Dr. Mads Liisberg: That's actually a funny question 'cause the last study of my thesis, which hasn't been published yet or even submitted for that fact, examines the correlation between use and the risk of arctic dissection. On a very specific level, the way that hypertension is treated mostly in Denmark is with your general practitioner. So the way that we examine in studies like these, is that we look at prescription drug use. So if we find that an individual has a TC codes corresponding with anti-hypertensive drugs, then we are able to code them as hypertensive patients. Dr. Mercedes Carnethon: Okay, thank you for that. I always... I'm an epidemiologist myself, so I really love to see great population science studies and this registry is large, you have long-term follow up and you've got a great deal of data, but those people who feel as though it's obviously not appropriate to make causal conclusions around epidemiology and that perhaps epidemiologic findings shouldn't be driving clinical decision-making. In response to that, I think I would pose the question to you, which is how do you see clinicians and providers using this information from your observational study? Dr. Mads Liisberg: I think that's rather difficult. One of the findings that we present is to pose it over mortality for type B dissections when we exclude the 30-day mortality, but then we show that type A dissections have almost a corresponding mortality rate compared to a hypertensive cohort... And this finding is difficult to draw any clinical conclusions from, but there's actually a Danish randomized controlled trial just starting up in the next year. I think it's called the Sunday trial, where they will include all uncomplicated type B dissections and randomize them for treatment or no treatment, and the issue here is that you'll probably be over-treating some patients and under-treating others, but this discussion with the uncomplicated type B dissection has been ongoing for so many years. So it's difficult for me to just give one golden answer. Dr. Mercedes Carnethon: Certainly, and I appreciate the caution as we certainly don't want to overstep our findings. You did make a recommendation in the conclusion that it might be beneficial to treat type B aortic dissections more aggressively. Is this what you're alluding to, based on the other study that you're referencing? Dr. Mads Liisberg: Yes, yes, definitely. We see some clinicians being more cautious treating type B dissections with a TIVA or a surgery. So it's a difficult thing when they're uncomplicated, why treat them? But they can't be or become complicated quite easily and fast and then it's a difficult thing, because should you have treated them earlier? Or do you need to treat them now in their acute phase? Or wait for a chronic phase? It's a really good question. Dr. Mercedes Carnethon: No, I appreciate that and what I really love are the types of research studies that leave you with many more questions and next steps, and so I would like to really sort of bring us to a close with the big picture question, which is what do you see as the next steps in this line of research, given really what we all agree on is a very significant clinical problem. Dr. Mads Liisberg: We would really like to expand our database with even more clinical data as of now and include any image diagnostics for our cohort. So we're might be able to see any trends in our modulation before the dissection occurs. If any of our patients have any diagnostics done prior to being diagnosed. Dr. Mercedes Carnethon: I really want to thank you today for spending time talking with us. I know that our readers rarely have an opportunity to hear from everybody behind the scenes views on what the rationale was for carrying out a paper and really how the authors themselves hope that the paper will be used. So I really thank you for sharing that with us today, Matts, on behalf of your co-authors, this has been really a wonderful conversation, and thank you again for sharing your research with the journal, Circulation. Dr. Mads Liisberg: Oh, thank you for having me in for accepting our paper. Dr. Mercedes Carnethon: So thank you so much to our listeners for listening to us on this episode of Circulation on the Run. Please tune in next week as we will have more exciting insights. Dr. Greg Hundley: This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more, please visit ahajournals.org.

  Vidcast:  https://youtu.be/nsq6RTRCqbk   Here are the quickie reports about cutting edge medical and healthcare discoveries this 1st Week of August, 2022.   Neuroblastoma, the development of cancer in the involuntary nervous system, is a deadly disease for children 5 years and younger.  Now a group of biologists at Virginia Commonwealth University have identified a compound called SHP099 that blocks the enzymatic activation of the MEK/ERK proteins that potentiate the growth of these tumors.  The best news is that even high doses of SHP099 have little effect on normal cells.  The compound shrinks neuroblastoma tumors in mice and, hopefully someday, will also do so in children.   https://bit.ly/3PNhduj   MIT mechanical bioengineers have developed a wearable sticker capable of providing continuous, live ultrasound images.  These stamp-sized devices mate arrays of ultrasound transducers with adhesives and capably create images of underlying organs for up to 48 hours.  Currently, the patches must be wired to the imaging processors but, in the future, wireless versions may transmit to mobile processors to permit continuous ultrasound monitoring of body organs and developing babies.   https://www.science.org/doi/10.1126/science.abo2542     Those awaiting heart transplants often need a boost in the pumping function provided by their own flagging hearts.  Traditionally, that requires an implantable pump, a so-called left ventricular assist device, to be surgically inserted into the left ventricle during an open heart procedure.  The Mayo Clinic now offers another option, a heart pumping a catheter called the Impella 5.5 that is non-invasively introduced into the heart via an armpit vessel.  The device pumps blood out of the left ventricle into the aorta taking the load off the heart muscle.   An added bonus: having this catheter pump pushes a transplant candidate higher up on the list.   https://www.mayoclinic.org/medical-professionals/transplant-medicine/news/mayo-clinic-in-florida-first-hospital-in-the-world-to-use-a-new-heart-pump-as-bridge-to-transplant/mac-20519622     A prickly skin patch not only delivers CoVid vaccine without the dreaded hypodermic syringe feared by the needle-phobic, but it also triggers a higher level of CoVid protection.  Australian researchers report that a protein-subunit CoVid vaccine triggers 11 fold higher levels of neutralizing antibodies against the Omicron variant when administered via a high density needle microarray patch compared with a conventional intradermal needle injection.   https://www.sciencedirect.com/science/article/pii/S0264410X2200888X?via%3Dihub       There you have the latest health reveals for this 1st Week of August, 2022, 2022.  When additional information about these developments becomes available, I'll pass it on to you.     #neuroblastoma #mekerk #shp099 #ultrasound #sticker #impella #lvad #CoVid #omicron #microarray    

O Array, ou microarray, é um exame cujo objetivo é investigar desbalanços cromossômicos que o cariótipo não consegue identificar, por serem pequenas deleções e pequenas duplicações. Essas variantes do número de cópias do genoma podem estar associadas à diversas síndromes genéticas, sendo um exame muito importante para investigação etiológica de sindromes malformativas, deficiência intelectual e transtorno do espectro autista. Vamos começar a dessenrolar nosso DNA e se aprofundar no Microarray! Acessem nossas redes sociais e deixem suas mensagens: Facebook: @genecastpodcast Twitter: @genecastpodcast Instagram: @genecastpodcast YouTube: @genecastpodcast Wordpress: genecastpodcast.wordpress.com E-mail: genecastpodcast@gmail.com Equipe: Host: Rodrigo Fock Geneticistas: Rayana Maia, Joselito Sobreira

Mariana Perepitchka, Yekaterina Galat, and Dr. Vasiliy Galat discuss their 2020 research paper published by Oncotarget, entitled, "Down syndrome iPSC model: endothelial perspective on tumor development." DOI - https://doi.org/10.18632/oncotarget.27712 Correspondence to - Mariana Perepitchka - mperepitchka@u.northwestern.edu, Yekaterina Galat - ygalat@luriechildrens.org, and Vasiliy Galat - v-galat@northwestern.edu Abstract Trisomy 21 (T21), known as Down syndrome (DS), is a widely studied chromosomal abnormality. Previous studies have shown that DS individuals have a unique cancer profile. While exhibiting low solid tumor prevalence, DS patients are at risk for hematologic cancers, such as acute megakaryocytic leukemia and acute lymphoblastic leukemia. We speculated that endothelial cells are active players in this clinical background. To this end, we hypothesized that impaired DS endothelial development and functionality, impacted by genome-wide T21 alterations, potentially results in a suboptimal endothelial microenvironment with the capability to prevent solid tumor growth. To test this hypothesis, we assessed molecular and phenotypic differences of endothelial cells differentiated from Down syndrome and euploid iPS cells. Microarray, RNA-Seq, and bioinformatic analyses revealed that most significantly expressed genes belong to angiogenic, cytoskeletal rearrangement, extracellular matrix remodeling, and inflammatory pathways. Interestingly, the majority of these genes are not located on Chromosome 21. To substantiate these findings, we carried out functional assays. The obtained phenotypic results correlated with the molecular data and showed that Down syndrome endothelial cells exhibit decreased proliferation, reduced migration, and a weak TNF-α inflammatory response. Based on this data, we provide a set of genes potentially associated with Down syndrome's elevated leukemic incidence and its unfavorable solid tumor microenvironment—highlighting the potential use of these genes as therapeutic targets in translational cancer research. Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.27712 Press release - https://www.oncotarget.com/news/pr/down-syndrome-ipsc-model-an-endothelial-perspective-on-tumor-development/ Keywords - Down syndrome, iPSC-derived endothelial model, T21 genome-wide Implications, meta-analysis, tumor microenvironment About Oncotarget Oncotarget is a peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/OncotargetYouTube LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC: https://www.ImpactJournals.com Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

In this podcast, sponsored by Thermo Fisher Scientific, discover the continuing advantages of chromosomal microarray in cytogenetics. Jonathan sits down with Dr Shashi Shetty, Director of Cytogenetics Laboratory, Cleveland Medical Centre, Cleveland, Ohio, to explore at what point microarrays are the preferred choice for genetic research testing in the age of next generation sequencing (NGS). They will also explore the multiple factors that can influence the choice to implement microarrays in a research testing regime.

「遺伝暗号表の構造が環境中のリソースから影響受けており、コドンの使用頻度にも反映されている」という説の論文を読みました。Show notes Resource conservation manifests in the genetic code. Science 2020…今回読んだ論文です。 Resource conservation manifests in the genetic code. BioRxiv 2020…Scienceに出版される前にプレプリントサーバーにも原稿があるので、無料で読めます。出版されたバージョンとは図表・番号などが違うので注意。 コドン (Wikipedia) … 今回の題材である、みんな大好き三文字のあいつ。今回コドンの詳しい説明は↓に丸投げしました。 37. Biological Enigma…dessanをゲストに迎え、分子細胞生物学の入門知識について話しました。タンパク質やコドンについて(dessanが）丁寧に話しています。 7. In the golden age of molecular biology… アミノ酸をコードするコドン発見に至る歴史を当時の論文と背景を交えて3人で話しました。 Ep19. Neuron Musk…Elon MuskがオーナーをつとめるNeuralinkがはじめて詳細にその技術を公開した時期に（当時）、bioRxivに投稿された論文と発表イベントの内容、Brain Machine Interfaceを含む脳情報関連技術の動向について話しました。 Ep50. My first journal club presentation…coela（たまき）が始めて紹介した論文。今回は2本目。 ResearchatLT vol.1 (YouTube)…Researchat.fmのリスナーから有志で、ライトニングトーク大会をやりました。その配信アーカイブです。 Researchat.fm Lightning Talk Vol.01 … ResearchatLTの要旨集を含む開催概要。 Last universal common ancestor, LUCA (Wikipedia) 窒素固定 (Wikipedia) Sequence Homology (Wikipedia)… 今回取り上げたOrthologyの意味、混同しやすいParalogy・Ohnology・Xenology・Homoeology・Gametologyの違いについて。 Single-nucleotide polymorphism (Wikipedia) … podcast中でSNP（スニップ）って言っているもの。日本語では一塩基多型。 KEGG: Kyoto Encyclopedia of Genes and Genomes KEGG ORTHOLOGY EggNOG v5.0…“A database of orthology relationships, functional annotation, and gene evolutionary histories” 同義置換と非同義置換: 計算方法、意味、論文での示し方 … podcast中でなんの説明もなく「dN/dS」と言っていた内容の説明（ごめんなさい）。 正の自然選択圧・負の自然選択圧 … ここらへんは限られた文字数で要約するのが怖いので割愛！！↑のリンクでも説明があります。 GLMM (Wikipedia) データ解析のための統計モデリング入門――一般化線形モデル・階層ベイズモデル・MCMC … GLMM(論文中ではLMM）の入門におすすめな書籍。 GEO (Gene Expression Omnibus)…“GEO is a public functional genomics data repository supporting MIAME-compliant data submissions. Array- and sequence-based data are accepted.” Microarray (Wikipedia) アミノ酸 生合成経路 細胞の物理生物学 … coelaがはじめて、アミノ酸を合成するのに必要なエネルギー（ATP数）に関する記述をみかけた本。他にも様々な生命現象を取り上げている素晴らしい書籍。鈍器になるレベルで分厚い。 BioNumbers … 直接は関係ないですが、今回議論された内容を調べるとっかかりとして便利とcoelaが思っているデータベース。 Editorial notes Researchat.fmは研究者3人が話すポッドキャストです！！(coela) また一年後にお願いします (soh) う、今回は反省の多い回でした。次回の論文回は最後まで聞いてから質問します(悪い癖が出ちまった…)(tadasu)

We review common genetic tests available for diagnosis of chromosomal anomalies: karyotype, FISH, and microarray. We are joined by Angela Scheuerle, MD, Professor of Pediatric Genetics at the University of Texas Southwestern Medical Center.

Episode 347 is an updated guide to somatropic hormone and GOD did I go crazy on this one! I honestly want to know more about growth hormone than anyone alive and thus, begins this string of GH based guides! I DID finally discuss the MoA for how GH causes localized fat loss which really had me excited since no one in our industry has EVER talked about this so that definitely was an interesting avenue to go down! Below I am going to reference a lot of the literature for this hormone that I was read through over the past few years on this topic so please DO NOT TAKE MY WORD FOR THIS - READ THESE YOURSELF! Keep in mind this is a brief snippet of every bit of literature on the topic however. REFERENCES   Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev. 1989;10:68–91. [PubMed] [Google Scholar] Jones JI, Clemmons DR. 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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.30.273870v1?rss=1 Authors: Subramanian, U., Devarajan, B. Abstract: Retinal angiogenesis is a common neovascularization mechanism that causes severe irreversible vision loss in the number of retinal diseases worldwide. Patients often do not respond to the current antiangiogenic therapies and have a vision loss. Understanding the various angiogenic pathways and factors involved in the pathogenic mechanism is vital for disease management. In this study, to identify dysregulated angiogenic pathways and specific angiogenic factors involved in vision-threatening diseases namely proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP) and neovascular age related macular degeneration (nAMD), we downloaded microarray metadata of samples and obtained the differentially expressed genes (DEGs) in all the disease and each disease samples compared to controls. Subsequently, we performed Gene Set Enrichment (GESA) analysis for pathways, a protein-protein interaction (PPI), and angiome network analysis using R and Cytoscape software. We identified highly enriched dysregulated pathways that were neuroactive ligand receptor interaction and cytokine cytokine receptor interaction. The angiogenic associated DEGs were predominately related to the cytokine cytokine receptor interaction pathway, which we further confirmed with RNA-seq data of PDR samples. Together, our analysis of these data elucidated the molecular mechanisms of retinal angiogenesis and provided potential angiogenic targets for therapeutics. Copy rights belong to original authors. Visit the link for more info

Advances in 1st trimester aneuploidy screening have increased the need for early prenatal diagnostic testing. There are only 2 prenatal diagnostic tests : CVS and amniocentesis. In this podcast, we will review these procedures and rates of potential complications. We will also summarize the difference between microarray chromosomal analysis and conventional karyotype.

In the mid-1990s, the idea of a genome-scale study seemed impossible or even crazy to some. Dr. Lou Staudt (director of NCI’s Center for Cancer Genomics) talks about putting together one of the first cancer genomic studies for diffuse large B-cell lymphoma and building the “Lymphochip” microarray.

Darin Zehrung, PATH's Global Program Leader for Medical Devices and Health Technologies, talks about the development of microarray technology.

Il sesto episodio di Ragù di Scienza è dedicato al becero tecno-ottimismo esponenziale, anche per compensare gli ultimi due episodi vagamente depressi di fila. Con *leggermente* meno tangenti insensate del solito, tra le altre cose in questo episodio si parla di: -Genoma da mille dollaros -Lampi gamma, lampi gamma ovunque -ELT, VLT e nomi intelligenti dati a telescopi da astronomi molto intelligenti. -Sequencing. In. SPAAAAACE. -Sequenziamento de novo e de vecio -Adrian si entusiasma per i microsatelliti sbagliati -Microarray e lab-on-chip -Elisa si entusiasma per gli Array sbagliati -SKA e il lato positivo di tanti rilevatori in paesi emergenti -Pulcini di T.rex! Mammuth domestici! -SKA, Event Horizon, e sfere stroboscopiche -Montagne di dati, rumore di fondo, cose che smettono di essere interessanti perché travolti da troppe cose buone Insomma, il solito pentolone di cose sulla carta interessanti spiegate in maniera improvvisata. Come piace a voi (?). Potete lasciarci qualsiasi commento/consiglio/recensione/insulto/ricette-per-il-ragù seguendoci sulla pagina Facebook, qui nei commenti o negli altri innumerevoli posti dove ci troviamo. Se ci seguite tramite Itunes, lasciateci una recensione sullo store per gli altri utenti della Mela. Se volete spargere questo podcast come fosse DNA codificante per #qualchegioia, la nostra fitness ne guadagnerebbe e ne saremmo contenti.

Barrett Bready is back on the program. He’s the CEO of Nabsys, a company with some new technology for genome mapping. Originally Nabsys had been working to develop nanopore sequencing, but after a recent reboot has become focused on scaling up scientists' ability to read structural genomic information. Barrett compares Nabsys’ new multiplex technology for genome mapping to the improvement of arrays over single nucleotide (SNP) detection.

Chromosomal microarray technology (CMA) facilitates the genetic diagnosis of intellectual disabilities, autism spectrum disorder, and congenital abnormalities in children. Previously, G-band karyotyping was the test performed for this purpose but it could only identify very large chromosomal abnormalities and was not very sensitive. Being a molecular rather than microscopic technique, CMA is far more sensitive for identifying genetic abnormalities and is now the test of choice. We interview David H. Ledbetter, MD, and Christa Lese Martin, PhD, from Geisinger Health System, authors of this JAMA Insights article. Articles discussed in this episode: Chromosomal Microarray Testing for Children With Unexplained Neurodevelopmental Disorders New Approaches to Molecular Diagnosis

1) Microarray screening of Guillain-Barré syndrome sera for antibodies to glycolipid complexes2) What's Trending: video gaming in children 3) Topic of the Month: Neurology Today® paper on mitochondrial replacementThis podcast begins and closes with Dr. Robert Gross, Editor-in-Chief, briefly discussing highlighted articles from the February 21, 2017 issue of Neurology. In the first segment, Dr. Ted Burns talks with Dr. Hugh Willison about his Neurology® Neuroimmunology & Neuroinflammation paper on microarray screening of autoantibodies to glycolipid complexes in Guillain-Barre syndrome. Dr. Ted Burns talks with Dr. Gerard Martínez Vilavella about video gaming in children for our “What's Trending” feature of the week. In the next part of the podcast Dr. Ted Burns focuses his interview with Dr. Shoukhrat Mitalipov on mitochondrial replacement for genetic diseases. Disclosures can be found at Neurology.org.DISCLOSURES: Dr. Burns serves as Podcast Editor for Neurology®; and has received research support for consulting activities with UCB, CSL Behring, Walgreens and Alexion Pharmaceuticals, Inc.Dr. Willison serves on the scientific advisory board for Wellcome Trust; serves on editorial boards for Nature Clinical Practice Neurology; Journal of Neuroimmunology, Muscle and Nerve, J Peripheral Nervous System, BMC Neurology (Associate Editor), Clinical and Experimental Neuroimmunology, and Experimental Neurology; holds a patent for combinatorial glycoarray technology; and receives research support from Ipsen, Annexon Biosciences, Alexion Pharmaceuticals, Inc., Medical Research Council, and Wellcome Trust. Dr. Mitalipov holds patents for Primate Totipotent and Pluripotent Stem Cells Produced by Somatic Cell Nuclear Transfer (U.S. patent No. 7,972,849), Human Pluripotent Stem Cells Produced by Somatic Cell Nuclear Transfer (U.S. Patent No. 9,546,383), and Methods for Mitochondrial DNA Replacement in Oocytes (U.S. Patent No. 9434921); has patents pending for Metabolic rescue in pluripotent cells from subjects with mitochondrial DNA disease (Application Number: 62192358), Generation of human oocytes by polar body transfer” (Application 62/419,638), and Mitochondrial Replacement in Human Oocytes Carrying Pathogenic Mitochondrial DNA Mutations (Application 62427546); has consulted for Flagship VentureLabs; and has received research support from NIH/NIA, Burroughs Wellcome Fund, and Foundation Leducq.All other participants report no disclosures.

Pediatrix

Presented by Ronald Wapner, MD

Muscles of all higher animals comprise different muscle types adapted to perform distinct functions in the body. These express different sets of genes controlled by distinct combinations of transcriptional programs and extracellular signals, and thus differ in their myofibrillar organization and contractile properties. Despite major progress in our understanding of myogenesis, the genetic pathways controlling the formation and function of different muscle types are still largely uncharacterized. Flying insects possess specialized flight muscles enabling wing oscillations with frequencies of up to 1000 Hz together with high power outputs of 80 W per kg muscle. To achieve these parameters, flight muscles contain stretch-activated myofibrils with a unique fibrillar organization, whereas all other, more slowly contracting muscles, such as leg muscles, display a tubular morphology. To delineate the genetic regulation of muscle development and function, and, in particular, muscle type specification, we performed a genome-wide RNA interference (RNAi) screen in Drosophila, in which we systematically inactivate genes exclusively in muscle tissue. We uncovered more than 2000 genes with putative roles in muscles, many of which we were able to assign to specific functions in muscle, myofibril or sarcomere organization by phenotypic characterization. Muscle-specific knockdown of 315 genes resulted in viable, but completely flightless animals, indicating a specific function of those genes in fibrillar flight muscles. Detailed morphological analysis of these 315 genes revealed a striking phenotype upon knockdown of the zinc finger transcription factor spalt major (salm): the fibrillar flight muscles are switched to tubular muscles, whereas tubular leg muscles are wild type, demonstrating that salm is a key determinant of fibrillar muscle fate. We could show that the transcription factor vestigial (vg) acts upstream of salm to induce its expression specifically in fibrillar flight muscles. Importantly, salm is not only required but also sufficient to induce the fibrillar muscle fate upon ectopic expression in other muscle types. Microarray analysis, comparing mRNA expression from adult wild-type flight and leg muscles to salm knockdown flight muscles, indicates that salm instructs most features of fibrillar muscles by regulating both gene expression as well as alternative splicing. Remarkably, we could show that spalt’s function in programming stretch-activated fibrillar muscles is conserved in insect species separated by 280 million years of evolution. Interestingly, in mouse two of the four spalt-like (sall) genes are expressed in heart, a stretch-activated muscle, sharing some features with insect fibrillar flight muscles. Since heart abnormalities observed in patients suffering from the Towns-Brocks syndrome are caused by a mutation in SALL1, it is possible that Spalt’s function to determine a fibrillar, stretch-modulated muscle type is conserved to vertebrates.

Introduction: Circulating microRNAs (miRNAs) are easily accessible and have already proven to be useful as prognostic markers in cancer patients. However, their origin and function in the circulation is still under discussion. In the present study we analyzed changes in the miRNAs in blood plasma of head and neck squamous cell carcinoma (HNSCC) patients in response to radiochemotherapy and compared them to the changes in a cell culture model of primary HNSCC cells undergoing simulated anti-cancer therapy. Materials and methods: MiRNA-profiles were analyzed by qRT-PCR arrays in paired blood plasma samples of HNSCC patients before therapy and after two days of treatment. Candidate miRNAs were validated by single qRT-PCR assays. An in vitro radiochemotherapy model using primary HNSCC cell cultures was established to test the possible tumor origin of the circulating miRNAs. Microarray analysis was performed on primary HNSCC cell cultures followed by validation of deregulated miRNAs via qRT-PCR. Results: Unsupervised clustering of the expression profiles using the six most regulated miRNAs (miR-425-5p, miR-21-5p, miR-106b-5p, miR-590-5p, miR-574-3p, miR-885-3p) significantly (p = 0.012) separated plasma samples collected prior to treatment from plasma samples collected after two days of radiochemotherapy. MiRNA profiling of primary HNSCC cell cultures treated in vitro with radiochemotherapy revealed differentially expressed miRNAs that were also observed to be therapy-responsive in blood plasma of the patients (miR-425-5p, miR-21-5p, miR-106b-5p, miR-93-5p) and are therefore likely to stem from the tumor. Of these candidate marker miRNAs we were able to validate by qRT-PCR a deregulation of eight plasma miRNAs as well as miR-425-5p and miR-93-5p in primary HNSCC cultures after radiochemotherapy. Conclusion: Changes in the abundance of circulating miRNAs during radiochemotherapy reflect the therapy response of primary HNSCC cells after an in vitro treatment. Therefore, the responsive miRNAs (miR-425-5p, miR-93-5p) may represent novel biomarkers for therapy monitoring. The prognostic value of this exciting observation requires confirmation using an independent patient cohort that includes clinical follow-up data.

Dr. Stephen Harrison talks to Drs. Thomas Wirth and Yoshiaki Suname about the paper: Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation Abstract: Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117–1128)

During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained at different times during the first six hours of MCMV infection revealed discrete functional clusters of cellular genes regulated with distinct kinetics at surprising temporal resolution. Immediately upon virus entry, a cluster of NF-κB- and interferon-regulated genes was induced. Rapid viral counter-regulation of this coincided with a very transient DNA-damage response, followed by a delayed ER-stress response. Rapid counter-regulation of all three clusters indicated the involvement of novel viral regulators targeting these pathways. In addition, down-regulation of two clusters involved in cell-differentiation (rapid repression) and cell-cycle (delayed repression) was observed. Promoter analysis revealed all five clusters to be associated with distinct transcription factors, of which NF-κB and c-Myc were validated to precisely match the respective transcriptional changes observed in newly transcribed RNA. 4sU-tagging also allowed us to study the real-time kinetics of viral gene expression in the absence of any interfering virion-associated-RNA. Both qRT-PCR and next-generation sequencing demonstrated a sharp peak of viral gene expression during the first two hours of infection including transcription of immediate-early, early and even well characterized late genes. Interestingly, this was subject to rapid gene silencing by 5-6 hours post infection. Despite the rapid increase in viral DNA load during viral DNA replication, transcriptional activity of some viral genes remained remarkably constant until late-stage infection, or was subject to further continuous decline. In summary, this study pioneers real-time transcriptional analysis during a lytic herpesvirus infection and highlights numerous novel regulatory aspects of virus-host-cell interaction.

Salmonella infections in humans arise through chicken-based food such as eggs, egg-products, or chicken meat. The most common cause for these infections is Salmonella enteritidis, and the aim of this study has been to analyze the early innate immune response of chickens induced via this pathogen. Note that S. enteritidis is a host-adapted serovar, which only causes clinical findings in young chickens during their first week of life; adult chickens do not get sick, but may nevertheless act as inapparent infected carriers. We studied the reaction from the chicken immune system on S. enteritidis, using macrophage cultures as well as tissue samples of infected adult chickens. The gene expression studies were carried out by an “Agilent 4x44K chicken microarray” method. In our in vitro studies, we infected primary macrophages with S. enteritidis for 4 hours, using a MOI of 10. The gene expression studies resulted in the inductions of interleukins (IL1β, IL6, IL12p40, IL18), of chemokines (CCL1, CCL4 (K203), CCL20, CXCL8 (IL8), CXCL13), of some members of the tumor-nekrose-factor-superfamily (TNFSF), and of some toll-like receptors (TLR). Hence the cells have an inflammatory reaction. Particularly prominent were the expression changes of K60 (IL8 homolog), K203 (chCCLi2, MIP-1β), CCL20, and TL1a (TNFSF15). Finally, infected macrophages expressed a group of typical Th1-cytokines, including IL12p40, IL18, and IFN-γ. In further analysis of our data, we focused on cytokines, chemokines, and members of the TNF-superfamily. In the ceca we found similar expression patterns within these three groups as was previously found for them in the macrophages study. In our in vivo studies, we infected chickens that were 8 weeks old and already had a well developed immune system. They were infected in the crop using a dose of 107 salmonella. At 5, 12, 24, and 48 hours of infection, we sampled the ceca and cecal tonsils for the bacterial, histological, and gene expression analyses. Already at 5 hours p.i., we were (for all but one animal) able to isolate bacteria from the ceca-tissue. The bacterial load reached its maximum at 12 hours p.i.. The infection of the cecal-tissues was confirmed in the histology, both by the detection of bacteria and by the occurrence of inflammatory cells. However, using histology, we could not detect any bacteria in cecal tonsils, which suggests that no infection was present in these organs. This suggestion was confirmed in gene expression analyses. When comparing the gene expression studies of cecal tonsils and ceca, the former showed lower counts of differential regulated genes (Tab. 11). Both their count maxima occurred at 12 hour p.i though. Moreover, at this time 41 significant regulated pathways had been identified.. In summary, the in vitro and the in vivo experiment both resulted in an initial inflammatory reaction, as well as in a typical Th1-cytokines reaction. To investigate functional characterisation of named candidate genes, in the first instance CCL20, CXCL8, K60, K203, and TL1a, future analyses of the innate immune response should involve them. This may contribute to a better understanding of the successful defense mechanisms against S. enteritidis in chicken, which may help to contain the amount of salmonellosis in humans.

Sat, 12 Feb 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13214/ https://edoc.ub.uni-muenchen.de/13214/1/Merkl_Maximiliane.pdf Merkl, Maximiliane ddc:590, ddc:500, Tierärztliche Fa

Background: Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. Results: Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e. g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1. The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. Conclusions: This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

CD25+ regulatory T cells develop in the thymus (nTregs), but may also be generated in the periphery upon stimulation of naive CD4 T cells under appropriate conditions (iTregs). To gain insight into the mechanisms governing iTreg development, we performed longitudinal transcriptional profiling of CD25+ T cells during their differentiation from uncommitted naive CD4 T cells. Microarray analysis of mRNA from CD25+ iTregs early after stimulation revealed expression of genes involved in cell cycle progression and T cell activation, which largely overlapped with genes expressed in CD25+ effector T cells (Teffs) used as a control. Whereas expression of these genes remained elevated in Teffs, it declined gradually in developing iTregs, resulting in a more quiescent phenotype in mature iTregs. A similar pattern of kinetics was observed for biological processes and for intracellular pathways over-represented within the expressed genes. A maximum dichotomy of transcriptional activity between iTregs and Teffs was reached at late stages of their maturation. Of interest, members of the FoxO and FoxM1 transcription factor family pathways exhibited a reciprocal expression pattern in iTregs and Teffs, suggesting a role of these transcription factors in determining T cell fate.

PC4 is a small protein with unique DNA-binding properties that affects transcription and has presumptive roles in DNA repair and genome stability. It was originally isolated from a cofactor fraction termed the ‘‘upstream stimulatory activity’’ (USA) of HeLa cell nuclear extracts. The cofactor has been shown to broadly enhance RNA polymerase II-driven gene transcription in the presence of activators (e.g., hormone receptors, viral activators, cell-specific and ubiquitous activators). Although such data imply that PC4 is a very important factor in vivo, human tumor cell lines with PC4 knockdowns are without obvious phenotypes. To further study the in vivo role of PC4, we constructed constitutive and conditional knockout mouse models as well as knockout embryonic stem cells. Mammalian PC4 is here shown to be an essential factor during early embryogenesis. PC4-/- embryos develop normally until E5.5, but then degenerated around E7.5. PC4 knockout ES cell lines were generated from PC4-/- blastocysts (E3.5), which develop normally from 2-cell stage embryos. All PC4 knockout ES cell lines displayed a severe proliferation deficit phenotype, which could be partially rescued by re-expression of human PC4. The reduced proliferation was not due to an increase in cell apoptosis. Occasionally, PC4 knockout ES cells undergo tetraploidy apparently as a survival mechanism to circumvent the loss of PC4. Knocking down PC4 in mouse embryonic fibroblasts also resulted in reduced proliferation rates. These data indicate that PC4 is important for cell proliferation in embryos. Moreover, in vitro embryoid body formation and in vivo teratoma formation assays provided preliminary evidence for an important role of PC4 in differentiation. Differentiated ES cells displayed alterations in germ-layer specific gene expression, that are in agreement with morphological abnormalities observed in histological analyses of PC4-/- embryos at E6.5 and E7.5. Thus, depletion of PC4 results in reduced proliferation and impaired differentiation, the consequence of which appears to be of gastrulation arrest in early embryos. In an attempt to understand the underlying mechanisms of this phenotype, differential gene expression in ES knockout and wild-type cells was studied. Microarray and qRT-PCR analyses revealed more than 2 fold alterations in expression of many genes in knockout ES cells as compared with wild-type cells. These include enhanced expression of p21, Rb1, and Ddit4l, and lower expression of Sfmbt2, Tdrd12, and Dppa3, suggesting a specific direct or indirect physiological role of the cofactor. Nevertheless, the previously proposed role of PC4 in p53 expression and function was not confirmed using the knockout model. Taken together, this work represents the first description of the physiological functions of PC4 during mammalian embryogenesis.

Chronic allograft dysfunction (CAD) following kidney transplantation is characterized by progressive fibrosis and a smoldering inflammatory infiltrate. A modified Fischer 344 (RT1lvl) to Lewis (RT1l) rat renal allograft model was used to study transcriptomic changes during the initiation and progression of CAD and to identify potential therapeutic modes of action of treatment with 13cRA previously shown to limit the development of CAD. Transcriptomic profiling was performed using Affymetrix DNA arrays at time points 0, 7, 14 and 56 days after transplantation. The animal model showed development of significant chronic fibrotic damage with accompanying inflammatory infiltrate by day 56 after transplantation. Regulatory pathways were identified by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and modulated, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database. Microarray analysis revealed dramatic changes in the mRNA expression levels of genes associated with inflammation and fibrosis, as well as the hedgehog and WNT pathways, with a gradual increase in the number of differentially regulated genes during progression of tissue damage. Disease phenotype, as well as differential regulation of select components of the hedgehog, canonical WNT and WNT-Ca2+ signaling pathways could be verified by quantitative PCR (qPCR) and immunohistochemistry. Treatment with 13cRA, not only attenuated disease progression, but even reversed early effects of CAD. The overall effects of the treatment are mediated by a potentially direct influence on fibrosis and inflammation associated gene expression, as well as by a specific modulation, observed for hedgehog and WNT pathway activations. The results identify a series of potential pathways that may represent therapeutic targets in chronic allograft dysfunction.

In the 1990s a number of technological innovations appeared that revolutionized biology, and 'Bioinformatics' became a new scientific discipline. Microarrays can measure the abundance of tens of thousands of mRNA species, data on the complete genomic sequences of many different organisms are available, and other technologies make it possible to study various processes at the molecular level. In Bioinformatics and Biostatistics, current research and computations are limited by the available computer hardware. However, this problem can be solved using high-performance computing resources. There are several reasons for the increased focus on high-performance computing: larger data sets, increased computational requirements stemming from more sophisticated methodologies, and latest developments in computer chip production. The open-source programming language 'R' was developed to provide a powerful and extensible environment for statistical and graphical techniques. There are many good reasons for preferring R to other software or programming languages for scientific computations (in statistics and biology). However, the development of the R language was not aimed at providing a software for parallel or high-performance computing. Nonetheless, during the last decade, a great deal of research has been conducted on using parallel computing techniques with R. This PhD thesis demonstrates the usefulness of the R language and parallel computing for biological research. It introduces parallel computing with R, and reviews and evaluates existing techniques and R packages for parallel computing on Computer Clusters, on Multi-Core Systems, and in Grid Computing. From a computer-scientific point of view the packages were examined as to their reusability in biological applications, and some upgrades were proposed. Furthermore, parallel applications for next-generation sequence data and preprocessing of microarray data were developed. Microarray data are characterized by high levels of noise and bias. As these perturbations have to be removed, preprocessing of raw data has been a research topic of high priority over the past few years. A new Bioconductor package called affyPara for parallelized preprocessing of high-density oligonucleotide microarray data was developed and published. The partition of data can be performed on arrays using a block cyclic partition, and, as a result, parallelization of algorithms becomes directly possible. Existing statistical algorithms and data structures had to be adjusted and reformulated for the use in parallel computing. Using the new parallel infrastructure, normalization methods can be enhanced and new methods became available. The partition of data and distribution to several nodes or processors solves the main memory problem and accelerates the methods by up to the factor fifteen for 300 arrays or more. The final part of the thesis contains a huge cancer study analysing more than 7000 microarrays from a publicly available database, and estimating gene interaction networks. For this purpose, a new R package for microarray data management was developed, and various challenges regarding the analysis of this amount of data are discussed. The comparison of gene networks for different pathways and different cancer entities in the new amount of data partly confirms already established forms of gene interaction.

Thu, 23 Jul 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10435/ https://edoc.ub.uni-muenchen.de/10435/1/Brendel_Michael.pdf Brendel, Michael

In this thesis, a novel sequential genes selection and classification (k-SS) method is proposed. The method is analogous to the classical non-linear stepwise variable selection (SVS) methods but unlike any of the SVS methods, this new method uses the misclassification error rates (MERs) as its search criteria for informative marker genes in any given microarray data. Here, the importance of any selected gene is determined based on its marginal contribution at improving the prediction accuracy of the classification rule. This method ensures continuous selection of more genes in as much as the improvements brought into the decision models by the selected genes are considered to be significant enough by some established test criteria. However, further gene selection terminates when none of the remaining genes is capable at improving the prediction accuracy (lowering the MER) of the current model. Therefore, our approach only seeks to select the best combination of k marker genes that are most predictive of the biological samples in any given microarray data sets. An important feature of our new k-SS method is that the size α used by its test is not arbitrarily fixed by the user as common to some of the classical SVS methods. Rather, the value of α at which the best prediction accuracy is achieved (or the best combination of genes is selected) is determined by cross-validation. The new k-SS classifier competes favourably with selected eight existing classification methods using eleven published microarray data sets. The k-SS classifier is very simple to apply and does not require any rigid assumption for its implementation. Another merit of this method lies in its ability to select only those genes that are of biological relevance to the existing cancer sub-groups in microarray data sets. Lastly, we proposed a new preliminary feature selection procedure that employs the cross-validated area under the ROC curve (CVAUC) for gene selection. This method is capable at removing all the irrelevant genes at the preliminary selection stage before any standard classifier like the k-SS method is employed on the remaining data set for final optimum gene selection and classification of mRNA samples. Unlike some other data pruning methods, the new method employs the sub-sampling technique of the v-fold cross-validation to ensure consistency and efficiency of selections made at the preliminary selection stage.

Archaea combine bacterial with eukaryotic features to regulate cellular processes. While initiation of transcription resembles the eukaryotic RNA-Polymerase II apparatus, transcriptional regulation is predominantly bacteria-like. In this work the gene expression profile of amino acid metabolism and metal dependent processes in Halobacterium salinarum R1 was elucidated. To gain insights into transcriptional regulatory processes, microarray technology was used as a global approach. Genes encoding certain DNA binding proteins were deleted and/or overexpressed, to compare the expression pattern of the deletion- and overexpression strains, respectively, with the parental strain R1. For a better understanding of metal dependent processes H. salinarum was grown under iron starvation and compared to cells grown under normal conditions. For the investigation of metal dependent processes the DNA binding proteins SirR and TroR were chosen. To determine a possible function of the regulator protein, conclusions were drawn from a comparison of the deletion mutants ∆sirR and ∆troR, respectively, with the parental strain. SirR (staphylococcal iron regulator repressor) was shown to repress the expression of a Fe(II)/Mn(II) dependent ABC-transporter in the presence of iron. In accordance with this data the same transport operon was shown to be induced under iron starvation. Furthermore, TroR (transport related operon) was shown to repress the expression of a Mn(II)-dependent ABC-transporter. In addition, TroR induces the gene expression of the metal dependent regulator gene idr2, which represses together with iron siderophor synthesis genes. To study the amino acid metabolism in H. salinarum Lrp (leucine-responsive regulatory protein) proteins were chosen, because in both archaea and bacteria Lrp is connected to the coordination of amino acid metabolism. To take a closer look on Lrp-homologs further investigation were performed with lrp and lrpA1. Both genes are located next to genes, encoding proteins involved in amino acid metabolism. Possible Lrp target genes were identified by either constructing lrp and lrpA1 deletion mutants or overexpressing the two genes. Microarray analysis revealed that Lrp functions as a global regulator of transcription. Lrp activates the gene expression of the glutamine synthetase gene glnA, regulates the peptide- and phosphate transport, as well as the central intermediary metabolism, and activates the expression of the transcriptional regulator sirR. By the control of sirR gene expression through Lrp correlation between amino acid metabolism and metal dependent processes could be demonstrated. In contrast to Lrp, LrpA1 regulates gene expression of less genes, amongst them the aspartate transaminase gene aspB3, so that further studies were focussed on the gene regulation of aspB3. The second part of this work examines with specific protein-DNA interactions. Prior to interaction studies, RACE-analysis was used to determine 5´UTR and 3´UTR of certain transcripts. To perform protein-DNA binding studies LrpA1 and TroR were recombinantly expressed in Escherichia coli. A DNA-binding assay adapted to halophilic conditions revealed manganese dependent binding of TroR to its own promoter region. LrpA1 was also shown to bind to the lrpA1 promoter region, as well as an aspartate dependent binding to the aspB3 promoter region. CD-spectroscopy experiments could prove that the interaction between L-aspartate and LrpA1 stabilizes the secondary structure of the protein. To gain more insights into the LrpA1 and L-aspartate dependent aspB3 gene expression, northern blot analysis were performed, that showed an induction of the aspB3 transcription in the absence of L- aspartate. This occurs either in a medium lacking aspartate or after aspartate is metabolized in the stationary phase. At the same time, an induction of the lrpA1 gene expression was observed. This can be illustrated in a model that postulates a reciprocal regulation of the lrpA1 and aspB3 gene expression.

Background: Alterations in brain-derived neurotrophic factor (BDNF) gene expression contribute to serious pathologies such as depression, epilepsy, cancer, Alzheimer's, Huntington and Parkinson's disease. Therefore, exploring the mechanisms of BDNF regulation represents a great clinical importance. Studying BDNF expression remains difficult due to its multiple neural activity-dependent and tissue-specific promoters. Thus, microarray data could provide insight into the regulation of this complex gene. Conventional microarray co-expression analysis is usually carried out by merging the datasets or by confirming the re-occurrence of significant correlations across datasets. However, co-expression patterns can be different under various conditions that are represented by subsets in a dataset. Therefore, assessing co-expression by measuring correlation coefficient across merged samples of a dataset or by merging datasets might not capture all correlation patterns. Results: In our study, we performed meta-coexpression analysis of publicly available microarray data using BDNF as a "guide-gene" introducing a "subset" approach. The key steps of the analysis included: dividing datasets into subsets with biologically meaningful sample content (e. g. tissue, gender or disease state subsets); analyzing co-expression with the BDNF gene in each subset separately; and confirming co-expression links across subsets. Finally, we analyzed conservation in co-expression with BDNF between human, mouse and rat, and sought for conserved over-represented TFBSs in BDNF and BDNF-correlated genes. Correlated genes discovered in this study regulate nervous system development, and are associated with various types of cancer and neurological disorders. Also, several transcription factor identified here have been reported to regulate BDNF expression in vitro and in vivo. Conclusion: The study demonstrates the potential of the "subset" approach in co-expression conservation analysis for studying the regulation of single genes and proposes novel regulators of BDNF gene expression.

Background: Interpretation of comprehensive DNA microarray data sets is a challenging task for biologists and process engineers where scientific assistance of statistics and bioinformatics is essential. Interdisciplinary cooperation and concerted development of software-tools for simplified and accelerated data analysis and interpretation is the key to overcome the bottleneck in data-analysis workflows. This approach is exemplified by gcExplorer an interactive visualization toolbox based on cluster analysis. Clustering is an important tool in gene expression data analysis to find groups of co-expressed genes which can finally suggest functional pathways and interactions between genes. The visualization of gene clusters gives practitioners an understanding of the cluster structure of their data and makes it easier to interpret the cluster results. Results: In this study the interactive visualization toolbox gcExplorer is applied to the interpretation of E. coli microarray data. The data sets derive from two fedbatch experiments conducted in order to investigate the impact of different induction strategies on the host metabolism and product yield. The software enables direct graphical comparison of these two experiments. The identification of potentially interesting gene candidates or functional groups is substantially accelerated and eased. Conclusion: It was shown that gcExplorer is a very helpful tool to gain a general overview of microarray experiments. Interesting gene expression patterns can easily be found, compared among different experiments and combined with information about gene function from publicly available databases.

Background: The increasing number of gene expression microarray studies represents an important resource in biomedical research. As a result, gene expression based diagnosis has entered clinical practice for patient stratification in breast cancer. However, the integration and combined analysis of microarray studies remains still a challenge. We assessed the potential benefit of data integration on the classification accuracy and systematically evaluated the generalization performance of selected methods on four breast cancer studies comprising almost 1000 independent samples. To this end, we introduced an evaluation framework which aims to establish good statistical practice and a graphical way to monitor differences. The classification goal was to correctly predict estrogen receptor status (negative/positive) and histological grade (low/high) of each tumor sample in an independent study which was not used for the training. For the classification we chose support vector machines (SVM), predictive analysis of microarrays (PAM), random forest (RF) and k-top scoring pairs (kTSP). Guided by considerations relevant for classification across studies we developed a generalization of kTSP which we evaluated in addition. Our derived version (DV) aims to improve the robustness of the intrinsic invariance of kTSP with respect to technologies and preprocessing. Results: For each individual study the generalization error was benchmarked via complete cross-validation and was found to be similar for all classification methods. The misclassification rates were substantially higher in classification across studies, when each single study was used as an independent test set while all remaining studies were combined for the training of the classifier. However, with increasing number of independent microarray studies used in the training, the overall classification performance improved. DV performed better than the average and showed slightly less variance. In particular, the better predictive results of DV in across platform classification indicate higher robustness of the classifier when trained on single channel data and applied to gene expression ratios. Conclusions: We present a systematic evaluation of strategies for the integration of independent microarray studies in a classification task. Our findings in across studies classification may guide further research aiming on the construction of more robust and reliable methods for stratification and diagnosis in clinical practice.

Radial glial cells are a widespread non-neuronal cell type in the developing central nervous system (CNS) of all vertebrates. In the cortex, distinct subsets of radial glial cells coexist that are either multipotent or specified towards the generation of neurons or glial cells (Malatesta et al., 2000). Radial glial cells in the cerebral cortex are also the source of a second type of neurogenic progenitors, called basal progenitors. However, whether the generation of basal progenitors occurs in a stochastic manner or whether a specific lineage of radial glial cells is specified towards the generation of these progenitors has not been previously known. To identify functionally distinct lineages of cortical radial glial cells, I developed a new strategy using fluorescence-activated cell sorting (FACS) to isolate them and study their progeny. I isolated radial glial cells by FACS from a transgenic mouse line in which green fluorescent protein (GFP) expression is under the control of the human GFAP promoter. Strikingly, GFP intensity was correlated with cell fate. Selective enrichment of cells with a higher GFP intensity separated a largely non-neurogenic from a neurogenic (low GFP-intensity) subsets of radial glial cells. Notable differences on the progeny of these distinct sets of radial glia were found. The neurogenic radial glial cells subset generated neurons directly and those that are largely non-neurogenic also gave rise to a small proportion of Tbr2-positive basal progenitors that are then neurogenic. Thus, this last subset comprises an indirect neurogenic population of radial glial cells present in the developing cortex. Microarray analysis of these distinct sets of radial glial cells revealed profound differences in their gene expression. Genes related to gliogenesis, proliferation and cell-cycle regulation were expressed at higher levels in the largely non-neurogenic set of radial glia while genes related to neurogenesis, cell adhesion, neurotransmitter secretion and axon guidance were expressed mostly in the neurogenic subset. Moreover, the set of genes expressed at higher levels in the neurogenic radial glia was down-regulated at later stages (cortical radial glia at E18). Thus, this analysis reveals differences at the transcriptional level between direct neurogenic and largely non-neurogenic radial glial cells, supporting their intrinsic lineage differences. The functional analysis of a key fate determinant for neurogenesis from radial glia discovered in this transcriptome analysis will also be presented. This gene is the transcription factor AP2γ which was expressed at significantly higher levels in radial glial cells generating basal progenitors. AP2γ is restricted to the ventricular zone (VZ)/subventricular zone (SVZ) regions of the developing cerebral cortex in the entire nervous system and is also highly expressed in primate and human cortical progenitors. Its genetic deletion within the mouse cerebral cortex results in the molecular misspecification of basal progenitors with decreased levels of Tbr2 and Math3 expression, as well as their overproliferation associated with increased cell death specifically in the occipital cortex. This causes a reduction in upper layer neuron generation with intriguing functional defects in visual acuity. Gain-of-function studies also revealed the important role of AP2γ for the adequate specification and development of basal progenitors in the cerebral cortex, while apical progenitors were not affected by the loss- and gain-of-function of this transcription factor. Thus, I show for the first time the prospective isolation of distinct radial glia subtypes in the mouse developing cortex demonstrated at the molecular and functional level.

Microarray-Daten werden in letzter Zeit häufig genutzt, um mit Hilfe verschiedener Verfahren Netzwerke der Gen-Gen-Interaktion zu generieren. Die vorliegende Arbeit beschäftigt sich mit Validierungsstudien solcher Verfahren. Der Startpunkt einer Validierungsstudie ist ein ungerichteter Graph, der biologische Strukturen repräsentieren soll. In dieser Arbeit wird motiviert, Graphen zu benutzen, die aus Microarray-Daten geschätzt worden sind. Nachdem ein Graph gewählt worden ist, werden Daten einer multivariaten Normalverteilung erzeugt, die durch eine zufällige Kovarianzmatrix charakterisiert ist. Diese Matrix muss symmetrisch und positiv definit sein, aber zusätzlich wird für eine nicht vorhandene Kante im Graphen gefordert, dass der zugehörige Eintrag in der Matrix Null ist. In dieser Arbeit wird ein neuer Ansatz vorgestellt, der es ermöglicht, symmetrische, positiv definite Matrizen mit Nebenbedingungen zu erzeugen. Diese Methode beruht auf der Moralisierung eines Graphen. Ein gerichteter, azyklischer Graph wird moralisiert, indem die gerichteten Kanten durch ungerichtete Kanten ersetzt werden und zusätzlich die Eltern eines jeden Knotens paarweise miteinander verbunden werden. Der zentrale Schritt bei der Erstellung der Matrizen mit Nebenbedingungen liegt in der Umkehrung des Moralisierungsvorganges. In dieser Arbeit wird die Klasse der Graphen eingeführt, die Resultat einer Moralisierung sein könnten - die prämoralisierbaren Graphen - und es wird ein Verfahren definiert, welches entscheidet, ob ein Graph prämoralisierbar ist und gegebenenfalls eine Umkehrung der Moralisierung durchführt. Die erzeugten Matrizen sollen als Korrelationsmatrizen für die Validierungsstudien genutzt werden. Dazu wird das vorgestellte Verfahren an einen Optimierungsalgorithmus gekoppelt, um die gewünschten Matrizen zu erzeugen, deren Diagonalelemente identisch 1 sind und für die die nicht als Null vorgegebenen Werte nahe 1 bzw. -1 liegen. Nicht jeder Graph ist prämoralisierbar. Da diese Eigenschaft notwendig ist für das Verfahren zur Erzeugung der Matrizen mit Nebenbedingungen, wird eine empirische Studie durchgeführt, die zeigt, dass ein Großteil der aus Microarray-Daten geschätzten Graphen auch prämoralisierbar ist. Die Arbeit schließt mit praktischen Anwendungen. Die Validierung eines bekannten Algorithmus zum Schätzen von Netzwerken wird durchgeführt und es wird ein Ansatz vorgestellt, mit dem man graphische Strukturen, die aus Microarray-Daten geschätzt worden sind, vergleichen kann, um signifikante Unterschiede zu finden.

ESF Courses

Diabetic nephropathy (DN) is a complex and chronic metabolic disease that evolves into a progressive fibrosing renal disorder. Effective transcriptomic profiling of slowly evolving disease processes such as DN can be problematic. The changes that occur are often subtle and can escape detection by conventional oligonucleotide DNA array analyses. We examined microdissected human renal tissue with or without DN using Affymetrix oligonucleotide microarrays (HG-U133A) by standard Robust Multi-array Analysis (RMA). Subsequent gene ontology analysis by Database for Annotation, Visualization and Integrated Discovery (DAVID) showed limited detection of biological processes previously identified as central mechanisms in the development of DN (e.g. inflammation and angiogenesis). This apparent lack of sensitivity may be associated with the gene-oriented averaging of oligonucleotide probe signals, as this includes signals from cross-hybridizing probes and gene annotation that is based on out of date genomic data. We then examined the same CEL file data using a different methodology to determine how well it could correlate transcriptomic data with observed biology. ChipInspector (CI) is based on single probe analysis and de novo gene annotation that bypasses probe set definitions. Both methods, RMA and CI, used at default settings yielded comparable numbers of differentially regulated genes. However, when verified by RT-PCR, the single probe based analysis demonstrated reduced background noise with enhanced sensitivity and fewer false positives. Using a single probe based analysis approach with de novo gene annotation allowed an improved representation of the biological processes linked to the development and progression of DN. The improved analysis was exemplified by the detection of Wnt signaling pathway activation in DN, a process not previously reported to be involved in this disease.

Gene duplication is an opportunity for evolving new functions from the newer gene, but also has a disadvantage due to local gene-rearrangement effects and, if duplications are numerous, through alterations of genome size. Therefore, selection is playing a central role in determining the fate of a duplicate gene. Plants are known to harbor numerous gene families, and are thus an ideal system to test the fate of gene duplicates. This thesis tackles the tropinone-reductase like enzymes (further TRL) and the tau GSTs located upstream from this gene family. TRL enzymes are short-chain dehydrogenases that are involved in a reduction step downstream in the synthesis of tropane alkaloids in Solanaceae, important defense compounds of plants. The function of TRLs in Brassicaceae is not clear, since most of the plants in this family do not produce tropane alkaloids, but some have been associated with the oxidative-stress response. This gene family contains 80% of its members duplicated in tandem in Arabidopsis thaliana. We profited from this fact to isolate 12 TRL (+ pseudogenes) from this species, further six species of Brassicaceae (A. thaliana, A. lyrata, A. cebennensis, Capsella rubella, Boechera divaricarpa and Brassica rapa), and one species from a closely related plant family, Cleome spinosa. We tested the role that selection plays in maintaining large numbers of this gene family. We used phylogenetic methods to analyze non-coding sequence evolution and identified regulatory motifs. We analyzed non-coding sequence evolution. Microarray expression data from A. thaliana and qPCR for A. thaliana and A. lyrata were analyzed to detect divergence in the expression patterns of orthologs and paralogs. TRL genes follow a gene birth and death dynamics. More probable, they originated from non-equal recombination of tandem duplicated genes. Positive selection at the origin of the duplicated genes allowed these to acquire differential expression patterns, leading to the preservation of numerous TRLs. The analysis of coding and non-coding sequences shows them to display correlated evolution, particularly in species recently separated by speciation. We further tested for selection on the tau glutathione-S-transferases (GST) enzymes, adjacent 3' in the genome to TRLs. Tau GSTs are unique to plants and are involved in detoxification. Multiple copies of these enzymes will allow flexibility in substrate specificity, which is important for the detoxification function. We detected positive selection among paralogs of tau GSTs supporting their potential of functional diversity, but we also detected negative selection among paralogs and groups of orthologs, indicating that more often their functions are conserved.

Placental proteomes in preeclampsia, Report: The 2006 Human Liver Proteome Project (HLPP) Workshops, Microarray for autoantibody profiling of prostate cancer sera, "goProteomics" website.

Placental proteomes in preeclampsia, Report: The 2006 Human Liver Proteome Project (HLPP) Workshops, Microarray for autoantibody profiling of prostate cancer sera, "goProteomics" website.

This course reviews the key genomic technologies and computational approaches that are driving advances in prognostics, diagnostics, and treatment. Throughout the semester, emphasis will return to issues surrounding the context of genomics in medicine including: what does a physician need to know? what sorts of questions will s/he likely encounter from patients? how should s/he respond? Lecturers will guide the student through real world patient-doctor interactions. Outcome considerations and socioeconomic implications of personalized medicine are also discussed. The first part of the course introduces key basic concepts of molecular biology, computational biology, and genomics. Continuing in the informatics applications portion of the course, lecturers begin each lecture block with a scenario, in order to set the stage and engage the student by showing: why is this important to know? how will the information presented be brought to bear on medical practice? The final section presents the ethical, legal, and social issues surrounding genomic medicine. A vision of how genomic medicine relates to preventative care and public health is presented in a discussion forum with the students where the following questions are explored: what is your level of preparedness now? what challenges must be met by the healthcare industry to get to where it needs to be?

This course reviews the key genomic technologies and computational approaches that are driving advances in prognostics, diagnostics, and treatment. Throughout the semester, emphasis will return to issues surrounding the context of genomics in medicine including: what does a physician need to know? what sorts of questions will s/he likely encounter from patients? how should s/he respond? Lecturers will guide the student through real world patient-doctor interactions. Outcome considerations and socioeconomic implications of personalized medicine are also discussed. The first part of the course introduces key basic concepts of molecular biology, computational biology, and genomics. Continuing in the informatics applications portion of the course, lecturers begin each lecture block with a scenario, in order to set the stage and engage the student by showing: why is this important to know? how will the information presented be brought to bear on medical practice? The final section presents the ethical, legal, and social issues surrounding genomic medicine. A vision of how genomic medicine relates to preventative care and public health is presented in a discussion forum with the students where the following questions are explored: what is your level of preparedness now? what challenges must be met by the healthcare industry to get to where it needs to be?

This course reviews the key genomic technologies and computational approaches that are driving advances in prognostics, diagnostics, and treatment. Throughout the semester, emphasis will return to issues surrounding the context of genomics in medicine including: what does a physician need to know? what sorts of questions will s/he likely encounter from patients? how should s/he respond? Lecturers will guide the student through real world patient-doctor interactions. Outcome considerations and socioeconomic implications of personalized medicine are also discussed. The first part of the course introduces key basic concepts of molecular biology, computational biology, and genomics. Continuing in the informatics applications portion of the course, lecturers begin each lecture block with a scenario, in order to set the stage and engage the student by showing: why is this important to know? how will the information presented be brought to bear on medical practice? The final section presents the ethical, legal, and social issues surrounding genomic medicine. A vision of how genomic medicine relates to preventative care and public health is presented in a discussion forum with the students where the following questions are explored: what is your level of preparedness now? what challenges must be met by the healthcare industry to get to where it needs to be?

Click to Play A quick look at a paper which allows me to launch into my favorite criticisms of microarray data. In short, most people doing Affy arrays don't have any idea what they're doing. Good for Affy. Bad for science. Bad for the public. These guys even try to pull in autism into their analysis. Blech. Running time ~7minFormats available: Quicktime (.mov), Flash Video (.flv)Tags: science, autism, rna, microarray, blood

Growing evidence supports the concept that polymorphonuclear neutrophils (PMN) are critically involved in inflammation-mediated angiogenesis which is important for wound healing and repair. We employed an oligonucleotide microarray technique to gain further insight into the molecular mechanisms underlying the proangiogenic potential of human PMN. In addition to 18 known angiogenesis-relevant genes, we detected the expression of 10 novel genes, namely midkine, erb-B2, ets-1, transforming growth factor receptor-beta(2) and -beta(3), thrombospondin, tissue inhibitor of metalloproteinase 2, ephrin A2, ephrin B2 and restin in human PMN freshly isolated from the circulation. Gene expression was confi rmed by the RT-PCR technique. In vivo evidence for the role of PMN in neovascularization was provided by studying neovascularization in a skin model of wound healing using CD18-deficient mice which lack PMN infi ltration to sites of lesion. In CD18-deficient animals, neo- vascularization was found to be signifi cantly compromised when compared with wild- type control animals which showed profound neovascularization within the granulation tissue during the wound healing process. Thus, PMN infiltration seems to facilitate inflammation mediated angiogenesis which may be a consequence of the broad spectrum of proangiogenic factors expressed by these cells. Copyright (c) 2006 S. Karger AG, Basel.

The application of global gene expression profiling allows to obtain detailed molecular fingerprints of underlying gene expression in any cell of interest. In this work gene expression profiles were generated from a comprehensive cohort of leukemia patients and healthy donors referred to and diagnosed in the Laboratory for Leukemia Diagnostics, Munich, Germany, which is a nation-wide reference center for the diagnosis of hematologic malignancies. Thoroughly characterized clinical samples were analyzed by high-density microarrays interrogating the expression status of more than 33,000 transcripts. In one specific aspect of this work the potential application of gene expression signatures for the prediction and classification of specific leukemia subtypes was assessed. Today the diagnosis and subclassification of leukemias is based on a controlled application of various techniques including cytomorphology, cytogenetics, fluorescence in situ hybridization, multiparameter flow cytometry, and PCR-based methods. The diagnostic procedure is performed according to a specific algorithm, but is time-consuming, cost-intensive, and requires expert knowledge. Based on a very low number of candidate genes it is demonstrated in this work that prognostically relevant acute leukemia subtypes can be classified using microarray technology. Moreover, in an expanded analysis including 937 patient samples representing 12 distinct clinically relevant acute and chronic leukemia subtypes and healthy, non-leukemia bone marrow specimens a diagnostic prediction accuracy of ~95% was achieved. Thus, given these results it can be postulated that the occurring patterns in gene expression would be so robust that they would allow to predict the leukemia subtype using global gene expression profiling technology. This finding is further substantiated through the demonstration that reported differentially expressed genes from the literature, namely pediatric gene expression signatures representing various acute lymphoblastic leukemia (ALL) subtypes, can be used to independently predict the corresponding adult ALL subtypes. Furthermore, it could be demonstrated that microarrays both confirm and reproduce data from standard diagnostic procedures, but also provide very robust results. Parameters such as partial RNA degradation, shipment time of the samples, varying periods of storage of the samples, or target preparations at different time points from either bone marrow or peripheral blood specimens by different operators did not dramatically influence the diagnostic gene expression signatures. In another major aspect of this work gene expression signatures were examined in detail to obtain new insights into the underlying biology of acute promyelocytic leukemia (APL) and t(11q23)/MLL leukemias. In APL, microarrays led to a deeper understanding of morphological and clinical characteristics. Firstly, genes which have a functional relevance in blood coagulation were found to be differentially expressed when APL was compared to other acute myeloid leukemia (AML) subtypes. Secondly, a supervised pairwise comparison between the two different APL phenotypes, M3 and its variant M3v, for the first time revealed differentially expressed genes encoding for biological functions and pathways such as granulation and maturation. With respect to 11q23 leukemias it could be demonstrated that leukemias with rearrangements of the MLL gene are characterized by a common specific gene expression signature. Additionally, in unsupervised and supervised data analysis algorithms ALL and AML cases with t(11q23)/MLL segregated according to the lineage, i.e., myeloid or lymphoid, respectively. This segregation could be explained by a highly differing transcriptional program. Through the use of biological network analyses essential regulators of early B cell development, PAX5 and EBF, were shown to be associated with a clear B-lineage commitment in lymphoblastic t(11q23)/MLL leukemias. Also, the influence of the different MLL translocation partners on the transcriptional program was directly assessed. But interestingly, gene expression profiles did not reveal a clear distinct pattern associated with one of the analyzed partner genes. Taken together, the identified molecular expression pattern of MLL fusion gene samples and biological networks revealed new insights into the aberrant transcriptional program in t(11q23)/MLL leukemias. In addition, a series of analyses was targeted to obtain new insights into the underlying biology in heterogeneous B-lineage leukemias not positive for BCR/ABL or MLL gene rearrangements. It could be demonstrated that the genetically more heterogeneous precursor B-ALL samples intercalate with BCR/ABL-positive cases, but their profiles were clearly distinct from T-ALL and t(11q23)/MLL cases. In conclusion, various unsupervised and supervised data analysis strategies demonstrated that defined leukemia subtypes can be characterized on the basis of distinct gene expression signatures. Specific gene expression patterns reproduced the taxonomy of this hematologic malignancy, provided new insights into different disease subtypes, and identified critical pathway components that might be considered for future therapeutic intervention. Based on these results it is now further possible to develop a one-step diagnostic approach for the diagnosis of leukemias using a customized microarray.

Fri, 23 Sep 2005 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/4238/ https://edoc.ub.uni-muenchen.de/4238/1/Dolce_Luca.pdf Dolce, Luca ddc:570, ddc:500, Fakultät für B

Usual microarray data sets include only a handful of observations, but several thousands of predictor variables. Transforming the high-dimensional predictor space to make classification (for instance cancer diagnosis) possible is a major challenge. This thesis deals with various dimension reduction approaches which can handle such data. Chapter 2 gives an introduction into classification with microarray data as well as an overview of a few specific problems such as variable selection and comparison of classification methods. In Chapter 3, I discuss a particular class of interaction structures in the classification framework: "emerging patterns". I propose a new and more general definition referring to underlying probabilities and present a new simple method which is based on the CART algorithm to find the corresponding empirical patterns in concrete data sets. In addition, the detected patterns can be used to define new variables for classification. Thus, I propose a simple scheme to use the patterns to improve the performance of classification procedures. I implemented the search algorithm as well as the classification procedure in the language R. Some of these programs are publicly available from my homepage. Chapter 4 deals with classical linear dimension reduction methods. In the context of binary classification with continuous predictors, I prove two properties concerning the connections between Partial Least Squares (PLS) dimension reduction, between-group PCA and between linear discriminant analysis and between-group PCA. PLS dimension reduction for classification is examined thoroughly in Chapter 5. The classification procedure consisting of PLS dimension reduction and linear discriminant analysis on the new components is compared favorably with some of the best state-of-the-art classification methods using nine real microarray cancer data sets. Moreover, I apply a boosting algorithm to this classification method, which is a novel approach. In addition, I suggest a simple procedure to choose the number of PLS components. At last, I examine the connection between PLS dimension reduction and variable selection and prove a property concerning the equivalence between a common univariate selection criterion and a variable selection approach based on the first PLS component.

The study of the network between transcription factors and their targets is important for understanding the complex regulatory mechanisms in a cell. However, due to post-translational modifications the regulator transcription levels (as measured, e.g., by microarray expression arrays) generally provide only little information about the true transcription factor activities (TFAs). Here we propose an approach based on partial least squares (PLS) regression to infer true TFAs from expression data integrated with information from DNA-protein binding experiments (e.g., ChIP). This method is statistically sound also for a small number of samples and enables to detect functional interaction among the transcription factors themselves via the inference of 'meta'-transcription factors. In addition, it allows to identify false positives in ChIP data as well as to predict activation and suppression activities (which is not possible from ChIP data alone). Subsequent to PLS inference, the estimated transcription factor activities may be subject to further analysis such as tests of periodicity or differential regulation. This method overcomes the limitations of previously used approaches, and is illustrated by analyzing expression and ChIP data from Yeast and E.Coli experiments.

PLS dimension reduction is known to give good prediction accuracy in the context of classification with high-dimensional microarray data. In this paper, PLS is compared with some of the best state-of-the-art classification methods. In addition, a simple procedure to choose the number of components is suggested. The connection between PLS dimension reduction and gene selection is examined and a property of the first PLS component for binary classification is proven. PLS can also be used as a visualization tool for high-dimensional data in the classification framework. The whole study is based on 9 real microarray cancer data sets.

Emerging patterns represent a class of interaction structures which has been recently proposed as a tool in data mining. In this paper, a new and more general definition refering to underlying probabilities is proposed. The defined interaction patterns carry information about the relevance of combinations of variables for distinguishing between classes. Since they are formally quite similar to the leaves of a classification tree, we propose a fast and simple method which is based on the CART algorithm to find the corresponding empirical patterns in data sets. In simulations, it can be shown that the method is quite effective in identifying patterns. In addition, the detected patterns can be used to define new variables for classification. Thus, we propose a simple scheme to use the patterns to improve the performance of classification procedures. The method may also be seen as a scheme to improve the performance of CARTs concerning the identification of interaction patterns as well as the accuracy of prediction.

Various supervised learning and gene selection methods have been used for cancer diagnosis. Most of these methods do not consider interactions between genes, although this might be interesting biologically and improve classification accuracy. Here we introduce a new CART-based method to discover emerging patterns. Emerging patterns are structures of the form (X1>a1)AND(X2