Podcasts about maldi tof

Dr. Suzanne Silbert, Microbiology Director for Tampa General Hospital, presents this introductory talk on the basics of the microbiology lab for 2025. Dr. Silbert begins with an overview of clinical microbiology, and then reviews the basic classification standards for bacterial organisms, viruses and fungi. She then goes on to discuss culture cultivation techniques, including the use of liquid, solid, or semi-solid media. The speaker then covers identification systems for bacterial organisms, including MALDI-TOF, Disk-Diffusion, and molecular diagnostic techniques.

In this episode, hosts Sophia Chandrasekar and Doryan Redding are joined by Emily Woten and ASCLS President-Elect Dr. Kyle Riding. The four explore the question that's plagued a few medical laboratory geeks—how do science fiction instrumentation work? How reliable are they? And why does MALDI-TOF keep coming up? Tune in and comment below on how you think this stuff works! References to the science fiction in this episode. Thanks to our episode sponsor, Memorial Healthcare System.

Dr. Richard Oehler, Professor of Medicine at the Division of Infectious Diseases at the University of South Florida Morsani College of Medicine, presents a review of bacteremia. Dr. Oehler begins by reviewing the epidemiology, personal and institutional costs of bacteremia. He then discusses blood culture collection and how false positive blood cultures occur. With the recent blood culture bottle shortage of 2024, he emphasizes the new concept of “blood culture stewardship”–when it is most appropriate to order a blood culture versus when it may be unnecessary. Next, Dr. Oehler reviews automated blood culture systems and other related identification technology, with a focus on new methods of blood culture isolate identification such as multiplex PCR and Maldi-TOF. The speaker then contrasts gram positive and gram negative bacteremia in general and by organism. The management of a positive blood culture with regard to treatment and source control is discussed. Lastly, ways to reduce the incidence of bacteremia are shared.

Welcome to our inaugural podcast episode where we dive into the captivating realm of clinical microbiology and beyond. This time, we're exploring the enigmatic presence of Legionella in hot springs, unraveling the nature of chopped meat broth, and revealing the magic behind the MALDI-TOF technology in bacteria detection. Ponder with us: Is more, more, more bacteria going to get you better MALDI-TOF scores? Tune in as we dissect these fascinating subjects with a mix of expertise and lighthearted banter, bringing the microscopic world to life right before your ears.

In this episode I continue talking about MALDI-TOF MS. This time I go over the advantages and disadvantages. How has MALDI-TOF helped Clinical Microbiology? How does it compare to traditional identification methods and the workflow? What about disadvantages? Tune in to find out.

Following the webinar on MALDI-TOF MS, we think that now it is a good time to go over MALDI-TOF MS. This topic was discussed two years ago, but since there are new listeners, now is a good time to go over it again. Are you familiar with MALDI-TOF? How does it work? How does it help Clinical Microbiology? Tune in to find out.

Dr. Suzanne Silbert, Microbiology Director for Tampa General Hospital, presents this introductory talk on the basics of the microbiology lab. Dr. Silbert begins with an overview of clinical microbiology, and then reviews the basic classification standards for bacterial organisms, viruses and fungi. She then goes on to discuss culture cultivation techniques, including the use of liquid, solid, or semi-solid media. The speaker then covers identification systems for bacterial organisms, including MALDI-TOF, Disk-Diffusion, and molecular diagnostic techniques. A robust question and answer session concludes the session

 AABP Executive Director Dr. Fred Gingrich is joined by Drs. Andy Lefeld, Justine Britten, and Allan Britten in this podcast brought to you by the AABP Milk Quality and Udder Health Committee. You can find information about the Milk Quality and Udder Health Committee on this page and find all AABP committee resources at this link. The Brittens manage Udder Health Systems with labs located in Washington, Utah and Idaho. Lefeld is a member of the Milk Quality and Udder Health Committee and a veterinarian at Maria Stein Animal Clinic in Maria Stein, Ohio, providing milk quality services to dairy farms.  Mastitis is the most costly disease in the dairy industry with an economic impact estimated at $2 billion annually. Our guests discuss the types of mastitis pathogens and the importance of identifying the pathogens causing mastitis on dairy farms to know if the infections are due to contagious or environmental pathogens. It is also important to obtain diagnostics to the species level for some pathogens. Monitoring pathogens can include bulk tank cultures, individual cow cultures, or string sampling. We discuss how veterinarians can get involved in mastitis diagnostic programs, including setting up and monitoring on-farm culture programs, in-clinic milk quality laboratories or utilizing an outside diagnostic lab. There are several newer technologies that labs now provide including PCR, MALDI-TOF, and chromogenic agars. Veterinarians have the opportunity to assist producers in developing diagnostic programs to manage mastitis on the dairy farm. We also discuss that a diagnostic test should be utilized if the results will alter an intervention, either treatment or prevention. Antimicrobial sensitivities on mastitis pathogens are not routinely recommended since an antimicrobial sensitivity test is unlikely to change the intervention on the farm due to the limited number of intramammary tubes available and the lack of break points for most intramammary antimicrobials. Finally, it is important for veterinarians to develop quality control programs for both in-house cultures as well as cultures from on-farm programs. One such program is the QMPS program from Cornell University. Veterinarians should discuss with their dairy farmers how they can utilize diagnostic testing as a part of a total milk quality control program.

Back after a summer break, Jame and Callum discuss the fascinating machine that is the MALDI-TOF. Matrix Assisted Laser Desorption Ionisation Time Of Flight mass spectrometry. We discuss the basics of how it functions, its application in the lab and some pros and cons of its use along with what the future may hold. Here are some relevant videos and papers:- Exciting video of MALDI-TOFhttps://www.youtube.com/watch?v=0jeFpXHZ8W0&ab_channel=BrukerDaltonics- MALDI-TOF to detect MRSA https://www.frontiersin.org/articles/10.3389/fmicb.2020.00232/full- Direct MALDI-TOF on blood cultures https://www.spandidos-publications.com/10.3892/etm.2020.9365- MALDI-TOF to detect CPEhttps://www.frontiersin.org/articles/10.3389/fmicb.2021.789731/full 

Antibiotic susceptibility testing is too slow. Faster identification of microorganisms is now common, as many laboratories use MALDI-TOF or molecular technologies for quick and definitive identification of bacteria. Improvements in susceptibility testing have lagged, as we continue to use tests that take a day for results, and which have not significantly changed in decades. Rapid phenotypic testing has can only be done on limited sample types, using a dedicated platform, and it has not been widely adopted. Tests for rapid genotypic testing usually include only a few genes and require confirmation by phenotypic testing. What are the prospects for fast susceptibility testing? Guests: Dr. Trish Simner. Trish, Associate Professor of Pathology at Johns Hopkins Medicine, where she is also Director of the Medical Bacteriology and Infectious Disease Sequencing. Dr. Dan Rhoads. Dan is the Section Head of Microbiology at the Cleveland Clinic, where he holds The Belinda Yen-Lieberman, PhD, and James M. Lieberman, MD, Endowed Chair in Clinical Microbiology. Trish and Dan are first and last authors on a paper in press at JCM. The title is “Multicenter Evaluation of the Acuitas AMR Gene Panel for Detection of an Extended Panel of Antimicrobial Resistance Genes among Bacterial Isolates.” Topics of Discussion • Scope of the AST problem • Conventional AST – how long does it take? • General approaches to reducing the time for AST – targeted genotypic (PCR), whole genome sequencing, and faster phenotypic methods. What do you see as potential for each? • What is the Acuitas AMR Gene Panel and how does it work? • Study design • Summary of results • Discrepant results • Workflow • Where do you see this fitting into current laboratory testing Links • Multicenter Evaluation of the Acuitas AMR Gene Panel for Detection of an Extended Panel of Antimicrobial Resistance Genes among Bacterial Isolates. https://journals.asm.org/doi/10.1128/JCM.02098-21 This episode of Editors in Conversation is brought to you by the Journal of Clinical Microbiology and hosted by JCM Editor in Chief, Alex McAdam and Dr. Elli Theel. JCM is available at https://asm.org/jcm. Follow EIC Alex McAdam on twitter for JCM updates via https://twitter.com/JClinMicro and co-host, Elli Theel at https://twitter.com/ellitheelphd. Subscribe to the podcast at https://asm.org/eic

Happy 2022 to you all! We are glad start a new year with you with this episode. This time, we bring you an interview with our first UAC PhD graduate, Christer Malmberg, who defended his industry-PhD thesis last year, where he developed a new rapid method of testing antibiotic susceptibility. We talk about his experience throughout his studies, what this new method brings to the table, and what he wishes for the future. In the news section, we continue talking about diagnostics, looking into a recent study that explores machine learning and the readily available MALDI-TOF system to provide early information that can help guide treatment recommendations for infections. We also talk about the new seminal paper published last month in The Lancet, presenting the most up-to-date and comprehensive data on the global burden of AMR. We hope you enjoy! Check relevant links in the show notes at www.uac.uu.se/the-amr-studio/episode35/. Follow our updates on twitter on www.twitter.com/uac_uu with #theAMRstudio hashtag! Theme music by Henrik Niss: www.tinyurl.com/henriknissspotify.

Dr Riati Scarboroughis is a researcher at the National Centre for Antimicrobial Stewardship and a PhD fellow at the University of Melbourne. Her PhD focuses on improving our understanding of the conscious and subconscious motivations behind suboptimal antimicrobial prescribing in Australian veterinary practices, and designing sustainable strategies to support better antimicrobial prescribing in veterinarians. Ri is particularly interested in the use of social norms and nudges to modify behaviour. And it's these behaviours that we discuss in this episode. Ri highlights some common areas where many vet practices could rethink their antibiotic prescribing protocols, with a great discussion on WHY it's so important. We cover topics like Why the old antibiotic mantra 'finish the course' is giving way to 'shorter is better" Antimicrobial dosing: you can't always trust the label UTIs - choose Amoxycillin over Amoxyclav or Convenia Bacteriuria does not always require antimicrobials Catfight abscesses - antimicrobials rarely needed Surgical antimicrobial prophylaxis - get the timing right A word of warning: some of these topics might wake up an annoying little voice in the back of your head that will bother you every time you head into the dispensary for those AB's, but we think it's worth it. See it as a little nudge! Here are the resources as promised in the episode. Thanks to the SVS Pathology Network, who our Australian listeners will know as Vetnostics in NSW, QML Vetnostics in Queensland, TML Vetnostics in Tasmania, ASAP in Victoria and Vetpath in WA, for supporting the podcast and introducing us to Ri and her work. Have a look at this video about Maldi TOF spectrometry - the technology that explains why SVS clients will now get super-fast turnaround times for their microbiology testing. Go to thevetvault.com for show notes and to check out our guests' favourite books, podcasts and everything else we talk about in the show. If you want to lift your clinical game, go to vvn.supercast.com for a free 2-week trial of our short and sharp high-value clinical podcasts. We love to hear from you. If you have a question for us or you'd like to give us some feedback please leave us a voice message by going to our episode page on the anchor app and hitting the record button, via email at thevetvaultpodcast@gmail.com, or just catch up with us on Instagram. And if you like what you heard then please share the love by clicking on the share button wherever you're listening and sending a link to someone who you know will enjoy listening. --- Send in a voice message: https://anchor.fm/vet-vault/message

For such a seemingly simple sample there's a lot you can learn from a urinalysis. Many of us also have a fair amount of uncertainty around much of the 'how' of urine sample handling, analysis and interpretation. It's also the one bit of lab work where being good at in-house testing can make a big difference to the reliability of your results. We KNOW you'll have had some disagreements at some point in your career about at least a few of the questions we answer in this episode, like fridge vs benchtop, how old is too old for a urine sample, how long after starting antibiotics can you still culture, is it even worth culturing a free-flow sample, WHEN should I culture, can you trust your dipstick, can you trust AI, why some urinary bugs just won't die, and what the heck is the deal with casts?! I also suspect that, like us, you'll learn a few things that you didn't even KNOW you should know. Our guest is Dr Kristen Todhunter, a pathologist for the SVS Pathology Network who confesses to having a bit of a soft spot for all things microbiology. She answers all of the questions we've ever had around how to get the most from your urinalysis. Thanks to the SVS Pathology Network, who our Aussie listeners will know as Vetnostics in NSW, QML Vetnostics in Queensland, TML Vetnostics in Tasmania, ASAP in Victoria and Vetpath in WA, for providing us with the brainpower for our pathology series and for supporting the podcast. If you love new toys and tech (or if you like lasers!) you should definitely check out this video about Maldi TOF spectrometry - the amazing new technology that will explain why SVS clients will now get super-fast turnaround times for their microbiology testing. Go to thevetvault.com for show notes and to check out our guests' favourite books, podcasts and everything else we talk about in the show. If you want to lift your clinical game, go to vvn.supercast.com for a free 2-week trial of our short and sharp high-value clinical podcasts. We love to hear from you. If you have a question for us or you'd like to give us some feedback please leave us a voice message by going to our episode page on the anchor app and hitting the record button, via email at thevetvaultpodcast@gmail.com, or just catch up with us on Instagram. And if you like what you heard then please share the love by clicking on the share button wherever you're listening and sending a link to someone who you know will enjoy listening. --- Send in a voice message: https://anchor.fm/vet-vault/message

This episode is part two of a two-part episode. What are the limitations of the Vitek MS? In this episode I go over the limitations of the Vitek MS regarding identification of organisms. I also go over the pros and cons of having this system in the laboratory.

What is MALDI-TOF? How is it used in Clinical Microbiology?. This is the first part of a two part episode where I discuss MALDI-TOF, and its applications in Clinical Microbiology.

Susceptibility testing for staphylococci other than S. aureus, or SOSA, has become increasingly complicated, as more laboratories use MALDI-TOF to routinely identify these bacteria to the species level. In particular, accurate identification of methicillin resistance has become more complex as the different species are distinguished by the accuracy of different susceptibility testing methods and breakpoints for interpreting MICs and zone sizes.  Some of the questions we’ll discuss include: What is the gold standard for detecting methicillin resistance in SOSA? How will the recommended breakpoints for detection of methicillin-resistant SOSA change? Why should we call these bacteria SOSA instead of coagulase-negative staphylococci? Guests: Dr. Romney Humphries, Dr. Lars Westblade Links mentioned: Evaluation of Surrogate Tests for the Presence of mecA-Mediated Methicillin Resistance in Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus warneri The End of Coagulase-Negative Staphylococci? A Micro-Comic Strip Subscribe to Editors in Conversation (free) on Apple Podcasts, Google Podcasts, Android, Spotify.

Los fructanos de agave son reconocidos como nutracéuticos y son ya un producto en el mercado, principalmente los de agave azul Agave tequilana W. Sin embargo no son los únicos y en México se estudian otras especies del género como A. salmiana ssp. crassispina también con esta finalidad. Una investigadora que ha contribuido a su estudio es la Dra. Loreno Moreno V. quien nos comparte su historia. Ella es investigadora de la Cátedra CONACYT adscrita al área de tecnología alimentaria del CIATEJ, unidad Guadalajara, desde el 2016 al 2020. Además es miembro del Sistema Nacional de Investigadores: nivel 1. Formación: Ingeniero en alimentos y Doctorado en Ciencias en Bioprocesos por parte de la Universidad Autónoma de San Luis Potosí. Egresada en el 2013. Posdoctorado en la Unidad de Biotecnología Industrial del CIATEJ. Estancias: Ha realizado estancias de investigación en la Universidad Autónoma de Coahuila, Universidad Autónoma del Estado de Hidalgo, Université Laval en Quebec, Canadá y Université d’Orléans en Francia. Publicaciones: Es autora de 17 publicaciones científicas (incluidos artículos originales, revisiones y capítulos de libro) y cuenta con alrededor de 130 citas. Ha dirigido 3 tesis de licenciatura y 1 de posgrado. Líneas de investigación: 1) Aprovechamiento de materias primas para la obtención de alimentos funcionales 2) Mejoramiento de procesos alimentarios con tecnologías emergentes Artículos recientes: 1. Luiz-Santos, y col. Performance Evaluation of Tight Ultrafiltration Membrane Systems at Pilot Scale for Agave Fructans Fractionation and Purification. Membranes 2020, 10, 261. 2. H.M. Hernández-Hernández y col. Current status of emerging food processing technologies in Latin America: Novel Non-thermal processing. Innovative Food Science & Emerging Technologies, 2019. Accepted 3. Moreno-Vilet L., y col. Comparative data of molecular weight distribution of agave fructans fractions using MALDI-ToF and HPLC-SEC. Data in brief, Volume 24, June 2019, 103984. 4. L. Moreno-Vilet y col. Current status of emerging food processing technologies in Latin America: Novel thermal processing. Innovative Food Science & Emerging Technologies, 2018. 50, pages: 196-206. 5. Moreno-Vilet L., y col. Size-exclusion chromatography (HPLC-SEC) technique optimization by simplex method to estimate molecular weight distribution of agave fructans. Analytical Methods. Food Chemistry, 2017. 237, pages 833-840. 6. L. Moreno-Vilet, y col. Assessment of sugars separation from a model carbohydrates solution by nanofiltration using a design of experiments (DoE) methodology, Separation and Purification Technology. 2014, 131, 84-93. 7. L. Moreno-Vilet, ycol. In vitro Assessment of Agave Fructans (Agave salmiana) as Prebiotics and Immune System Activators, International Journal of Biological Macromolecules, 2014, 63: 181-187. (No. 8 in the list of most downloaded of the journal) 8. L. Moreno-Vilet, y col. Sugars and Fructans Separation by Nanofiltration from Model Sugar Solution and Comparative Study with Natural Agave Juice, Separation Science and Technology. 2013, 48:12, 1768-1776. 9. C. Michel-Cuello, y col. Study of Enzymatic Hydrolysis of Fructans from Agave salmiana Characterization and Kinetic Assessment, The scientific world journal. 2012, DOI: 10.1080/01496395.2013.786729 --- Send in a voice message: https://anchor.fm/ana-g-valenzuela-zapata/message Support this podcast: https://anchor.fm/ana-g-valenzuela-zapata/support

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.13.337162v1?rss=1 Authors: Bridel, S. M., Watts, S. C., Judd, L. M., Harshegyi, T., Passet, V., Rodrigues, C., Holt, K. E., Brisse, S. Abstract: Motivation: Klebsiella species are increasingly multidrug resistant pathogens affecting human and animal health and are widely distributed in the environment. Among these, the Klebsiella pneumoniae species complex (KpSC), which includes seven phylogroups, is an important cause of community and hospital infections. In addition, the Klebsiella oxytoca species complex (KoSC) also causes hospital infections and antibiotic-associated haemorrhagic colitis. The unsuitability of widely used clinical microbiology methods to distinguish species within each of these species complexes leads to high rates of misidentifications that are masking the true clinical significance and potential epidemiological specificities of individual species. Results: We developed a web-based tool, Klebsiella MALDI TypeR, a platform-independent and user-friendly application that enables uploading raw data from MALDI-TOF mass spectrometer to identify Klebsiella isolates at the species complex and phylogroup levels. The tool is based on a database of previously identified biomarkers that are specific for either the species complex, individual phylogroups, or related phylogroups, and is available at https://maldityper.pasteur.fr. Copy rights belong to original authors. Visit the link for more info

As weird as it may sound, yeast is everywhere. It’s mostly found in food, but sometimes we also get it from some of our favorite beverages. We also have yeast inside our body, and for the most part, that’s healthy. To help us understand yeasts better is Jim Kelton, Lab Manager of Microbiology at Genova. He has over 20 years of experience in supervising and managing laboratories and is an expert in microbiological test procedures, including a method called MALDI-TOF.   Today, we start things off by describing what yeast is, why we think it’s trending, and its links to chronic diseases. Jim explains how yeast research has affected the health industry and the MALDI-TOF test method. We discuss the various ways clinicians can detect yeast growth in their patients. Jim also shares his thoughts on Candida albicans and the effects of yeast overgrowth on the human body.   “The microbiome has opened up this whole world of what is clinically significant.” - Jim Kelton   This week on The Lab Report Podcast: Why talking about yeast is interesting. The role of yeast in everyday health and common diseases. The most common "bad yeast" in the world. The different ways to detect yeast. Why the whole concept of yeast testing is tricky. Jim's professional experience and his work at Genova. What MALDI-TOF is and its use in microbiology. Using yeast cultures as a testing method The next step after the identification and recovery of yeast. Jim’s thoughts on Candida albicans and yeast overgrowth.   Subscribe, Rate & Review The Lab Report Thanks for tuning into this week’s episode of The Lab Report, presented by Genova Diagnostics, with your hosts, Michael Chapman and Patti Devers. If you enjoyed this episode, please head over to Apple Podcasts to subscribe to the show and give us a rating and review. Don’t forget to visit our website, like us on Facebook, and follow us on Twitter, Instagram and LinkedIn. Email Patti and Michael with your most pressing questions on functional medicine. And be sure to share your favorite Lab Report episodes with your friends and colleagues on social media to help others learn more about Genova and all things related to functional medicine and specialty lab testing.   Disclaimer: The content and information shared in The Lab Report is for educational purposes only and should not be taken as medical advice. The views and opinions expressed in The Lab Report represent the opinions of Michael Chapman and Patti Devers and their guests. For medical advice, diagnosis, and/or treatment, please consult a medical professional. See omnystudio.com/listener for privacy information.

O artigo que discutimos nesse episódio se chama “Response of Gut Microbiota to Metabolite Changes Induced by Endurance Exercise”, ou seja “Resposta da Microbiota intestinal a mudanças metabólicas induzidas por exercícios intensos”, mais especificamente uma meia-maratona e foi publicado em abril de 2018 na Frontiers in Microbiology, por um grupo chinês da Third Military Medical University. Resumidamente o que os autores fizeram foi comparar a microbiota intestinal de corredores antes e depois de correr uma meia-maratona. No Microlitros de Notícias vamos saber sobre as novidades na identificação de microrganismos pela técnica de MALDI TOF; As dificuldades de se estudar infecções nas membranas fetais com uma breve entrevista do pesquisador Dr. Dave Aronoff da Vanderbilt University; e a descoberta de resistência em Aeromonas à baixas concentrações de antibióticos. Na Filogenia da Ciência, teremos a vida e pesquisa do microbiologista Robert Koch, esse mesmo… o do Postulado de Koch.   Tópicos comentados nesse episódio Microbiota intestinal Alteração da Microbiota induzida por exercícios intensos Meia-maratona Identificação de microrganismos MALDI TOF Membranas fetais Dave Aronoff Instrumented Fetal Membrane on a Chip (IFMOC) Resistência à baixas concentrações de antibióticos Robert Koch Postulado de Koch Antraz   Referências desse episódio 2018.Zhao X, Zhang Z, Hu B, Huang W, Yuan C e Zou L. Response of Gut Microbiota to Metabolite Changes Induced by Endurance Exercise. Front Microbiol. 2018.  Vrioni G, Tsiamis C, Oikonomidis G, Theodoridou K, Kapsimali V e Tsakris A. MALDI-TOF mass spectrometry technology for detecting biomarkers of antimicrobial resistance: current achievements and future perspectives. ATM. 2009. Piseth Seng, Michel Drancourt, Frédérique Gouriet, Bernard La Scola, Pierre-Edouard Fournier, Jean Marc Rolain e Didier Raoult. Ongoing Revolution in Bacteriology: Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry. Clin Infect Dis. 2017. Gnecco JS, Anders AP, Cliffel D, Pensabene V, Rogers LM, Osteen K e Aronoff DM.  Instrumenting a Fetal Membrane on a Chip as Emerging Technology for Preterm Birth Research. Curr Pharm Des. 2010. Blevins SM, Bronze MS. Robert Koch and the 'golden age' of bacteriology. 2010. Sociedade Brasileira de Microbiologia. Robert Koch: grande descobridor de pequenas bactérias. Biblioteca Virtual em Saúde Adolpho Lutz. Robert Koch. Noel Prize.org. Robert Koch - Biographical   Sobre o Podcast Microbiando A ideia do Microbiando é discutir artigos científicos de ponta em todas as áreas da microbiologia e imunologia. Vamos utilizar uma linguagem bem acessível para destrinchar esses artigos para vocês, mas sem perder o rigor científico e analítico necessário para essa tarefa. Além de discutir artigos nós teremos o quadro Microlitros de Notícias, onde nossos microbiologistas e imunologistas de plantão irão abordar pequenas reportagens e trazer novidades para vocês. No quadro filogenia da Ciência vamos contar um pouco sobre a vida de grandes personalidades que revolucionaram a Microbiologia e Imunologia com suas descobertas. Você pode nos ouvir no Spotify, Google Podcast, Player FM, Podcast Addict, CastBox, Blubrry Podcasting, iTunes e outros agregadores de podcasts.   Contatos E-mail: microbiando@micro.ufrj.br Twitter Facebook Instagram   Expediente Produção Geral: Rosana Ferreira Hosts: Rosana Ferreira Equipe de Pauta/Gravação: Cláudia M. d'Avila-Levy, Juliana Echevarria, Leandro Lobo, Mateus Godoy, Rosana Ferreira, Sidcley Lyra, Cecília Vieira, Gabriel Martins, Gustavo Meira, Michel Leon e Úrsula Lopes. Edição: Hugo Marins/NNOTEM (Núcleo de Novas Tecnologias e Mídias/IBCCF) Trilha Sonora: Daniel Vasquez   O podcast Microbiando tem o apoio do Instituto de Microbiologia Professor Paulo de Góes e do Instituto de Biofísica...

Electrophoretic separation of serum and urine proteins has played a central role in diagnosing and monitoring plasma cell disorders. Despite limitations in resolution and analytical sensitivity, plus the necessity for adjunct methods, protein gel electrophoresis and immunofixation electrophoresis (IFE) remain front-line tests.

In this seventh installment of the Open Forum Infectious Diseases podcast, Editor in Chief Paul Sax, MD, interviews Angela Caliendo, MD, PhD, about the latest advances in the microbiology laboratory and where the future will lead as clinicians demand faster and more accurate pathogen diagnostics. The pair explores the implementation of MALDI-TOF mass spectometry in the U.S., the time it saves in the laboratory, and how it can help prevent the overuse of antibiotics.

The application of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry or MALDI-TOF MS, to microbial identification is revolutionizing clinical microbiology, providing rapid identification with minimal sample preparation and potential cost savings.

Desoxyribonuklerinsäure (DNA), die Erbinformation, definiert die Struktur und Funktion jeder Zelle. In Eukaryoten ist sie um Oktamere aus den Histonen H2A, H2B, H3 und H4 gewunden. Sie bilden mit der DNA höhere Strukturen, das sog. Chromatin. Die Struktur des Chromatins beeinflusst direkt die Aktivität der gebundenen DNA. Eukaryoten besitzen daher viele molekulare Mechanismen zu ihrer Veränderung, z.B. posttranslationale Modifizierungen (PTMs) von Histonproteinen oder den Austausch von kanonischen Histonen mit Histonvarianten (z.B. H3.1, H3.2, H3.3, u.a.). Für einige Varianten sind ihnen eigene, charakteristische PTMs beschrieben, z.B. die Phosphorylierung des Serins 31 der Variante H3.3 (H3.3S31ph). Die Histone Code Hypothesis postuliert, dass PTMs von Histonen in festen Mustern vorliegen können. Die Switch Hypothesis beschreibt die Regulation von Bindemolekülen an Histone durch benachbarte PTMs. Auf ihrer Grundlage wurde die These der Doppelmodifizierung einer bekannten Trimethylierung der Aminosäure (AS) Lysin 79 mit einer mutmaßlichen Phosphorylierung der AS Threonin 80 auf Histon H3 aufgestellt (H3K79me3T80ph). Neben der Etablierung eines einfachen Systems zur Identifizierung bisher unbeschriebener Phosphorylierungen bestand ein zweites Ziel dieser Arbeit im Nachweis der Phosphorylierung von H3 Threonin 80 in vivo. Eine weitere Zielsetzung lag in der genaueren Charakterisierung der bereits beschriebenen Varianten-spezifischen PTM H3.3S31ph, deren Expression zwar eng umschrieben ist, über deren spezifische Funktionen, Kinase und mögliche Effektorproteine aber wenig bekannt ist. Um einen Überblick über Phosphorylierungen verschiedener Histonroteine zu gewinnen, wurden unterschiedliche Polyacrylamidgel-Elektrophoresen Verfahren (PAGE) etabliert. Zum Einsatz kamen A/U-, T/A/U- und 2D-T/A/U-PAGE Verfahren. Sie ermöglichten in Übereinstimmung mit der Literatur die Auftrennung bekannter PTM und stehen nun für weiterführende Studien zur Verfügung. Der massenspektrometrische Nachweis der putativen PTM H3K79me3T80ph in vivo gelang nicht. Trotz optimierter Versuchsbedingungen konnte die Phosphorylierung weder in der MALDI-ToF, noch in der Orbitrap MS/MS nachgewiesen werden. Initiale Antikörperdaten wurde aufgrund einer aufgedeckten Kreuzreaktivität in Frage gestellt. Obgleich nicht mit letzter Sicherheit gesagt werden kann, dass H3K79me3T80ph in vivo nicht existiert, wurde die These letztlich verworfen. Die molekularbiologische Untersuchung der Varianten-spezifischen PTM H3.3S31ph ergab bei verifizierter Überexpression und Chromatin-Integration punktmutierter Histone übereinstimmend Hinweise auf einen Effekt der PTM auf die Zellteilung. Es konnte gezeigt werden, dass Serin 31 bzw. ihre PTM H3.3S31ph sowohl Zellzyklus, als auch Wachstums- und Proliferationsgeschwindigkeiten von HeLa Zellen beeinflusst. Dies argumentiert für eine aktivierende Funktion von H3.3S31ph in der Mitose. Weiter konnten mithilfe eines ELISAs fünf potentielle Proteinkinasen für H3.3S31ph identifiziert werden. Vorrangig kommt dabei die nukleäre Kinase PIM1 in Betracht. Zur weiteren Untersuchung potentieller Effektorproteine wurde in vitro ein molekularbiologisches Modellsystem etabliert. Es steht nun für weiterführende Studien zur Verfügung. Erste Vorarbeiten konnten bereits zeigen, dass hier evtl. Interaktionen mit dem Linkerhiston H1 eine wichtige Rolle spielen.

Mass spectrometry has revolutionized many areas of clinical chemistry, but this technology is not just limited to chemistry. Introduction of MALDI-TOF mass spectrometry into the clinical microbiology laboratory has markedly altered workflow allowing bacterial and fungal colonies to be accurately, rapidly, and inexpensively identified.

The aim of this study was to examine whether the classical biochemical differentiation methods in relation to clostridial analysis can be replaced by newer molecular biological techniques such as real-time PCR and by MALDI-TOF MS. For this purpose, 118 potentially pathogenic Clostridium spp. isolates were compared at the Bavarian Health and Food Safety Authority using the above mentioned methods. On the basis of the obtained results a recommendation concerning the general approach to the detection of Clostridium spp. in the food industry has been elaborated.

Vertebrate embryos are derived from a transitory pool of pluripotent embryonic cells. By the process of induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While at gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue histones isolated from four different developmental stages of Xenopus laevis were analysed by mass spectrometry. Initally, a variety of different protocols for histone extraction from Xenopus laevis embryos and stable cell lines was tested and evaluated. Since non of the available methods worked sufficiently, a new reliable and effective protocol for nuclei preparation and histone extraction was established. Using mass spectrometry, core histone modifications were unambiguously determined. The techniques for identification and quantification of histone modifications by tandem mass spectrometry were improved as well. In total, an average sequence coverage of 68% of modification sites for the four core histones was achived by tryptic digestion after covalent modification of lysine residues with propionic anhydride. Using both LC-MS/MS and MALDI-TOF mass spectrometry, a total number of 2 modifications of H2A and 3 modifications H2B, 39 modifications of H3 and 20 modifications of H4 were identified and quantified. During this developmental period, an increase in the unmodified states, and a shift from histone modifications associated with transcriptionally active to transcriptionally repressive histone marks, was observed. Furthermore, these naturally occurring histone modifications were compared to the histone modifications of murine ES cells, detecting large differences in the methylation patterns of lysines 27 and 36 of histone H3 between pluripotent cells from Xenopus blastulae and murine ES cells. By combining all detected modification transitions, their patterns could be clustered according to their embryonic origin, defining specific histone modification profiles for each developmental stage. These specific histone modification profiles indicated a stepwise maturation of the embryonic epigenome, which may be cause to the progressing restriction of cellular potency during development. This thesis has revealed major quantitative shifts for several histone modifications known to be involved in gene regulation and furthermore enabled the definition of stage specific histone modification profiles accompanying and potentially regulating the transition from pluripotent to determined cell states using an antibody-independent method.

Background: Gastric inhibitory polypeptide ( GIP) is postulated to be involved in type 2 diabetes mellitus and obesity. It exerts its function through its receptor, GIPR. We genotyped three GIPR SNPs (rs8111428, rs2302382 and rs1800437) in German families with at least one obese index patient, two case-control studies and two cross-sectional population-based studies. Methods: Genotyping was performed by MALDI-TOF, ARMS-PCR and RFLP. The family-study: 761 German families with at least one extremely obese child or adolescent (n = 1,041) and both parents ( n = 1,522). Case-control study: ( a) German obese children ( n = 333) and (b) obese adults ( n = 987) in comparison to 588 adult lean controls. The two cross-sectional population-based studies: KORA ( n = 8,269) and SHIP ( n = 4,310). Results: We detected over-transmission of the A-allele of rs2302382 in the German families (pTDT-Test = 0.0089). In the combined case-control sample, we estimated an odd ratio of 1.54 (95% CI 1.09; 2.19, pCA-Test = 0.014) for homozygotes of the rs2302382 A-allele compared to individuals with no A-allele. A similar trend was found in KORA where the rs2302382 A-allele led to an increase of 0.12 BMI units (p = 0.136). In SHIP, however, the A-allele of rs2302382 was estimated to contribute an average decrease of 0.27 BMI units (p-value = 0.031). Conclusion: Our data suggest a potential relevance of GIPR variants for obesity. However, additional studies are warranted in light of the conflicting results obtained in one of the two population-based studies.

Protein expression profiling of human glatiramer acetate (GA) and myelin basic protein (MBP) specific T cell lines from a multiple sclerosis (MS) patient and healthy donors Investigations in MS and the animal model experimental autoimmune encephalomyelitis (EAE) indicate that autoreactive T cells play a pivotal role in the pathogenesis. Putative auto antigens include e.g. MBP. One approved therapy of patients with relapsing-remitting MS is GA (Copaxone®), which induces a shift in the phenotype of CD4+ T cells from TH1- to TH2-type. As the molecular mechanisms involved are largely unknown, we were interested in the regulated proteome of GA- and MBP-specific human T cell lines. We generated GA- and MBP-specific T cell lines from three healthy donors and GA- specific T cell lines from a MS patient before and after 6 months of GA therapy. Proteome analysis of activated and resting GA- and MBP-specific, CD3+ CD4+ CD8- T cells was done by SDS-PAGE and MALDI-TOF mass spectrometry. A total of 120 regulated protein spots were detected on the gels. 69 were identified as different proteins by mass spectrometry. A single protein pathway analysis of these proteins led us to five proteins. One of them is known to be a negative regulator of growth factors. Three proteins known for their anti-inflammatory properties were upregulated in GA-specific T cells, one protein with pro-inflammatory characteristics was found upregulated in encephalitogenic MBP-specific T cells. Based on their expression profile and their characteristics these proteins may be involved in immunological mechanisms relevant in MS and/or possibly during GA treatment.

Following oxidative stress often two kinds of DNA-lesions can be found: 8-OxodG and the 2,6-Diamino-4-hydroxy-5-formamidopyrimidine (FaPydG). Both derive from the DNA-Nucleobase guanine. The FaPydG-lesion can exist both in the beta-configuration and the alpha configuration. To clarify biological questions concerning the basepairing and the encoding features of both forms it was necessary to synthesise of the alpha-anomer of the carbocyclic FaPydG-DNA-lesion. The synthesis of the beta-cFaPydG-lesion was already finished in 2005 in the Carell group. In a sixteen-step synthesis the alpha-lesion could be prepared in the form of its phosphoramidite component. Oligonucleotides could be prepared on solid support using automated DNA-synthesis. Some of the synthesised DNA-Nucleotides contained the inserted FaPydG-lesion. Others showed an oxidation to alpha-c8-OxodG. This reaction is not known for natural lesion. Just a de-hydration to guanosine under harsh conditions is described. The observations can be ex-plained by formulation of a cyclic intermediate, which is possibly oxidized by the oxidation di-lution during the DNA-synthesis. If the sequence-dependent equilibrium between alpha cFaPydG and alpha c8-Hydro,hydroxydG is at the side of the closed form, an oxidation from this interme-diate directly to 8-OxodG is imaginable. This assumption is proven by the fact that at the same time synthesised and completely identically treated DNA-strands of different sequence didn´t show oxidation products. This could be ascertained by high resolution ESI-FTICR-mesurement. With this analytical method even smallest variations are detectable in a reliable way, whereas the often used MALDI-TOF-mesurements not always show these variations. Thermodynamic studies showed that alpha-cFaPydG doesn´t form a preferred DNA-basepair. All possible basepairs showed a strong destabilisation in comparison to the undamaged, cor-rect basepair. In vitro elongation experiments with S. cerevisiae polymerase Pol eta, G. stearothermophilus DNA-polymerase I and DinB from G. stearothermophilus showed that the alpha-lesion is a definitive block for all mentioned polymerases. Only by applying rigorous re-action conditions an elongation of the primer with BstPol I could be detected. This elongation showed a favored incorporation of dCTP, followed by an incorporation of dATP. In the time to come experiments to avoid the unwanted oxidation of alpha-cFaPydG will be es-sential to promote the cocristallisation of the alpha-cFaPydG-oligomere with the Fpg-protein from Lactococcus lactis and the BstPol I. In the second part of the thesis attempts were taken to prepare tetrafunctional amino acid derivatives, which were planned to be used in a PNA-template directed ligation reaction. Liga-tion takes place between two amino acids, so that for a ligation attempt with this method there is always needed a matching pair of amino acids. This pair requires a connection of the aminoacid to the PNA over the side chain of the amino acid. The N- and C-terminus of the amino acid had to be unprotected for the ultimate ligation reaction. These termini must not react during the solid support PNA-synthesis. After ligation the template should be separated selectively. Thus, the following requirements for the pair of amino acids result: The amino acids have to possess functional side chains, which exist naturally after cleavage of the PNA-template. For the selective cleavage of the template a con-nection orthogonal to acid and base unstable protecting groups is necessary, because these are already needed for the solid support synthesis on the residual three termini of the compound. Imaginable orthogonal cleavable links are allylic compounds, which are cleavable with Pd(0), but also silicon based compounds, which are cleavable with fluoridions, would be appropriate. In the area of the Pd(0)-cleavable link a retrosynthetic cut at this funtionality should be adequate. Here, e.g. a Horner-Wadsworth-Emmons-reaction is imaginable. The aldehydes and phosphorylides needed for this key step could be prepared with good yields, however only one of the two amino acid derivatives was obtained. The synthesis of the other compound didn´t succeed under diverse reaction conditions. Also efforts to prepare analogue Pd(0)-cleavable link molecules through classic Wittig or metathese reactions with Grubbs catalysts of the first and second generation were unsuccessful. The most successful efforts to make a target molecule synthetically accessible for a first liga-tion attempt involved the preparing of a fluorid-cleavable silyl link. Here all requirements stay the same, just instead of allylic compounds silyl ethers are needed. The acid stability of the silyl protecting group depends on the size of the alkyl groups (tert-butyl>isopropyl>ethyl). Firstly, substitution experiments with the compounds Di-tert-butylchlorosilane, Di-tert-butyl-dichlorosilane and Di-tert-butylsilylbis(trifluoromethanesulfonate) were undertaken to estab-lish a connection between PNA and the amino acid through a Di-tert-butyldisilylether. How-ever, the substitutions produced just monosubstituted silanoles, irrespective of the choice of reaction conditions and substitution order. The experiments with the commercially available (3-Cyanopropyl)-diisopropyl-chlorosilane were more successfull. Here the synthesis of one of the target molecules could be achieved.

Functional inactivation of the transcription factor CAAT Enhancer Binding Protein Alpha (C/EBPα) either by mutation or direct protein-protein interaction leads to acute myeloid leukemia (AML), whereas the activation of C/EBPα restores normal myeloid cell differentiation. We and others have shown that protein-protein interactions of C/EBPα play a pivotal role in myeloid differentiation and AML. In the present study we applied proteomics based mass spectrometry to identify C/EBPα interacting proteins on a proteome-wide scale. For this, the GST and GST-DNA binding domain of C/EBP (GST-DBD) was incubated with nuclear extracts of U937 cells. Interacting proteins separated by two-dimensional gel electrophoresis were identified by MALDI-TOF mass spectrometry. Using this approach we could identify PAK6, MADP-1, ZNF45 and the c-Jun N-terminal kinase 1 (JNK1) as C/EBPα interacting proteins. Since JNK1 activates c-Jun, the contra-player for C/EBPα, we hypothesized that the JNK1 and C/EBPα interaction might have some biological relevance. We could show that JNK1 binds to the DNA binding domain of C/EBP in GST-pull-down and to the C/EBPα in co-immunoprecipitation assays in-vitro and in-vivo respectively. JNK1 phosphorylates and increases the half life of the C/EBPα protein via inhibiting its ubiquitination and thereby enhances its transactivation and DNA binding activity. Furthermore, JNK mRNA expression as well as its kinase activity is lower in the AML patients in comparison to normal bone marrow mononuclear cells which implicates a possible mechanism of C/EBPα inactivation in certain acute myeloid leukemia subtypes. Thus our data suggest that a JNK activity is required for C/EBPα activation in myeloid cells and a loss of JNK regulated C/EBPα expression may contribute to leukemogenesis.

The development of relevant cellular model systems for colorectal cancer is of utmost importance for an improved in vitro assessment of therapeutic strategies against colorectal cancer. Recently published low passage colon cancer cell lines that closely reflect the characteristics of the respective parental in vivo tumor cells represent very promising cell culture models and were therefore used for the investigations in the present thesis. To provide an in vitro model system that also recapitulates the three-dimensional structure of in vivo tumors, these low passage cell lines were cultivated as multicellular spheroids. Compared to monolayer cultures the multicellular spheroids exhibited a wide variety of changes in their expression patterns. The differential expression includes proteins that are involved in growth signaling (15-hydroxyprostaglandin dehydrogenase), protein biosynthesis (acidic ribosomal protein P0), and regulation of the cyto- or nucleoskeleton (acidic calponin and LMNA protein). These proteins were identified by 2D electrophoresis and subsequent MALDI-TOF mass spectrometry. Both methods were established in the lab in the context of this work. Chemotherapy with 5-fluorouracil (5-FU) represents the traditional treatment of colorectal cancer. However, in many patients the efficiency of this therapeutic strategy is often limited by the development of chemoresistance against 5-FU. Therefore, it was an aim of this thesis to detect novel proteins involved in 5-FU chemoresistance that were previously not ascribed to resistance against this chemotherapeutic drug. A chemoresistant subline of a colon cancer cell line was generated by long-term treatment with 5-FU and served as a model for the investigation of 5-FU chemoresistance. This subline exhibited resistance against both 5-FU-induced inhibition of proliferation and apoptosis. Differences in the expression of cytokeratin 18, heat shock protein 27 and aldehyde dehydrogenase 1B1 between the chemoresistant subline and parental cells were detected by 2D electrophoresis. These findings imply that the cytoskeleton plays a role in the development of chemoresistance against 5-FU. Furthermore, processes located to the mitochondria seem to be involved in this resistance, since heat shock protein 27 and aldehyde dehydrogenase 1B1 are associated with this subcellular organelle. The biological relevance of the findings made in the present PhD thesis has to be determined in further studies. Gene therapy represents a promising alternative strategy for the treatment of colorectal cancer. A novel nonviral gene transfer system was developed by combination of DNA with the polycation PEI25br and the cationic lipids DOCSPER or DOSPER to form lipopolyplexes. These lipopolyplexes enabled enhanced gene transfer in vitro and are promising for in vivo applications, since the established lipopolyplexes preserved their small size at physiological conditions; a property essential for a successful in vivo application. Furthermore, the lipopolyplexes exhibited the capability to efficiently transfect three-dimensional multicellular spheroids. The potential of lipopolyplexes for therapeutic applications was further increased by the utilization of the artificial promoter CTP4 which enables highly specific gene expression in cancer cells with mutations in the Wnt signaling pathway by transcriptional targeting. In addition to its high specificity, this promoter enabled high gene expression levels that were comparable to expression levels obtained by the strong, but unspecific CMV promoter. The efficiency of the CTP4 promoter was demonstrated in seven low passage colon cancer cell lines and also in multicellular spheroids. The transcriptional targeted lipopolyplexes not only enabled high tumor specific expression of reporter genes like luciferase or EGFP but also the expression of a therapeutic gene, interleukin-2 (IL-2). Furthermore, tumor specific expression of cytotoxic protease 2A in combination with IL-2 was possible by using a novel bicistronic construct. The expression of the rhinoviral protease 2A led to efficient reduction of overall cap-dependent gene expression levels and therefore also the proliferation of the transfected cells, while continued IL-2 expression was guaranteed by an IRES element enabling cap-independent gene expression in the presence of protease 2A. In summary, the present results provide a promising basis for the development of novel potent strategies in the treatment of colorectal cancer.

Background: A single nucleotide polymorphism (SNP) in the coding region of the prion protein gene (PRNP) at codon 129 has been repeatedly shown to be an associated factor to sporadic Creutzfeldt-Jakob disease (sCJD), but additional major predisposing DNA variants for sCJD are still unknown. Several previous studies focused on the characterisation of polymorphisms in PRNP and the prion-like doppel gene (PRND), generating contradictory results on relatively small sample sets. Thus, extensive studies are required for validation of the polymorphisms in PRNP and PRND.Methods: We evaluated a set of nine SNPs of PRNP and one SNP of PRND in 593 German sCJD patients and 748 German healthy controls. Genotyping was performed using MALDI-TOF mass spectrometry.Results: In addition to PRNP 129, we detected a significant association between sCJD and allele frequencies of six further PRNP SNPs. No significant association of PRND T174M with sCJD was shown. We observed strong linkage disequilibrium within eight adjacent PRNP SNPs, including PRNP 129. However, the association of sCJD with PRNP 1368 and PRNP 34296 appeared to be independent on the genotype of PRNP 129. We additionally identified the most common haplotypes of PRNP to be over-represented or under-represented in our cohort of patients with sCJD.Conclusion: Our study evaluated previous findings of the association of SNPs in the PRNP and PRND genes in the largest cohorts for association study in sCJD to date, and extends previous findings by defining for the first time the haplotypes associated with sCJD in a large population of the German CJD surveillance study.

Background: For a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis. Results: A transcribed mutation such as a single nucleotide polymorphism ( SNP) is used as the marker for allele-specific expression analysis. A synthetic mutation created in the competitor is close to a natural mutation site in the cDNA sequence. PCR is used to amplify the two cDNA sequences from the two alleles and the competitor. A base extension reaction with a mixture of ddNTPs/ dNTP is used to generate three oligonucleotides for the two cDNAs and the competitor. The three products are identified and their ratios are calculated based on their peak areas in the MALDI-TOF mass spectrum. Several examples are given to illustrate how allele-specific gene expression can be applied in different biological studies. Conclusions: This technique can quantify the absolute expression level of each individual allele of a gene with high precision and throughput.