Podcasts about dsdna

With prior Mol Bio Minutes episodes covering DNA form migration and staining considerations for nucleic acid gel electrophoresis, we tie it all together with this great set of overall tips, tricks and resources for the topic. Anyone that's ever run a gel has undoubtedly produced gels with smeared, faint or poorly separated bands. What causes these and how can you avoid them?  Well, Aistė Polikaitytė, Scientist at Thermo Fisher Scientific is here to cover the likely causes and troubleshooting tips to help avoid the most common gel issues. She touches on how much sample to load, the importance of reagent selection, gel preparation, separation conditions, staining, as well as purification and contamination considerations. Helpful resource links mentioned in this episode:Selection guide for electrophoresis dyes and buffersLearn how using precast E-Gel agarose gel can help avoid common issuesA helpful troubleshooting guide for nucleic acid gel electrophoresisView an on-demand webinar covering these topics in more detail Subscribe to get future episodes as they drop and if you like what you're hearing we hope you'll share a review or recommend the series to a colleague.  Visit the Invitrogen School of Molecular Biology to access helpful molecular biology resources and educational content, and please share this resource with anyone you know working in molecular biology. For Research Use Only. Not for use in diagnostic procedures.

You can run the perfect agarose gel to separate your nucleic acid fragments but if you don't stain and image the gel properly, it's all for not. In this second installment of Mol Bio Minutes we take a look at the staining considerations for nucleic acid gel electrophoresis with Paulius Palaima, Product Manager at Thermo Fisher Scientific. He covers the range of stains and staining approaches available while calling out pros, cons and considerations for each. How these recommendations change, depending on your sample, is also covered in this approachable but informative episode. Helpful resource links mentioned in this episode:A helpful DNA stain selection guideRNA stain options and detailsEffects of dyes on gel electrophoresis properties Subscribe to get future episodes as they drop and if you like what you're hearing we hope you'll share a review or recommend the series to a colleague.  Visit the Invitrogen School of Molecular Biology to access helpful molecular biology resources and educational content, and please share this resource with anyone you know working in molecular biology. For Research Use Only. Not for use in diagnostic procedures.

Agarose gel electrophoresis is a staple method in almost all biology and biochemistry lab where separation and analysis of nucleic acids is needed. In this Mol Bio Minutes mini episode Augustė Užuotaitė, Scientist III at Thermo Fisher Scientific, covers the basics of electrophoresis with a spotlight on how different forms of DNA migrate differently in agarose gel electrophoresis.In less than 10 quick minutes, you'll learn about the many factors that affect DNA migration rate. Augustė reviews how DNA size, sequence, and conformation all affect migration rate, and she gives some beautifully simple examples to help it all make sense. Helpful resource links mentioned in this episode:Nucleic acid gel electrophoresis – summarized in 5 easy stepsFive important considerations for the nucleic acid gel electrophoresis Subscribe to get future episodes as they drop and if you like what you're hearing we hope you'll share a review or recommend the series to a colleague.  Visit the Invitrogen School of Molecular Biology to access helpful molecular biology resources and educational content, and please share this resource with anyone you know working in molecular biology. For Research Use Only. Not for use in diagnostic procedures.

Unfortunately, there simply isn't a one-size-fits-all treatment protocol for patients infected with Lyme disease and/or co-infections. This is why it's critical for physicians treating Lyme disease to invest time with patients, thoroughly understand their medical history, and closely monitor symptoms and treatment response. With that in mind, there are currently two different treatment approaches for Lyme disease. The Infectious Disease Society of America (IDSA) and the International Lyme and Associated Diseases Society (ILADS) have each published their own set of evidence-based treatment guidelines. IDSA guidelines recommend a short course of antibiotics, typically 14 to 30 days. IDSA argues that the Borrelia burgdorferi bacteria do not persist in a patient beyond this timeframe and that lingering symptoms are the result of an ongoing immune response and not an active infection. It also cites scientific evidence claiming treatments beyond 30 days are ineffective, unnecessary, and even dangerous. IDSA physicians will stop treatment after 30 days, even if symptoms remain. They advise an additional 30 days of treatment recommended for patients with Lyme arthritis.  On the contrary, ILADS offers its own scientific data to show that a additional treatment with antibiotics is required to eradicate the bacteria. ILADS recognizes that a month of treatment may be sufficient for patients in the acute stage of Lyme disease, but in cases where the spirochete has disseminated and the disease has advanced, a 30-day treatment regimen is inadequate. ILADS guidelines recommend additional antibiotics until a patient's symptoms have been resolved. Treating Lyme disease in its advanced stage can be complicated based on the complexity of the organism itself, differences in each patient's immune system, the length of time infected, and the possible presence of other co-infections transmitted by the same tick. There are several choices in treating Lyme disease, which include oral, intravenous, and intramuscular antibiotic options. Other options may include sequential antibiotic therapy, higher doses of antibiotics, taking antibiotics for a longer period of time, a combination of antibiotics, retreatment, as well as diagnosing and treating co-infections. Some specific antibiotics used in treating Lyme disease are doxycycline, minocycline, amoxicillin, cefuroxime, azithromycin, and clarithromycin. Other tests include measures of blood counts, chemistries, liver function tests, ANA, dsDNA, RF, TSH, free T3, free T4, ESR may be helpful at ruling out other conditions.  Referral to specialist might help to rule out other conditions.  I find shared decision with my patient helpful. I also find follow-up helpful to assess my patient's response to treatment to rule out other conditions. There are additional protocols that may also aid in treating Lyme disease, such as avoiding alcohol, simple and processed sugars, exercising as tolerated, counseling for a Jarisch-Herxheimer reaction, managing symptoms, monitoring and reducing the risk of an adverse event, and reducing stress. However, there is a chance of side effects such as Clostridium difficile-associated diarrhea (CDAD). Probiotic have been prescribed with the hope of reducing the risk of developing CDAD.   

What is a common-sense approach to testing for Tick-borne infection. I focus on the most common infections that I see a Lyme disease infection. I order a Lyme disease test as well as test for infections like Ehrlichia, Anaplasmosis, and Babesia. I don't typically order Mycoplasma or Chlamydia unless there is evidence that there's active infection.  I order an ELISA test, which is also called Lyme titer.  I also order a western blot and IgG and IgM test. These are test where you need two out of three bands IgM bands. You need five out of 10 IgG bands to be called positive by the CDC criteria. These are bands that were identified and in 1994 at a consensus meeting in Dearborn Michigan.  These markers are protein that have been identified in Lyme disease infections.  For example, the 41 kDa band represents a protein contained in the tail of a spirochete. I have not been ordering a C6 peptide or VlsE protein tests for Lyme disease as they are not as reliable as I would like.  None of these tests for Lyme disease are all that sensitive in my experience.  I have often had to use clinical judgement to diagnose and treat Lyme disease. I also order IgG and IgM tests for co-infections with Babesia, Bartonella, Anaplasmosis, and Ehrlichia. I have not found PCR tests for these co-infections as helpful as I would like. I have found a blood smear for Babesia not helpful if a patient has been sick more than 2 weeks.   Some doctors have assumed Bartonella tests have been positive due to exposure to fecal matter from mites living on cats. I can't be sure the cause of positive tests for Bartonella in patients with Lyme disease.  I don't typically ordered labs for infections such as tularemia or Brucellosis despite concerns by some of my colleagues. I have found treatment for Lyme disease would take care of these infections if they were present. I typically do not sent bloods to a specialty lab if someone's on a budget. I also do not send bloods to these labs if I am going to treat clinically.   I also order extensive testing to rule other illnesses like a CBC, comprehensive metabolic profile, ANA, RA, thyroid, sed rate, vitamin B12 and D.    I may order a free T4 and free T3 if I am considering a thyroid condition.  I have found ANA frustrating as most of the ANA tests are false positive. A positive dsDNA supports the diagnosis of lupus. My patients don't typically have three other conditions that would support the diagnosis of Lupus. I refer my patients to see a rheumatologist if there is a need to rule out lupus. I typical order blood test for a tick-borne illness four weeks or 4 to 6 weeks after onset of their illness to increase the chances that I might get a positive test.  I have had to use clinical judgement to treat a tick-borne infection if my patient is sick for less than 4 weeks or if I suspect a false negative test,

CardioNerds join Dr. Inbar Raber and Dr. Susan Mcilvaine from the Beth Israel Deaconess Medical Center for a Fenway game. They discuss the following case: A 72-year-old man presents with two weeks of progressive dyspnea, orthopnea, nausea, vomiting, diarrhea, and right upper quadrant pain. He has a history of essential thrombocytosis, Barrett's esophagus, basal cell skin cancer, and hypertension treated with hydralazine. He is found to have bilateral pleural effusions and a pericardial effusion. He undergoes a work-up, including pericardial cytology, which is negative, and blood tests reveal a positive ANA and positive anti-histone antibody. He is diagnosed with drug-induced lupus due to hydralazine and starts treatment with intravenous steroids, resulting in an improvement in his symptoms. Expert commentary is provided by UT Southwestern internal medicine residency program director Dr. Salahuddin (“Dino”) Kazi. US Cardiology Review is now the official journal of CardioNerds! Submit your manuscript here. CardioNerds Case Reports PageCardioNerds Episode PageCardioNerds AcademyCardionerds Healy Honor Roll CardioNerds Journal ClubSubscribe to The Heartbeat Newsletter!Check out CardioNerds SWAG!Become a CardioNerds Patron! Case Media Pearls - A Drug's Adverse Effect Unleashes the Wolf The differential diagnosis for pericardial effusion includes metabolic, malignant, medication-induced, traumatic, rheumatologic, and infectious etiologies. While pericardial cytology can aid in securing a diagnosis of cancer in patients with malignant pericardial effusions, the sensitivity of the test is limited at around 50%.  Common symptoms of drug-induced lupus include fever, arthralgias, myalgias, rash, and/or serositis. Anti-histone antibodies are typically present in drug-induced lupus, while anti-dsDNA antibodies are typically absent (unlike in systemic lupus erythematosus, SLE). Hydralazine-induced lupus has a prevalence of 5-10%, with a higher risk for patients on higher doses or longer durations of drug exposure. Onset is usually months to years after drug initiation. Show Notes - A Drug's Adverse Effect Unleashes the Wolf There is a broad differential diagnosis for pericardial effusion which includes metabolic, malignant, medication-induced, traumatic, rheumatologic, and infectious etiologies. Metabolic etiologies include renal failure and thyroid disease. Certain malignancies are more likely to cause pericardial effusions, including lung cancer, lymphoma, breast cancer, sarcoma, and melanoma. Radiation therapy to treat chest malignancies can also result in a pericardial effusion. Medications can cause pericardial effusion, including immune checkpoint inhibitors, which can cause myocarditis or pericarditis, and medications associated with drug-induced lupus, such as procainamide, hydralazine, phenytoin, minoxidil, or isoniazid. Trauma can cause pericardial effusions, including blunt chest trauma, cardiac surgery, or cardiac catheterization. Rheumatologic etiologies include lupus, rheumatoid arthritis, systemic sclerosis, sarcoid, and vasculitis. Many different types of infections can cause pericardial effusions, including viruses (e.g., coxsackievirus, echovirus, adenovirus, human immunodeficiency virus, and influenza), bacteria (TB, staphylococcus, streptococcus, and pneumococcus), and fungi. Other must-not-miss etiologies include emergencies like type A aortic dissection and myocardial infarction. In a retrospective study of all patients who presented with a hemodynamically significant pericardial effusion and underwent pericardiocentesis, 33% of patients were found to have an underlying malignancy(Ben-Horin et al). Bloody effusion and frank tamponade were significantly more common among patients with malignant effusion, but the overlap was significant, and no epidemiologic or clinical parameter was found useful to differentiate between cancerous and noncancerous effus...

Dr. Anna Wolska speaks to Dr. Michael Belmont and Devyn Zaminski to discuss the importance of dual dsDNA testing in order to full understand disease activity. In this study, they made the observation that there is a large amount of discordance in anti-dsDNA antibodies assays. Misinterpretation or errors in anti- dsDNA antibody testing could have negative implications on long term outcomes in people living with SLE.   Read the related paper - https://doi.org/10.1136/lupus-2023-001012

4.11 Antibody Review Rheumatology review for the USMLE Step 1 Exam. ANA Principles ANA (Anti-Nuclear Antibody): Non-specific antibody. Reacts against nuclear antigens, including proteins, DNA, RNA, and nucleic acid-protein complexes. Includes a group of antibodies such as anti-dsDNA, anti-histone, SSA/Ro, SSB/La, Scl-70, anti-aminoacyl-tRNA synthetase (Jo-1). Found in 20-30% of the general public without connective tissue disorder symptoms. ANA+ individuals may or may not have a rheumatologic disorder. Further workup is needed in ANA+ cases to determine the specific disorder. Antibodies by Disease Process Systemic Lupus Erythematosus (SLE) Anti-dsDNA antibody. Anti-Smith antibody. Drug-Induced Lupus Anti-histone antibody. Diffuse vs. Limited Scleroderma Diffuse: Anti-Scl-70 (anti-topoisomerase I). Limited: Anti-centromere (often called CREST syndrome, with CREST standing for centromere). Sjogren's Syndrome Anti-SSA (Ro). Anti-SSB (La), which usually occurs in the presence of SSA. SSA is considered the Sjogren-specific antibody, leading to the presence of SSB. Rheumatoid Arthritis (RA) Anti-CCP (Cyclic Citrullinated Peptide). RF (Rheumatoid Factor) is non-specific. Thanks for listening!

Find Monica:  Website: https://monicasdeepdives.com Twitter: https://twitter.com/monicaperezshow Rokfin: https://rokfin.com/deepdives Rumble: https://rumble.com/user/monicaperezshow YouTube: https://www.youtube.com/c/MonicaPerez  Shownotes: 1) EMA Quality Report - Rapporteurs-Rolling-Review-Report-Quality-COVID-19-mRNA-Vaccine- BioNTec.doc (live.com) 2) SARS-CoV-2 spike mRNA vaccine sequences circulate in blood up to 28 days after COVID-19 vaccination: https://onlinelibrary.wiley.com/doi/10.1111/apm.13294 3) Biodistribution of mRNA COVID-19 vaccines in human breast milk https://www.thelancet.com/action/showPdf?pii=S2352-3964%2823%2900366-3 4) FDA - Emergency Use Authorization (EUA) for an Unapproved Product Review Memorandum https://www.fda.gov/media/144416/download 5) EMA Comirnaty Assessment Report - https://www.ema.europa.eu/en/documents/assessment- report/comirnaty-epar-public-assessment-report_en.pdf 6) Three decades of messenger RNA vaccine development - ScienceDirect 7) Frontiers | Are There Hidden Genes in DNA/RNA Vaccines? (frontiersin.org) 8) Effect of mRNA Vaccine Manufacturing Processes on Efficacy and Safety Still an Open Question | The BMJ 9) Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line (nih.gov) 10) dsDNA variance in Pfizer Docs - by Anandamide (substack.com) 11) COVID-19 primary series and booster vaccination and immune imprinting https://www.medrxiv.org/content/10.1101/2022.10.31.22281756v1.full.pdf 12) Circulating Spike Protein Detected in Post–COVID-19 mRNA Vaccine Myocarditis: https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.122.061025 13) A SYSTEMATIC REVIEW OF AUTOPSY FINDINGS IN DEATHS AFTER COVID-19 VACCINATION: https://zenodo.org/record/8120771 14) Effectiveness of the Coronavirus Disease 2019 Bivalent Vaccine (Cleveland Clinic Study): https://academic.oup.com/ofid/article/10/6/ofad209/7131292?login=false 15) Cardiovascular Manifestation of the BNT162b2 mRNA COVID-19 Vaccine in Adolescents: https://pubmed.ncbi.nlm.nih.gov/36006288/ 16) Deep sequencing of the Moderna and Pfizer bivalent vaccines identifies contamination of expression vectors designed for plasmid amplification in bacteria | Kevin McKernan: https://rumble.com/v2c785k-march-8-2023.html?mref=umbzp&mc=dprv6 17) BNT162b2 Vaccine-Associated Myo/Pericarditis in Adolescents: A Stratified Risk-Benefit Analysis: https://onlinelibrary.wiley.com/doi/10.1111/eci.13759 18) SARS-CoV-2 Vaccination and Myocarditis in a Nordic Cohort Study of 23 Million Residents: https://jamanetwork.com/journals/jamacardiology/fullarticle/2791253 19) Serious adverse events of special interest following mRNA COVID-19 vaccination in randomized trials in adults: https://www.sciencedirect.com/science/article/pii/S0264410X22010283 20) N1 methylpseudouridine is incorporated with higher fdelity than pseudouridine in synthetic RNAs (demonstrates high error rates) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9335462/pdf/41598_2022_Article_17249.pdf 22) LNP packaging of dsDNA: https://anandamide.substack.com/p/lnp-packaging-of-dsdna 23) IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10222767/ 24) COVID-19 vaccine-associated mortality in the Southern Hemisphere: https://correlation- canada.org/covid-19-vaccine-associated-mortality-in-the-southern-hemisphere/ Learn more about your ad choices. Visit megaphone.fm/adchoices

Find Monica:  Website: https://monicasdeepdives.com Twitter: https://twitter.com/monicaperezshow Rokfin: https://rokfin.com/deepdives Rumble: https://rumble.com/user/monicaperezshow YouTube: https://www.youtube.com/c/MonicaPerez  Shownotes: 1) EMA Quality Report - Rapporteurs-Rolling-Review-Report-Quality-COVID-19-mRNA-Vaccine- BioNTec.doc (live.com) 2) SARS-CoV-2 spike mRNA vaccine sequences circulate in blood up to 28 days after COVID-19 vaccination: https://onlinelibrary.wiley.com/doi/10.1111/apm.13294 3) Biodistribution of mRNA COVID-19 vaccines in human breast milk https://www.thelancet.com/action/showPdf?pii=S2352-3964%2823%2900366-3 4) FDA - Emergency Use Authorization (EUA) for an Unapproved Product Review Memorandum https://www.fda.gov/media/144416/download 5) EMA Comirnaty Assessment Report - https://www.ema.europa.eu/en/documents/assessment- report/comirnaty-epar-public-assessment-report_en.pdf 6) Three decades of messenger RNA vaccine development - ScienceDirect 7) Frontiers | Are There Hidden Genes in DNA/RNA Vaccines? (frontiersin.org) 8) Effect of mRNA Vaccine Manufacturing Processes on Efficacy and Safety Still an Open Question | The BMJ 9) Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line (nih.gov) 10) dsDNA variance in Pfizer Docs - by Anandamide (substack.com) 11) COVID-19 primary series and booster vaccination and immune imprinting https://www.medrxiv.org/content/10.1101/2022.10.31.22281756v1.full.pdf 12) Circulating Spike Protein Detected in Post–COVID-19 mRNA Vaccine Myocarditis: https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.122.061025 13) A SYSTEMATIC REVIEW OF AUTOPSY FINDINGS IN DEATHS AFTER COVID-19 VACCINATION: https://zenodo.org/record/8120771 14) Effectiveness of the Coronavirus Disease 2019 Bivalent Vaccine (Cleveland Clinic Study): https://academic.oup.com/ofid/article/10/6/ofad209/7131292?login=false 15) Cardiovascular Manifestation of the BNT162b2 mRNA COVID-19 Vaccine in Adolescents: https://pubmed.ncbi.nlm.nih.gov/36006288/ 16) Deep sequencing of the Moderna and Pfizer bivalent vaccines identifies contamination of expression vectors designed for plasmid amplification in bacteria | Kevin McKernan: https://rumble.com/v2c785k-march-8-2023.html?mref=umbzp&mc=dprv6 17) BNT162b2 Vaccine-Associated Myo/Pericarditis in Adolescents: A Stratified Risk-Benefit Analysis: https://onlinelibrary.wiley.com/doi/10.1111/eci.13759 18) SARS-CoV-2 Vaccination and Myocarditis in a Nordic Cohort Study of 23 Million Residents: https://jamanetwork.com/journals/jamacardiology/fullarticle/2791253 19) Serious adverse events of special interest following mRNA COVID-19 vaccination in randomized trials in adults: https://www.sciencedirect.com/science/article/pii/S0264410X22010283 20) N1 methylpseudouridine is incorporated with higher fdelity than pseudouridine in synthetic RNAs (demonstrates high error rates) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9335462/pdf/41598_2022_Article_17249.pdf 22) LNP packaging of dsDNA: https://anandamide.substack.com/p/lnp-packaging-of-dsdna 23) IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10222767/ 24) COVID-19 vaccine-associated mortality in the Southern Hemisphere: https://correlation- canada.org/covid-19-vaccine-associated-mortality-in-the-southern-hemisphere/ Learn more about your ad choices. Visit megaphone.fm/adchoices

Dan Wilson returns to TWiV to debunk vaccine misinformation by RFK Jr. during his recent appearance on the Joe Rogan Experience. Hosts: Vincent Racaniello, Dickson Despommier, Rich Condit, Kathy Spindler, and Brianne Barker Guest: Dan Wilson Subscribe (free): Apple Podcasts, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode MicrobeTV Discord Server Joe Rogan's worst misinformation yet, with RFK Jr. (YouTube) Every first vaccine has been tested in placebo-controlled trials before going to market. Polio Measles HPV COVID-19 HepB Haemophilus influenzae B Pertussis Diphtheria Tetanus Pneumococcal Mumps Meningococcal Chickenpox HepA KiGGS Study results on atopic diseases after vaccination (Vaccine) Vaccine history by year (CHOP) Financing vaccines in the 21st Century (Nat Acad Press) DALY rates from non-communicable diseases (Our World in Data) Science loses one to creationism (WaPo) Children's Health Defense (Wikipedia) COVID-19 vaccines saved 3 million US lives (CIDRAP) Beyond the Noise with Paul Offit (YouTube) Letters read on TWiV 1026 Timestamps by Jolene. Thanks! Weekly Picks Dickson – Jazz G.O.A.T.S – Louis Armstrong, Duke Ellington, Ella Fitzgerald Brianne – Defining the Anthropocene? Kathy – How to use your thermostat on A/C Rich – Star Trek: Strange New Worlds Vincent – Step Aside, Joe Biden Listener Picks Kim – Virus found in a boreal lake links ssDNA and dsDNA viruses and Structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv

In this episode our host, Dr Raquel Faria, is joined by Professors Ricard Cervera and Zahir Amoura who will be providing expert insights from the key lupus research in 2022.  For the full written report '2022 Year in Review' please refer to our online library: https://www.lupus-academy.org/library/congress-reports Disclaimer: ‘During Lupus Academy podcast episodes, participants may refer to off label use of medicines for patients with lupus. Lupus Academy does not make any recommendations about using a medicine outside the terms of its approved licence for use.' References for this episode: Bruce IN, Furie RA, Morand EF, et al. Concordance and discordance in SLE clinical trial outcome measures: analysis of three anifrolumab phase 2/3 trials. Ann Rheum Dis. 2022;81(7):962-969. Dörner T, van Vollenhoven RF, Doria A, et al. Baricitinib decreases anti-dsDNA in patients with systemic lupus erythematosus: results from a phase II double-blind, randomized, placebo-controlled trial. Arthritis Res Ther. 2022;24(1):112. Furie RA, Aroca G, Cascino MD, et al. B-cell depletion with obinutuzumab for the treatment of proliferative lupus nephritis: a randomised, double-blind, placebo-controlled trial. Ann Rheum Dis. 2022;81(1):100-107. Furie RA, Hough DR, Gaudy A, et al. Iberdomide in patients with systemic lupus erythematosus: a randomised, double-blind, placebo-controlled, ascending-dose, phase 2a study. Lupus Sci Med. 2022;9(1). Furie RA, van Vollenhoven RF, Kalunian K, Navarra S, Romero-Diaz J, Werth VP, Huang X, Clark G, Carroll H, Meyers A, Musselli C, Barbey C, Franchimont N; LILAC Trial Investigators. Trial of Anti-BDCA2 Antibody Litifilimab for Systemic Lupus Erythematosus. N Engl J Med. 2022 Sep 8;387(10):894-904. Ginzler E, Guedes Barbosa LS, D'Cruz D, et al. Phase III/IV, Randomized, Fifty-Two-Week Study of the Efficacy and Safety of Belimumab in Patients of Black African Ancestry With Systemic Lupus Erythematosus. Arthritis Rheumatol. 2022;74(1):112-123. Jayne D, Rovin B, Mysler EF, et al. Phase II randomised trial of type I interferon inhibitor anifrolumab in patients with active lupus nephritis. Ann Rheum Dis. 2022;81(4):496-506. Sun L, Shen Q, Gong Y, Li Y, Lv Q, Liu H, Zhao F, Yu H, Qiu L, Li X, He X, Chen Y, Xu Z, Xu H. Safety and efficacy of telitacicept in refractory childhood-onset systemic lupus erythematosus: A self-controlled before-after trial. Lupus. 2022 Jul;31(8):998-1006. van Vollenhoven RF, Hahn BH, Tsokos GC, et al. Efficacy and Safety of Ustekinumab in Patients With Active Systemic Lupus Erythematosus: Results of a Phase II Open-label Extension Study. J Rheumatol. 2022;49(4):380-387.

Die Themen in den Wissensnachrichten: +++ Archäologie-Team interpretiert Linien und Pünktchen in Höhlenmalereien als eine Art Jäger-Kalender +++ Neue Variante der CRISPR-Cas-Genschere könnte gegen Krebszellen helfen +++ Kartoffelförmige Steine flitschen höher +++ **********Weiterführende Quellen zu dieser Folge:An Upper Palaeolithic Proto-writing System and Phenological Calendar, Cambridge Archaeological Journal, 05.01.2023Cas12a2 elicits abortive infection via RNA-triggered destruction of dsDNA. Nature, 04.1.2023The role of body shape and mass in skimming on water, Proceedings of the Royal Society A, 04.01.2023Decoupling the skull and skeleton in a Cretaceous bird with unique appendicular morphologies, Nature Eology and Evolution, 02.01.2023I Love It, I'll Never Use It: Exploring Factors of Product Attachment and Their Effects on Sustainable Product Usage Behaviors, International Journal of Design, Dezember 2022**********Ihr könnt uns auch auf diesen Kanälen folgen: Tiktok und Instagram.**********Weitere Wissensnachrichten zum Nachlesen: https://www.deutschlandfunknova.de/nachrichten

Lumpy skin disease (LSD) is a WOAH notifiable transboundary cattle disease caused by lumpy skin disease virus (LSDV), a dsDNA virus member of the Capripoxvirus genus, belonging to the Poxviridae family. LSD primarily affects cattle, causing fever, enlarged lymph nodes, lesions of the oral mucous membranes, of the respiratory, and digestive tract, abortions and a reduction in milk production. Considering the important economic impact on livestock, innovative and effective tools to detect the virus in fields would be of the greatest interest to speed up the diagnosis. Given its feasibility in field conditions and its easiness to use, lateral flow immunoassay (LFIA) could fulfil this goal. This study aimed at developing LFIA using monoclonal antibodies (mAb) produced against p32 LSDV structural protein and evaluating the preliminary specificity and sensitivity.

Lumpy skin disease (LSD) is a WOAH notifiable transboundary cattle disease caused by lumpy skin disease virus (LSDV), a dsDNA virus member of the Capripoxvirus genus, belonging to the Poxviridae family. LSD primarily affects cattle, causing fever, enlarged lymph nodes, lesions of the oral mucous membranes, of the respiratory, and digestive tract, abortions and a reduction in milk production. Considering the important economic impact on livestock, innovative and effective tools to detect the virus in fields would be of the greatest interest to speed up the diagnosis. Given its feasibility in field conditions and its easiness to use, lateral flow immunoassay (LFIA) could fulfil this goal. This study aimed at developing LFIA using monoclonal antibodies (mAb) produced against p32 LSDV structural protein and evaluating the preliminary specificity and sensitivity.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.383745v1?rss=1 Authors: Reyes-Aldrete, E., Dill, E., Bussetta, C., Szymanski, M. R., Diemer, G. S., Maindola, P., White, M., Choi, K., Morais, M. Abstract: Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here we describe biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage ascc{varphi}28. Size-exclusion chromatography, analytical ultracentrifugation, small angle x-ray scattering, and negative stain TEM indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Angstroms and radius of gyration of ~54 Angstroms. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other dsDNA phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high resolution structural studies and rigorous biophysical/biochemical analysis. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.375329v1?rss=1 Authors: Tomko, E. J., Luyties, O., Rimel, J. K., Tsai, C.-L., Fuss, J. O., Fishburn, J., Hahn, S., Tsutakawa, S. E., Taatjes, D. J., Galburt, E. A. Abstract: The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates the C-terminal domain to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.27.316166v1?rss=1 Authors: Xiang, Y., Chen, S., ROng, M., Zhu, D., Lv, Y. Abstract: Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA (dsDNA) sensor that functions in the innate immune system. Upon binding dsDNA in the cytoplasm, cGAS and dsDNA form phase-separated aggregates in which cGAS catalyzes synthesis of 2'3'-cyclic GMP-AMP that subsequently triggers a STING-dependent, type I IFN response. Here, we showed that cytoplasmic RNAs, especially tRNAs, regulate cGAS activity. We discovered that RNAs did not activate cGAS but rather promoted phase separation in vitro. In cells, cGAS colocalized with RNAs and formed phase-separated granules even in the absence of cytoplasmic dsDNA. An Opti-prep gradient analysis of cell lysates showed that the endogenous cGAS was associated with cytoplasmic RNAs in an aggregative form. Further in vitro assays showed that RNAs compete for binding of cGAS with dsDNA and inhibit cGAS activity when the dsDNA concentration is high and promote the formation of phase separations and enhance cGAS activity when the dsDNA concentration is low. Thus, cytoplasmic RNAs regulate cGAS activity by interfering with formation of cGAS-containing aggregates. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.19.304816v1?rss=1 Authors: Balci, H., Globyte, V., Joo, C. Abstract: Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into non-canonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of CRISPR-Cas9 complex would be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR-Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the non-target strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson-Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.11.293639v1?rss=1 Authors: Purkait, D., Bandyopadhyay, D., Mishra, P. P. Abstract: Integration Host Factor (IHF) is a heterodimeric site-specific nucleoid-associated protein (NAP) well known for its DNA bending ability. The binding is mediated through the narrow minor grooves of the consensus sequence, involving van der-Waals interaction and hydrogen bonding. Although the DNA bend state of IHF has been captured by both X-ray Crystallography and Atomic Force Microscopy (AFM), the range of flexibility and degree of heterogeneity in terms of quantitative analysis of the nucleoprotein complex has largely remained unexplored. Here we have monitored and compared the trajectories of the conformational dynamics of a dsDNA upon binding of wild-type (wt) and single-chain (sc) IHF at millisecond resolution through single-molecule FRET (smFRET). Our findings reveal that the nucleoprotein complex exists in a Slacked-Dynamic state throughout the observation window where many of them have switched between multiple Wobbling States in the course of attainment of packaged form. A range of DNA Flexure Angles has been calculated that give us vital insights regarding the nucleoid organization and transcriptional regulation in prokaryotes. This study opens up an opportunity to improve the understanding of the functions of other nucleoid-associated proteins (NAPs) by complementing the previous detailed atomic-level structural analysis, which eventually will allow accessibility towards a better hypothesis. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.26.269084v1?rss=1 Authors: El-Kamand, S., Jergic, S., Lawson, T., Kariawasam, R., Richard, D. J., Cubeddu, L., Gamsjaeger, R. Abstract: The oxidative modification of DNA can result in the loss of genome integrity and must be repaired to maintain overall genomic stability. We have recently demonstrated that human single stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays a crucial role in the removal of 8-oxo-7,8-dihydro-guanine (8-oxoG), the most common form of oxidative DNA damage. The ability of hSSB1 to form disulphide-bonded tetramers and higher oligomers in an oxidative environment is critical for this process. In this study, we have used nuclear magnetic resonance (NMR) spectroscopy and surface plasmon resonance (SPR) experiments to determine the molecular details of ssDNA binding by oligomeric hSSB1. We reveal that hSSB1 oligomers can open up damaged dsDNA and interact with individual single strands; however, our data also show that oxidised bases are recognised in the same manner as undamaged DNA bases. NMR experiments provide evidence that oligomeric hSSB1 is able to bind longer ssDNA in both binding polarities using a distinct set of residues different to those of the related SSB from Escherichia coli. We further demonstrate that oligomeric hSSB1 recognises ssDNA with a significantly higher affinity than its monomeric form and propose structural models for oligomeric hSSB1-ssDNA interaction. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.22.262667v1?rss=1 Authors: Zacharias, M. Abstract: Double-strand (ds)DNA formation and dissociation are of fundamental biological importance. The negatively DNA charge influences the dsDNA stability. However, the base pairing and the stacking between neighboring bases are responsible for the sequence dependent stability of dsDNA. The stability of a dsDNA molecule can be estimated from empirical nearest-neighbor models based on contributions assigned to base pair steps along the DNA and additional parameters due to DNA termini. In efforts to separate contributions it has been concluded that base-stacking dominates dsDNA stability whereas base-pairing contributes negligibly. Using a different model for dsDNA formation we re-analyze dsDNA stability contributions and conclude that base stacking contributes already at the level of separate ssDNAs but that pairing contributions drive the dsDNA formation. The theoretical model also predicts that stability contributions of base pair steps that contain only guanine/cytosine, mixed steps and steps with only adenine/thymine follows the order 6:5:4, respectively, as expected based on the formed hydrogen bonds. The model is fully consistent with available stacking data and nearest-neighbor dsDNA parameters. It allows to assign a narrowly distributed value for the effective free energy contribution per formed hydrogen bond during dsDNA formation of -0.72 kcal/mol based entirely on experimental data. Copy rights belong to original authors. Visit the link for more info

Prot —> mRNA —> cDNA —> dsDNA. (Step 2 via reverse transcriptase and step 3 via DNA Polymerase). Now the dsDNA is injected into a virus which infects bacteria to make lots of the DNA.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.03.235036v1?rss=1 Authors: Ghanta, K. S., Mello, C. C. Abstract: CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.21.214445v1?rss=1 Authors: Losakul, R., Tobar, D. E., Pimenov, A., Gutierrez, A., Schipper, R., Jehle, W. A., Postma, H. W. C. Abstract: Nanopores are an established paradigm in genome sequencing technology, with remarkable advances still being made today. All efforts continually address the challenges associated with rapid, accurate, high-throughput, and low cost detection, particularly with long-read length DNA. We report on the in situ melting and unzipping of long, high molecular weight DNA. At varying salt concentration, we directly compare the translocation conductance and speeds between SiN and graphene nanopores at sub-10 nm pore diameters. We observe the force-induced unzipping of dsDNA at higher salt concentrations than previously reported in literature. We observe free running translocation without secondary structures of ssDNA that is an order of magnitude longer than reported before. We hypothesize that the frayed single strands at the molecules end get captured with a higher likelihood than both ends together. In understanding this phenomena for long-read lengths, we continue to address the challenges revolving around future generations of sequencing technology. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.20.213124v1?rss=1 Authors: Saeed, A. F. U. H., Chan, C., Guo, H., Gong, B., Guo, P., Cheng, X., Ouyang, S. Abstract: Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. Most of these biomotors belong to a group of enzymes known as ATPases, which adopt a multi-subunit ring-shaped structure and hydrolyze adenosine triphosphate (ATP) to generate forces. The gene product 16 (gp16), an ATPase in bacteriophage {square}29, is among the most powerful biomotors known. It can overcome substantial resisting forces from entropic, electrostatic, and DNA bending sources to package double-stranded DNA (dsDNA) into a preformed protein shell (procapsid). Despite numerous studies of the {square}29 packaging mechanism, a structure of the full-length gp16 is still lacking, let alone that of the packaging motor complex that includes two additional molecular components: a connector gp10 protein and a prohead RNA (pRNA). Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment of gp16-CTD with related RNase H-like nuclease domains revealed a nucleic acid binding surface in gp16-CTD, whereas no nuclease activity has been detected for gp16. Subsequent molecular dynamics (MD) simulations showed that this nucleic acid binding surface is likely essential for pRNA binding. Furthermore, our simulations of a full-length gp16 structural model highlighted a dynamic interplay between the N-terminal domain (NTD) and CTD of gp16, which may play a role in driving DNA movement into the procapsid, providing structural support to the previously proposed inchworm model. Lastly, we assembled an atomic structural model of the complete {square}29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage {square}29 and suggested how the individual domains of gp16 work together to power such translocation. ABSTRACT (SHORT)Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. The gene product 16 (gp16) in bacteriophage {square}29 is among the most powerful biomotors known, which adopts a multi-subunit ring-shaped structure and hydrolyzes ATP to package double-stranded DNA (dsDNA) into a preformed procapsid. Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment and molecular dynamics (MD) simulations revealed an essential binding surface of gp16-CTD for prohead RNA (pRNA), a unique component of the motor complex. Furthermore, our simulations highlighted a dynamic interplay between the N-terminal domain (NTD) and CTD of gp16, which may play a role in driving DNA movement into the procapsid. Lastly, we assembled an atomic structural model of the complete {square}29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage {square}29 and suggested how the individual domains of gp16 work together to power such translocation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.13.200352v1?rss=1 Authors: Fatima, N., Gromnicova, R., Loughlin, J., Sharrack, B., Male, D. Abstract: Treatment of diseases that affect the CNS by gene therapy requires delivery of oligonucleotides to target cells within the brain. As the blood brain barrier prevents movement of large biomolecules, current approaches involve direct injection of the oligonucleotides, which is invasive and may have only a localised effect. The aim of this study was to investigate the potential of 2 nm galactose-coated gold nanoparticles (NP-Gal) as a delivery system of oligonucleotides across brain endothelium. DNA oligonucleotides of different types were attached to NP-Gal by the place exchange reaction and were characterised by EMSA (electrophoretic mobility shift assay). Several nanoparticle formulations were created, with single- or double-stranded (20nt or 40nt) DNA oligonucleotides, or with different amounts of DNA attached to the carriers. These nanocarriers were applied to transwell cultures of human brain endothelium in vitro (hCMEC/D3 cell-line) or to a 3D-hydrogel model of the blood-brain barrier including astrocytes. Transfer rates were measured by quantitative electron microscopy for the nanoparticles and qPCR for DNA. Despite the increase in nanoparticle size caused by attachment of oligonucleotides to the NP-Gal carrier, the rates of endocytosis and transcytosis of nanoparticles were both considerably increased when they carried an oligonucleotide cargo. Carriers with 40nt dsDNA were most efficient, accumulating in vesicles, in the cytosol and beneath the basal membrane of the endothelium. The oligonucleotide cargo remained attached to the nanocarriers during transcytosis and the transport rate across the endothelial cells was increased at least 50fold compared with free DNA. The nanoparticles entered the extracellular matrix and were taken up by the astrocytes in biologically functional amounts. Attachment of DNA confers a strong negative charge to the nanoparticles which may explain the enhanced binding to the endothelium and transcytosis by both vesicular transport and the transmembrane/cytosol pathway. These gold nanoparticles have the potential to transport therapeutic amounts of nucleic acids into the CNS. Copy rights belong to original authors. Visit the link for more info

Between the latest online fads and the crazy media headlines, it’s easier than ever to get confused about your health. If you want to make better decisions about your health today so you can feel better and live longer, you’ve come to the right place. Today is the first episode of a three part series that examines the effect of grains on health. I’m joined today by the Gluten-Free Warrior — Dr. Peter Osborne, the founder of the Gluten-Free Society who works to help people with autoimmune and degenerative diseases. He shares some of the experiences and citations that have led him to work so passionately to educate people about the effect of grains on our overall health. Dr. Osborne has helped over 5,000 patients regain overall health and wellness with a grain-free diet, and his approach to healing is focused on individuals with auto-immune diseases.  If you’ve ever suspected that you might have gluten intolerance but aren’t sure if you have Celiac disease, if you’re wondering what environmental factors might be affecting your health, or if you are looking for ways to eliminate painful reactions to gluten, you need to listen to this conversation to help you decide which steps you need to take next.   Key Takeaways: [1:10] Today’s topic is grains — are they harmful to people who don’t have Celiac’s disease? [2:00] Introducing Dr. Peter Osborne, who shares his overall thoughts on the effects of grains on human health, the quality of studies that have been conducted about grains, and the importance of identifying autoimmune diseases when healing for chronic disease. [5:48] Dr. Osborne reacts to the statement that reduced-grain diets contribute to worse health outcomes, including cardiovascular death, and explains how the quality of the diet is a greater factor than the gluten intake itself. [9:52] The correlation between a grain-free diet and autoimmune disease, and a snapshot of the overall history and changing health factors that have an influence on a person's weakened health status. [15:00] The danger of not controlling for variables in a patient, the importance of believing that you can feel better again, and the value of overall body-fuel balance. [18:10] How can you tell if you have non-Celiac gluten sensitivity, and what can you do about it? Dr. Osborne suggests starting with diet change and supporting nutritional deficit. [22:30] Understanding the prevalence of non-Celiac gluten sensitivity and the environmental factors that accelerate it including exposure to pesticides, plastics, antibiotics, chemicals and medications. [27:04] Should all people with a gluten sensitivity be tested for Celiac’s disease?  Dr. Osborne says that it is not necessary, and he explains why. [29:40] What are the pitfalls of making dietary changes that are not accurate relative to the symptoms that are actually present? A look at the variables that affect overall health regardless of gluten-sensitivity. [37:04] If variables such as molds, pesticides and hybridization are present, it becomes increasingly difficult to separate individual aspects of the diet. Here’s what to do about it. [39:24] A list of conditions for non-Celiac gluten sensitivities that are actually caused by gluten and the next steps you should take to begin healing. [43:41] Dr. Osborne’s number one tip for enhancing overall health starts with self observation — pay attention to how your body responds to activity, exercise and food before you decide that your condition is irreversible. [47:14] Do you have a topic you’d like me to cover? Contact me on Facebook or Instagram using #medicalmyths.   To learn more: www.drchristianson.com Dr. Christianson on Instagram Dr. Christianson on Facebook Integrative Healthcare No Grain, No Pain: A 30-Day Diet for Eliminating the Root of Chronic Pain by Dr. Peter Osborne Gluten-Free Society Glutenology on YouTube   Article References: 1. Arbuckle MR, James JA, Kohlhase KF, et al. Development of anti-dsDNA auto-antibodies prior to clinical diagnosis of systemic lupus erythematosus. Scand J Immunol. 2001;54(1–2):211–219. [PubMed] [Google Scholar] 2. Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. 2003;349(16):1526–1533. [PubMed] [Google Scholar] 3. Arslan S, Erkut B, Ates A, et al. Pseudoaneurysm of left ventricular following left ventricular apical venting. Clin Res Cardiol. 2009;98(4):280–282. [PubMed] [Google Scholar] 4. Majka DS, Deane KD, Parrish LA, et al. Duration of preclinical rheumatoid arthritis-related autoantibody positivity increases in subjects with older age at time of disease diagnosis. Ann Rheum Dis. 2008;67(6):801–807. [PMC free article] [PubMed] [Google Scholar] 5. Majka DS, Holers VM. Can we accurately predict the development of rheumatoid arthritis in the preclinical phase? Arthritis Rheum. 2003;48(10):2701–2705. [PubMed] [Google Scholar] 6. Rantapaa-Dahlqvist S, de Jong BA, Berglin E, et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum. 2003;48(10):2741–2749. [PubMed] [Google Scholar] 7. Eriksson C, Kokkonen H, Johansson M, et al. Autoantibodies predate the onset of systemic lupus erythematosus in northern Sweden. Arthritis Res Ther. 2011;13(1):R30. [PMC free article] [PubMed] [Google Scholar]

BioTechniques Digital and Assistant Editors Abigail Sawyer and Tristan Free talk to Theo Roth from the University of California San Francisco Marson Lab (CA, USA). Having recently made headlines for his work in T-cell engineering, Theo takes us through his groundbreaking CRISPR research, its potential impacts in CAR-T cell therapy and further afield, whilst also tackling the evolving ethics in gene editing and the potential benefits of using ssDNA over dsDNA for CRISPR techniques.

Myocardial infarction (MI) in children is uncommon, but underdiagnosed.  This is due to two main factors: the etiologies are varied; and the presenting symptoms are “atypical”. We need a mental metal detector!  Case examples Congenital Two main presentations of MI due to congenital lesions: novel and known.  The novel presentation is at risk for underdiagnosis, due to its uncommonness and vague, atypical symptoms.  There are usually some red flags with a careful H&P.  The known presentation is a child with a history of congenital heart disease, addressed by corrective or palliative surgery.  This child is at risk for expected complications, as well as overdiagnosis and iatrogenia.  Risk stratify, collaborate with specialists. The fussy, sweaty feeder: ALCAPA Anomalous Left Coronary Artery from the Pulmonary Artery (ALCAPA) is an example of what can go wrong during fetal development: any abnormality in the number, origin, course, or morphology of the coronary arteries can present as a neonate with sweating during feeds (steal syndrome), an infant in CHF, or an older child with failure to thrive or poor exercise tolerance. The stable child with chest pain: myocardial bridge Normal coronary arteries run along the epicardial surface of the heart, with projections into the myocardium.  If part of the artery’s course runs within the myocardium (i.e. the artery weaves into and/or out of the myocardium), then there is a myocardial bridge of the coronary artery.  With every systolic contraction, the artery is occluded.  Although a myocardial bridge may not cause symptoms (especially at distal portions), the area it supplies is at risk. With any minor trauma or exertion, demand may outpace supply, resulting in ischemia.  Diagnosis is made on coronary angiography. The unwell child post-cardiac surgery: Fontan problems The child with single ventricle physiology may have a Norwood procedure at birth (creation of a neoaorta, atrial septectomy, and Blalock-Taussig shunt), a Bidirectional Glenn procedure at 3-6 months (shunt removed, superior vena cava connected to pulmonary arteries), and a Fontan procedure at about 2-3 years of age (inferior vena cava blood flow is shunted into the pulmonary arteries). These children depend on their preload to run blood passively into the pulmonary circuit; afterload reduction is also important to compensate for a poor left ejection fraction, as well as to avoid the development of pulmonary hypertension.  They are typically on an anticoagulant (often aspirin), a diuretic (e.g. furosemide), and an afterload reduction agent (e.g. enalapril).  Any disturbance in volume status (hyper- or hypovolemia), anticoagulation, or afterload may cause myocardial strain or infarction.  Take the child s/p Fontan seriously and involve his specialists early with any concerns. Autoimmune The body’s inflammatory-mediated reaction to a real or perceived insult can cause short- and long-term cardiac sequelae.  Find out how well the underlying disease is controlled, and what complications the child has had in the past. The red, hot, crispy, flaky child: acute Kawasaki disease Kawasaki disease (KD) is an acute systemic vasculitis, diagnosed by the presence of fever for five or more days accompanied by four or more criteria:  bilateral conjunctival injection, mucositis, cervical lymphadenopathy, polymorphous rash, and palmar or sole desquamation.  The criteria may occur (and disappear) at any time during the illness. Infants are under double jeopardy with Kawasaki Disease.  They are more likely to have incomplete KD (i.e. not fulfill strict criteria) and if they have KD, they are more likely to suffer the dangerous consequences of aneurysm formation (chiefly coronary arteries, but also brain, kidney).  Have a low threshold for investigation. Treatment includes 2 g/kg/day IVIG and high-dose aspirin (30-50 mg/kg/day) acutely, then low-dose aspirin (5 mg/kg/day) for weeks to months.  Regular and long-term follow-up with Cardiology is required. The aftermath: sequelae of Kawasaki disease The family and child with a history of KD may have psychological trauma and continuous anxiety about the child’s risk of MI.  Approximately 4.7% of children who were promptly diagnosed and correctly treated will go on to have cardiac sequelae. Children who have no detected cardiac sequelae by 8 weeks, typically continue to be asymptomatic up to 20 years later.  Smaller aneurysms tend to regress over time, especially those < 6 mm. Thrombi may calcify, or the lumen may become stenotic due to myofibroblast proliferation.  Children with any coronary artery dilatation from KD should be followed indefinitely. Giant aneurysms (≥8 mm) connote the highest risk for MI.  Parents often are concerned about recurrence, and any subsequent fever can be distressing.  There is a low rate of recurrence for KD: approximately 2%.  Infants who have coronary aneurysms are at the highest risk for recurrence. The older child with vague chest complaints and hypercoagulability: Systemic Lupus Erythematosus and Anti-Phospholipid Syndrome Up to 15% of cases of SLE begin in childhood.  Adult criteria are used, with the caveat that the diagnosis of SLE in children can be challenging; many children only manifest a few of the criteria initially before going on to develop further systemic involvement. The Systemic Lupus International Collaborating Clinics (SLICC) revised the criteria in 2012.  The patient should have ≥4/17 clinical and/or immunologic criteria.  The clinical criteria are: acute cutaneous (malar); chronic cutaneous (discoid); oral; alopecia; synovitis; serositis; renal; neurologic; hemolytic anemia; leukopenia; or thrombocytopenia.  The immunologic criteria are: ANA; anti-dsDNA; anti-Sm; antiphospholipid; low complement; and/or Direct Coombs (in absence of hemolytic anemia).  At least one criterion should be clinical, and at least one should be immunologic.  Children with antiphospholipid syndrome (APS) may occur with or without SLE.  Patients are at risk for venous and arterial thrombi formation.  APS may also cause structural damage, such as valvular thickening and valvular nodes (Libman-Sacks endocarditis).  Mitral and aortic valves are at the highest risk. Although most children with chest pain will not have MI, those with comorbidities should be investigated carefully. Trauma Direct, blunt trauma to the chest can cause myocardial stunning, dysrhythmias, or an asymptomatic rise in Troponin I.  However, some children are at risk for disproportionate harm due to a previously unknown risk factor.  Clinically significant cardiac injury occurs in up to 20% of patients with non-penetrating thoracic trauma. The motor vehicle collision: blunt myocardial injury Direct trauma (steering wheel, airbag, seatbelt), especially in fast acceleration-deceleration injury, may cause compression of the heart between the sternum and the thoracic spine. Electrocardiography (ECG) should be performed on any patient with significant blunt chest injury.  A negative ECG is highly consistent with no significant blunt myocardial injury. Any patient with a new abnormality on ECG (dysrhythmia, heart block, or signs of ischemia) should be admitted for continuous ECG monitoring. Elevation in troponin is common, but not predicted.  A solitary elevated troponin without ECG abnormality is of unclear significance.  Author’s advice: obtain troponin testing if there is an abnormal ECG, more than fleeting suspicion of BCI, and/or the child will be admitted for monitoring. Hemodynamically labile children should be resuscitated and a stat transesophageal echocardiogram obtained. The high-velocity object: coronary artery dissection or thrombus Direct trauma (e.g. MVC, baseball, high-velocity soccer ball) may cause damage to the left anterior descending artery or left circumflex artery, at the highest risk due to their proximity to the chest wall.  Thrombosis and/or dissection may result, often presenting in a focal pattern of ischemia on the ECG. Echocardiography may reveal valvular damage related to the injury, as well as effusion and ejection fraction.  Since there is often a need to investigate the coronary anatomy, percutaneous coronary intervention (PCI) is recommended. The minor trauma with disproportionate complaint: myocardial bridge As mentioned in the congenital section (above), a known variation of a coronary artery’s course involves weaving in and out of the myocardium, creating a baseline risk for ischemia.  Even minor trauma in a child with a myocardial bridge may cause acute thrombus, or slow stenosis from resulting edema.  Unfortunately, the presence of myocardial bridging is often unknown at the time of injury.  Approximately 25% of the population may have myocardial bridging, based on autopsy studies. Take the child seriously who has disproportionate symptoms to what should be a minor injury. Hematologic Coagulopathic and thrombophilic states may predispose children to focal cardiac ischemia.  The best documented cormorbidity is sickle cell disease, although other pro-thrombotic conditions also put the child at risk. The child with sickle cell disease and chest pain: when it’s not acute chest syndrome Sickle cell disease (SCD) can affect any organ system, although the heart is traditionally considered a lower-risk target organ for direct sickling and ischemia.  The major cardiac morbidity in sickle cell is from strain, high-output failure and multiple, serial increases in myocardial demand, causing left ventricular hypertrophy and congestive heart failure. However, there is mounting evidence that acute myocardial ischemia in sickle cell disease may be underappreciated and/or attributed to other causes of chest pain. Other cardiac sequelae from SCD include pulmonary hypertension, left ventricular dysfunction, right ventricular dysfunction, and chronic iron overload. Evidence of myocardial ischemia/infarction in children with SCD has been demonstrated on single-photon emission computed tomography (SPECT) scan. The puffy faced child with chest pain: nephrotic syndrome hypercoagulability Children who suffer from nephrotic syndrome lose proteins that contribute to the coagulation cascade.  In addition, lipoprotein profiles are altered: there is a rise in the very low-density lipoproteins (LDL), contributing to accelerated atherosclerosis.  Typically nephrotic patients have normal levels of high-density lipoproteins (HDL), unless there is profuse proteinuria. Children with difficult-to-control nephrotic syndrome (typically steroid-resistant) may form accelerated plaques that rupture, causing focal MI, as early as school age. The previously well child now decompensated: undiagnosed thrombophilia Asymptomatic patent foramen ovale (PFO) is the cause of some cases of cryptogenic vascular disease, such as stroke and MI.  However, the presence of PFO alone does not connote higher risk.  When paired with an inherited or acquired thrombogenic condition, the venous thrombus may travel from the right-sided circulation to the left, causing distal ischemia.  Many of these cases are unknown until a complication arises. The chronically worried, now with a reason: hypercholesterolemia A family history of adult-onset hypercholesterolemia is not necessarily a risk factor for early complications in children, provided the child does not have the same acquired risk factors as adults (e.g. obesity, sedentary lifestyle, smoking, etc).  Parents may seek help in the ED for children with chest pain and no risk factors, but adult parents who have poor cholesterol profiles. The exception is the child with familial hypercholesterolemia, who is at risk for accelerated atherosclerosis and MI. Infectious Myocarditis has varied etiologies, including infectious, medications (chemotherapy agents), immunologic (rheumatologic, transplant rejection), toxins (arsenic, carbon monoxide, heavy metals such as iron or copper), or physical stress (electrical injury, heat illness, radiation). In children, the most common cause of myocarditis is infectious (viruses, protozoa, bacteria, fungal, parasites).  Of these, viral causes are the most common (adenovirus, enterovirus, echovirus, rubella, HHV6). The verbal child may complain of typical chest complaints, or may come in with flu-like illness and tachycardia or ill appearance out of proportion to presumed viral illness. The most common presenting features in children with myocarditis are: shortness of breath, vomiting, poor feeding, hepatomegaly, respiratory distress, and fever. The infant in shock after a ‘cold’: myocarditis Beware of the poor feeding, tachycardic, ill appearing infant who “has a cold” because everyone else around him has a ‘cold’.  That may very well be true, but any virus can be invasive with myocardial involvement.  Infants are only able to increase their cardiac output through increasing their heart rate; they cannot respond to increased demands through ionotropy.  Look for signs of acute heart failure, such as hepatomegaly, respiratory distress, and sacral edema. The child with tachycardia out of proportion to complaint: myocarditis The previously healthy child with “a bad flu” may simply be very symptomatic from influenza-like illness, or he may be developing myocarditis.  Look for chest pain and tachycardia out of proportion to presumed illness, and constant chest pain, not just associated with cough. The “pneumonia” with suspicious chest x-ray: myocarditis Acute heart failure may mimic viral pneumonia.  Look for disproportionate signs and symptoms. Toxins Younger children may get into others’ medications, be given dangerous home remedies, take drugs recreationally, have environmental exposures (heavy metals), suffer from a consequence of a comorbidity (iron or copper overload) or have adverse events from generally safe medications. The hyperactive boy with a hyperactive precordium: methylphenidate Attention deficit hyperactivity disorder (ADHD) is growing in rate of diagnosis and use of medications.  As the only medical diagnosis based on self-reported criteria, many children are given stimulants regardless of actual neurologic disorder; with a higher proportion of children exposed to stimulants, adverse effects are seen more commonly. Methylphenidate is related to amphetamine, and they both are dopaminergic drugs.  Their mechanisms of action are different, however.  Methylphenidate increases neuronal firing rate.  Methamphetamine reduces neuronal firing rate; cardiovascular sequelae such as MI and CHF are more common in chronic methamphetamine use. Although methylphenidate is typically well tolerated, risks include dysrhythmias such as ventricular tachycardia. The child with seizure disorder and chest pain: anti-epileptics Some anti-epileptic agents, such as carbamazepine, promote a poor lipid profile, leading to atherosclerosis and early MI.  Case reports include school-aged children on carbamazepine who have foamy cells in the coronary arteries, aorta, and vasa vasorum on autopsy.  It is unclear whether this is a strong association. The spice trader: synthetic cannabinoids Synthetic cannabinoids are notoriously difficult to regulate and study, as the manufacturers label them as “not for human consumption”.  Once reports surface of abuse of a certain compound, the formula is altered slightly and repackaged, often in a colorful or mysterious way that is attractive to teenagers. The misperceptions are: are a) synthetics are related to marijuana and therefore safe and b) marijuana is inherently “safe”. Both tend to steer unwitting teens to take these unknown entities.  Some suffer MI as a result. Exposure to tetrahydrocannabinol (THC) in high-potency marijuana has been linked to myocardial ischemia, ventricular tachycardia, and ventricular fibrillation.  Marijuana can increase the heart rate from 20-100%, depending on the amount ingested. K2 (“kush 2.0”) or Spice (Zohai, Genie, K3, Bliss, Nice, Black Mamba, fake weed, etc) is a mixture of plant leaves doused in synthetic chemicals, including cannabinoids and fertilizer (JWH-108), none of which are tested or safe for human consumption.  Synthetic cannabinoids have a higher affinity to cannabinoid receptors, conferring higher potency, and therefore worse adverse effects.  They are thought to be 100 to 800 times more potent as marijuana. Bath salts (Purple Wave, Zoom, Cloud Nine, etc) can be ingested, snorted, or injected.  They typically include some form of cathinone, such as mephedrone, similar to the substance found in the naturally occurring khat plant. Hallucinations, palpitations, tachycardia, MI, and dysrhythmias have been reported from their use as a recreational drug. Chest pain with marijuana, synthetic cannabinoid, or bath salt ingestion should be investigated and/or monitored. Riding that train: high on cocaine Cocaine is a well-known cause of acute MI in young people.  In addition to the direct stimulant causes acutely, such as hypertension, tachycardia, and impaired judgement (coingestions, risky behavior), chronic cocaine use has long-term sequelae.  Cocaine causes accelerated atherosclerosis.  That, in conjunction with arterial vasospasm and platelet activation, is a recipe for acute MI in the young. Cranky: methamphetamine Methamphetamine is a highly addictive stimulant that is relatively inexpensive and widely available.  Repeated use causes multiple psychiatric, personality, and neurologic changes.  Risky behavior, violence, and motor vehicle accidents are all linked to this drug.  Like cocaine, methamphetamine may cause fatal dysrhythmias, acute MI from demand ischemia, and long-term sequelae such as congestive heart failure. Summary Acute MI is a challenging presentation in children: Easily missed: uncommon and atypical Varied etiology Respect vague symptoms with a non-reassuring H&P Try to detect it: CATH IT! References Congenital AboulHosn JA et al. Fontan Operation and the Single Ventricle. Congenit Heart Dis. 2007; 2:2-11. Aliku TO et al. A case of anomalous origin of the left coronary artery presenting with acute myocardial infarction and cardiovascular collapse. African Health Sci. 2014; 14(1): 23-227. Andrews RE et al. Acute myocardial infarction as a cause of death in palliated hypoplastic left heart syndrome. Heart. 2004; 90:e17. Canale LS et al. Surgical treatment of anomalous coronary artery arising from the pulmonary artery. Interactive Cardiovascaulr and Thoracic Surgery. 2009; 8:67-69. Güvenç O et al. Correctable Cause of Dilated Cardiomyopathy in an Infant with Heart Failure: ALCAPA Syndrome. J Curr Pediatr. 2017; 15:47-50. Hastings RS et al. Embolic Myocardial Infarction in a Patient with a Fontan Circulation. World Journal for Pediatric Congenital Heart Surgery. 2014; 5(4)L631-634. Hoffman JIE et al. Electrocardiogram of Anomalous Left Coronary Artery From the Pulmonary Artery in Infants. Pediatr Cardiol. 2013; 34(3):489-491. Kei et al. Rare Case of Myocardial Infarction in a 19-Year-Old Caused by a Paradoxical Coronary Artery Embolism. Perm J.2015; 19(2):e107-e109. Liu Y, Miller BW. ALCAPA Presents in an Adult with Exercise Inlerance but Preserved Cardiac Function. Case Reports Cardiol. 2012; AID 471759. Möhlenkamp S et al. Update on Myocardial Bridging.Circulation. 2002;106:2616-2622. Murgan SJ et al. Acute myocardial infraction n the neonatal period. Cardiol Young. 2002; 12:411-413. Sieweke JT et al. Myocardial infarction in grown up patients with congenital heart disease: an emergening high-risk combination. International Journal of Cardiology. 2016; 203:138-140. Schwerzmann M et al. Anomalous Origin of the Left Coronary Artery From the Main Pulmonary Artery in Adults. Circulation. 2004; 110:e511-e513. Tomkewicz-Pajak L et al. Arterial stiffness in adult patients after Fontan procedure. Cardiovasculr Ultrasound. 2014; 12:15. Varghese MJ et al. The caveats in the diagnosis of anomalous origin of left coronary artery from pulmonary artery (ALCAPA). Images Paediatr Cardiol. 2010; 12(3): 3–8. Autoimmune Ayala et al. Acute Myocardial Infarction in a Child with Systemic Lupus Erythematosus and Antiphospholipid Syndrome. Turk J Rheumatol. 2009; 24:156-8. Nakano H et al. Clinical characteristics of myocardial infarction following Kawasaki disease: Report of 11 cases. J Pediatr. 1986; 108(2):198-203. Pongratz G et al. Myocardial infarction in an adult resulting from coronary aneurysms previously documented in childhood after an acute episode of Kawasaki’s disease. European Heart J. 1994. 15:1002-1004. Newburger JW et al.  Diagnosis, Treatment, and Long-Term Management of Kawasaki Disease. A Statement for Health Professionals From the Committee on Rheumatic Fever, Endocarditis and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association. Circulation. 2004;110:2747-2771. Son MB et al. Kawaski Disease. Pediatr Rev. 2013; 34(4). Yuan S. Cardiac surgical procedures for the coronary sequelae of Kawasaki disease. Libyan J Med. 2012; 7:19796. Trauma Abdolrahim SA et al. Acute Myocardial Infarction Following Blunt Chest Trauma and Coronary Artery Dissection. J Clin Diagnost Res. 2016; 10(6):14-15. Galiuto L et al. Post-traumatic myocardial infarction with hemorrhage and microvascular damage in a child with myocardial bridge: is coronary anatomy actor or bystander. Signa Vitae. 2013; 8(2):61-63. Janella BL et al. Acute Myocardial Infarction related to Blunt Thoracic Trauma. Arq Bras Cardiol. 2006; 87:e168-e171. Liu X et al. Acute myocardial infarction in a child with myocardial bridge World J Emerg Med. 2011; 2(1):70-72. Long WA et al. Childhood Traumatic Infarction Causing Left Ventricular Aneurysm: Diagnosis by Two-Dimensional Echocardiography. JACC. 1985; 5(6):1478-83. Smith S. Right Bundle Branch Block after Blunt Trauma: A Tragic Case. [Blog Post] July 22, 2012. Retrievable at: http://hqmeded-ecg.blogspot.com/2012/07/right-bundle-branch-block-after-blunt.html. Hematologic Carano N et al. Acute Myocardial Infarction in a Child: Possible Pathogenic Role of Patent Foramen Ovale Associated with Heritable Thrombophilia. Pediatr. 2004; 114(2):255-258.      Chacko P et al. Myocardial Infarction in Sickle Cell Disease. J Cardiovascl Transl Res. 2013; 6(5):752-761. De Montalembert M et al. Myocardial ischaemia in children with sickle cell disease. Arch Dis Child. 2004; 89:359-362. Gladwin MT et al. Cardiovascular Abnormalities in Sickle Cell Disease. JACC. 2012; 59(13):1123-1133. Osula S et al. Acute myocardial infarction in young adults: causes and management. Postgrad Med J. 2002; 78:27-30. Silva JMP et al. Premature acute myocardial infarction in a child with nephrotic syndrome. Pediatr Nephrol. 2002; 17:169-172. Suryawanshi SP. Myocardial infarction in children: Two interesting cases. Ann Pediatr Cardiol. 2011 Jan-Jun; 4(1): 81–83. Infectious Cunningham R et al. Viral myocarditis Presenting with Seizure and Electrocardiographic Findings of Acute Myocardial Infarction in a 14-Month-Old Child. Ann Emerg Med. 2000; 35(6):618-622. De Vettten L et al. Neonatal Myocardial Infarction or Myocarditis? Pediatr Cardiol. 2011; 32:492-497. Durani Y et al. Pediatric myocarditis: presenting clinical characteristics. Am J Emerg Med. 2009; 27:942-947. Erden I et al. Acute myocarditis mimicking acute myocardial infarction associated with pandemic 2009 (H1N1) influenza virus. Cardiol J. 2011; 552-555. Hover MH et al. Acute Myocarditis Simulating Myocardial Infarction in a Child. Pediatr. 1191; 87(2):250-252. Lachant D et al. Meningococcemia Presenting as a Myocardial Infarction. Case Reports in Critical Care. 2015; AID 953826. Laissy JP et al. Differentating Myocardial Infarction from Myocarditis. Radiology. 2005; 237(1):75-82. Miranda CH et al. Evaluation of Cardiac Involvement During Dengue Viral Infection. CID. 2013; 57:812-819. Rettig JS et al. Myocarditis in Children Requiring Critical Care Transport. In:  "Diagnosis and Treatment of Myocarditis", Milei J, Ambrosio G (Eds). DOI: 10.5772/56177. Toxins De Chadarévian JP et al. Epilepsy, Atherosclerosis, Myocardial Infarction, and Carbamazepine. J Child Neurol. 2003; 18(2):150-151. McIlroy G et al. Acute myocardial infarction, associated with the use of a synthetic adamantly-canabinoid: a case report. BMC Pharmacology and Toxicology. 2016; 17:2. Mir A et al. Myocardial Infarction Associated with Use of the Synthetic Cannabinoid K2. Pediatr. 2011; 128(6):1-6 Munk K et al. Cardiac Arrest following a Myocardial Infarction in a Child Treated with Methylphenidate. Case Reports Pediatr. 2015; AID 905097. Rezkalla SH et al. Cocaine-Induced Acte Mycardial Infarction. Clin Med Res. 2007; 5(3):172-176. Schelleman H et al. Methylphenidate and risk of serious cardiovascular events in adults. Am J Psychiatry. 2012 Feb;169(2):178-85. Sheridan J et al. Injury associated with methamphetamine use: a review of the literature. Harm Reduction Journal, 2006; 3(14):1-18. Stiefel G et al. Cardiovascular effects of methylphenidate, amphetamines and atomoxetine in the treatment of attention-deficit hyperactivity disorder. Drug Saf. 2010 Oct 1;33(10):821-42.   This post and podcast are dedicated to Edwin Leap, MD for his sanity and humanity in the practice of Emergency Medicine.  Thank you, Dr Leap for all that you do.

Obwohl die immunstimulatorischen Effekte viraler Nukleinsäursen, wie auch IFN -α und IFN-β, während Virusinfektionen eine wichtige Rolle spielen, ist wenig über ihre Funktion bei viraler Glomerulonephritis, wie beispielsweise HIV Nephropathie, bekannt. Virusinfektionen aktivieren, vor allem mittels IFN-α und IFN-β Produktion eine systemische antivirale Immunantwort. Es wurde gezeigt, dass diese inflammatorischen Zytokine einen pleiotropen immunmodulatorischen Effekt auf renale Mesangialzellen ausüben, was direkt zu glomerulären Krankheiten führt. Aber es ist bisher nicht bekannt, ob die viralen Nukleinsäuren und Typ I IFN einen Effekt auf die glomerulären Epithelzellen haben. (z.B. Podozyten und PECs). Um den Effekt von Nukleinsäuren auf Podozyten und PECs zu erforschen, stimulierten wir diese Zellen mit synthetischen dsDNA-(poly-dAdT) Komplexen mit lipofectamine, um eine virale Infektion zu imitieren. Wir haben herausgefunden, dass dsDNA stetig viele IFN-stimulierte Gene in Podozyten und PECs induziert. Desweitern haben wir herausgefunden, dass dsDNA die PECs Proliferation mindert und die CD24+/CD133+PECs Differenzierung zu ausgereiften Podozyten inhibiert. Um unsere Hypothese, dass deis aufgrund von der Sekretion von IFN-α und IFN-β passiert ist, zu bestätigen, haben wir den Effekt von diesen anitviralen Zytokinen auf PECs- und Podozyten-Homöostase etabliert. Wir haben herausgefunden, dass beide IFNs stetig Podozyten und PECs dazu anregen, stetig mehrere IFN-stimulierte Gene zu exprimieren. Trotzdem hat nur IFN-β das Podozytensterben induziert und die Permeabilität der Podozyten-Monolayer erhöht. In der Adriamycin-induzierter Nephropathie bei SCID Mäusen haben Injektionen mit IFN-α oder IFN-β die Proteinurie, den Makrophagen Influx und die Glomerulosklerose verstärkt. Trotzdem induziert nur IFN-β das mitotische Podozytensterben (katastrophale Mitose), welches zu einer reduzierten Podozytenanzahl führt. Wir haben führt, dass IFN-α einen Zellzyklusarrest in-vivo bei PECs induziert, der zur glomerulären Schädigung führt. Balb/c Mäuse, die Adriamycin gespritzt bekommen haben und täglich mit IFN-α und IFN-β behandelt wurden zeigten einen aggravierten Phänotyp mit vermehrter Proteinurie. Im Gegensatz zu dem, was an Studien in SCID Mausen gezeigt wurde, war der Effekt auf die Proteinurie nach IFN-α Behandlung prominenter bei Balb/c Mäusen, verglichen mit IFN-β. Deshalb haben Typ I IFNs einen deutlichen Effekt auf Podozyten und Parietalzellen. Zusammen fördern die Typ I IFNs die Glomerulosklerose durch verstärkten Untergang der Podozyten sowie durch Unterdrückung ihrer Regeneration aus Vorläuferzellen.

ObjectiveThe objective of this article is to evaluate the safety and clinical outcome of rituximab treatment in systemic lupus erythematosus (SLE) patients refractory to standard of care therapy in a real-life setting in Germany. MethodsThe GRAID registry included patients with different autoimmune diseases who were given off-label treatment with rituximab. Data on safety and clinical response were collected retrospectively. In SLE patients, clinical parameters included tender and swollen joint counts, fatigue, myalgia, general wellbeing, Raynaud's and the SLEDAI index. Laboratory tests included dsDNA antibody titres, complement factors, hematologic parameters and proteinuria. Finally, the investigators rated their patients as non-, partial or complete responders based on clinical grounds. ResultsData from 85 SLE patients were collected, 69 female and 16 male, with a mean disease duration of 9.8 years. The mean follow-up period was 9.67.4 months, resulting in 66.8 patient years of observation. A complete response was reported in 37 patients (46.8%), partial response in 27 (34.2%), no response in 15 (19.0%). On average, major clinical as well as laboratory efficacy parameters improved substantially, with the SLEDAI decreasing significantly from 12.2 to 3.3 points. Concerning safety, one infusion reaction leading to discontinuation of treatment occurred. Infections were reported with a rate of 19.5 (including six severe infections) per 100 patient years. ConclusionWith the restrictions of a retrospective data collection, the results of this study confirm data of other registries, which suggest a favourable benefit-risk ratio of rituximab in patients with treatment-refractory SLE.

ASA Meeting 2012 on Science, Faith, and the Media: Communication Beyond Books at Point Loma Nazarene University in San Diego, CA

The integrity of the genome displays a central role for all living organisms. Double strand breaks (DSBs) are probably the most cytotoxic and hazardous type of DNA lesion and are linked to cancerogenic chromosome aberrations in humans. To maintain genome stability, cells use various repair mechanisms, including homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. The Mre11:Rad50 (MR) complex plays a crucial role in DSB repair processes including DSB sensing and processing but also tethering of DNA ends. The complex consists of the evolutionarily conserved core of two Rad50 ATPases from which a long coiled-coil region protrudes and a dimer of the Mre11 nuclease. Even though various enzymatic and also structural functions of MR(N) could be determined, so far the molecular interplay of Rad50´s ATPase together with DNA binding and processing by Mre11 is rather unclear. The crystal structure of the bacterial MR complex in its nucleotide free state revealed an elongated conformation with accessible Mre11 nuclease sites in the center and a Rad50 monomer on each outer tip, thus suggesting conformational changes upon ATP and/or DNA binding. However, so far high resolution structures of MR in its ATP and/or DNA bound state are lacking. The aim of this work was to understand the ATP-dependent engagement-disengagement cycle of Rad50´s nucleotide binding domains (NBDs) and thereby the ATP-controlled interaction between Mre11 and Rad50. For this purpose high resolution crystal structures of the bacterial Thermotoga maritima (Tm) MR complex with engaged Rad50 NBDs were determined. Small angle x-ray scattering proved the conformation of the nucleotide bound complex in solution. DNA affinity was also analyzed to investigate MR´s DNA binding mechanism. ATP binding to TmRad50 induces a large structural change and surprisingly, the NBD dimer binds directly in the Mre11 DNA binding cleft, thereby blocking Mre11’s dsDNA binding sites. DNA binding studies show that MR does not entrap DNA in a ring-like structure and that within the complex Rad50 likely forms a dsDNA binding site in response to ATP, while the Mre11 nuclease module retains ssDNA binding ability. Finally, a possible mechanism for ATP dependent DNA tethering and DSB processing by MR is proposed.

Non-CpG-DNA and 3P-RNA activated mesangial cells to produce inflammatory cytokines and type 1 interferons in TLR-independent manner. Both ligands induced similar gene expression patterns in mesangial cells. This data unravelled the existence of TLR-independent pathways and production of type1 interferons in mesangial cells. These results substantiate the idea that, immune complexes that are associated with nucleic acids can activate glomerular cells via TLR-independent manner, which leads to glomerular inflammation. Furthermore, exposure of non-CpG-DNA and 3P-RNA in MRLlpr/lpr mice aggravated the disease pathology in different manner. Even though 3P-RNA and non-CpG-DNA induced similar gene expression in mesangial cells but in vivo both ligands aggravated the disease in different fashion. 3P-RNA induced type I IFN signaling and decreased the number of regulatory T cells while non-CpG-DNA induced plasma cell expansion, lymphoproliferation and splenomegaly. However, both ligands induced the production of dsDNA autoantibodies and increased the glomerular deposition of IgG. These data suggest that viral 3P-RNA and non-CpG-DNA differently modulate autoimmunity but still both aggravate autoimmune tissue injury by activating non-immune cells at the tissue level

Die vorliegende Studie trägt zum besseren Verständnis des breiten klinischen Spektrums und des Fehlens universeller Serum- Marker der infektionsassoziierten Krankheitsaktivität der Lupus- Nephritis und möglicherweise anderer Formen der Immunkomplexnephritis bei. Exogener Kontakt mit TLR 7- Liganden hat die Entwicklung der Lupus- Nephritis bei jungen, gesunden MRL- Wildtyp und MRLlpr/lpr Mäusen nicht getriggert und hatte keinen signifikanten Effekt auf die Krankheitsaktivität bei diesen jungen Mäusen. Bei alten Lupus- Mäusen führte eine ähnliche Exposition jedoch zu einem merklichen Anstieg der Serumspiegel proinflammatorischer Zytokine und von IFNα, sowie zu einer Infiltration der Nierenglomeruli 18 Wochen alter MRLlpr/lpr Mäuse mit Makrophagen und einer (nicht signifikanten) Erhöhung der Autoantikörperspiegel. Diese Daten unterstützen die Theorie, dass dem Toll- like Rezeptor 7 eine Rolle bei den Mechanismen, die das Fortschreiten der autoimmunen Gewebsschädigung in MRLlpr/lpr Mäusen fördern, zukommt. Basierend auf den Ergebnissen der funktionellen Rolle von TLR 7 in Lupus- Mäusen und in Primärzellen, die aus Lupusmäusen isoliert wurden, sahen wir in der TLR 7- Blockade ein mögliches neues Ziel, um die schädlichen Effekte des Signalings über Immunkomplexe und endogene Liganden zu begrenzen. Es zeigte sich, dass die Blockade von TLR 7 und TLR 7 + TLR 9 die Gewebsschäden in Nieren und Lunge signifikant reduzieren konnte. Eine TLR 7 Antagonisierung mit dem Oligodeoxyribonukleotid IRS661 senkte die Menge an Autoantikörpern (insbesondere anti- SM, anti- dsDNA, IgG2a, IgG2b), entzündlichen Zytokine und Chemokinen im Serum, glomerulären Ablagerungen von IgG2a und Komplementfaktor C3c deutlich. Die Hemmung von TLR 7 reduzierte ebenfalls die CC- Chemokin- gesteuerten Makrophagen- und T- Zell- Infiltrate in den Nieren. Dies zeigte sich durch erniedrigte Spiegel von Ccr2, Ccr5, Ccl2 und Ccl5 in den Nieren behandelter Tiere. Diese Ergebnisse unterstützen das Konzept, dass endogene TLR 7- Liganden zur Pathogenese der Autoantikörperproduktion und der autoimmun vermittelten Gewebsschädigung des SLE beitragen. Die TLR 7- Blockade könnte ein neues therapeutisches Konzept beim Lupus erythematodes sein.

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.

In der vorliegenden Arbeit wurden die diffusiven und elektrophoretischen Eigenschaften von Desoxyribonuklein-säure (DNA) und die Selbstassemblierung von DNA-Chromophor-Hybridmolekülen untersucht. Hierzu wurden, neben Gelelektrophorese und temperaturabhängigen Absorptionsmessungen, vor allem Fluoreszenz-Korre-lationsmethoden angewandt. Um quantitative Aussagen mittels Fluoreszenz-Korrelations-Spektroskopie (FCS) über die freie Diffusion von doppelsträngigen (ds) DNA-Fragmenten (75 bp - 1019 bp) in wässrigen Lösungen zu treffen, wurden laserleis-tungsabhängige Messungen durchgeführt. Diese Experimente ergaben, dass photophysikalische Effekte wie Triplettrelaxation, Isomerisations- oder Bleichprozesse die Autokorrelation entscheidend beeinflussen. Die Län-genabhängigkeit der gemessenen Diffusionskonstanten kann mit einem Stabmodell beschrieben werden, das für alle verwendeten dsDNA-Fragmente, die Konturlängen von bis zu 7 Persistenzlängen aufweisen, Gültigkeit besitzt. In diesem Zusammenhang konnte auch gezeigt werden, dass kleinere Diffusionskonstanten lediglich bei der Zuga-be von zweiwertigen Salzen und bei hohen Salzstärken (> 0,1 M) beobachtet werden. Durch eine Komplexierung der dsDNA-Fragmente mit kationischen und neutralen Lipiden war es möglich, dsDNA in ein unpolares Lösungs-mittel (n-Alkan) zu überführen. Durch Messung der freien Diffusion konnte die Monodispersität von Lipid-dekorierten dsDNA-Fragmenten festgestellt werden. In der unpolaren Phase wurde eine kritische DNA-Konzentration (≈ 10 nM) festgestellt, die für die Stabilität der DNA-Lipid-Komplexe notwendig ist. In der Arbeitsgruppe von Prof. Dr. Müllen am Max-Planck-Institut für Polymerforschung in Mainz wurde ein DNA-Chromophor-Hybrid synthetisiert, bei dem an ein zentrales Farbstoffmolekül (Perylen) beidseitig jeweils ein kurzes ca. 20 Basen langes Oligonukleotid (ODN) kovalent angebunden wurde. FCS-Messungen, die die intrinsi-schen Fluoreszenzeigenschaften des Hybrids ausnutzten, konnten die Löslichkeit und Monodispersität der Hybride bzw. der Lipid-Hybrid-Komplexe sowohl in wässriger Phase als auch in Alkanen nachweisen. Durch eine geeigne-te Wahl der ODN-Sequenzen und der Anknüpfungsstelle der ODN an den Farbstoffkern entstanden durch Basen-paarung unterschiedliche, supramolekulare Strukturen, die in Gelelektrophorese-Experimenten nachgewiesen wur-den. Symmetrische DNA-Chromophor-Hybride können neben beliebig langen, linearen Ketten bei entsprechender Modifikation der Bausteine sandwichartige Dimere ausbilden. Asymmetrische Hybride ermöglichen den Aufbau linearer Strukturen definierter Länge (z. B. Dimere). Die thermodynamischen Eigenschaften der unterschiedlichen, supramolekularen Konstrukte wurden durch tempe-raturabhängige Absorptionsexperimente untersucht. Die Denaturierung der linearen kettenartigen Strukturen kann durch ein Zwei-Zustands-Modell beschrieben werden, dessen energetische Eigenschaften sehr gut mit denen der verwendeten ODN übereinstimmen. Im Fall der sandwichartigen Strukturen musste für den Schmelzübergang ein Drei-Zustands-Modell angenommen werden, wobei die eingebauten Farbstoffkerne eine energetische Wechselwir-kung vermittelten. Neben der freien Diffusion von dsDNA wurde deren elektrophoretische Drift untersucht. Dazu wurde ein Mikroe-lektrophorese-System entwickelt, bei dem die Drift im elektrischen Feld mittels zweier Laserfoki detektiert wird, die einen Abstand von ca. 5 µm aufweisen. Hierbei wirken die beiden Foki wie eine mikroskopische „Lichtschran-ke“; die Driftzeit wird dabei durch eine Orts-Orts-Kreuzkorrelation der beiden Fluoreszenzsignale zugänglich ge-macht. Auf Grund der methodisch bedingten sehr hohen Ortsauflösung ist es möglich, detaillierte Aussagen über die elektrophoretischen und elektroosmotischen Anteile an der Drift zu treffen. Experimente mit unterschiedlichen Feldstärken zeigen, dass eine Temperaturänderung durch den Eintrag von Joulscher Wärme nicht vernachlässigbar ist. Die elektrophoretische Mobilität ist in freier Lösung bei der verwendeten dsDNA unabhängig von der Frag-mentlänge und beträgt im Mittel 4,5∙10-4 cm2/Vs. Durch die gleichzeitige Messung von Drift und Diffusion konnte neben der elektrophoretischen Mobilität der dsDNA-Fragmente auch der Einfluss der hydrodynamischen Reibung ermittelt werden. Dadurch zeigt sich, dass neben der elektrostatischen Kraft und der Reibungskraft auch hydrody-namische Abschirmeffekte berücksichtigt werden müssen, um mit einem entsprechenden Kraftbild die Elektropho-rese-Experimente zu erklären. Im Gegensatz zu den Messungen in freier Lösung zeigt die elektrophoretische Drift der dsDNA-Fragmente in ei-nem physikalischen Polyethylenoxid-Netzwerk eine Längenabhängigkeit, die einem Potenzgesetz folgt (exp=-0,3). Berechnet man das Auflösungspotential der Elektrophorese-Experimente und vergleicht dies mit theoretischen Vorhersagen, so ergibt sich, dass die Diffusion im Vergleich zur Detektorausdehnung den deutlich stärker limitie-renden Faktor bezüglich des Auftrennpotentials unterschiedlich langer DNA darstellt. Die Auftrennung unter-schiedlich langer dsDNA-Fragmente in der Lösung konnte experimentell nachgewiesen werden, wobei die Auflö-sungsgrenze ungefähr 400 bp betrug. Die vorgestellten Ergebnisse belegen, dass FCS zur quantitativen Charakterisierung der Diffusion eingesetzt wer-den kann. Darüber hinaus erlaubt die simultane Messung von elektrophoretischer Drift und Diffusion mit Hilfe von Doppelfokus-FCS, die Bildung von supramolekularen Konstrukten im Bezug auf Ladung und Geometrie mit einer Zeitauflösung im Minutenbereich zu verfolgen.

Dynamic remodeling of chromatin or other persistent protein:DNA complexes is essential for genome expression and maintenance. Proteins of the SWI2/SNF2 family catalyze rearrangements of diverse protein:DNA complexes. Although SWI2/SNF2 enzymes exhibit a diverse domain organisation, they share a conserved catalytic ATPase domain that is related to superfamily II helicases through the presence of seven conserved sequence motifs. In contrast to helicases, SWI2/SNF2 enzymes lack helicase activity, but use ATP hydrolysis to translocate on DNA and to generate superhelical torsion into DNA. How these features implicate remodeling function or how ATP hydrolysis is coupled to these rearrangements is poorly understood and suffers from the lack of structural information regarding the catalytic domain of SWI2/SNF2 ATPase In this PhD thesis I characterized the catalytic domain of Sulfolobus solfataricus Rad54 homolog (SsoRad54cd). Like the eukaryotic SWI2/SNF2 ATPases, SsoRad54cd exhibits dsDNA stimulated ATPase activity, lacks helicase activity and has dsDNA translocation and distortion activity. These activities are thereby features of the conserved catalytic ATPase domain itself. Furthermore, the crystal structures of SsoRad54cd in absence and in complex with its dsDNA substrate were determined. The Sulfolobus solfataricus Rad54 homolog catalytic domain consists of two RecA-like domains with two helical SWI2/SNF2 specific subdomains, one inserted in each domain. A deep cleft separates the two domains. Fully base paired duplex DNA binds along the domain 1: domain 2 interface in a position, where rearrangements of the two RecA-like domains can directly be translated in DNA manipulation. The binding mode of DNA to SsoRad54cd is consistent with an enzyme that translocate along the minor groove of DNA. The structure revealed a remarkable similarity to superfamily II helicases. The related composite ATPase active site as well as the mode of DNA recognition suggests that ATP-driven transport of dsDNA in the active site of SWI2/SNF2 enzymes is mechanistically related to ATP-driven ssDNA in the active site of helicases. Based on structure-function analysis a specific model for SWI2/SNF2 function is suggested that links ATP hydrolysis to dsDNA translocation and DNA distortion. The represented results have structural implications for the core mechanism of remodeling factors. If SWI2/SNF2 ATPases are anchored to the substrate protein:DNA complex by additional substrate interacting domains or subunits, ATP-driven cycles of translocation could transport DNA towards or away from the substrate or generate torsional stress at the substrate:DNA interface. Finally, I provide a molecular framework for understanding mutations in Cockayne and X-linked mental retardation syndromes. Mapping of the mutations on the structure of SsoRad54cd reveal that the mutations colocalize in two surface clusters: Cluster I is located adjacent to a hydrophobic surface patch that may provide a macromolecular interaction site. Cluster II is situated in the domain 1 : domain 2 interface near the proposed pivot region and may interfere with ATP driven conformational changes between domain 1 and domain 2.