POPULARITY
Ko starši ali drugi bližnji, vzgojiteljica, vzgojitelj, učiteljica opazijo, da otrok diha težko in zelo hitro Za Ultrazvok smo šli po sledi poročil, da slovenski pediatri letos zaznavajo več primerov atipičnih pljučnic pri otrocih in mladostnikih. Podatek smo preverili pri pediatrinji Alenki Stepišnik, ki vodi Oddelek za pediatrijo Splošne bolnišnice v Izoli. Ker so okužbe dihal najpogostejše bolezensko stanje otrok in ker lahko pri otroku povzročijo dihalno stisko, je strokovnjakinja med pogovorom opozorila še na okužbo z respiratornim sincicijskim virusom, na prepoznavanje znakov in simptomov oteženega dihanja ter na problematiko tujkov v dihalnih poteh. Foto: Elektronska mikrografija virionov človeškega respiratornega sincicijskega virusa (RSV; obarvani modro) in označenih s protitelesi anti-RSV F protein/ zlato (obarvani rumeno), ki se izločajo s površine človeških pljučnih epitelijskih celic A549. NIAID/ Wiki, cc Povzročiteljice atipičnih pljučnic - mikoplazma, legionela, šigela TUKAJ, TUKAJ Nujna stanja v pediatriji ZD Ljubljana TUKAJ
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.549904v1?rss=1 Authors: Clancy, A., Rusilowicz-Jones, E. V., Wallace, I., Swatek, K. N., Urbe, S., Clague, M. J. Abstract: Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signaling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.16.537099v1?rss=1 Authors: Pei, X., Zheng, F., Li, Y., Lin, Z., Zhang, Y., Han, X., Feng, Y., Li, F., Yang, J., Li, T., Tian, Z., Cao, K., Ren, D., Li, C. Abstract: Idiopathic pulmonary fibrosis (IPF) is marked with the replacement of normal alveolar tissue by thicker and harder fibrous material, damaged exchange ability. Currently, nintedanib and pirfenidone, are the only FDA-approved drugs with limited efficacy for IPF, which indicated an urgent need to explore new therapies. Disulfiram (DSF), an acetaldehyde dehydrogenase inhibitor, used as anti-alcohol treatment. Despite reported with anti-hepatic fibrosis effect of DSF, the underlying mechanism remains unclear. In our study, DSF exhibited regulative impact on abnormal proliferation, EMT and ECM production in cell models of IPF including primary DHLF-IPF cells and TGF-{beta}1-stimulated A549 cells. The absence of COX-2 was restored by DSF treatment, together with elevated prostaglandin biosynthesis both in vitro and in vivo models of IPF. Furthermore, the anti-fibrotic effect of DSF was impeded with COX-2 knockdown or pharmacological inhibition in TGF-{beta}1-stimulated A549 cells, however, exogenous PGE2 reclaimed with anti-EMT function. In established animal model of IPF, DSF ameliorated declined lung function and histopathological changes, and restrained the lung hydroxyproline content. Together, these findings suggest that the anti-fibrotic effect of DSF was achieved through re-activation of COX-2 mediated PGE2 biosynthesis. The above results suggest that DSF can be applied therapeutically in fibrotic conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
A new research paper was published in Oncotarget's Volume 14 on March 24, 2023, entitled, “Downregulation of angulin-1/LSR induces malignancy via upregulation of EGF-dependent claudin-2 and TGF-β-dependent cell metabolism in human lung adenocarcinoma A549 cells.” Abnormal expression of bicellular tight junction claudins, including claudin-2 are observed during carcinogenesis in human lung adenocarcinoma. However, little is known about the role of tricellular tight junction molecule angulin-1/lipolysis-stimulated lipoprotein receptor (LSR). In the present study, researchers Wataru Arai, Takumi Konno, Takayuki Kohno, Yuki Kodera, Mitsuhiro Tsujiwaki, Yuma Shindo, Hirofumi Chiba, Masahiro Miyajima, Yuji Sakuma, Atsushi Watanabe, and Takashi Kojima from Sapporo Medical University School of Medicine examined expression of claudin-2 in the lung adenocarcinoma tissues and found it was higher than in normal lung tissues, while angulin-1/LSR was poorly or faintly expressed. “We investigated how loss of angulin-1/LSR affects the malignancy of lung adenocarcinoma cell line A549 and normal human lung epithelial (HLE) cells.” The researchers found that the EGF receptor tyrosine kinase inhibitor AG1478 prevented the increase of claudin-2 expression induced by EGF in A549 cells. Knockdown of LSR induced expression of claudin-2 at the protein and mRNA levels and AG1478 prevented the upregulation of claudin-2 in A549 cells. Knockdown of LSR induced cell proliferation, cell migration and cell metabolism in A549 cells. Knockdown of claudin-2 inhibited the cell proliferation but did not affect the cell migration or cell metabolism of A549 cells. The TGF-β type I receptor inhibitor EW-7197 prevented the decrease of LSR and claudin-2 induced by TGF-β1 in A549 cells and 2D culture of normal HLE cells. EW-7197 prevented the increase of cell migration and cell metabolism induced by TGF-β1 in A549 cells. EW-7197 prevented the increase of epithelial permeability of FITC-4kD dextran induced by TGF-β1 in 2.5D culture of normal HLE cells. In conclusion, downregulation of angulin-1/LSR induces malignancy via EGF-dependent claudin-2 and TGF-β-dependent cell metabolism in human lung adenocarcinoma. “In conclusion, AG1478 and EW-7197 demonstrated potent in vitro anti-lung adenocarcinoma therapeutic activities via LSR/CLDN-2 and the cell metabolism. The use of both AG1478 and EW-7197 may provide a clinical therapeutic approach for lung adenocarcinoma caused by loss of angulin-1/LSR.” Full research paper: DOI: https://doi.org/10.18632/oncotarget.27728 Correspondence to: Takashi Kojima - ktakashi@sapmed.ac.jp Keywords: angulin-1/LSR, claudin-2, cell metabolism, malignancy, lung adenocarcinoma About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957
A new research paper was published in Oncotarget's Volume 14 on March 24, 2023, entitled, “Polyisoprenylated cysteinyl amide inhibitors deplete singly polyisoprenylated monomeric G-proteins in lung and breast cancer cell lines.” Finding effective therapies against cancers driven by mutant and/or overexpressed hyperactive G-proteins remains an area of active research. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) are agents that mimic the essential posttranslational modifications of G-proteins. It is hypothesized that PCAIs work as anticancer agents by disrupting polyisoprenylation-dependent functional interactions of the G-Proteins. In their new study, researchers Nada Tawfeeq, Jassy Mary S. Lazarte, Yonghao Jin, Matthew D. Gregory, and Nazarius S. Lamango from Florida A&M University College of Pharmacy Pharmaceutical Sciences and Imam Abdulrahman bin Faisal University tested this hypothesis by determining the effect of the PCAIs on the levels of RAS and related monomeric G-proteins. “To investigate the hypothesized anticancer mechanisms of the PCAIs through disruption of G-protein function, we checked the effects of the PCAIs on the G-protein levels in lung cancer (A549 and NCI-H1299) and breast cancer (MDA-MB-231 and MDA-MB-468) cell lines.” Following 48 hours of exposure, they found significant decreases in the levels of KRAS, RHOA, RAC1, and CDC42 ranging within 20–66% after NSL-YHJ-2-27 (5 μM) treatment in all four cell lines tested, A549, NCI-H1299, MDA-MB-231, and MDA-MB-468. However, no significant difference was observed on the G-protein, RAB5A. Interestingly, 38 and 44% decreases in the levels of the farnesylated and acylated NRAS were observed in the two breast cancer cell lines, MDA-MB-231, and MDA-MB-468, respectively, while HRAS levels showed a 36% decrease only in MDA-MB-468 cells. Moreover, after PCAIs treatment, migration, and invasion of A549 cells were inhibited by 72 and 70%, respectively while the levels of vinculin and fascin dropped by 33 and 43%, respectively. Their results show that PCAIs deplete the protein levels of some significant G-proteins which are known to be involved in the migration and invasion of cells (i.e., metastasis) such as RAC1, RHOA, and CDC42. These findings implicate the potential role of PCAIs as anticancer agents through their direct interaction with monomeric G-proteins. “The initial findings presented here indicate how PCAIs can be used as potent agents in developing new anticancer therapeutics, therefore, more extensive studies need to be done to elucidate on its potency. Although we cannot conclusively explain the exact mechanism of action of PCAIs on how they affect the levels of some G-proteins yet, but we can say that these PCAIs have the ability to affect the progression of cancer.” Research paper: DOI: https://doi.org/10.18632/oncotarget.28390 Correspondence to: Nazarius S. Lamango - nazarius.lamango@famu.edu Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords: PCAIs, G-proteins, KRAS, RHOA, RAC1 About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957
Date: May 1, 2022 Speaker: Ps. Dr. K.J.Wesley Venue: Bethesda Church Hyderabad
No episódio de hoje, Sula explica como se dá a infecção pelo nove SARS-Cov-2 e quais são as novidades em termos de descobertas de substâncias marinhas que combatem o vírus. Para isso utilizou como base quatro diferentes artigos: 1- Atividade antiviral de lambda-carragenina contra os vírus da gripe e coronavírus da síndrome respiratória aguda grave 2; 2- Morfogenética (expressão de mucina) bem como potencial atividade anti-corona viral do metabólito secundário marinho polifosfato em células A549; 3- Novas esperanças para drogas contra COVID-19 vêm do mar; e, 4- A plitidepsina tem uma eficácia pré-clínica potente contra SARS-CoV-2 ao direcionar a proteína hospedeira eEF1A. Referências: https://covid19.who.int/ (número de infectados e mortes no mundo) Jang, Y; Shin, H.; Lee, M.K. et al. Antiviral activity of lambda-carrageenan against influenza viruses and severe acute respiratory syndrome coronavirus 2. Scientific Reports, 2021. Müller, W.E.G.; Neufurth, M.; Wang, S. et al. Morphogenetic (mucin expression) as well as potential anti-corona viral activity of the marine secondary metabolite polyphosphate on A549 cells. Marine Drugs, 2020. Taglialatela-Scafati, O. New Hopes for Drugs against COVID-19 Come from the Sea. Marine Drugs, 2021. White, K.M.; Rosales, R.; Yildiz, S. et al. Plitidepsin has a potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A. Science, 2021.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.28.358432v1?rss=1 Authors: Akram, S., Thakur, J., Shree, M., Masakapalli, S. K., Nanda, R. K. Abstract: In vitro studies involving cell lines or primary cells, provide critical insights into their physiology under normal and perturbed conditions like cancer and infection. Given that there are multiple sources of carbon, nitrogen, and other nutrients available in routinely used standard media (such as DMEM, RPMI), it is vital to quantify their contribution to cellular metabolism. 13C based Isotopic tracers of the media components can be used to kinetically track their oxidation by the cell systems such as Human Lung Carcinoma (A549) cells. In this study, a universally labelled glucose tracer ([13C6]glucose) was used to quantify its metabolic contribution that provided further insights into the central carbon metabolism of A549 cells. Gas chromatography and mass spectrometry (GC-MS) based mass isotopomer analysis (average 13C) of methanolic extracts (glycerol: 5.46{+/-}3.53 % and lactate: 74.4{+/-}2.65 %), amino acids derived from acid hydrolysates of protein (Serine: 4.51{+/-}0.21 %, Glycine: 2.44{+/-}0.31 %, Alanine: 24.56{+/-}0.59 %, Glutamate: 8.81{+/-}0.85 %, Proline: 6.96{+/-}0.53 % and Aspartate: 10.72{+/-}0.95 %) and the metabolites of the culture filtrate (glycerol: 43.14{+/-}1.45 % and lactate: 81.67{+/-}0.91 %), allowed to capture the relative contribution of glucose. We observed the Warburg effect and a significant amount of lactate contributed from glucose, was released to the media. 13C glycerol of glucogenic origin was kinetically released to the culture filtrate and might be playing a critical role in metabolic reprogramming of A549 cells. Part of the protein biomass contributed from amino acids were of glucogenic origin. Besides, the workflow adopted for 13C analysis and derived average 13C of each metabolite provided a standard methodology that could be useful in defining the metabolic phenotypes of cells in normal and perturbed conditions. Understanding precisely the altered cellular metabolism to meet the biomass demand under a range of physiological conditions, kinetically, may identify pathways for targeted and effective therapeutic interventions. Copy rights belong to original authors. Visit the link for more info
The cover for Issue 41 of Oncotarget features Figure 7, "IPA ATM-signaling pathway in (A) EFV treated MRC-5 and (B) A549 cells," recently published in "Efavirenz induces DNA damage response pathway in lung cancer" by Marima, et al. which reported that the cell-cycle related genes are potential gene targets in understanding the effects of efavirenz in lung cancer. The present study aimed at investigating the expression changes of cell-cycle related genes in response to EFV drug treatment in human non-small cell lung carcinoma and normal lung fibroblast cells. The loss in nuclear integrity in response to EFV was detected by 4′, 6-diamidino-2-phenylindole staining. Gene expression profiling was performed using human cell cycle PathwayFinder RT2 Profiler™ PCR Array. The expression changes of 84 genes key to the cell cycle pathway in humans following EFV treatment was examined. Interestingly, the p53 signaling pathway was activated irrespective of the repressed ATM pathway in A549 cells as revealed by the Ingenuity Pathway Analysis. Dr. Rahaba Marima from The University of Pretoria as well as The University of the Witwatersrand said, "The non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) is frequently used in human immunodeficiency virus (HIV) treatment, and forms part of the first-line Highly Active Antiretroviral Treatment (HAART) treatment against HIV/AIDS." Xulu and Hosie showed that ARV drugs including EFV caused apoptosis in the Human Squamous Cell carcinoma from Uterine Cervix cells and observed a change in morphological features such as rounding-up of cells, retraction of filopodia, blebbing and maintenance of plasma membrane integrity- characteristic features of apoptosis. Due to the fact that the cell cycle is a tightly regulated process, eukaryotic cells respond to external stimuli such as DNA damage by activating signaling pathways that promote cell cycle arrest and DNA repair. A previous study performed by the Marima Research group, involved assessing the effects of EFV on lung cancer cells at the cellular level on the physiological health of treated cells. To date, several studies including Hecht et al., have revealed the cytotoxic effects of EFV against several cancer cells, but to our knowledge, no study yet has shown the anti-proliferative effects of EFV on lung epithelial cancer cells in relation to primary lung fibroblast cells. In conjunction with preceding studies on EFV′s cyto-and-genotoxicity, this Oncotarget study is the first to reveal EFV mediated ATM/ATR genotoxicity in lung cells. The Marima Research Team concluded in their Oncotarget Research Paper that the treatment of MRC-5 and A549 cells with EFV alters the gene expression of important factors that are essential in the maintenance of genomic stability in relation to the cell cycle. This is particularly observed in the cancerous cells, with the significant down-regulation of AURKB and MAD2L2. Even though the normal p53 expression was shown here, p27, CASP3, Cyclin G1 and G2, NBN, RAD1 and RAD17 were significantly up-regulated. Interestingly, the S-phase and DNA replication genes were downregulated; MCM4 in particular was –3.65 significantly down-regulated. Depending on the severity of these effects in the physiological health of normal cells, EFV poses as a promising drug that can be used in synergy with chemo/radiotherapy. Posttranscriptional gene regulation targeted by EFV in lung cells would also be interesting to pursue. Sign up for free Altmetric alerts about this article DOI - https://doi.org/10.18632/oncotarget.27725 Full text - https://www.oncotarget.com/article/27725/text/ Correspondence to - Rahaba Marima - rahaba.marima@up.ac.za Keywords - efavirenz, cell cycle, differential gene expression, DNA damage response pathway, lung cancer About Oncotarget Oncotarget is a weekly, peer-reviewed, open-access biomedical journal covering research on all aspects of oncology.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.24.311985v1?rss=1 Authors: Snyder, N. W., Singh, B., Buchan, G., O'Brien, J., Arroyo, A. D., Liu, X., Sobol, R. W., Blair, I. A., Mesaros, C. A., Wendell, S. G. Abstract: Metabolism of PUFA results in the formation of hydroxylated fatty acids that can be further oxidized by dehydrogenases, often resulting in the formation of electrophilic, ,{beta}-unsaturated ketone containing fatty acids. As electrophiles are associated with redox signaling, we sought to investigate the metabolism of the oxo-fatty acid products in relation to their double bond architecture. Using an untargeted liquid chromatography mass spectrometry approach, we identified mono- and di-saturated products of the arachidonic acid-derived 11-oxoeicosatetraenoic acid (11-oxoETEs) and mono-saturated metabolites of 15-oxoETE and 17-oxoDHA in both human A549 lung carcinoma and umbilical vein endothelial cells. Notably, mono-saturated oxo-fatty acids maintained their electrophilicity as determined by nucleophilic conjugation to glutathione while a second saturation of 11-oxoETE resulted in a loss of electrophilicity. These results would suggest that prostaglandin reductase, known for its reduction of the ,{beta}-unsaturated double bond, was not responsible for saturation of oxo-fatty acids. Surprisingly, the knock down of prostaglandin reductase by shRNA confirmed its participation in the formation of 15-oxoETE and 17-oxoDHA mono-saturated metabolites. These findings will further facilitate the study of electrophilic fatty acid metabolism and signaling in the context of inflammatory diseases and cancer where they have been shown to have anti-inflammatory and anti-proliferative signaling properties. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.28.223784v1?rss=1 Authors: Liu, W. R., Yang, K. S., Ma, X. R., Ma, Y., Alugubelli, Y. R., Scott, D. A., Vatansever, E. C., Drelich, A. K., Geng, Z. Z., Blankenship, L. R., Sankaran, B., Ward, H. E., Sheng, Y. J., Hsu, J. C., Kratch, K. K., Zhao, B., Liu, J., Li, P., Fierke, C. A., Tseng, C.-T. K., Xu, S., Hayatshahi, H. S. Abstract: The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain beta-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active site cysteine C145. All inhibitors display high potency with IC50 values at or below 100 nM. The most potent compound MPI3 has as an IC50 value as 8.5 nM. Crystallographic analyses of SC2MPro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 uM and A549 cells at 0.16-0.31 uM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19. Copy rights belong to original authors. Visit the link for more info
General immune maintenance & prevention What about the flu vaccine? Food-as-medicine tactics, recipes & herbs If you’ve been exposed/are around someone who has it: How to prevent the spread of influenza What to do when you first start feeling symptoms Treatment for early onset flu Ways to ease symptoms What to do with a fever Get the Flu Prevention & Care GUIDE: https://mailchi.mp/erinholthealth/flu Please leave a review on iTunes! https://podcasts.apple.com/us/podcast/the-funktional-nutrition-podcast/id1292089107 Coyote River Hemp Co. FUNK10 for the 10% off code at goodandcompany.co Micronutrient Panel: https://www.erinholthealth.com/tests-and-consults Find me on Instagram! https://www.instagram.com/erinholthealth/ ALIGN: a day experience for dreamers Use code ERIN2020 to save $20 off tickets by 2/20 Dr. Ben Lynch talking about the coronavirus: https://www.facebook.com/drbenjaminlynch/videos/177458776838512/ Vaccines for preventing influenza in healthy adults: https://www.ncbi.nlm.nih.gov/pubmed/20614424 N-acetyl-L-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus. https://www.ncbi.nlm.nih.gov/pubmed/19732754 Cannabinoids as novel anti-inflammatory drugs. https://www.ncbi.nlm.nih.gov/pubmed/20191092 How Fevers Help Our Immune System Hunt Down Infections https://www.discovermagazine.com/health/how-fevers-help-our-immune-system-hunt-down-infections Fever Phobia Revisited: Have Parental Misconceptions About Fever Changed in 20 Years? https://www.ncbi.nlm.nih.gov/pubmed/11389237 Clinical Report—Fever and Antipyretic Use in Children https://pediatrics.aappublications.org/content/pediatrics/early/2011/02/28/peds.2010-3852.full.pdf Natural Fever Treatments for Kids From a Mommy-MD https://avivaromm.com/natural-fever-treatments/ Herbal Antivirals: Natural Remedies for Emerging & Resistant Viral Infections Stephen Buhner https://amzn.to/37R335k A Simple Homeopathic Alternative to Influenza https://joettecalabrese.com/downloads/Ahh_Choo_The_Flu.pdf?fbclid=IwAR2VUga8LZB_RhjgIDirQHMrpir-94VNN9fjinSb9fuSsUglDtUsi8ZB6kE Miro Humidifer: https://amzn.to/37YXcLr
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 05/06
Idiopathic pulmonary fibrosis (IPF) is an irreversible and progressive disease of the lungs, which is characterized by aberrant tissue remodeling and massive deposition of extracellular matrix proteins. This process is mainly conducted by myofibroblasts, an activated fibroblast phenotype. During the pathogenesis of IPF, the fine alveolar structure is destroyed and gas exchange declines, finally resulting in organ failure. So far, pharmacological treatment options are very limited and lung transplantation still remains the only curative therapy. Pathologic tissue remodeling in IPF is closely connected to altered cell and protein homeostasis. The ubiquitin-proteasome system is critical for degradation of polyubiquitinated proteins in a spatially and timely controlled manner, thereby regulating protein levels. The proteasome is a multicatalytic enzyme complex consisting of a barrel shaped 20S catalytic core particle (CP) and one or two 19S regulatory particles (RP), thus forming active 26S/30S proteasomes. Dysregulation of the proteasome has been reported for several chronic diseases of the heart, brain, and also lung. Furthermore, inhibition of the proteasome has been shown to provide antifibrotic effects in different organs, including the lung. As nothing is known about proteasome function in the pathogenesis of IPF, the first aim of the present study was to analyze proteasomal regulation during tissue remodeling and myofibroblast differentiation. For that, lung fibroblasts were treated with transforming growth factor-β (TGF-β) and proteasome activity as well as composition was examined. For in vivo testing, the bleomycin mouse model of lung fibrosis was used and human lung tissue of IPF patients was analyzed. It was found that induction of myofibroblast differentiation by TGF-β mediated assembly of 19S RPs with 20S CPs, thereby forming 26S/30S complexes, which was critically dependent on the regulatory particle non ATPase 6 subunit (Rpn6). In addition, silencing of Rpn6 in primary human lung fibroblasts counteracted TGF β induced myofibroblast differentiation. During bleomycin-induced fibrotic remodeling of mouse lungs, increased formation of 26S/30S proteasomes was accompanied by augmented expression of Rpn6 in fibrotic lungs. Here, Rpn6 was highly expressed in hyperplastic alveolar epithelial cells and Clara cells. Overexpression of Rpn6 was also observed in myofibroblasts and hyperplastic bronchiolar basal cells of fibrotic lung tissue of IPF patients and accompanied by enhanced polyubiquitination of proteins. As therapeutic application of proteasome inhibitors in pulmonary fibrosis showed controversial results including beneficial antifibrotic effects but also toxicity, the second aim of this study was to test whether site specific inhibition of the proteasome, using the novel second generation inhibitor oprozomib, provides antifibrotic effects in the absence of systemic side effects after local pulmonary application. Oprozomib was compared to the FDA-approved proteasome inhibitor bortezomib and tested on the human alveolar epithelial cancer cell line A549 and on primary mouse alveolar epithelial type II cells regarding its cytotoxic effects. Oprozomib was less toxic than bortezomib and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, oprozomib showed significant antifibrotic effects like reduction of collagen I and α-smooth muscle actin expression at non-toxic doses. When applied locally into the lungs of healthy mice via instillation, oprozomib was well tolerated and effectively reduced pulmonary proteasome activity. In bleomycin-challenged mice, however, locally applied oprozomib resulted in accelerated weight loss and increased mortality. Furthermore, oprozomib failed to reduce fibrosis in these mice, but rather augmented fibrotic lung remodeling in bleomycin-challenged animals. To conclude, this study identified a novel mechanism for fibrotic remodeling of the lungs involving 26S/30S proteasome activation via Rpn6 upon TGF-β-mediated myofibroblast differentiation. Increased levels of Rpn6 and polyubiquitinated proteins in IPF lungs further suggest an important contribution of the ubiquitin-proteasome system to the pathogenesis of this disease. Inhibition of the proteasome with the novel site-specific proteasome inhibitor oprozomib provided low toxicity and antifibrotic effects in alveolar epithelial cells and pulmonary fibroblasts. These results could not be confirmed in pulmonary fibrosis of bleomycin-treated mice, as oprozomib treatment showed high toxicity in fibrotic animals. In light of these data, current proteasome inhibitors, which block the catalytic core, might be too toxic as therapeutic agents for the treatment of fibrotic lung diseases. However, interference with the formation of 26S/30S proteasomes, as shown by Rpn6 knockdown, might provide a novel concept for therapeutic regulation of proteasome activity in lung fibrosis.
Micro-patterned surfaces are frequently used in high-throughput single-cell studies, as they allow one to image isolated cells in defined geometries. Commonly, cells are seeded in excess onto the entire chip, and non-adherent cells are removed from the unpatterned sectors by rinsing. Here, we report on the phenomenon of cellular self-organization, which allows for autonomous positioning of cells on micro-patterned surfaces over time. We prepared substrates with a regular lattice of protein-coated adhesion sites surrounded by PLL-g-PEG passivated areas, and studied the time course of cell ordering. After seeding, cells randomly migrate over the passivated surface until they find and permanently attach to adhesion sites. Efficient cellular self-organization was observed for three commonly used cell lines (HuH7, A549, and MDA-MB-436), with occupancy levels typically reaching 40-60% after 3-5 h. The time required for sorting was found to increase with increasing distance between adhesion sites, and is well described by the time-to-capture in a random-search model. Our approach thus paves the way for automated filling of cell arrays, enabling high-throughput single-cell analysis of cell samples without losses.
Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 13/19
Mutations in the gene coding for the ATP binding cassette protein A3 (ABCA3) are known as the most frequent genetic cause of fatal neonatal respiratory distress syndrome and chronic interstitial lung disease (ILD) of children. ABCA3 transporter is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells. It transports surfactant phospholipids into lamellar bodies and is essential for their biogenesis. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER). This study showed previously not examined cellular dysfunction in cultured lung epithelial A549 cells overexpressing the three ABCA3 mutations R43L, R280C and L101P. All three mutations were found in children with ABCA3-associated lung disease either with fatal neonatal respiratory distress syndrome (L101P and R43L) or chronic pediatric ILD (R280C). Cell biology of R43L and R280C mutations was studied here for the first time. L101P mutation was used as a known example of the trafficking/folding defect leading to the ER retention of ABCA3 protein. Human lung epithelial A549 cells were transfected with vectors containing wild type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress- and apoptosis-proteins, Annexin V surface staining and determination of the intracellular glutathion level. Induction of epithelial-mesenchymal transition (EMT) was assessed by immunoblotting. It was demonstrated that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptosis in A549 cells. A549 cells expressing L101P additionally gain a mesenchymal phenotype. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. These data suggest that expression of partially or completely ER localized ABCA3 mutant proteins induce raised intracellular stress and apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD via a fatal ER-stress/apoptosis/fibrogenesis-axis.
Background: ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER). Methods: Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level. Results: We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. Conclusion: Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.
Despite numerous efforts, drug based treatments for patients suffering from lung cancer remains poor. As a promising alternative, we investigated the therapeutic potential of BC-819 for the treatment of lung cancer in mouse tumor models. BC-819 is a novel plasmid DNA which encodes for the A-fragment of Diphtheria toxin and has previously been shown to successfully inhibit tumor growth in human clinical study of bladder carcinoma. In a first set of experiments, we examined in vitro efficacy of BC-819 in human lung cancer cell-lines NCI-H460, NCI-H358 and A549, which revealed >90% reduction of cell growth. In vivo efficacy was examined in an orthotopic mouse xenograft lung cancer model and in a lung metastasis model using luminescent A549-C8-luc adenocarcinoma cells. These cells resulted in peri- and intra-bronchiolar tumors upon intrabronchial application and parenchymal tumors upon intravenous injection, respectively. Mice suffering from these lung tumors were treated with BC-819, complexed to branched polyethylenimine (PEI) and aerosolized to the mice once per week for a period of 10 weeks. Using this regimen, growth of intrabronchially induced lung tumors was significantly inhibited (p = 0.01), whereas no effect could be observed in mice suffering from lung metastasis. In summary, we suggest that aerosolized PEI/BC-819 is capable of reducing growth only in tumors arising from the luminal part of the airways and are therefore directly accessible for inhaled BC-819.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 07/19
Thu, 10 Jan 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/8224/ https://edoc.ub.uni-muenchen.de/8224/1/Reinelt_Kerstin.pdf Reinelt, Kerstin ddc:600, ddc:610, Medizinische Fakultät
Background: Production of interferon (IFN)-gamma is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFN. on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells. Methods: A549 cells were cultured and stimulated with interleukin (IL)-1 beta alone or in combination with IFN gamma. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA). mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA), respectively. Expression of inhibitor-kappa B alpha, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis. Results: Here we demonstrate that IFN gamma efficiently reduced IL-8 secretion under the influence of IL-1 beta. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFN gamma on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFN gamma on IL-1 beta-induced NF-kappa B activation as assessed by cellular I kappa B levels. Moreover, analysis of intracellular IL-8 suggests that IFN gamma modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1 beta-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFN gamma indicating that modulation of IL-1 beta action by this cytokine displays specificity. Conclusion: Data presented herein agree with an angiostatic role of IFN gamma as seen in rodent models of solid tumors and suggest that increasing T helper type I (Th1)-like functions in lung cancer patients e.g. by local delivery of IFN gamma may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 06/19
Die apparativen Probleme, die sich bei einer Exposition von Zellkulturen gegenüber volatilen Giftstoffen und -gasen ergeben, werden im Zuge der Vorstellung einer neuen Expositionsapparatur dargelegt. Anhand von Extraktionsprofilen, die sich bei der Extraktion von Stickstoffdioxid (NO2) ins Kulturmedium ergeben, wird die Apparatur zunächst in zellfreien Versuchen auf deren Reproduzierbarkeit untersucht. Expositionsversuche mit A549 Zellen über unterschiedliche Zeitdauern zeigen die sich verändernde Zellvitalität anhand des Gesamtglutathiongehalts. Anhand der 4-stündigen Begasungsversuche wird eine Dosis-Wirkungskurve aufgestellt. Im Anschluss an diese Expositionsversuche wird in einer weiteren Versuchsreihe nach der Begasung mit NO2 der Nutzen einer Glucocorticoidgabe (Beclomethason und Budesonid) auf die Zellvitalität untersucht.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 02/07
Untersuchungen strahleninduzierter Änderungen der Proliferation und des Zelltodes stellen Schwerpunkte der radiobiologischen Forschung dar. Aus strahlentherapeutischer Sicht interessieren hier im Besonderen die in Tumoren nach Strahlenexposition zu findenden Genexpressionsänderungen, die assoziiert mit strahleninduzierten Änderungen der Proliferation und des Zelltods auftreten. Detaillierte Kenntnisse der diesen biologischen Prozessen zugrundeliegenden Änderungen auf Genexpressionsebene könnten dazu beitragen, die Effizienz der Strahlentherapie humaner und tierischer Tumoren zu verbessern. So ist eine große Anzahl an für Proliferation und Apoptose kodierenden Genen bekannt. Es sind bisher jedoch nicht alle an der Proliferationskontrolle beteiligten Gene gefunden worden. Ebenso wird postuliert, dass auch andere Formen des Zelltodes als Apoptose auf Genexpressionsebene reguliert werden. Deshalb wurde mithilfe eines Mikroarrays mit 11.835 Genen ein Screening nach differentiell exprimierten Genen an strahlenexponierten A549 Zellen (humanen Lungenkarzinomzellen) vorgenommen. Hierzu wurden die Zellen synchronisiert, in der S-Phase mit 5 Gy bestrahlt und an den Zeitpunkten, die der Ausbildung des G2-Blocks und dem Anstieg mikrokernhaltiger und abnormaler Zellen zeitlich vorausgingen, das Screening durchgeführt. Die geeigneten Zeitpunkte wurden zuvor anhand durchflusszytometrischer Zellzyklusuntersuchungen und der Messung des Zelltodes (MAA-Assay) bestimmt. Die hybridisierten Mikroarrays wurden nach dem Digitalisieren unter Zuhilfenahme einer geeigneten Software interaktiv ausgewertet. Es konnten maximal 987 exprimierte Gene gefunden werden, was 12 % aller Gene des Mikroarrays entsprach. Setzte man die Genexpression der mit 5 Gy bestrahlten Zellen ins Verhältnis zu der Kontrolle, konnten 101 Gene als differentiell exprimiert ermittelt werden. Die Anzahl der herunterregulierten differentiell exprimierten Gene übertraf die Anzahl der hochregulierten differentiell exprimierten Gene zu jedem gemessenen Zeitpunkt immer ca. um den Faktor 4. Des Weiteren wurden die differentiellen Genexpressionen relativ zur Kontrolle der unterschiedlichen Zeitpunkte miteinander verglichen, wobei eine auffällige homogene Herunterregulation der Gene festzustellen war. Nach Einteilung der differentiell exprimierten Gene in funktionelle Gruppen konnten viele Gene, die für den Aufbau des Zytoskeletts kodierten, ermittelt werden. Hierbei standen im Vordergrund vor allem Gene für Tubulinproteine und Aktin. Des Weiteren konnten 8 Gene, die für ribosomale Proteine kodieren, identifiziert werden. Der Anteil bekannter, die Proliferation ("cyclin-dependent kinase inhibitor 1A" (p21, Cip1), "prothymosin, alpha") bzw. die Apoptose ("Caplain" und "TNF receptor-associated factor 1", "Caspase recruitment domain protein 14") regulierender Gene war gering. In Übereinstimmung mit zuvor durchgeführten Untersuchungen in anderen in vitro Modellen konnte eine aktive Herunterregulation bestimmter biologischer Funktionen (z.B. Zytoskelett, Proteinbiosynthese) bei gleichzeitiger Inhibition anderer Funktionen (Proliferation)gezeigt werden ("active silencing"). Da die Aussagen des Mikroarrays nur semiquantitativ sind, müssen die Ergebnisse noch durch ein quantitatives Verfahren (RTQ-PCR) validiert und ergänzt werden. Die vorläufigen Ergebnisse dieser Arbeit geben Hinweise darauf, dass neben den bekannten Zellproliferation und Zelltod kodierenden Genen in einem erheblichen Maß auch andere funktionelle Gengruppen wie z.B. Zytoskelett- und ribosomale Proteine kodierende Gene beteiligt sind und die Zelle im Sinne eines "active silencings" durch Abschalten verschiedener Zellfunktionen ihren eigenen Untergang vorbereitet.