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- ItemSurface Plasmon Resonance Sensitivity Enhancement Based on Protonated Polyaniline Films Doped by Aluminum Nitrate(Basel : MDPI, 2022) Al-Bataineh, Qais M.; Shpacovitch, Victoria; Sadiq, Diyar; Telfah, Ahmad; Hergenröder, RolandComplex composite films based on polyaniline (PANI) doped hydrochloric acid (HCl) incorporated with aluminum nitrate (Al(NO3)3) on Au-layer were designed and synthesized as a surface plasmon resonance (SPR) sensing device. The physicochemical properties of (PANI-HCl)/Al(NO3)3 complex composite films were studied for various Al(NO3)3 concentrations (0, 2, 4, 8, 16, and 32 wt.%). The refractive index of the (PANI-HCl)/Al(NO3)3 complex composite films increased continuously as Al(NO3)3 concentrations increased. The electrical conductivity values increased from 5.10 µS/cm to 10.00 µS/cm as Al(NO3)3 concentration increased to 32 wt.%. The sensitivity of the SPR sensing device was investigated using a theoretical approach and experimental measurements. The theoretical system of SPR measurement confirmed that increasing Al(NO3)3 in (PANI-HCl)/Al(NO3)3 complex composite films enhanced the sensitivity from about 114.5 [Deg/RIU] for Au-layer to 159.0 [Deg/RIU] for Au-((PANI-HCl)/Al(NO3)3 (32 wt.%)). In addition, the signal-to-noise ratio for Au-layer was 3.95, which increased after coating by (PANI-HCl)/Al(NO3)3 (32 wt.%) complex composite layer to 8.82. Finally, we conclude that coating Au-layer by (PANI-HCl)/Al(NO3)3 complex composite films enhances the sensitivity of the SPR sensing device.
- ItemThe mTOR and PP2A pathways regulate PHD2 phosphorylation to Fine-Tune HIF1α levels and colorectal cancer cell survival under hypoxia(Amsterdam : Elsevier, 2017) Di Conza, Giusy; Cafarello, Sarah Trusso; Loroch, Stefan; Mennerich, Daniela; Deschoemaeker, Sofie; Di Matteo, Mario; Ehling, Manuel; Gevaert, Kris; Prenen, Hans; Zahedi, Rene Peiman; Sickmann, Albert; Kietzmann, Thomas; Moretti, Fabiola; Mazzone, MassimilianoOxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth.
- ItemDuring early stages of cancer, neutrophils initiate anti-tumor immune responses in tumor-draining lymph nodes(Maryland Heights, MO : Cell Press, 2022) Pylaeva, Ekaterina; Korschunow, Georg; Spyra, Ilona; Bordbari, Sharareh; Siakaeva, Elena; Ozel, Irem; Domnich, Maksim; Squire, Anthony; Hasenberg, Anja; Thangavelu, Kruthika; Hussain, Timon; Goetz, Moritz; Lang, Karl S; Gunzer, Matthias; Hansen, Wiebke; Buer, Jan; Bankfalvi, Agnes; Lang, Stephan; Jablonska, JadwigaTumor-draining lymph nodes (LNs) play a crucial role during cancer spread and in initiation of anti-cancer adaptive immunity. Neutrophils form a substantial population of cells in LNs with poorly understood functions. Here, we demonstrate that, during head and neck cancer (HNC) progression, tumor-associated neutrophils transmigrate to LNs and shape anti-tumor responses in a stage-dependent manner. In metastasis-free stages (N0), neutrophils develop an antigen-presenting phenotype (HLA-DR+CD80+CD86+ICAM1+PD-L1-) and stimulate T cells (CD27+Ki67highPD-1-). LN metastases release GM-CSF and via STAT3 trigger development of PD-L1+ immunosuppressive neutrophils, which repress T cell responses. The accumulation of neutrophils in T cell-rich zones of LNs in N0 constitutes a positive predictor for 5-year survival, while increased numbers of neutrophils in LNs of N1-3 stages predict poor prognosis in HNC. These results suggest a dual role of neutrophils as essential regulators of anti-cancer immunity in LNs and argue for approaches fostering immunostimulatory activity of these cells during cancer therapy.
- ItemExpression stability of commonly used reference genes in canine articular connective tissues(London : BioMed Central, 2007) Ayers, Duncan; Clements, Dylan N.; Salway, Fiona; Day, Philip J.R.Background: The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Realtime reverse transcription PCR and a mathematical algorithm were used to establish which reference genes were most stably expressed in normal and diseased canine articular tissues and two canine cell lines stimulated with lipolysaccaride (LPS). Results: The optimal reference genes for comparing gene expression data between normal and diseased infrapatella fat pad were RPL13A and YWHAZ (M = 0.56). The ideal reference genes for comparing normal and osteoarthritic (OA) cartilage were RPL13A and SDHA (M = 0.57). The best reference genes for comparing normal and ruptured canine cranial cruciate ligament were B2M and TBP (M = 0.59). The best reference genes for normalising gene expression data from normal and LPS stimulated cell lines were SDHA and YWHAZ (K6) or SDHA and HMBS (DH82), which had expression stability (M) values of 0.05 (K6) and 0.07 (DH82) respectively. The number of reference genes required to reduce pairwise variation (V) to <0.20 was 4 for cell lines, 5 for cartilage, 7 for cranial cruciate ligament and 8 for fat tissue. Reference gene stability was not related to the level of gene expression. Conclusion: The reference genes demonstrating the most stable expression within each different canine articular tissue were identified, but no single reference gene was identified as having stable expression in all different tissue types. This study underlines the necessity to select reference genes on the basis of tissue and disease specific expression profile evaluation and highlights the requirement for the identification of new reference genes with greater expression stability for use in canine articular tissue gene expression studies.
- ItemThe DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Corynebacterium glutamicum(London : BioMed Central, 2007) Gaigalat, Lars; Schlüter, Jan-Philip; Hartmann, Michelle; Mormann, Sascha; Tauch, Andreas; Pühler, Alfred; Kalinowski, JörnBackground: The major uptake system responsible for the transport of fructose, glucose, and sucrose in Corynebacterium glutamicum ATCC 13032 is the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The genes encoding PTS components, namely ptsI, ptsH, and ptsF belong to the fructose-PTS gene cluster, whereas ptsG and ptsS are located in two separate regions of the C. glutamicum genome. Due to the localization within and adjacent to the fructose-PTS gene cluster, two genes coding for DeoR-type transcriptional regulators, cg2118 and sugR, are putative candidates involved in the transcriptional regulation of the fructose-PTS cluster genes. Results: Four transcripts of the extended fructose-PTS gene cluster that comprise the genes sugRcg2116, ptsI, cg2118-fruK-ptsF, and ptsH, respectively, were characterized. In addition, it was shown that transcription of the fructose-PTS gene cluster is enhanced during growth on glucose or fructose when compared to acetate. Subsequently, the two genes sugR and cg2118 encoding for DeoR-type regulators were mutated and PTS gene transcription was found to be strongly enhanced in the presence of acetate only in the sugR deletion mutant. The SugR regulon was further characterized by microarray hybridizations using the sugR mutant and its parental strain, revealing that also the PTS genes ptsG and ptsS belong to this regulon. Binding of purified SugR repressor protein to a 21 bp sequence identified the SugR binding site as an AC-rich motif. The two experimentally identified SugR binding sites in the fructose-PTS gene cluster are located within or downstream of the mapped promoters, typical for transcriptional repressors. Effector studies using electrophoretic mobility shift assays (EMSA) revealed the fructose PTS-specific metabolite fructose-1-phosphate (F-1-P) as a highly efficient, negative effector of the SugR repressor, acting in the micromolar range. Beside F-1-P, other sugar-phosphates like fructose-1,6-bisphosphate (F-1,6- P) and glucose-6-phosphate (G-6-P) also negatively affect SugR-binding, but in millimolar concentrations.
- ItemIdentification of herbal teas and their compounds eliciting antiviral activity against SARS-CoV-2 in vitro(Heidelberg : Springer, 2022) Le-Trilling, Vu Thuy Khanh; Mennerich, Denise; Schuler, Corinna; Sakson, Roman; Lill, Julia K.; Kasarla, Siva Swapna; Kopczynski, Dominik; Loroch, Stefan; Flores-Martinez, Yulia; Katschinski, Benjamin; Wohlgemuth, Kerstin; Gunzer, Matthias; Meyer, Folker; Phapale, Prasad; Dittmer, Ulf; Sickmann, Albert; Trilling, MirkoBackground: The SARS-CoV-2/COVID-19 pandemic has inflicted medical and socioeconomic havoc, and despite the current availability of vaccines and broad implementation of vaccination programs, more easily accessible and cost-effective acute treatment options preventing morbidity and mortality are urgently needed. Herbal teas have historically and recurrently been applied as self-medication for prophylaxis, therapy, and symptom alleviation in diverse diseases, including those caused by respiratory viruses, and have provided sources of natural products as basis for the development of therapeutic agents. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2. Results: Aqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 when applied after infection as well as prior to infection of cells. The herbal infusions exerted in vitro antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin. Conclusions: In conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron, and we identified HMOX-1 as potential therapeutic target. Given that perilla and sage have been suggested as treatment options for various diseases, our dataset may constitute a valuable resource also for future research beyond virology.
- ItemFunctional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells(London : BioMed Central, 2013) Kinzebach, Sven; Dietz, Lisa; Klüter, Harald; Thierse, Hermann-Josef; Bieback, KarenBackground: Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. Results: Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation. In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. Conclusions: The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.
- ItemGli protein activity is controlled by multisite phosphorylation in vertebrate hedgehog signaling(Amsterdam : Elsevier, 2013) Niewiadomski, Pawel; Kong, Jennifer H.; Ahrends, Robert; Ma, Yan; Humke, Eric W.; Khan, Sohini; Teruel, Mary N.; Novitch, Bennett G.; Rohatgi, RajatGli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity.
- ItemMalignant transformation in a defined genetic background: Proteome changes displayed by 2D-PAGE(London : BioMed Central, 2010) Pütz, Stephanie M.; Vogiatzi, Fotini; Stiewe, Thorsten; Sickmann, AlbertBackground: Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER). Results: The stepwise malignant transformation of human fibroblasts was analyzed on the protein level by differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins. The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen (PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90 were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2 (PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we confirmed our 2D-PAGE results by Western Blot. Conclusion: These findings give further hints for intriguing interactions between the p16-RB pathway, the mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of tumorigenesis
- ItemIdentification of cleavage sites and substrate proteins for two mitochondrial intermediate peptidases in Arabidopsis thaliana(Oxford : Oxford University Press, 2015) Carrie, Chris; Venne, A. Saskia; Zahedi, René P.; Soll, JürgenMost mitochondrial proteins contain an N-terminal targeting signal that is removed by specific proteases following import. In plant mitochondria, only mitochondrial processing peptidase (MPP) has been characterized to date. Therefore, we sought to determine the substrates and cleavage sites of the Arabidopsis thaliana homologues to the yeast Icp55 and Oct1 proteins, using the newly developed ChaFRADIC method for N-terminal protein sequencing. We identified 88 and seven putative substrates for Arabidopsis ICP55 and OCT1, respectively. It was determined that the Arabidopsis ICP55 contains an almost identical cleavage site to that of Icp55 from yeast. However, it can also remove a far greater range of amino acids. The OCT1 substrates from Arabidopsis displayed no consensus cleavage motif, and do not contain the classical –10R motif identified in other eukaryotes. Arabidopsis OCT1 can also cleave presequences independently, without the prior cleavage of MPP. It was concluded that while both OCT1 and ICP55 were probably acquired early on in the evolution of mitochondria, their substrate profiles and cleavage sites have either remained very similar or diverged completely.
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