Browsing by Subject "SUPPRESSION"

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  • Okutachi, Sunday; Manoharan, Ganesh Babu; Kiriazis, Alexandros; Laurini, Christina; Catillon, Marie; McCormick, Frank; Yli-Kauhaluoma, Jari; Abankwa, Daniel (2021)
    Recently, the highly mutated oncoprotein K-Ras4B (hereafter K-Ras) was shown to drive cancer cell stemness in conjunction with calmodulin (CaM). We previously showed that the covalent CaM inhibitor ophiobolin A (OphA) can potently inhibit K-Ras stemness activity. However, OphA, a fungus-derived natural product, exhibits an unspecific, broad toxicity across all phyla. Here we identified a less toxic, functional analog of OphA that can efficiently inactivate CaM by covalent inhibition. We analyzed a small series of benzazulenones, which bear some structural similarity to OphA and can be synthesized in only six steps. We identified the formyl aminobenzazulenone 1, here named Calmirasone1, as a novel and potent covalent CaM inhibitor. Calmirasone1 has a 4-fold increased affinity for CaM as compared to OphA and was active against K-Ras in cells within minutes, as compared to hours required by OphA. Calmirasone1 displayed a 2.5-4.5-fold higher selectivity for KRAS over BRAF mutant 3D spheroid growth than OphA, suggesting improved relative on-target activity. Importantly, Calmirasone1 has a 40-260-fold lower unspecific toxic effect on HRAS mutant cells, while it reaches almost 50% of the activity of novel K-RasG12C specific inhibitors in 3D spheroid assays. Our results suggest that Calmirasone1 can serve as a new tool compound to further investigate the cancer cell biology of the K-Ras and CaM associated stemness activities.
  • Zöttl, Markus; Vullioud, Philippe; Goddard, Katy; Torrents-Ticó, Miquel; Gaynor, David; Bennett, Nigel C.; Clutton-Brock, Tim (2018)
    Abstract In Damaraland mole-rats (Fukomys damarensis), non-breeding subordinates contribute to the care of offspring born to the breeding pair in their group by carrying and retrieving young to the nest. In social mole-rats and some cooperative breeders, dominant females show unusually high testosterone levels and it has been suggested that high testosterone levels may increase reproductive and aggressive behavior and reduce investment in allo-parental and parental care, generating age and state-dependent variation in behavior. Here we show that, in Damaraland mole-rats, allo-parental care in males and females is unaffected by experimental increases in testosterone levels. Pup carrying decreases with age of the non-breeding helper while the change in social status from non-breeder to breeder has contrasting effects in the two sexes. Female breeders were more likely than female non-breeders to carry pups but male breeders were less likely to carry pups than male non-breeders, increasing the sex bias in parental care compared to allo-parental care. Our results indicate that testosterone is unlikely to be an important regulator of allo-parental care in mole-rats.
  • Hamari, S.; Kirveskoski, T.; Glumoff, V.; Kulmala, P.; Simell, O.; Knip, M.; Veijola, R. (2016)
    Our aim was to study whether the aberrant amount or function of regulatory T cells is related to the development of type 1 diabetes (T1D) in children. We also set out to investigate the balance of different T cell subtype markers during the T1D autoimmune process. Treg cells were quantified with flow cytometric assay, and the suppression capacity was analysed with a carboxyfluorescein succinimidyl ester (CFSE)-based T cell suppression assay in children in various phases of T1D disease process and in healthy autoantibody-negative control children. The mRNA expression of different T cell subpopulation markers was analysed with real-time qPCR method. The proportion and suppression capacity of regulatory T cells were similar in seroconverted children at an early stage of beta cell autoimmunity and also in children with T1D when compared to healthy and autoantibody-negative children. Significant differences were observed in the mRNA expression of different T cell subpopulation markers in prediabetic children with multiple (2) autoantibodies and in children with newly diagnosed T1D when compared to the control children. In conclusion, there were no quantitative or functional differences in regulatory T cells between the case and control groups in any phase of the autoimmune process. Decreased mRNA expression levels of T cell subtype markers were observed in children with multiple islet autoantibodies and in those with newly diagnosed T1D, probably reflecting an exhaustion of the immune system after the strong immune activation during the autoimmune process or a generally aberrant immune response related to the progression of the disease.
  • The ALICE collaboration; Acharya, S.; Brücken, E. J.; Chang, B.; Hilden, T. E.; Kim, D. J.; Litichevskyi, V.; Mieskolainen, M. M.; Orava, R.; Parkkila, J. E.; Rak, J.; Räsänen, S. S.; Saarinen, S.; Slupecki, M.; Snellman, T. W.; Trzaska, W. H.; Vargyas, M.; Viinikainen, J. (2019)
    Charged-particle spectra at midrapidity are measured in Pb-Pb collisions at the centre-of-mass energy per nucleon-nucleon pair root s(NN) = 5.02 TeV and presented in centrality classes ranging from most central (0-5%) to most peripheral (95-100%) collisions. Possible medium effects are quantified using the nuclear modification factor (R-AA) by comparing the measured spectra with those from proton-proton collisions, scaled by the number of independent nucleon-nucleon collisions obtained from a Glauber model. At large transverse momenta (8 < p(T) < 20 GeV/c), the average R-AA is found to increase from about 0.15 in 0-5% central to a maximum value of about 0.8 in 75-85% peripheral collisions, beyond which it falls off strongly to below 0.2 for the most peripheral collisions. Furthermore, R-AA initially exhibits a positive slope as a function of p(T) in the 8-20 GeV/c interval, while for collisions beyond the 80% class the slope is negative. To reduce uncertainties related to event selection and normalization, we also provide the ratio of R-AA in adjacent centrality intervals. Our results in peripheral collisions are consistent with a PYTHIA-based model without nuclear modification, demonstrating that biases caused by the event selection and collision geometry can lead to the apparent suppression in peripheral collisions. This explains the unintuitive observation that R-AA is below unity in peripheral Pb-Pb, but equal to unity in minimum-bias p-Pb collisions despite similar charged-particle multiplicities. (C) 2019 Conseil Europeen pour la Recherche Nucleaire. Published by Elsevier B.V.
  • Puranen, Jooseppi; Koponen, Sanna; Nieminen, Tiina; Kanerva, Iiris; Kokki, Emmi; Toivanen, Pyry; Urtti, Arto; Ylä-Herttuala, Seppo; Ruponen, Marika (2022)
    Pathological angiogenesis related to neovascularization in the eye is mediated through vascular endothelial growth factors (VEGFs) and their receptors. Ocular neovascular-related diseases are mainly treated with anti-VEGF agents. In this study we evaluated the efficacy and safety of novel gene therapy using adeno associated virus 2 vector expressing a truncated form of soluble VEGF receptor-2 fused to the Fc-part of human IgG1 (AAV2-sVEGFR-2-Fc) to inhibit ocular neovascularization in laser induced choroidal neovascularization (CNV) in mice. The biological activity of sVEGFR-2-Fc was determined in vitro. It was shown that sVEGFR-2-Fc secreted from ARPE-19 cells was able to bind to VEGF-A165 and reduce VEGF-A165 induced cell growth and survival. A single intravitreal injection (IVT) of AAV2-sVEGFR-2-Fc (1 mu l, 4.7 x 1012 vg/ml) one-month prior laser photocoagu-lation did not cause any changes in the retinal morphology and significantly suppressed fluorescein leakage at 7, 14, 21 and 28 days post-lasering compared to controls. Macrophage infiltration was observed after the injection of both AAV2-sVEGFR-2-Fc and PBS. Our findings indicate that AAV2 mediated gene delivery of the sVEGFR-2-Fc efficiently reduces formation of CNV and could be developed to a therapeutic tool for the treatment of retinal diseases associated with neovascularization.
  • Mittag, Maria; Inauri, Karina; Huovilainen, Tatu; Leminen, Miika; Salo, Emma; Rinne, Teemu; Kujala, Teija; Alho, Kimmo (2013)
  • Adam, J.; Brucken, E. J.; Chang, B.; Kim, D. J.; Mieskolainen, M. M.; Orava, R.; Rak, J.; Räsänen, S. S.; Snellman, T. W.; Trzaska, W. H.; The ALICE collaboration (2016)
    We present measurements of the azimuthal dependence of charged jet production in central and semi-central root s(NN) = 2.76 TeV Pb-Pb collisions with respect to the second harmonic event plane, quantified as nu(ch)(2) (jet). Jet finding is performed employing the anti-k(T) algorithm with a resolution parameter R = 0.2 using charged tracks from the ALICE tracking system. The contribution of the azimuthal anisotropy of the underlying event is taken into account event-by-event. The remaining (statistical) region-to-region fluctuations are removed on an ensemble basis by unfolding the jet spectra for different event plane orientations independently. Significant non-zero nu(ch)(2) (jet) is observed in semi-central collisions (30-50% centrality) for 20 <p(T)(ch) (jet) <90 GeV/c. The azimuthal dependence of the charged jet production is similar to the dependence observed for jets comprising both charged and neutral fragments, and compatible with measurements of the nu(2) of single charged particles at high p(T). Good agreement between the data and predictions from JEWEL, an event generator simulating parton shower evolution in the presence of a dense QCD medium, is found in semi-central collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
  • The CMS collaboration; Sirunyan, A. M.; Eerola, P.; Pekkanen, J.; Voutilainen, M.; Härkönen, J.; Järvinen, T.; Karimäki, V.; Kinnunen, R.; Lampén, T.; Lassila-Perini, K.; Lehti, S.; Lindén, T.; Luukka, P.; Tuominiemi, J.; Tuovinen, E.; Wendland, L.; Talvitie, J.; Tuuva, T. (2018)
    The Fourier coefficients v(2) and v(3) characterizing the anisotropy of the azimuthal distribution of charged particles produced in PbPb collisions at root S-NN = 5.02 TeV are measured with data collected by the CMS experiment. The measurements cover a broad transverse momentum range, 1 <p(T) <100 GeV/c. The analysis focuses on the p(T) > 10 GeV/c range, where anisotropic azimuthal distributions should reflect the path-length dependence of parton energy loss in the created medium. Results are presented in several bins of PbPb collision centrality, spanning the 60% most central events. The v(2) coefficient is measured with the scalar product and the multiparticle cumulant methods, which have different sensitivities to initial-state fluctuations. The values from both methods remain positive up to p(T) similar to 60-80 GeV/c, in all examined centrality classes. The v(3) coefficient, only measured with the scalar product method, tends to zero for p(T) greater than or similar to 20 GeV/c. Comparisons between theoretical calculations and data provide new constraints on the path-length dependence of parton energy loss in heavy ion collisions and highlight the importance of the initial-state fluctuations. (C) 2017 The Author. Published by Elsevier B.V.
  • Alsufyani, Abdulmajeed; Harris, Kathryn; Zoumpoulaki, Alexia; Filetti, Marco; Bowman, Howard (2021)
    Studies have shown that presenting own-name stimuli on the fringe of awareness in Rapid Serial Visual Presentation (RSVP) generates a P3 component and provides an accurate and countermeasure resistant method for detecting identity deception (Bowman et al., 2013, 2014). The current study investigates how effective this Fringe-P3 method is at detecting recognition of familiar name stimuli with lower salience (i.e., famous names) than own name stimuli, as well as its accuracy with multi-item stimuli (i.e., first and second name pairs presented sequentially). The results demonstrated a highly significant ERP difference between famous and non-famous names at the group level and a detectable P3 for famous names for 86% of participants at the individual level. This demonstrates that the Fringe-P3 method can be used for detecting name stimuli other than own-names and for multi-item stimuli, thus further supporting the method's potential usefulness in forensic applications such as in detecting recognition of accomplices. (c) 2021 Elsevier Ltd. All rights reserved.
  • Adam, J.; Brucken, E. J.; Chang, B.; Hilden, T. E.; Kim, D. J.; Kral, J.; Mieskolainen, M. M.; Orava, R.; Rak, J.; Rasanen, S. S.; Snellman, T. W.; Trzaska, W. H.; The ALICE collaboration (2015)
    We present a measurement of inclusive J/psi production in p-Pb collisions at root S-NN = 5.02 TeV as a function of the centrality of the collision, as estimated from the energy deposited in the Zero Degree Calorimeters. The measurement is performed with the ALICE detector down to zero transverse momentum, p(T), in the backward (-4.46 <y(cms) <-2.96) and forward (2.03 <y(cms) <3.53) rapidity intervals in the dimuon decay channel and in the mid-rapidity region (-1.37 <y(cms) <0.43) in the dielectron decay channel. The backward and forward rapidity intervals correspond to the Pb-going and p-going direction, respectively. The p(T)-differential J/psi production cross section at backward and forward rapidity is measured for several centrality classes, together with the corresponding average p(T) and p(T)(2) values. The nuclear modification factor is presented as a function of centrality for the three rapidity intervals, and as a function of p(T) for several centrality classes at backward and forward rapidity. At mid-and forward rapidity, the J/psi yield is suppressed up to 40% compared to that in pp interactions scaled by the number of binary collisions. The degree of suppression increases towards central p-Pb collisions at forward rapidity, and with decreasing p(T) of the J/psi. At backward rapidity, the nuclear modification factor is compatible with unity within the total uncertainties, with an increasing trend from peripheral to central p-Pb collisions.
  • CENTER-TBI High Resolution ICU; Zeiler, Frederick A.; Ercole, Ari; Beqiri, Erta; Stocchetti, Nino; Smielewski, Peter; Czosnyka, Marek; Piippo-Karjalainen, Anna; Raj, Rahul (2019)
    Background Impaired cerebrovascular reactivity in adult traumatic brain injury (TBI) is known to be associated with poor outcome. However, there has yet to be an analysis of the association between the comprehensively assessed intracranial hypertension therapeutic intensity level (TIL) and cerebrovascular reactivity. Methods Using the Collaborative European Neuro Trauma Effectiveness Research in TBI (CENTER-TBI) high-resolution intensive care unit (ICU) cohort, we derived pressure reactivity index (PRx) as the moving correlation coefficient between slow-wave in ICP and mean arterial pressure, updated every minute. Mean daily PRx, and daily % time above PRx of 0 were calculated for the first 7 days of injury and ICU stay. This data was linked with the daily TIL-Intermediate scores, including total and individual treatment sub-scores. Daily mean PRx variable values were compared for each TIL treatment score via mean, standard deviation, and the Mann U test (Bonferroni correction for multiple comparisons). General fixed effects and mixed effects models for total TIL versus PRx were created to display the relation between TIL and cerebrovascular reactivity. Results A total of 249 patients with 1230 ICU days of high frequency physiology matched with daily TIL, were assessed. Total TIL was unrelated to daily PRx. Most TIL sub-scores failed to display a significant relationship with the PRx variables. Mild hyperventilation (p <0.0001), mild hypothermia (p = 0.0001), high levels of sedation for ICP control (p = 0.0001), and use vasopressors for CPP management (p <0.0001) were found to be associated with only a modest decrease in mean daily PRx or % time with PRx above 0. Conclusions Cerebrovascular reactivity remains relatively independent of intracranial hypertension therapeutic intensity, suggesting inadequacy of current TBI therapies in modulating impaired autoregulation. These findings support the need for investigation into the molecular mechanisms involved, or individualized physiologic targets (ICP, CPP, or Co2) in order to treat dysautoregulation actively.
  • Linden, Elin; te Beest, Mariska; Aubreu, Ilka; Moritz, Thomas; Sundqvist, Maja K.; Barrio, Isabel C.; Boike, Julia; Bryant, John P.; Brathen, Kari Anne; Buchwal, Agata; Bueno, C. Guillermo; Currier, Alain; Egelkraut, Dagmar D.; Forbes, Bruce C.; Hallinger, Martin; Heijmans, Monique; Hermanutz, Luise; Hik, David S.; Hofgaard, Annika; Holmgren, Milena; Huebner, Diane C.; Hoye, Toke T.; Jonsdottir, Ingibjorg S.; Kaarlejärvi, Elina; Kissler, Emilie; Kumpula, Timo; Limpens, Juul; Myers-Smith, Isla H.; Normand, Signe; Post, Eric; Rocha, Adrian; Schmidt, Niels Martin; Skarin, Anna; Soininen, Eeva M.; Sokolov, Aleksandr; Sokolova, Natalia; Speed, James D. M.; Street, Lorna; Tananaev, Nikita; Tremblay, Jean-Pierre; Urbanowicz, Christine; Watts, David A.; Zimmermann, Heike; Olofsson, Johan (2022)
    Spatial variation in plant chemical defence towards herbivores can help us understand variation in herbivore top-down control of shrubs in the Arctic and possibly also shrub responses to global warming. Less defended, non-resinous shrubs could be more influenced by herbivores than more defended, resinous shrubs. However, sparse field measurements limit our current understanding of how much of the circum-Arctic variation in defence compounds is explained by taxa or defence functional groups (resinous/non-resinous). We measured circum-Arctic chemical defence and leaf digestibility in resinous (Betula glandulosa, B. nana ssp. exilis) and non-resinous (B. nana ssp. nana, B. pumila) shrub birches to see how they vary among and within taxa and functional groups. Using liquid chromatography-mass spectrometry (LC-MS) metabolomic analyses and in vitro leaf digestibility via incubation in cattle rumen fluid, we analysed defence composition and leaf digestibility in 128 samples from 44 tundra locations. We found biogeographical patterns in anti-herbivore defence where mean leaf triterpene concentrations and twig resin gland density were greater in resinous taxa and mean concentrations of condensing tannins were greater in non-resinous taxa. This indicates a biome-wide trade-off between triterpene- or tannin-dominated defences. However, we also found variations in chemical defence composition and resin gland density both within and among functional groups (resinous/non-resinous) and taxa, suggesting these categorisations only partly predict chemical herbivore defence. Complex tannins were the only defence compounds negatively related to in vitro digestibility, identifying this previously neglected tannin group as having a potential key role in birch anti-herbivore defence. We conclude that circum-Arctic variation in birch anti-herbivore defence can be partly derived from biogeographical distributions of birch taxa, although our detailed mapping of plant defence provides more information on this variation and can be used for better predictions of herbivore effects on Arctic vegetation.
  • Tervo, Aino E.; Nieminen, Jaakko O.; Lioumis, Pantelis; Metsomaa, Johanna; Souza, Victor H.; Sinisalo, Heikki; Stenroos, Matti; Sarvas, Jukka; Ilmoniemi, Risto J. (2022)
    Background: Transcranial magnetic stimulation (TMS) is widely used in brain research and treatment of various brain dysfunctions. However, the optimal way to target stimulation and administer TMS therapies, for example, where and in which electric field direction the stimuli should be given, is yet to be determined. Objective: To develop an automated closed-loop system for adjusting TMS parameters (in this work, the stimulus orientation) online based on TMS-evoked brain activity measured with electroencephalography (EEG). Methods: We developed an automated closed-loop TMS-EEG set-up. In this set-up, the stimulus parameters are electronically adjusted with multi-locus TMS. As a proof of concept, we developed an algorithm that automatically optimizes the stimulation orientation based on single-trial EEG responses. We applied the algorithm to determine the electric field orientation that maximizes the amplitude of the TMS-EEG responses. The validation of the algorithm was performed with six healthy volunteers, repeating the search twenty times for each subject. Results: The validation demonstrated that the closed-loop control worked as desired despite the large variation in the single-trial EEG responses. We were often able to get close to the orientation that maximizes the EEG amplitude with only a few tens of pulses. Conclusion: Optimizing stimulation with EEG feedback in a closed-loop manner is feasible and enables effective coupling to brain activity. (C) 2022 The Author(s). Published by Elsevier Inc.
  • Trokovic, Ras; Weltner, Jere; Noisa, Parinya; Raivio, Taneli; Otonkoski, Timo (2015)
    Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced upregulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time. (C) 2015 The Authors. Published by Elsevier B.V.
  • Cervera-Carrascon, Victor; Quixabeira, Dafne C.A.; Havunen, Riikka; Santos, Joao M.; Kutvonen, Emma; Clubb, James H.A.; Siurala, Mikko; Heiniö, Camilla; Zafar, Sadia; Koivula, Teija; Lumen, Dave; Vaha, Marjo; Garcia-Horsman, Arturo; Airaksinen, Anu J.; Sorsa, Suvi; Anttila, Marjukka; Hukkanen, Veijo; Kanerva, Anna; Hemminki, Akseli (2020)
    Despite some promising results, the majority of patients do not benefit from T-cell therapies, as tumors prevent T-cells from entering the tumor, shut down their activity, or downregulate key antigens. Due to their nature and mechanism of action, oncolytic viruses have features that can help overcome many of the barriers currently facing T-cell therapies of solid tumors. This study aims to understand how four different oncolytic viruses (adenovirus, vaccinia virus, herpes simplex virus and reovirus) perform in that task. For that purpose, an immunocompetent in vivo tumor model featuring adoptive tumor-infiltrating lymphocyte (TIL) therapy was used. Tumor growth control (p
  • Kögler, Martin; Paul, Andrea; Anane, Emmanuel; Birkholz, Mario; Bunker, Alex; Viitala, Tapani; Maiwald, Michael; Junne, Stefan; Neubauer, Peter (2018)
    The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here, we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman), and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples. We investigated concentration transients of glucose, acetate, AMP, and cAMP at alternating substrate availability, from deficiency to excess. Raman and SERS signals were compared to off-line metabolite analysis of carbohydrates, carboxylic acids, and nucleotides. Results demonstrate that SERS, in almost all cases, led to a higher number of identifiable signals and better resolved spectra. Spectra derived from the TG-Raman were comparable to those of micro-Raman resulting in well-discernable Raman peaks, which allowed for the identification of a higher number of compounds. In contrast, NIR-Raman provided a superior performance for the quantitative evaluation of analytes, both with and without SERS nanoparticles when using multivariate data analysis. (c) 2018 American Institute of Chemical Engineers
  • d'Enterria, David; Eskola, Kari J.; Helenius, Ilkka; Paukkunen, Hannu (2014)
  • O'Toole, John M.; Boylan, Geraldine B.; Lloyd, Rhodri O.; Goulding, Robert M.; Vanhatalo, Sampsa; Stevenson, Nathan J. (2017)
    Aim: To develop a method that segments preterm EEG into bursts and inter-bursts by extracting and combining multiple EEG features. Methods: Two EEG experts annotated bursts in individual EEG channels for 36 preterm infants with gestational age <30 weeks. The feature set included spectral, amplitude, and frequency-weighted energy features. Using a consensus annotation, feature selection removed redundant features and a support vector machine combined features. Area under the receiver operator characteristic (AUC) and Cohen's kappa (K) evaluated performance within a cross-validation procedure. Results: The proposed channel-independent method improves AUC by 4-5% over existing methods (p <0.001, n = 36), with median (95% confidence interval) AUC of 0.989 (0.973-0.997) and sensitivity -specificity of 95.8-94.4%. Agreement rates between the detector and experts' annotations, K = 0.72 (0.36-0.83) and K = 0.65 (0.32-0.81), are comparable to inter-rater agreement, K = 0.60 (0.21-0.74). Conclusions: Automating the visual identification of bursts in preterm EEG is achievable with a high level of accuracy. Multiple features, combined using a data-driven approach, improves on existing single-feature methods. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of IPEM.
  • De, Swarnalok; Chavez-Calvillo, Gabriela; Wahlsten, Matti; Mäkinen, Kristiina (2018)
    Infection caused by the synergistic interaction of two plant viruses is typically manifested by severe symptoms and increased accumulation of either virus. In potex-potyviral synergism, the potyviral RNA silencing suppressor helper component proteinase (HCPro) is known to enhance the pathogenicity of the potexvirus counterpart. In line with this, Potato virus X (PVX; genus Potexvirus) genomic RNA (gRNA) accumulation and gene expression from subgenomic RNA (sgRNA) are increased in Nicotiana benthamiana by Potato virus A (PVA; genus Potyvirus) HCPro expression. Recently, we have demonstrated that PVA HCPro interferes with the host cell methionine cycle by interacting with its key enzymes S-adenosyl-l-methionine synthetase (SAMS) and S-adenosyl-l-homocysteine hydrolase (SAHH). To study the involvement of methionine cycle enzymes in PVX infection, we knocked down SAMS and SAHH. Increased PVX sgRNA expression between 3 and 9 days post-infiltration (dpi) and upregulation of (-)-strand gRNA accumulation at 9 dpi were observed in the SAHH-silenced background. We found that SAMS and SAHH silencing also caused a significant reduction in glutathione (GSH) concentration, specifically in PVX-infected plants between 2 and 9 dpi. Interestingly, HCPro expression in PVX-infected plants caused an even stronger reduction in GSH levels than did SAMS+SAHH silencing and a similar level of reduction was also achieved by knocking down GSH synthetase. PVX sgRNA expression was increased in the GSH synthetase-silenced background. GSH is a major antioxidant of plant cells and therefore GSH shortage may explain the strong oxidative stress and severe symptoms observed during potex-potyvirus mixed infection.
  • Zini, Jacopo; Kekkonen, Jere; Kaikkonen, Ville A.; Laaksonen, Timo; Keränen, Pekka; Talala, Tuomo; Mäkynen, Anssi J.; Yliperttula, Marjo; Nissinen, Ilkka (2021)
    Hydrogels, natural and synthetic origin, are actively studied for their use for implants and payload carriers. These biomaterials for delivery systems have enormous potential in basic biomedical research, drug development, and long-term delivery of biologics. Nanofibrillated cellulose (NFC) hydrogels, both natural and anionic (ANFC) ones, allow drug loading for immediate and controlled release via the slow drug dissolution of solid drug crystals into hydrogel and its subsequent release. This property makes NFC originated hydrogels an interesting non-toxic and non-human origin material as drug reservoir for long-term controlled release formulation or implant for patient care. A compelling tool for studying NFC hydrogels is Raman spectroscopy, which enables to resolve the chemical structures of different molecules in a high-water content like hydrogels, since Raman spectroscopy is insensitive to water molecules. That offers real time investigation of label-free drugs and their release in high-water-content media. Despite the huge potential of Raman spectroscopy in bio-pharmaceutical applications, the strong fluorescence background of many drug samples masking the faint Raman signal has restricted the widespread use of it. In this study we used a Raman spectrometer capable of suppressing the unpleasant fluorescence background by combining a pulsed laser and time-resolved complementary metal-oxide-semiconductor (CMOS) singlephoton avalanche diode (SPAD) line sensor for the label-free investigation of Metronidazole and Vitamin C diffusivities in ANFC. The results show the possibility to modulate the ANFC-based implants and drug delivery systems, when the release rate needs to be set to a desired value. More importantly, the now developed label free real-time method is universal and can be adapted to any hydrogel/drug combination for producing reliable drug diffusion coefficient data in complex and heterogeneous systems, where traditional sampling-based methods are cumbersome to use. The wide temporal range of the time-resolved CMOS SPAD sensors makes it possible to capture also the fluorescence decay of samples, giving rise to a combined time-resolved Raman and fluorescence spectroscopy, which provides additional information on the chemical, functional and structural changes in samples.