Browsing by Subject "1182 Biochemistry, cell and molecular biology"

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  • Virjamo, Virpi; Fyhrquist, Pia; Koskinen, Akseli; Lavola, Anu; Nissinen, Katri; Julkunen-Tiitto, Riitta (2020)
    Knowledge about the defensive chemistry of coniferous trees has increased in recent years regarding a number of alkaloid compounds; in addition to phenolics and terpenes. Here, we show that Norway spruce (Picea abies (L.) H. Karst.), an important boreal zone tree species; accumulates 1,6-dehydropinidine (2-methyl-6-(2-propenyl)-1,6-piperideine) in its needles and bark. We reanalyzed previously published GC-MS data to obtain a full picture of 1,6-dehydropinidine in P. abies. 1,6-dehydropinidine appeared to especially accumulate in developing spring shoots. We used solid-phase partitioning to collect the alkaloid fraction of the sprouts and thin-layer chromatography to purify 1,6-dehydropinidine. The antibacterial properties of the 1,6-dehydropinidine fraction were tested using a broth microdilution method; with Streptococcus equi subsp. equi as a model organism. Based on our results 1,6-dehydropinidine is common in alkaloid extractions from P. abies (0.4 +/- 0.03 mg g(-1) dw in mature needles) and it is especially abundant in young spruce shoots (2.7 +/- 0.5 mg g(-1) dw). Moreover; 1,6-dehydropinidine extracted from P. abies sprouts showed mild antibacterial potential against Streptococcus equi subsp. equi (MIC 55 mu g mL(-1)). The antibacterial activity of a plant compound thought of as an intermediate rather than an end-product of biosynthesis calls for more detailed studies regarding the biological function of these coniferous alkaloids
  • Kahila, Hanna; Marjonen, Heidi; Auvinen, Pauliina; Avela, Kristiina; Riikonen, Raili; Kaminen-Ahola, Nina (2020)
    Abstract Background A pair of dizygotic twins discordantly affected by heavy prenatal alcohol exposure (PAE) was reported previously by Riikonen, suggesting the role of genetic risk or protective factors in the etiology of alcohol-induced developmental disorders. Now, we have re-examined these 25-year-old twins and explored genetic origin of the phenotypic discordancy reminiscent with fetal alcohol syndrome (FAS). Furthermore, we explored alterations in DNA methylation profile of imprinting control region at growth-related insulin-like growth factor 2 (IGF2)/H19 locus in twins' white blood cells (WBC), which have been associated earlier with alcohol-induced genotype-specific changes in placental tissue. Methods Microarray-based comparative genomic hybridization (aCGH) was used to detect potential submicroscopic chromosomal abnormalities, and developmental as well as phenotypic information about twins were collected. Traditional bisulfite sequencing was used for DNA methylation analysis. Results Microarray-based comparative genomic hybridization revealed a microdeletion 18q12.3-q21.1. in affected twin, residing in a known 18q deletion syndrome region. This syndrome has been associated with growth restriction, developmental delay or intellectual deficiency, and abnormal facial features in previous studies, and thus likely explains the phenotypic discordancy between the twins. We did not observe association between WBCs? DNA methylation profile and PAE, but interestingly, a trend of decreased DNA methylation at the imprinting control region was seen in the twin with prenatal growth retardation at birth. Conclusions The microdeletion emphasizes the importance of adequate chromosomal testing in examining the etiology of complex alcohol-induced developmental disorders. Furthermore, the genotype-specific decreased DNA methylation at the IGF2/H19 locus cannot be considered as a biological mark for PAE in adult WBCs.
  • Villaseñor-Altamirano, Ana B.; Watson, John D.; Prokopec, Stephenie D.; Yao, Cindy Q.; Boutros, Paul C.; Pohjanvirta, Raimo; Valdés-Flores, Jesús; Elizondo, Guillermo (2019)
    Alternative splicing is a co-transcriptional mechanism that generates protein diversity by including or excluding exons in different combinations, thereby expanding the diversity of protein isoforms of a single gene. Abnormalities in this process can result in deleterious effects to human health, and several xenobiotics are known to interfere with splicing regulation through multiple mechanisms. These changes could lead to human diseases such as cancer, neurological disorders, autoimmune diseases, and developmental disorders. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant generated as a byproduct of various industrial activities. Exposure to this dioxin has been linked to a wide range of pathologies through the alteration of multiple cellular processes. However, the effects of TCDD exposure on alternative splicing have not yet been studied. Here, we investigated whether a single po. dose of 5 μg/kg or 500 μg/kg TCDD influence hepatic alternative splicing in adult male C57BL/6Kou mouse. We identified several genes whose alternative splicing of precursor messenger RNAs was modified following TCDD exposure. In particular, we demonstrated that alternative splicing of Cyp1a1, Ahrr, and Actn1 was significantly altered after TCDD treatment. These findings show that the exposure to TCDD has an impact on alternative-splicing, and suggest a new avenue for understanding TCDD-mediated toxicity and pathogenesis.
  • Lou, Yan-Ru; Toh, Tai Chong; Tee, Yee Han; Yu, Hanry (2017)
    25-Hydroxyvitamin D-3 [25(OH)D-3] has recently been found to be an active hormone. Its biological actions are demonstrated in various cell types. 25(OH)D-3 deficiency results in failure in bone formation and skeletal deformation. Here, we investigated the effect of 25(OH)D-3 on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We also studied the effect of 1 alpha, 25-dihydroxyvitamin D-3[1 alpha,25-(OH)(2)D-3], a metabolite of 25(OH)D-3. One of the vitamin D responsive genes, 25(OH)D-3-24-hydroxylase (cytochrome P450 family 24 subfamily A member 1) mRNA expression is up-regulated by 25(OH)D-3 at 250-500 nM and by 1 alpha, 25-(OH)(2)D-3 at 1-10 nM. 25(OH)D-3 and 1 alpha, 25-(OH)(2)D-3 at a time-dependent manner alter cell morphology towards osteoblast-associated characteristics. The osteogenic markers, alkaline phosphatase, secreted phosphoprotein 1 (osteopontin), and bone gamma-carboxyglutamate protein (osteocalcin) are increased by 25(OH)D-3 and 1 alpha,25-(OH)(2)D-3 in a dose-dependent manner. Finally, mineralisation is significantly increased by 25(OH)D-3 but not by 1 alpha, 25-(OH)(2)D-3. Moreover, we found that hMSCs express very low level of 25(OH)D-3-1 alpha-hydroxylase (cytochrome P450 family 27 subfamily B member 1), and there is no detectable 1 alpha, 25-(OH)(2)D-3 product. Taken together, our findings provide evidence that 25(OH)D-3 at 250-500 nM can induce osteogenic differentiation and that 25(OH)D-3 has great potential for cell-based bone tissue engineering.
  • Beauchamp, Philippe; Jackson, Christopher B.; Ozhathil, Lijo Cherian; Agarkova, Irina; Galindo, Cristi L.; Sawyer, Douglas B.; Suter, Thomas M.; Zuppinger, Christian (2020)
    Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions.Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR).Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture.Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.
  • Tasnadi, Ervin A.; Toth, Timea; Kovacs, Maria; Diosdi, Akos; Pampaloni, Francesco; Molnar, Jozsef; Piccinini, Filippo; Horvath, Peter (2020)
    aSummary: Segmentation of single cells in microscopy images is one of the major challenges in computational biology. It is the first step of most bioimage analysis tasks, and essential to create training sets for more advanced deep learning approaches. Here, we propose 3D-Cell-Annotator to solve this task using 3D active surfaces together with shape descriptors as prior information in a semi-automated fashion. The software uses the convenient 3D interface of the widely used Medical Imaging Interaction Toolkit (MITK). Results on 3D biological structures (e.g. spheroids, organoids and embryos) show that the precision of the segmentation reaches the level of a human expert.
  • Buettner, Ralf; Le Xuan Truong Nguyen; Kumar, Bijender; Morales, Corey; Liu, Chao; Chen, Lisa S.; Pemovska, Tea; Synold, Timothy W.; Palmer, Joycelynne; Thompson, Ryan; Li, Ling; Dinh Hoa Hoang; Zhang, Bin; Ghoda, Lucy; Kowolik, Claudia; Kontro, Mika; Leitch, Calum; Wennerberg, Krister; Yu, Xiaochun; Chen, Ching-Cheng; Horne, David; Gandhi, Varsha; Pullarkat, Vinod; Marcucci, Guido; Rosen, Steven T. (2019)
    Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD (+) MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.
  • Linkens, Armand M. A.; van Best, Niels; Niessen, Petra M.; Wijckmans, Nicole E. G.; de Goei, Erica E. C.; Scheijen, Jean L. J. M.; van Dongen, Martien C. J. M.; van Gool, Christel C. J. A. W.; de Vos, Willem M.; Houben, Alfons J. H. M.; Stehouwer, Coen D. A.; Eussen, Simone J. M. P.; Penders, John; Schalkwijk, Casper G. (2022)
    Dietary advanced glycation endproducts (AGEs), abundantly present in Westernized diets, are linked to negative health outcomes, but their impact on the gut microbiota has not yet been well investigated in humans. We investigated the effects of a 4-week isocaloric and macronutrient-matched diet low or high in AGEs on the gut microbial composition of 70 abdominally obese individuals in a double-blind parallel-design randomized controlled trial (NCT03866343). Additionally, we investigated the cross-sectional associations between the habitual intake of dietary dicarbonyls, reactive precursors to AGEs, and the gut microbial composition, as assessed by 16S rRNA amplicon-based sequencing. Despite a marked percentage difference in AGE intake, we observed no differences in microbial richness and the general community structure. Only the Anaerostipes spp. had a relative abundance >0.5% and showed differential abundance (0.5 versus 1.11%; p = 0.028, after low- or high-AGE diet, respectively). While the habitual intake of dicarbonyls was not associated with microbial richness or a general community structure, the intake of 3-deoxyglucosone was especially associated with an abundance of several genera. Thus, a 4-week diet low or high in AGEs has a limited impact on the gut microbial composition of abdominally obese humans, paralleling its previously observed limited biological consequences. The effects of dietary dicarbonyls on the gut microbiota composition deserve further investigation.
  • Lutfullahoglu-Bal, Güleycan; Seferoglu, Ayse Bengisu; Keskin, Abdurrahman; Akdogan, Emel; Dunn, Cory D. (2018)
    Prokaryotes can provide new genetic information to eukaryotes by horizontal gene transfer (HGT), and such transfers are likely to have been particularly consequential in the era of eukaryogenesis. Since eukaryotes are highly compartmentalized, it is worthwhile to consider the mechanisms by which newly transferred proteins might reach diverse organellar destinations. Toward this goal, we have focused our attention upon the behavior of bacteria-derived tail anchors (TAs) expressed in the eukaryote Saccharomyces cerevisiae. In this study, we report that a predicted membrane-associated domain of the Escherichia coli YgiM protein is specifically trafficked to peroxisomes in budding yeast, can be found at a pre-peroxisomal compartment (PPC) upon disruption of peroxisomal biogenesis, and can functionally replace an endogenous, peroxisome-directed TA. Furthermore, the YgiM(TA) can localize to peroxisomes in mammalian cells. Since the YgiM(TA) plays no endogenous role in peroxisomal function or assembly, this domain is likely to serve as an excellent tool allowing further illumination of the mechanisms by which TAs can travel to peroxisomes. Moreover, our findings emphasize the ease with which bacteria-derived sequences might target to organelles in eukaryotic cells following HGT, and we discuss the importance of flexible recognition of organelle targeting information during and after eukaryogenesis.
  • Guenther, Carla; Faisal, Imrul; Uotila, Liisa; Llort Asens, Marc; Harjunpää, Heidi; Savinko, Terhi; Öhman, Tiina; Yao, Sean; Moser, Markus; Morris, Stephan W.; Tojkander, Sari; Fagerholm, Susanna (2019)
    beta2-integrins are essential for immune system function because they mediate immune cell adhesion and signaling. Consequently, a loss of beta2-integrin expression or function causes the immunodeficiency disorders, Leukocyte Adhesion Deficiency (LAD) type I and III. LAD-III is caused by mutations in an important integrin regulator, kindlin-3, but exactly how kindlin-3 regulates leukocyte adhesion has remained incompletely understood. Here we demonstrate that mutation of the kindlin-3 binding site in the b2-integrin (TTT/AAA-b2-integrin knock-in mouse/KI) abolishes activation of the actin-regulated myocardin related transcription factor A/serum response factor (MRTF-A/SRF) signaling pathway in dendritic cells and MRTF-A/SRF-dependent gene expression. We show that Ras homolog gene family, member A (RhoA) activation and filamentous-actin (F-actin) polymerization is abolished in murine TTT/AAA-b2-integrin KI dendritic cells, which leads to a failure ofMRTF-A to localize to the cell nucleus to coactivate genes together with SRF. In addition, we show that dendritic cell gene expression, adhesion and integrin-mediated traction forces on ligand coated surfaces is dependent on the MRTF-A/SRF signaling pathway. The participation of b2-integrin and kindlin-3-mediated cell adhesion in the regulation of the ubiquitous MRTF-A/SRF signaling pathway in immune cells may help explain the role of b2-integrin and kindlin-3 in integrin-mediated gene regulation and immune system function.
  • Zhou, Qi-Hang; Qin, Wei-Wei; Finel, Moshe; He, Qing-Qing; Tu, Dong-Zhu; Wang, Chao-Ran; Ge, Guang-Bo (2021)
    Strong inhibition of the human UDP-glucuronosyltransferase enzymes (UGTs) may lead to undesirable effects, including hyperbilirubinaemia and drugiherb-drug interactions. Currently, there is no good way to examine the inhibitory effects and specificities of compounds toward all the important human UGTs, side-by-side and under identical conditions. Herein, we report a new, broad-spectrum substrate for human UGTs and its uses in screening and characterizing of UGT inhibitors. Following screening a variety of phenolic compound(s), we have found that methylophiopogonanone A (MOA) can be readily O-glucuronidated by all tested human UGTs, including the typical N-glucuronidating enzymes UGT1A4 and UGT2B10. MOA-O-glucuronidation yielded a single mono-O-glucuronide that was biosynthesized and purified for structural characterization and for constructing an LC-UV based MOA-O-glucuronidation activity assay, which was then used for investigating MOA-O-glucuronidation kinetics in recombinant human UGTs. The derived K-m values were crucial for selecting the most suitable assay conditions for assessing inhibitory potentials and specificity of test compound(s). Furthermore, the inhibitory effects and specificities of four known UGT inhibitors were reinvestigated by using MOA as the substrate for all tested UGTs. Collectively, MOA is a broad-spectrum substrate for the human UGTs, which offers a new and practical tool for assessing inhibitory effects and specificities of UGT inhibitors. (C) 2021 Elsevier B.V. All rights reserved.
  • Douglass Jr., Eugene F.; Allaway, Robert J.; Szalai, Bence; Wang, Wenyu; Tian, Tingzhong; Fernández-Torras, Adrià; Realubit, Ron; Karan, Charles; Zheng, Shuyu; Pessia, Alberto; Tanoli, Ziaurrehman; Jafari, Mohieddin; Wan, Fangping; Li, Shuya; Xiong, Yuanpeng; Duran-Frigola, Miquel; Bertoni, Martino; Badia-i-Mompel, Pau; Mateo, Lídia; Guitart-Pla, Oriol; Chung, Verena; Tang, Jing; Zeng, Jianyang; Aloy, Patrick; Saez-Rodriguez, Julio; Guinney, Justin; Gerhard, Daniela S.; Califano, Andrea (2022)
    The Columbia Cancer Target Discovery and Development (CTD2) Center is developing PANACEA, a resource comprising dose-responses and RNA sequencing (RNA-seq) profiles of 25 cell lines perturbed with similar to 400 clinical oncology drugs, to study a tumor-specific drug mechanism of action. Here, this resource serves as the basis for a DREAM Challenge assessing the accuracy and sensitivity of computational algorithms for de novo drug polypharmacology predictions. Dose-response and perturbational profiles for 32 kinase inhibitors are provided to 21 teams who are blind to the identity of the compounds. The teams are asked to predict high-affinity binding targets of each compound among similar to 1,300 targets cataloged in DrugBank. The best performing methods leverage gene expression profile similarity analysis as well as deep-learning methodologies trained on individual datasets. This study lays the foundation for future integrative analyses of pharmacogenomic data, reconciliation of polypharmacology effects in different tumor contexts, and insights into network-based assessments of drug mechanisms of action.
  • Castelo-Branco, Raquel; Verhoeven, Stefan; Ridder, Lars; Huber, Florian; Acharya, Deepa D.; Aksenov, Alexander A.; Aleti, Gajender; Moghaddam, Jamshid Amiri; Aron, Allegra T.; Aziz, Saefuddin; Bauermeister, Anelize; Bauman, Katherine D.; Baunach, Martin; Beemelmanns, Christine; Beman, J. Michael; Berlanga-Clavero, Maria Victoria; Blacutt, Alex A.; Bode, Helge B.; Boullie, Anne; Brejnrod, Asker; Bugni, Tim S.; Calteau, Alexandra; Cao, Liu; Carrion, Victor J.; Castelo-Branco, Raquel; Chanana, Shaurya; Chase, Alexander B.; Chevrette, Marc G.; Costa-Lotufo, Leticia V.; Crawford, Jason M.; Currie, Cameron R.; Cuypers, Bart; Dang, Tam; de Rond, Tristan; Demko, Alyssa M.; Dittmann, Elke; Du, Chao; Drozd, Christopher; Dujardin, Jean-Claude; Dutton, Rachel J.; Edlund, Anna; Fewer, David P.; Garg, Neha; Gauglitz, Julia M.; Gentry, Emily C.; Gerwick, Lena; Glukhov, Evgenia; Gross, Harald; Gugger, Muriel; Guillen Matus, Dulce G.; Helfrich, Eric J. N.; Hempel, Benjamin-Florian; Hur, Jae-Seoun; Iorio, Marianna; Jensen, Paul R.; Kang, Kyo Bin; Kaysser, Leonard; Kelleher, Neil L.; Kim, Chung Sub; Kim, Ki Hyun; Koester, Irina; Koenig, Gabriele M.; Leao, Tiago; Lee, Seoung Rak; Lee, Yi-Yuan; Li, Xuanji; Little, Jessica C.; Maloney, Katherine N.; Maennle, Daniel; Martin H., Christian; McAvoy, Andrew C.; Metcalf, Willam W.; Mohimani, Hosein; Molina-Santiago, Carlos; Moore, Bradley S.; Mullowney, Michael W.; Muskat, Mitchell; Nothias, Louis-Felix; O'Neill, Ellis C.; Parkinson, Elizabeth I.; Petras, Daniel; Piel, Jorn; Pierce, Emily C.; Pires, Karine; Reher, Raphael; Romero, Diego; Roper, M. Caroline; Rust, Michael; Saad, Hamada; Saenz, Carmen; Sanchez, Laura M.; Sorensen, Soren Johannes; Sosio, Margherita; Sussmuth, Roderich D.; Sweeney, Douglas; Tahlan, Kapil; Thomson, Regan J.; Tobias, Nicholas J.; Trindade-Silva, Amaro E.; van Wezel, Gilles P.; Wang, Mingxun; Weldon, Kelly C.; Zhang, Fan; Ziemert, Nadine; Duncan, Katherine R.; Cruesemann, Max; Rogers, Simon; Dorrestein, Pieter C.; Medema, Marnix H.; van der Hooft, Justin J. J. (2021)
    Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.
  • Domanska, Ausra; Guryanov, Sergey; Butcher, Sarah J. (2021)
    Parechoviruses belong to the genus Parechovirus within the family Picornaviridae and are non-enveloped icosahedral viruses with a single-stranded RNA genome. Parechoviruses include human and animal pathogens classified into six species. Those that infect humans belong to the Parechovirus A species and can cause infections ranging from mild gastrointestinal or respiratory illness to severe neonatal sepsis. There are no approved antivirals available to treat parechovirus (nor any other picornavirus) infections. In this parechovirus review, we focus on the cleaved protein products resulting from the polyprotein processing after translation comparing and contrasting their known or predicted structures and functions to those of other picornaviruses. The review also includes our original analysis from sequence and structure prediction. This review highlights significant structural differences between parechoviral and other picornaviral proteins, suggesting that parechovirus drug development should specifically be directed to parechoviral targets.
  • Kjaerbolling, Inge; Vesth, Tammi; Frisvad, Jens C.; Nybo, Jane L.; Theobald, Sebastian; Kildgaard, Sara; Petersen, Thomas Isbrandt; Kuo, Alan; Sato, Atsushi; Lyhne, Ellen K.; Kogle, Martin E.; Wiebenga, Ad; Kun, Roland S.; Lubbers, Ronnie J. M.; Makela, Miia R.; Barry, Kerrie; Chovatia, Mansi; Clum, Alicia; Daum, Chris; Haridas, Sajeet; He, Guifen; LaButti, Kurt; Lipzen, Anna; Mondo, Stephen; Pangilinan, Jasmyn; Riley, Robert; Salamov, Asaf; Simmons, Blake A.; Magnuson, Jon K.; Henrissat, Bernard; Mortensen, Uffe H.; Larsen, Thomas O.; de Vries, Ronald P.; Grigoriev, Igor V.; Machida, Masayuki; Baker, Scott E.; Andersen, Mikael R. (2020)
    Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.
  • McWilliams, Thomas G.; Prescott, Alan R.; Villarejo-Zori, Beatriz; Ball, Graeme; Boya, Patricia; Ganleya, Ian G. (2019)
    Photoreception is pivotal to our experience and perception of the natural world; hence the eye is of prime importance for most vertebrate animals to sense light. Central to visual health is mitochondrial homeostasis, and the selective autophagic turnover of mitochondria (mitophagy) is predicted to play a key role here. Despite studies that link aberrant mitophagy to ocular dysfunction, little is known about the prevalence of basal mitophagy, or its relationship to general autophagy, in the visual system. In this study, we utilize the mito-QC mouse and a closely related general macroautophagy reporter model to profile basal mitophagy and macroautophagy in the adult and developing eye. We report that ocular macroautophagy is widespread, but surprisingly mitophagy does not always follow the same pattern of occurrence. We observe low levels of mitophagy in the lens and ciliary body, in stark contrast to the high levels of general MAP1LC3-dependent macroautophagy in these regions. We uncover a striking reversal of this process in the adult retina, where mitophagy accounts for a larger degree of the macroautophagy taking place, specifically in the photoreceptor neurons of the outer nuclear layer. We also show the developmental regulation of autophagy in a variety of ocular tissues. In particular, mitophagy in the adult mouse retina is reversed in localization during the latter stages of development. Our work thus defines the landscape of mitochondrial homeostasis in the mammalian eye, and in doing so highlights the selective nature of autophagy in vivo and the specificity of the reporters used.
  • 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.
  • Grubman, Alexandra; Vandekolk, Teresa H.; Schröder, Jan; Sun, Guizhi; Hatwell-Humble, Jessica; Chan, Jonathan; Oksanen, Minna; Lehtonen, Sarka; Hunt, Cameron; Koistinaho, Jan E.; Nilsson, Susan K.; Haynes, John M.; Pouton, Colin W.; Polo, Jose M. (2020)
    Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.
  • Cornetti, Luca; Fields, Peter D.; Van Damme, Kay; Ebert, Dieter (2019)
    In the post-genomic era, much of phylogenetic analyses still relies on mitochondrial DNA, either alone or in combination with few nuclear genes. Although this approach often makes it possible to construct well-supported trees, it is limited because mtDNA describes the history of a single locus, and nuclear phylogenies based on a few loci may be biased, leading to inaccurate tree topologies and biased estimations of species divergence time. In this study, we perform a phylogenomic analysis of the Daphniidae family (Crustacea: Branchiopoda: Anomopoda) including some of the most frequently studied model organisms (Daphnia magna and D. pulex) whose phylogenetic relationships have been based primarily on an assessment of a few mtDNA genes. Using high-throughput sequencing, we were able to assemble 38 whole mitochondrial genomes and draft nuclear genomes for 18 species, including at least one species for each known genus of the family Daphniidae. Here we present phylogenies based on 636 nuclear single-copy genes shared among all sampled taxa and based on whole mtDNA genomes. The phylogenies we obtained were highly supported and showed some discrepancies between nuclear and mtDNA based trees at deeper nodes. We also identified a new candidate sister lineage of Daphnia magna. Our time-calibrated genomic trees, which we constructed using both fossil records and substitution rates, yielded very different estimates of branching event times compared to those based on mtDNA. By providing multi-locus, fossil-calibrated trees of the Daphniidae, our study contributes to an improved phylogenetic framework for ecological and evolutionary studies that use water fleas as a model system.
  • Alanko, Jarno; Cunial, Fabio; Belazzougui, Djamal; Mäkinen, Veli (2017)
    Background: A metagenomic sample is a set of DNA fragments, randomly extracted from multiple cells in an environment, belonging to distinct, often unknown species. Unsupervised metagenomic clustering aims at partitioning a metagenomic sample into sets that approximate taxonomic units, without using reference genomes. Since samples are large and steadily growing, space-efficient clustering algorithms are strongly needed. Results: We design and implement a space-efficient algorithmic framework that solves a number of core primitives in unsupervised metagenomic clustering using just the bidirectional Burrows-Wheeler index and a union-find data structure on the set of reads. When run on a sample of total length n, with m reads of maximum length l each, on an alphabet of total size sigma, our algorithms take O(n(t + log sigma)) time and just 2n + o(n) + O(max{l sigma log n, K logm}) bits of space in addition to the index and to the union-find data structure, where K is a measure of the redundancy of the sample and t is the query time of the union-find data structure. Conclusions: Our experimental results show that our algorithms are practical, they can exploit multiple cores by a parallel traversal of the suffix-link tree, and they are competitive both in space and in time with the state of the art.