SCIENCE TRANSLATIONAL MEDICINE 10:(429) Paper eaal3973. (2018)
High-throughput metabolomic analysis predicts mode of action of uncharacterized antimicrobial compounds.
Zampieri M, Szappanos B, Buchieri MV, Trauner A, Piazza I, Picotti P, Gagneux S, Borrell S, Gicquel B, Lelievre J, Papp B, Sauer U
Rapidly spreading antibiotic resistance and the low discovery rate of new antimicrobial compounds demand more effective strategies for early drug discovery. One bottleneck in the drug discovery pipeline is the identification of the modes of action (MoAs) of new compounds. We have developed a rapid systematic metabolome profiling strategy to classify the MoAs of bioactive compounds. The method predicted MoA-specific metabolic responses in the nonpathogenic bacterium Mycobacterium smegmatis after treatment with 62 reference compounds with known MoAs and different metabolic and nonmetabolic targets. We then analyzed a library of 212 new antimycobacterial compounds with unknown MoAs from a drug discovery effort by the pharmaceutical company GlaxoSmithKline (GSK). More than 70% of these new compounds induced metabolic responses in M. smegmatis indicative of known MoAs, seven of which were experimentally validated. Only 8% (16) of the compounds appeared to target unconventional cellular processes, illustrating the difficulty in discovering new antibiotics with different MoAs among compounds used as monotherapies. For six of the GSK compounds with potentially new MoAs, the metabolome profiles suggested their ability to interfere with trehalose and lipid metabolism. This was supported by whole-genome sequencing of spontaneous drug-resistant mutants of the pathogen Mycobacterium tuberculosis and in vitro compound-proteome interaction analysis for one of these compounds. Our compendium of drug-metabolome profiles can be used to rapidly query the MoAs of uncharacterized antimicrobial compounds and should be a useful resource for the drug discovery community.
ELIFE 7: Paper e29845. 23 p. (2018)
Hsp70-associated chaperones have a critical role in buffering protein production costs.
Farkas Z, Kalapis D, Bodi Z, Szamecz B, Daraba A, Almasi K, Kovacs K, Boross G, Pal F, Horvath P, Balassa T, Molnar C, Pettko-Szandtner A, Klement E, Rutkai E, Szvetnik A, Papp B, Pal C
Proteins are necessary for cellular growth. Concurrently, however, protein production has high energetic demands associated with transcription and translation. Here, we propose that activity of molecular chaperones shape protein burden, that is the fitness costs associated with expression of unneeded proteins. To test this hypothesis, we performed a genome-wide genetic interaction screen in baker's yeast. Impairment of transcription, translation, and protein folding rendered cells hypersensitive to protein burden. Specifically, deletion of specific regulators of the Hsp70-associated chaperone network increased protein burden. In agreement with expectation, temperature stress, increased mistranslation and a chemical misfolding agent all substantially enhanced protein burden. Finally, unneeded protein perturbed interactions between key components of the Hsp70-Hsp90 network involved in folding of native proteins. We conclude that specific chaperones contribute to protein burden. Our work indicates that by minimizing the damaging impact of gratuitous protein overproduction, chaperones enable tolerance to massive changes in genomic expression.
NATURE COMMUNICATIONS 9:(1) p. 226. (2018)
Intelligent image-based in situ single-cell isolation.
Brasko C, Smith K, Molnar C, Farago N, Hegedus L, Balind A, Balassa T, Szkalisity A, Sukosd F, Kocsis K, Balint B, Paavolainen L, Enyedi MZ,Nagy I, Puskas LG, Haracska L, Tamas G, Horvath P
Quantifying heterogeneities within cell populations is important for many fields including cancer research and neurobiology; however, techniques to isolate individual cells are limited. Here, we describe a high-throughput, non-disruptive, and cost-effective isolation method that is capable of capturing individually targeted cells using widely available techniques. Using high-resolution microscopy, laser microcapture microscopy, image analysis, and machine learning, our technology enables scalable molecular genetic analysis of single cells, targetable by morphology or location within the sample.
REDOX BIOLOGY 14: pp. 439-449. (2018)
Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells
Nazıroğlu M, Blum W, Jósvay K, Çiğ B, Henzi T, Oláh Z, Vizler C, Schwaller B, Pecze L
Menthol is a naturally occurring monoterpene alcohol possessing remarkable biological properties including antipruritic, analgesic, antiseptic, anti-inflammatory and cooling effects. Here, we examined the menthol-evoked Ca2+ signals in breast and prostate cancer cell lines. The effect of menthol (50–500 µM) was predicted to be mediated by the transient receptor potential ion channel melastatin subtype 8 (TRPM8). However, the intensity of menthol-evoked Ca2+ signals did not correlate with the expression levels of TRPM8 in breast and prostate cancer cells indicating a TRPM8-independent signaling pathway. Menthol-evoked Ca2+ signals were analyzed in detail in Du 145 prostate cancer cells, as well as in CRISPR/Cas9 TRPM8-knockout Du 145 cells. Menthol (500 µM) induced Ca2+ oscillations in both cell lines, thus independent of TRPM8, which were however dependent on the production of inositol trisphosphate. Results based on pharmacological tools point to an involvement of the purinergic pathway in menthol-evoked Ca2+ responses. Finally, menthol (50–500 µM) decreased cell viability and induced oxidative stress independently of the presence of TRPM8 channels, despite that temperature-evoked TRPM8-mediated inward currents were significantly decreased in TRPM8-knockout Du 145 cells compared to wild type Du 145 cells. © 2017 The Authors
MOLECULAR & CELLULAR PROTEOMICS 17:(1) pp. 2-17. (2018)
Analysis of Mammalian O-Glycopeptides-We Have Made a Good Start, but There is a Long Way to Go.
Darula Z, Medzihradszky KF
Glycosylation is perhaps the most common post-translational modification. Recently there has been growing interest in cataloging the glycan structures, glycoproteins, and specific sites modified and deciphering the biological functions of glycosylation. Although the results are piling up for N-glycosylation, O-glycosylation is seriously trailing behind. In our review we reiterate the difficulties researchers have to overcome in order to characterize O-glycosylation. We describe how an ingenious cell engineering method delivered exciting results, and what could we gain from "wild-type" samples. Although we refer to the biological role(s) of O-glycosylation, we do not provide a complete inventory on this topic.
JOURNAL OF CELL BIOLOGY 217:(1) pp. 361-374. (2018)
Molecular mechanisms of developmentally programmed crinophagy in Drosophila.
Csizmadia T, Lorincz P, Hegedus K, Szeplaki S, Low P, Juhasz G
At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. We find that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.
ANTIOXIDANTS & REDOX SIGNALING Paper ahead of print. 53 p. (2017)
Concentration does matter: The beneficial and potentially harmful effects of ascorbate in humans and plants
Tóth SZ, Lőrincz T, Szarka A
Significance: Ascorbate (Asc) is an essential compound both in animals and plants, mostly due to its reducing properties, thereby playing a role in scavenging reactive oxygen species (ROS) and acting as a cofactor in various enzymatic reactions. Recent Advances: Growing number of evidence shows that excessive Asc accumulation may have negative effects on cellular functions both in humans and plants; inter alia it may negatively affect signaling mechanisms, cellular redox status, and contribute to the production of ROS via the Fenton reaction. Critical Issues: Both plants and humans tightly control cellular Asc levels, possibly via biosynthesis, transport, and degradation, to maintain them in an optimum concentration range, which, among other factors, is essential to minimize the potentially harmful effects of Asc. On the contrary, the Fenton reaction induced by a high-dose Asc treatment in humans enables a potential cancer-selective cell death pathway. Future Directions: The elucidation of Asc induced cancer selective cell death mechanisms may give us a tool to apply Asc in cancer therapy. On the contrary, the regulatory mechanisms controlling cellular Asc levels are also to be considered, for example, when aiming at generating crops with elevated Asc levels. Antioxid. Redox Signal. 00, 000-000.
PLANT CELL AND ENVIRONMENT 40:(11) pp. 2457-2468. (2017)
New insights of red light-induced development
Viczian A, Klose C, Adam E, Nagy F
The red/far-red light absorbing photoreceptors phytochromes regulate development and growth and thus play an essential role in optimizing adaptation of the sessile plants to the ever-changing environment. Our understanding of how absorption of a red/far-red photon by phytochromes initiates/modifies diverse physiological responses has been steadily improving. Research performed in the last 5 years has been especially productive and led to significant conceptual changes about the mode of action of these photoreceptors. In this review, we focus on the phytochrome B photoreceptor, the major phytochrome species active in light-grown plants. We discuss how its light-independent inactivation (termed dark/thermal reversion), post-translational modification, including ubiquitination, phosphorylation and sumoylation, as well as heterodimerization with other phytochrome species modify red light-controlled physiological responses. Finally, we discuss how photobiological properties of phytochrome B enable this photoreceptor to function also as a thermosensor.
NATURE CELL BIOLOGY 19:(11) pp. 1326-1335. (2017)
Synthetic hydrogels for human intestinal organoid generation and colonic wound repair
Cruz-Acuna R, Quiros M, Farkas AE, Dedhia PH, Huang S, Siuda D, Garcia-Hernandez V, Miller AJ, Spence JR, Nusrat A, Garcia AJ
In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs, such that three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.
ACS PHOTONICS 4:(9) pp. 2171-2178. (2017)
Large band-edge Photocurrent Peak in thick methylammonium lead iodide photosensors with symmetric metal electrodes
Hantz P, Sarlós F, Jalsovszky I, Plesz B
This study examines the photocurrent (photoconductivity) spectra of thick methylammonium lead iodide perovskite layers coupled to symmetric metal electrodes. A large photocurrent peak has been observed in the near-band-gap wavelength region. The origin of this current peak is related to an interplay between the penetration depths of light having a certain wavelength and its ability to raise the concentration of the charge carriers. A model including the generation, position-specific or uniform recombination, as well as diffusion of charge carrier pairs, made it possible to reproduce the experimental findings. Besides the microscopic approach, we also present a phenomenological model and a simulation providing the conductance for an arbitrary relative orientation of the illuminating beam and the field imposed by the electrodes. The results enabled us to outline the functioning principle of a sensor for determining the angle of incidence of electromagnetic radiation. Furthermore, a method for estimating optical absorption spectra from photocurrent spectra is delineated.
GENOME BIOLOGY 18: Paper 190. 17 p. (2017)
Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.
Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor E, Welker E
BACKGROUND: The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects. RESULTS: These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s. CONCLUSIONS: No single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high-fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple means for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.
FRONTIERS IN IMMUNOLOGY 8: Paper 1369. 9 p. (2017)
Interaction of THP-1 monocytes with conidia and hyphae of different Curvularia strains
Tóth EJ, Boros É, Hoffmann A, Szebenyi C, Homa M, Nagy G, Vágvölgyi C, Nagy I, Papp T
Interaction of the human monocytic cell line, THP-1 with clinical isolates of three Curvularia species were examined. Members of this filamentous fungal genus can cause deep mycoses emerging in both immunocompromised and immunocompetent patients. It was found that monocytes reacted only to the hyphal form of Curvularia lunata. Cells attached to the germ tubes and hyphae and production of elevated levels of interleukin (IL)-8 and IL-10 and a low level of TNF-alpha were measured. At the same time, monocytes failed to produce IL-6. This monocytic response, especially with the induction of the anti-inflammatory IL-10, correlates well to the observation that C. lunata frequently cause chronic infections even in immunocompetent persons. Despite the attachment to the hyphae, monocytes could not reduce the viability of the fungus and the significant decrease in the relative transcript level of HLA-DRA assumes the lack of antigen presentation of the fungus by this cell type. C. spicifera and C. hawaiiensis failed to induce the gathering of the cells or the production of any analyzed cytokines. Monocytes did not recognize conidia of Curvularia species, even when melanin was lacking in their cell wall.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114:(35) pp. 9481-9486. (2017)
Changes in aggregation states of light-harvesting complexes as a mechanism for modulating energy transfer in desert crust cyanobacteria
Bar Eyal L, Ranjbar Choubeh R, Cohen E, Eisenberg I, Tamburu C, Dorogi M, Unnep R, Appavou MS, Nevo R, Raviv U, Reich Z, Garab G, van Amerongen H, Paltiel Y, Keren N
In this paper we propose an energy dissipation mechanism that is completely reliant on changes in the aggregation state of the phycobilisome light-harvesting antenna components. All photosynthetic organisms regulate the efficiency of excitation energy transfer (EET) to fit light energy supply to biochemical demands. Not many do this to the extent required of desert crust cyanobacteria. Following predawn dew deposition, they harvest light energy with maximum efficiency until desiccating in the early morning hours. In the desiccated state, absorbed energy is completely quenched. Time and spectrally resolved fluorescence emission measurements of the desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced EET between phycobilisome components, (ii) shorter fluorescence lifetimes, and (iii) red shift in the emission spectra, compared with the hydrated state. These changes coincide with a loss of the ordered phycobilisome structure, evident from small-angle neutron and X-ray scattering and cryo-transmission electron microscopy data. Based on these observations we propose a model where in the hydrated state the organized rod structure of the phycobilisome supports directional EET to reaction centers with minimal losses due to thermal dissipation. In the desiccated state this structure is lost, giving way to more random aggregates. The resulting EET path will exhibit increased coupling to the environment and enhanced quenching.
PLANT PHYSIOLOGY 175:(1) pp. 555-567. (2017)
Proline accumulation is regulated by transcription factors associated with phosphate starvation
Aleksza D, Horvath GV, Sandor G, Szabados L
Proline accumulation in plants is a well-documented physiological response to osmotic stress caused by drought or salinity. In Arabidopsis thaliana the stress and ABA-induced Delta1-PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 (P5CS1) gene was previously shown to control proline biosynthesis in such adverse conditions. To identify regulatory factors which control the transcription of P5CS1, yeast one hybrid (Y1H) screens were performed with a genomic fragment of P5CS1, containing 1.2 kB promoter and 0.8 kB transcribed regions. The transcription factors PHOSPHATE STARVATION RESPONSE 1 (PHR1) and PHR1-LIKE 1 (PHL1) were identified to bind to P5CS1 sequences in the first intron, which carry a conserved PHR1-binding site (P1BS) motif. PHR1 and PHL1 binding to P1BS was confirmed by Y1H, electrophoretic mobility assay (EMSA) and chromatin immune precipitation (ChIP). Phosphate starvation led to gradual increase in proline content in wild type Arabidopsis plants as well as transcriptional activation of P5CS1 and PROLINE DEHYDROGENASE 2 (PDH2) genes. Induction of P5CS1 transcription and proline accumulation during phosphate deficiency was considerably reduced by phr1 and phl1 mutations and was impaired in the ABA deficient aba2-3 and ABA insensitive abi4-1 mutants. Growth and viability of phr1phl1 double mutant was reduced in phosphate-depleted medium, it was slightly diminished in the aba2-3 mutant, suggesting that ABA mediates growth retardation in such stress. Our results reveal a previously unknown link between proline metabolism and phosphate nutrition, and show that proline biosynthesis is target of crosstalk between ABA signaling and regulation of phosphate homeostasis through PHR1 and PHL1-mediated transcriptional activation of the P5CS1 gene.
PLOS GENETICS 13:(8) Paper e1006968. 35 p. (2017)
Kek-6: A truncated-Trk-like receptor for Drosophila neurotrophin 2 regulates structural synaptic plasticity
Ulian-Benitez S, Bishop S, Foldi I, Wentzell J, Okenwa C, Forero MG, Zhu BF, Moreira M, Phizacklea M, McIlroy G, Li GY, Gay NJ, Hidalgo A
Neurotrophism, structural plasticity, learning and long-term memory in mammals critically depend on neurotrophins binding Trk receptors to activate tyrosine kinase (TyrK) signaling, but Drosophila lacks full-length Trks, raising the question of how these processes occur in the fly. Paradoxically, truncated Trk isoforms lacking the TyrK predominate in the adult human brain, but whether they have neuronal functions independently of full-length Trks is unknown. Drosophila has TyrK-less Trk-family receptors, encoded by the kekkon (kek) genes, suggesting that evolutionarily conserved functions for this receptor class may exist. Here, we asked whether Keks function together with Drosophila neurotrophins (DNTs) at the larval glutamatergic neuromuscular junction (NMJ). We tested the eleven LRR and Igcontaining (LIG) proteins encoded in the Drosophila genome for expression in the central nervous system (CNS) and potential interaction with DNTs. Kek-6 is expressed in the CNS, interacts genetically with DNTs and can bind DNT2 in signaling assays and co-immunoprecipitations. Ligand binding is promiscuous, as Kek-6 can also bind DNT1, and Kek-2 and Kek-5 can also bind DNT2. In vivo, Kek-6 is found presynaptically in motoneurons, and DNT2 is produced by the muscle to function as a retrograde factor at the NMJ. Kek-6 and DNT2 regulate NMJ growth and synaptic structure. Evidence indicates that Kek-6 does not antagonise the alternative DNT2 receptor Toll-6. Instead, Kek-6 and Toll-6 interact physically, and together regulate structural synaptic plasticity and homeostasis. Using pull-down assays, we identified and validated CaMKII and VAP33A as intracellular partners of Kek-6, and show that they regulate NMJ growth and active zone formation downstream of DNT2 and Kek-6. The synaptic functions of Kek-6 could be evolutionarily conserved. This raises the intriguing possibility that a novel mechanism of structural synaptic plasticity involvingtruncated Trk-family receptors independently of TyrK signaling may also operate in the human brain.
ELIFE 6: Paper e29942. 3 p. (2017)
POST-TRANSLATIONAL MODIFICATIONS Reversing ADP-ribosylation
Moeller GK, Timinszky G
The modification of serines by molecules of ADP-ribose plays an important role in signaling that the DNA in a cell has been damaged and needs to be repaired.
EMBO JOURNAL 36:(13) pp. 1811-1836. (2017)
Molecular definitions of autophagy and related processes.
Galluzzi L, Baehrecke EH, Juhasz G, Kroemer G
Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.
JOURNAL OF CELL BIOLOGY 216:(7) pp. 1937-1947. (2017)
Rab2 promotes autophagic and endocytic lysosomal degradation.
Lorincz P, Toth S, Benko P, Lakatos Z, Boda A, Glatz G, Zobel M, Bisi S, Hegedus K, Takats S, Scita G, Juhasz G
Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes.
CELL SYSTEMS 4:(6) pp. 651-655. (2017)
Advanced Cell Classifier: User-Friendly Machine-Learning-Based Software for Discovering Phenotypes in High-Content Imaging Data.
Piccinini F, Balassa T, Szkalisity A, Molnar C, Paavolainen L, Kujala K, Buzas K, Sarazova M, Pietiainen V, Kutay U, Smith K, Horvath P
High-content, imaging-based screens now routinely generate data on a scale that precludes manual verification and interrogation. Software applying machine learning has become an essential tool to automate analysis, but these methods require annotated examples to learn from. Efficiently exploring large datasets to find relevant examples remains a challenging bottleneck. Here, we present Advanced Cell Classifier (ACC), a graphical software package for phenotypic analysis that addresses these difficulties. ACC applies machine-learning and image-analysis methods to high-content data generated by large-scale, cell-based experiments. It features methods to mine microscopic image data, discover new phenotypes, and improve recognition performance. We demonstrate that these features substantially expedite the training process, successfully uncover rare phenotypes, and improve the accuracy of the analysis. ACC is extensively documented, designed to be user-friendly for researchers without machine-learning expertise, and distributed as a free open-source tool at www.cellclassifier.org.
INTERNATIONAL JOURNAL OF CARDIOLOGY 241: pp. 364-372. (2017)
A Novel ‘Splice Site’ HCN4 Gene mutation, c.1737+1 G>T, Causes Familial Bradycardia, Reduced Heart Rate Response, Impaired Chronotropic Competence and Increased Short-Term Heart Rate Variability
Lidia Hategan, Beáta Csányi, Balázs Ördög, Kornél Kákonyi, Annamária Tringer, Orsolya Kiss, Andrea Orosz, László Sághy, István Nagy, Zoltán Hegedűs, László Rudas, Márta Széll, András Varró, Tamás Forster, Róbert Sepp
Background: The most important molecular determinant of heart rate regulation in sino-atrial pacemaker cells includes hyperpolarization-activated, cyclic nucleotide-gated ion channels, the major isoform of which is encoded by the HCN4 gene. Mutations affecting the HCN4 gene are associated primarily with sick sinus syndrome.
Methods and results: A novel c. 1737+ 1 G>T 'splice-site' HCN4 mutation was identified in a large family with familial bradycardia which co-segregated with the disease providing a two-point LOD score of 4.87. Twelve out of the 22 investigated familymembers [4 males, 8 females average age 36 (SD 6) years] were considered as clinically affected (heart rate < 60/min on resting ECG). Minimum[36 (SD 7) vs. 47 (SD 5) bpm, p = 0.0087) and average heart rates [62 (SD 8) vs. 73 (SD 8) bpm, p = 0.0168) were significantly lower in carriers on 24-hour Holter recordings. Under maximum exercise test carriers achieved significantly lower heart rates than non-carrier family members, and percent heart rate reserve and percent corrected heart rate reserve were significantly lower in carriers. Applying rigorous criteria for chronotropic incompetence a higher number of carriers exhibited chronotropic incompetence. Parameters, characterizing short-term variability of heart rate (i.e. rMSSD and pNN50%) were increased in carrier family members, even after normalization for heart rate, in the 24-hour ECG recordings with the same relative increase in 5-minute recordings.
Conclusions: The identified novel 'splice-site' HCN4 gene mutation, c. 1737+ 1 G>T, causes familial bradycardia and leads to reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability in the mutation carriers. (C) 2017 Elsevier Ireland Ltd. All rights reserved.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114:(26) pp. 6854-6859. (2017)
Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula.
Wang Q, Yang S, Liu J, Terecskei K, Abraham E, Gombar A, Domonkos A, Szucs A, Kormoczi P, Wang T, Fodor L, Mao L, Fei Z, Kondorosi E, Kalo P, Kereszt A, Zhu H
Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.
PLANT CELL AND ENVIRONMENT 40:(7) pp. 1104-1114. (2017)
Expression of the UVR8 photoreceptor in different tissues reveals tissue-autonomous features of UV-B signalling
Bernula P, Crocco CD, Arongaus AB, Ulm R, Nagy F, Viczian A
The Arabidopsis UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) orchestrates the expression of hundreds of genes, many of which can be associated with UV-B tolerance. UV-B does not efficiently penetrate into tissues, yet UV-B regulates complex growth and developmental responses. To unravel to what extent and how UVR8 located in different tissues contributes to UV-B-induced responses, we expressed UVR8 fused to the YELLOW FLUORESCENT PROTEIN (YFP) under the control of tissue-specific promoters in a uvr8 null mutant background. We show that (1) UVR8 localized in the epidermis plays a major role in regulating cotyledon expansion, and (2) expression of UVR8 in the mesophyll is important to protect adult plants from the damaging effects of UV-B. We found that UV-B induces transcription of selected genes, including the key transcriptional regulator ELONGATED HYPOCOTYL 5 (HY5), only in tissues that express UVR8. Thus, we suggest that tissue-autonomous and simultaneous UVR8 signalling in different tissues mediates, at least partly, developmental and defence responses to UV-B.
PLOS BIOLOGY 15:(5) Paper e2000644. 26 p. (2017)
Phenotypic heterogeneity promotes adaptive evolution
Bodi Z, Farkas Z, Nevozhay D, Kalapis D, Lazar V, Csorgo B, Nyerges A, Szamecz B, Fekete G, Papp B, Araujo H, Oliveira JL, Moura G, Santos MAS, Szekely T, Balazsi G, Pal C
Genetically identical cells frequently display substantial heterogeneity in gene expression, cellular morphology and physiology. It has been suggested that by rapidly generating a subpopulation with novel phenotypic traits, phenotypic heterogeneity (or plasticity) accelerates the rate of adaptive evolution in populations facing extreme environmental challenges. This issue is important as cell-to-cell phenotypic heterogeneity may initiate key steps in microbial evolution of drug resistance and cancer progression. Here, we study how stochastic transitions between cellular states influence evolutionary adaptation to a stressful environment in yeast Saccharomyces cerevisiae. We developed inducible synthetic gene circuits that generate varying degrees of expression stochasticity of an antifungal resistance gene. We initiated laboratory evolutionary experiments with genotypes carrying different versions of the genetic circuit by exposing the corresponding populations to gradually increasing antifungal stress. Phenotypic heterogeneity altered the evolutionary dynamics by transforming the adaptive landscape that relates genotype to fitness. Specifically, it enhanced the adaptive value of beneficial mutations through synergism between cell-to-cell variability and genetic variation. Our work demonstrates that phenotypic heterogeneity is an evolving trait when populations face a chronic selection pressure. It shapes evolutionary trajectories at the genomic level and facilitates evolutionary rescue from a deteriorating environmental stress.
BIOTECHNOLOGY FOR BIOFUELS 10: Paper 116. 5 p. (2017)
On the pathways feeding the H-2 production process in nutrient-replete, hypoxic conditions. Commentary on the article "Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures", by Jurado-Oller et al., Biotechnology for Biofuels, published September 7, 2015; 8: 149
Scoma A, Toth SZ
Background: Under low O-2 concentration ( hypoxia) and low light, Chlamydomonas cells can produce H-2 gas in nutrient-replete conditions. This process is hindered by the presence of O-2, which inactivates the [FeFe]-hydrogenase enzyme responsible for H-2 gas production shifting algal cultures back to normal growth. The main pathways accounting for H-2 production in hypoxia are not entirely understood, as much as culture conditions setting the optimal redox state in the chloroplast supporting long-lasting H-2 production. The reducing power for H-2 production can be provided by photosystem II (PSII) and photofermentative processes during which proteins are degraded via yet unknown pathways. In hetero- or mixotrophic conditions, acetate respiration was proposed to indirectly contribute to H-2 evolution, although this pathway has not been described in detail.
Main body: Recently, Jurado-Oller et al. (Biotechnol Biofuels 8: 149, 7) proposed that acetate respiration may substantially support H-2 production in nutrient-replete hypoxic conditions. Addition of low amounts of O-2 enhanced acetate respiration rate, particularly in the light, resulting in improved H-2 production. The authors surmised that acetate oxidation through the glyoxylate pathway generates intermediates such as succinate and malate, which would be in turn oxidized in the chloroplast generating FADH(2) and NADH. The latter would enter a PSII-independent pathway at the level of the plastoquinone pool, consistent with the light dependence of H-2 production. The authors concluded that the water-splitting activity of PSII has a minor role in H-2 evolution in nutrient-replete, mixotrophic cultures under hypoxia. However, their results with the PSII inhibitor DCMU also reveal that O-2 or acetate additions promoted acetate respiration over the usually dominant PSII-dependent pathway. The more oxidized state experienced by these cultures in combination with the relatively short experimental time prevented acclimation to hypoxia, thus precluding the PSII-dependent pathway from contributing to H-2 production.
Conclusions: In Chlamydomonas, continuous H-2 gas evolution is expected once low O-2 partial pressure and optimal reducing conditions are set. Under nutrient-replete conditions, the electrogenic processes involved in H-2 photoproduction may rely on various electron transport pathways. Understanding how physiological conditions select for specific metabolic routes is key to achieve economic viability of this renewable energy source.
PLANT PHYSIOLOGY 173:(3) pp. 1750-1762. (2017)
ABA Suppresses Root Hair Growth via the OBP4 Transcriptional Regulator
Rymen B, Kawamura A, Schafer S, Breuer C, Iwase A, Shibata M, Ikeda M, Mitsuda N, Koncz C, Ohme-Takagi M, Matsui M, Sugimoto K
Plants modify organ growth and tune morphogenesis in response to various endogenous and environmental cues. At the cellular level, organ growth is often adjusted by alterations in cell growth, but the molecular mechanisms underlying this control remain poorly understood. In this study, we identify the DNA BINDING WITH ONE FINGER (DOF)-type transcription regulator OBF BINDING PROTEIN4 (OBP4) as a repressor of cell growth. Ectopic expression of OBP4 in Arabidopsis (Arabidopsis thaliana) inhibits cell growth, resulting in severe dwarfism and the repression of genes involved in the regulation of water transport, root hair development, and stress responses. Among the basic helix-loop-helix transcription factors known to control root hair growth, OBP4 binds the ROOT HAIR DEFECTIVE6-LIKE2 (RSL2) promoter to repress its expression. The accumulation of OBP4 proteins is detected in expanding root epidermal cells, and its expression level is increased by the application of abscisic acid (ABA) at concentrations sufficient to inhibit root hair growth. ABA-dependent induction of OBP4 is associated with the reduced expression of RSL2. Furthermore, ectopic expression of OBP4 or loss of RSL2 function results in ABA-insensitive root hair growth. Taken together, our results suggest that OBP4-mediated transcriptional repression of RSL2 contributes to the ABA-dependent inhibition of root hair growth in Arabidopsis.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114:(19) pp. 5041-5046. (2017)
Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides
Montiel J, Downie JA, Farkas A, Bihari P, Herczeg R, Balint B, Mergaert P, Kereszt A, Kondorosi E
In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongatedbranched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.
JOURNAL OF CELL BIOLOGY 216:(5) pp. 1421-1438. (2017)
Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila
Foldi I, Anthoney N, Harrison N, Gangloff M, Verstak B, Nallasivan MP, AlAhmed S, Zhu BF, Phizacklea M, Losada-Perez M, Moreira M, Gay NJ, Hidalgo A
Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs) bind Toll receptors instead to promote neuronal survival, but whether they can also regulate cell death is unknown. In this study, we show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-kappa B and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts.
EMBO JOURNAL 36:(9) pp. 1261-1278. (2017)
Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control
Horvath BM, Kourova H, Nagy S, Nemeth E, Magyar Z, Papdi C, Ahmad Z, Sanchez-Perez GF, Perilli S, Blilou I, Pettkó-Szandtner A, Darula Z, Meszaros T, Binarova P, Bogre L, Scheres B
The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta. Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.
NATURE COMMUNICATIONS 8: Paper 15127. 12 p. (2017)
De novo evolved interference competition promotes the spread of biofilm defectors
Martin M, Dragos A, Holscher T, Maroti G, Balint B, Westermann M, Kovacs AT
Biofilms are social entities where bacteria live in tightly packed agglomerations, surrounded by self-secreted exopolymers. Since production of exopolymers is costly and potentially exploitable by non-producers, mechanisms that prevent invasion of non-producing mutants are hypothesized. Here we study long-term dynamics and evolution in Bacillus subtilis biofilm populations consisting of wild-type (WT) matrix producers and mutant non-producers. We show that non-producers initially fail to incorporate into biofilms formed by the WT cells, resulting in 100-fold lower final frequency compared to the WT. However, this is modulated in a long-term scenario, as non-producers evolve the ability to better incorporate into biofilms, thereby slightly decreasing the productivity of the whole population. Detailed molecular analysis reveals that the unexpected shift in the initially stable biofilm is coupled with newly evolved phage-mediated interference competition. Our work therefore demonstrates how collective behaviour can be disrupted as a result of rapid adaptation through mobile genetic elements.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114:(17) pp. 4543-4548. (2017)
Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression
Nagymihaly M, Veluchamy A, Gyorgypal Z, Ariel F, Jegu T, Benhamed M, Szucs A, Kereszt A, Mergaert P, Kondorosi E
The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells.
NEW PHYTOLOGIST 214:(2) pp. 668-681. (2017)
Expression of the eRF1 translation termination factor is controlled by an autoregulatory circuit involving readthrough and nonsense-mediated decay in plants
Nyiko T, Auber A, Szabadkai L, Benkovics A, Auth M, Merai Z, Kerenyi Z, Dinnyes A, Nagy F, Silhavy D
When a ribosome reaches a stop codon, the eukaryotic Release Factor 1 (eRF1) binds to the A site of the ribosome and terminates translation. In yeasts and plants, both over- and underexpression of eRF1 lead to altered phenotype indicating that eRF1 expression should be strictly controlled. However, regulation of eRF1 level is still poorly understood. Here we show that expression of plant eRF1 is controlled by a complex negative autoregulatory circuit, which is based on the unique features of the 3'untranslated region (3'UTR) of the eRF1-1 transcript. The stop codon of the eRF1-1 mRNA is in a translational readthrough promoting context, while its 3'UTR induces nonsense-mediated decay (NMD), a translation termination coupled mRNA degradation mechanism. We demonstrate that readthrough partially protects the eRF1-1 mRNA from its 3'UTR induced NMD, and that elevated eRF1 levels inhibit readthrough and stimulate NMD. Thus, high eRF1 level leads to reduced eRF1-1 expression, as weakened readthrough fails to protect the eRF1-1 mRNA from the more intense NMD. This eRF1 autoregulatory circuit might serve to finely balance general translation termination efficiency.
NEW PHYTOLOGIST 214:(2) pp. 668-681. (2017)
Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase
Vidal-Meireles A, Neupert J, Zsigmond L, Rosado-Souza L, Kovacs L, Nagy V, Galambos A, Fernie AR, Bock R, Toth SZ
Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and 1 O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.
MOLECULAR BIOLOGY AND EVOLUTION 34:(2) pp. 380-390. (2017)
No Evidence That Protein Noise-Induced Epigenetic Epistasis Constrains Gene Expression Evolution
Boross G, Papp B
Changes in gene expression can affect phenotypes and therefore both its level and stochastic variability are frequently under selection. It has recently been proposed that epistatic interactions influence gene expression evolution: gene pairs where simultaneous knockout is more deleterious than expected should evolve reduced expression noise to avoid concurrent low expression of both proteins. In apparent support, yeast genes with many epistatic partners have low expression variation both among isogenic individuals and between species. However, the specific predictions and basic assumptions of this verbal model remain untested. Using bioinformatics analysis, we first demonstrate that the model's predictions are unsupported by available large-scale data. Based on quantitative biochemical modeling, we then show that epistasis between expression reductions (epigenetic epistasis) is not expected to aggravate the fitness cost of stochastic expression, which is in sharp contrast to the verbal argument. This nonintuitive result can be readily explained by the typical diminishing return of fitness on gene activity and by the fact that expression noise not only decreases but also increases the abundance of proteins. Overall, we conclude that stochastic variation in epistatic partners is unlikely to drive noise minimization or constrain gene expression divergence on a genomic scale.
PLANT CELL AND ENVIRONMENT 40:(3) pp. 378-389. (2017)
Dissecting the Photoprotective Mechanism Encoded by the flv4-2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization
Bersanini L, Allahverdiyeva Y, Battchikova N, Heinz S, Lespinasse M, Ruohisto E, Mustila H, Nickelsen J, Vass I, Aro EM
In Synechocystis sp. PCC 6803, the flv4-2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4-2 operon mutants. In the sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with sll0218-flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the sll0218 and sll0218-flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218-lacking mutants. The YFP-tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA-defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the flv4-2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting. This work clarifies and dissects the roles of the flv4-2 operon-encoded proteins, Flv2/Flv4 heterodimer and the elusive Sll0218, in photoprotection of the photosynthetic apparatus in Synechosystis. While Flv2/Flv4 heterodimer is involved in an alternative electron transfer route, the Sll0218 protein is localized to specific cell compartments where photosynthetic complexes are assembled, and it is involved in the stabilization of Photosystem II complexes.
NUCLEIC ACIDS RESEARCH 45:(1) Paper gkw1315. 17 p. (2017)
DNA-dependent protease activity of human Spartan facilitates replication of DNA-protein crosslink-containing DNA.
Morocz M, Zsigmond E, Toth R, Enyedi MZ, Pinter L, Haracska L
Mutations in SPARTAN are associated with early onset hepatocellular carcinoma and progeroid features. A regulatory function of Spartan has been implicated in DNA damage tolerance pathways such as translesion synthesis, but the exact function of the protein remained unclear. Here, we reveal the role of human Spartan in facilitating replication of DNA-protein crosslink-containing DNA. We found that purified Spartan has a DNA-dependent protease activity degrading certain proteins bound to DNA. In concert, Spartan is required for direct DPC removal in vivo; we also show that the protease Spartan facilitates repair of formaldehyde-induced DNA-protein crosslinks in later phases of replication using the bromodeoxyuridin (BrdU) comet assay. Moreover, DNA fibre assay indicates that formaldehyde-induced replication stress dramatically decreases the speed of replication fork movement in Spartan-deficient cells, which accumulate in the G2/M cell cycle phase. Finally, epistasis analysis mapped these Spartan functions to the RAD6-RAD18 DNA damage tolerance pathway. Our results reveal that Spartan facilitates replication of DNA-protein crosslink-containing DNA enzymatically, as a protease, which may explain its role in preventing carcinogenesis and aging.
BLOOD 129:(11) pp. E26-E37. (2017)
Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia
Frismantas V, Dobay MP, Horvath P, Bourquin JP
Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.
NATURE 541:(7637) pp. 417-420. (2017)
Microenvironmental autophagy promotes tumour growth.
Katheder NS, Khezri R, O'Farrell F, Schultz SW, Jain A, Rahman MM, Schink KO, Theodossiou TA, Johansen T, Juhasz G, Bilder D, Brech A, Stenmark H, Rusten TE
As malignant tumours develop, they interact intimately with their microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial. Here we demonstrate, using a well characterized Drosophila melanogaster malignant tumour model, that non-cell-autonomous autophagy is induced both in the tumour microenvironment and systemically in distant tissues. Tumour growth can be pharmacologically restrained using autophagy inhibitors, and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment. Induction of autophagy is mediated by Drosophila tumour necrosis factor and interleukin-6-like signalling from metabolically stressed tumour cells, whereas tumour growth depends on active amino acid transport. We show that dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. We conclude that transformed cells engage surrounding normal cells as active and essential microenvironmental contributors to early tumour growth through nutrient-generating autophagy.
EUROPEAN UROLOGY 71:(3) pp. 319-327. (2017)
Comprehensive Drug Testing of Patient-derived Conditionally Reprogrammed Cells from Castration-resistant Prostate Cancer
Saeed K, Rahkama V, Eldfors S, Bychkov D, Mpindi JP, Yadav B, Paavolainen L, Aittokallio T, Heckman C, Wennerberg K, Peehl DM, Horvath P, Mirtti T, Rannikko A, Kallioniemi O, Östling P, af Hällström TM
Background: Technology development to enable the culture of human prostate cancer (PCa) progenitor cells is required for the identification of new, potentially curative therapies for PCa. Objective: We established and characterized patient-derived conditionally reprogrammed cells (CRCs) to assess their biological properties and to apply these to test the efficacies of drugs. Design, setting, and participants: CRCs were established from seven patient samples with disease ranging from primary PCa to advanced castration-resistant PCa (CRPC). The CRCs were characterized by genomic, transcriptomic, protein expression, and drug profiling. Outcome measurements and statistical analysis: The phenotypic quantification of the CRCs was done based on immunostaining followed by image analysis with Advanced Cell Classifier using Random Forest supervised machine learning. Copy number aberrations (CNAs) were called from whole-exome sequencing and transcriptomics using in-house pipelines. Dose-response measurements were used to generate multiparameter drug sensitivity scores using R-statistical language. Results and limitations: We generated six benign CRC cultures which all had an androgen receptor-negative, basal/transit-amplifying phenotype with few CNAs. In three-dimensional cell culture, these cells could re-express the androgen receptor. The CRCs from a CRPC patient (HUB.5) displayed multiple CNAs, many of which were shared with the parental tumor. We carried out high-throughput drug-response studies with 306 emerging and clinical cancer drugs. Using the benign CRCs as controls, we identified the Bcl-2 family inhibitor navitoclax as the most potent cancer-specific drug for the CRCs from a CRPC patient. Other drug efficacies included taxanes, mepacrine, and retinoids. Conclusions: Comprehensive cancer pharmacopeia-wide drug testing of CRCs from a CRPC patient highlighted both known and novel drug sensitivities in PCa, including navitoclax, which is currently being tested in clinical trials of CRPC. Patient summary: We describe an approach to generate patient-derived cancer cells from advanced prostate cancer and apply such cells to discover drugs that could be applied in clinical trials for castration-resistant prostate cancer. Proof-of-concept study with the aim of generating patient-derived ex vivo models of prostate cancer combined with high-throughput drug testing to identify potential efficacies among 306 existing and emerging cancer drugs highlighting the effect of navitoclax in an advanced disease model. © 2016 European Association of Urology.
JOURNAL OF PHYSICAL CHEMISTRY LETTERS 8:(1) pp. 257-263. (2017)
Two-Dimensional Spectroscopy of Chlorophyll a Excited-State Equilibration in Light-Harvesting Complex II
Akhtar P, Zhang C, Do TN, Garab G, Lambrev PH, Tan HS
Excited-state relaxation dynamics and energy-transfer processes in the chlorophyll a (Chl a) manifold of the light-harvesting complex II (LHCII) were examined at physiological temperature using femtosecond two-dimensional electronic spectroscopy (2DES). The experiments were done under conditions free from singlet-singlet annihilation and anisotropic decay. Energy transfer between the different domains of the Chl a manifold was found to proceed on time scales from hundreds of femtoseconds to five picoseconds, before reaching equilibration. No component slower than 10 ps was observed in the spectral equilibration dynamics. We clearly observe the bidirectional (uphill and downhill) energy transfer of the equilibration process between excited states. This bidirectional energy flow, although implicit in the modeling and simulation of the EET processes, has not been observed in any prior transient absorption studies. Furthermore, we identified the spectral forms associated with the different energy transfer lifetimes in the equilibration process.