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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.

CURRENT OPINION IN PLANT BIOLOGY 34: pp. 100-106. (2016)

DREAMs make plant cells to cycle or to become quiescent

Magyar Z, Bogre L, Ito M

Cell cycle phase specific oscillation of gene transcription has long been recognized as an underlying principle for ordered processes during cell proliferation. The G1/S-specific and G2/M-specific cohorts of genes in plants are regulated by the E2F and the MYB3R transcription factors. Mutant analysis suggests that activator E2F functions might not be fully required for cell cycle entry. In contrast, the two activator-type MYB3Rs are part of positive feedback loops to drive the burst of mitotic gene expression, which is necessary at least to accomplish cytokinesis. Repressor MYB3Rs act outside the mitotic time window during cell cycle progression, and are important for the shutdown of mitotic genes to impose quiescence in mature organs. The two distinct classes of E2Fs and MYB3Rs together with the RETINOBLATOMA RELATED are part of multiprotein complexes that may be evolutionary related to what is known as DREAM complex in animals. In plants, there are multiple such complexes with distinct compositions and functions that may be involved in the coordinated cell cycle and developmental regulation of E2F targets and mitotic genes.

PLANT PHYSIOLOGY 172:(3) pp. 1418-1431. (2016)

An NADPH-Oxidase/Polyamine Oxidase Feedback Loop Controls Oxidative Burst Under Salinity

Gemes K, Kim YJ, Park KY, Moschou PN, Andronis E, Valassaki C, Roussis A, Roubelakis-Angelakis KA

The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (H2O2) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of H2O2 and superoxides (O2.-) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The H2O2-specific probe benzene sulfonyl-H2O2 showed that, under salinity, H2O2 increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O2.--specific probe benzene sulfonyl-So showed that O2.- levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that H2O2 and O2.- accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.

NATURE REVIEWS DRUG DISCOVERY 15:(11) pp. 751-769. (2016)

Screening out irrelevant cell-based models of disease

Horvath P, Aulner N, Bickle M, Davies AM, Del Nery E, Ebner D, Montoya MC, Ostling P, Pietiainen V, Price LS, Shorte SL, Turcatti G, von Schantz C, Carragher NO

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell-and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.

NEW PHYTOLOGIST 212:(2) pp. 472-484. (2016)

Symbiodinium sp cells produce light-induced intra- and extracellular singlet oxygen, which mediates photodamage of the photosynthetic apparatus and has the potential to interact with the animal host in coral symbiosis

Rehman AU, Szabo M, Deak Z, Sass L, Larkum A, Ralph P, Vass I

Coral bleaching is an important environmental phenomenon, whose mechanism has not yet been clarified. The involvement of reactive oxygen species (ROS) has been implicated, but direct evidence of what species are involved, their location and their mechanisms of production remains unknown. Histidine-mediated chemical trapping and singlet oxygen sensor green (SOSG) were used to detect intra- and extracellular singlet oxygen (O-1(2)) in Symbiodinium cultures. Inhibition of the Calvin-Benson cycle by thermal stress or high light promotes intracellular O-1(2) formation. Histidine addition, which decreases the amount of intracellular O-1(2), provides partial protection against photosystem II photoinactivation and chlorophyll (Chl) bleaching. O-1(2) production also occurs in cell-free medium of Symbiodinium cultures, an effect that is enhanced under heat and light stress and can be attributed to the excretion of O-1(2)-sensitizing metabolites from the cells. Confocal microscopy imaging using SOSG showed most extracellular O-1(2) around the cell surface, but it is also produced across the medium distant from the cells. We demonstrate, for the first time, both intra- and extracellular O-1(2) production in Symbiodinium cultures. Intracellular O-1(2) is associated with photosystem II photodamage and pigment bleaching, whereas extracellular O-1(2) has the potential to mediate the breakdown of symbiotic interaction between zooxanthellae and their animal host during coral bleaching.

PLANT CELL AND ENVIRONMENT 39:(9) pp. 2074-2084. (2016)

Gene mining in halophytes: functional identification of stress tolerance genes in Lepidium crassifolium

Rigo G, Valkai I, Farago D, Kiss E, Van Houdt S, Van de Steene N, A Hannah M, Szabados L

Extremophile plants are valuable sources of genes conferring tolerance traits, which can be explored to improve stress tolerance of crops. Lepidium crassifolium is a halophytic relative of the model plant Arabidopsis thaliana, and displays tolerance to salt, osmotic and oxidative stresses. We have employed the modified Conditional cDNA Overexpression System to transfer a cDNA library from L. crassifolium to the glycophyte A. thaliana. By screening for salt, osmotic and oxidative stress tolerance through in vitro growth assays and non-destructive chlorophyll fluorescence imaging, 20 Arabidopsis lines were identified with superior performance under restrictive conditions. Several cDNA inserts were cloned and confirmed to be responsible for the enhanced tolerance by analysing independent transgenic lines. Examples include full-length cDNAs encoding proteins with high homologies to GDSL-lipase/esterase or acyl CoA-binding protein or proteins without known function, which could confer tolerance to one or several stress conditions. Our results confirm that random gene transfer from stress tolerant to sensitive plant species is a valuable tool to discover novel genes with potential for biotechnological applications.

CURRENT OPINION IN COLLOID & INTERFACE SCIENCE 23: pp. 29-40. (2016)

The interfacial tension concept, as revealed by fluctuations

Násztor Zoltán, Bogár Ferenc, Dér András

Abstract A simple, didactic model that could have conclusively interpreted the complexity of specific salt (Hofmeister-) effects on protein solubility and function, using a single physical quantity as a central parameter, has long been missing. Via surveying a row of recent papers we show in this review that a phenomenological formalism based on the salt-induced change of protein–water interfacial tension (∆γ) is able to account for a wide range of Hofmeister effects, including also such “exceptions”, where inverse or “V-shaped” Hofmeister series occurs. A close relationship between protein–water interfacial tension and conformational fluctuations is pinpointed on theoretical grounds, then it is shown how one can use a complex experimental arsenal to demonstrate conformational fluctuations on two prototypical proteins, the membrane protein bacteriorhodopsin and the cytoplasmic protein myoglobin. Finally, via the results of recent and new molecular dynamics simulations on a model peptide, the tryptophan-cage miniprotein, independent evidences are given in favor of the interfacial tension concept, at the same time demonstrating the predictive power of the theory. It is shown that salt-induced fluctuation changes of surface-exposed amino acid groups can be used as a sensitive measure for mapping the local features of Hofmeister effects on protein conformations. General implications of the interfacial tension concept are also discussed.

MOLECULAR THERAPY 24:(8) pp. 1369-1377. (2016)

Structural Determinants of Sleeping Beauty Transposase Activity

Abrusan G, Yant SR, Szilagyi A, Marsh JA, Mates L, Izsvak Z, Barabas O, Ivics Z

Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.

SCIENCE TRANSLATIONAL MEDICINE 8:(350) p. 350ra103. (2016)

The chaperone co-inducer BGP-15 alleviates ventilation-induced diaphragm dysfunction

Salah H, Li M, Cacciani N, Gastaldello S, Ogilvie H, Akkad H, Namuduri AV, Morbidoni V, Artemenko KA, Balogh G, Martinez-Redondo V, Jannig P, Hedstrom Y, Dworkin B, Bergquist J, Ruas J, Vigh L, Salviati L, Larsson L

Ventilation-induced diaphragm dysfunction (VIDD) is a marked decline in diaphragm function in response to mechanical ventilation, which has negative consequences for individual patients' quality of life and for the health care system, but specific treatment strategies are still lacking. We used an experimental intensive care unit (ICU) model, allowing time-resolved studies of diaphragm structure and function in response to long-term mechanical ventilation and the effects of a pharmacological intervention (the chaperone co-inducer BGP-15). The marked loss of diaphragm muscle fiber function in response to mechanical ventilation was caused by posttranslational modifications (PTMs) of myosin. In a rat model, 10 days of BGP-15 treatment greatly improved diaphragm muscle fiber function (by about 100%), although it did not reverse diaphragm atrophy. The treatment also provided protection from myosin PTMs associated with HSP72 induction and PARP-1 inhibition, resulting in improvement of mitochondrial function and content. Thus, BGP-15 may offer an intervention strategy for reducing VIDD in mechanically ventilated ICU patients.

NATURE COMMUNICATIONS 7: Paper 12454. 12 p. (2016)

In situ high-resolution structure of the baseplate antenna complex in Chlorobaculum tepidum

Nielsen JT, Kulminskaya NV, Bjerring M, Linnanto JM, Ratsep M, Pedersen MO, Lambrev PH, Dorogi M, Garab G, Thomsen K, Jegerschold C, Frigaard NU, Lindahl M, Nielsen NC

Photosynthetic antenna systems enable organisms harvesting light and transfer the energy to the photosynthetic reaction centre, where the conversion to chemical energy takes place. One of the most complex antenna systems, the chlorosome, found in the photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum contains a baseplate, which is a scaffolding super-structure, formed by the protein CsmA and bacteriochlorophyll a. Here we present the first high-resolution structure of the CsmA baseplate using intact fully functional, light-harvesting organelles from Cba. tepidum, following a hybrid approach combining five complementary methods: solid-state NMR spectroscopy, cryo-electron microscopy, isotropic and anisotropic circular dichroism and linear dichroism. The structure calculation was facilitated through development of new software, GASyCS for efficient geometry optimization of highly symmetric oligomeric structures. We show that the baseplate is composed of rods of repeated dimers of the strongly amphipathic CsmA with pigments sandwiched within the dimer at the hydrophobic side of the helix.

PLANT CELL AND ENVIRONMENT 39:(7) pp. 1460-1472. (2016)

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H production of the green alga Chlamydomonas reinhardtii

Nagy V, Vidal-Meireles A, Tengolics R, Rakhely G, Garab G, Kovacs L, Toth SZ

In nature, H2 production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia and it prevents the over-reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress-related genes, downregulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H2 production because it supplies electrons but the evolved O2 inhibits the hydrogenase. Here we show that upon sulphur deprivation the ascorbate content in C. reinhardtii increases about 100-fold, reaching the mM range; at this concentration ascorbate inactivates the Mn-cluster of PSII and afterwards it can donate electrons to tyrozin Z+ at a slow rate. This stage is followed by donor-side induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction center degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H2 production. We also show that ascorbate influenced H2 evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.

ACS SYNTHETIC BIOLOGY 5:(7) pp. 619-631. (2016)

Efflux Pump Control Alters Synthetic Gene Circuit Function

Diao JC, Charlebois DA, Neyozhay D, Bodi Z, Pal C, Balazsi G

Synthetic biology aims to design new biological systems for predefined purposes, such as the controlled secretion of biofuels, pharmaceuticals, or other chemicals. Synthetic gene circuits regulating an efflux pump from the ATP-binding cassette (ABC) protein family could achieve this. However, ABC efflux pumps can also drive out intracellular inducer molecules that control the gene circuits. This will introduce an implicit feedback that could alter gene circuit function in ways that are poorly understood. Here, we used two synthetic gene circuits inducible by tetracycline family molecules to regulate the expression of a yeast ABC pump (Pdr5p) that pumps out the inducer. Pdr5p altered the dose-responses of the original gene circuits substantially in Saccharomyces cerevisiae. While one aspect of the change could be attributed to the efflux pumping function of Pdr5p, another aspect remained unexplained. Quantitative modeling indicated that reduced regulator gene expression in addition to efflux pump function could fully explain the altered dose-responses. These predictions were validated experimentally. Overall, we highlight how efflux pumps can alter gene circuit dynamics and demonstrate the utility of mathematical modeling in understanding synthetic gene circuit function in new circumstances.

ELIFE 5: Paper e14226. 27 p. (2016)

MiniCORVET is a Vps8-containing hemocyte- and nephrocyte-specific early endosomal tether in Drosophila

Lorincz P, Lakatos Z, Varga A, Maruzs T, Simon-Vecsei Z, Darula Z, Benko P, Csordas G, Lippai M, Ando I, Hegedus K, Medzihradszky K, Takats S, Juhasz G

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Nps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila.

NEW PHYTOLOGIST 211:(2) pp. 584-598. (2016)

Characterization of photomorphogenic responses and signaling cascades controlled by phytochrome-A expressed in different tissues

Kirchenbauer D, Viczian A, Adam E, Hegedus Z, Klose C, Lepper M, Hiltbrunner A, Kircher S, Schaefer E, Nagy F

The photoreceptor phytochrome A acts as a light-dependent molecular switch and regulates responses initiated by very low fluences of light (VLFR) and high fluences (HIR) of far-red light. PhyA is expressed ubiquitously, but how phyA signaling is orchestrated to regulate photo-morphogenesis is poorly understood. To address this issue, we generated transgenic Arabidopsis thaliana phyA-201 mutant lines expressing the biologically active phyA-YFP photoreceptor in different tissues, and analyzed the expression of several reporter genes, including ProHY5: HY5-GFP and Pro35S: CFP-PIF1, and various FR-HIR-dependent physiological responses. We show that phyA action in one tissue is critical and sufficient to regulate flowering time and root growth; control of cotyledon and hypocotyl growth requires simultaneous phyA activity in different tissues; and changes detected in the expression of reporters are not restricted to phyA-containing cells. We conclude that FR-HIR-controlled morphogenesis in Arabidopsis is mediated partly by tissue-specific and partly by intercellular signaling initiated by phyA. Intercellular signaling is critical for many FR-HIR induced responses, yet it appears that phyA modulates the abundance and activity of key regulatory transcription factors in a tissue-autonomous fashion.

TRENDS IN PLANT SCIENCE 21:(7) pp. 594-608. (2016)

The Role of SWI/SNF Chromatin Remodeling Complexes in Hormone Crosstalk

Sarnowska E, Gratkowska DM, Sacharowski SP, Cwiek P, Tohge T, Fernie AR, Siedlecki JA, Koncz C, Sarnowski TJ

SWI/SNF-type ATP-dependent chromatin remodeling complexes (CRCs) are evolutionarily conserved multiprotein machineries controlling DNA accessibility by regulating chromatin structure. We summarize here recent advances highlighting the role of SWI/SNF in the regulation of hormone signaling pathways and their crosstalk in Arabidopsis thaliana. We discuss the functional interdependences of SWI/SNF complexes and key elements regulating developmental and hormone signaling pathways by indicating intriguing similarities and differences in plants and humans, and summarize proposed mechanisms of SWI/SNF action on target loci. We postulate that, given their viability, several plant SWI/SNF mutants may serve as an attractive model for searching for conserved functions of SWI/SNF CRCs in hormone signaling, cell cycle control, and other regulatory pathways.

PLOS GENETICS 12:(5) Paper e1006022. 22 p. (2016)

BOD1 Is Required for Cognitive Function in Humans and Drosophila

Esmaeeli-Nieh S, Fenckova M, Porter IM, Motazacker MM, Nijhof B, Castells-Nobau A, Asztalos Z, Weissmann R, Behjati F, Tzschach A, Felbor U, Scherthan H, Sayfati SM, Ropers HH, Kahrizi K, Najmabadi H, Swedlow JR, Schenck A, Kuss AW

Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features.

NATURE COMMUNICATIONS 7: Paper 11654. (2016)

A voltage-dependent chloride channel fine-tunes photosynthesis in plants

Herdean A, Teardo E, Nilsson AK, Pfeil BE, Johansson ON, Ünnep R, Nagy G, Zsiros O, Dana S, Solymosi K, Garab G, Szabó I, Spetea C, Lundin B

In natural habitats, plants frequently experience rapid changes in the intensity of sunlight. To cope with these changes and maximize growth, plants adjust photosynthetic light utilization in electron transport and photoprotective mechanisms. This involves a proton motive force (PMF) across the thylakoid membrane, postulated to be affected by unknown anion (Cl-) channels. Here we report that a bestrophin-like protein from Arabidopsis thaliana functions as a voltage-dependent Cl- channel in electrophysiological experiments. AtVCCN1 localizes to the thylakoid membrane, and fine-tunes PMF by anion influx into the lumen during illumination, adjusting electron transport and the photoprotective mechanisms. The activity of AtVCCN1 accelerates the activation of photoprotective mechanisms on sudden shifts to high light. Our results reveal that AtVCCN1, a member of a conserved anion channel family, acts as an early component in the rapid adjustment of photosynthesis in variable light environments.

BIOTECHNOLOGY FOR BIOFUELS 9: Paper 102. 14 p. (2016)

Conversion of H2 and CO2 to CH4 and acetate in fed-batch biogas reactors by mixed biogas community: a novel route for the power-to-gas concept

Márk Szuhaj, Norbert Ács, Roland Tengölics, Attila Bodor, Gábor Rákhely, Kornél L Kovács, Zoltán Bagi

Background: Applications of the power-to-gas principle for the handling of surplus renewable electricity have been proposed. The feasibility of using hydrogenotrophic methanogens as CH4 generating catalysts has been demonstrated. Laboratory and scale-up experiments have corroborated the benefits of the CO2 mitigation via biotechnological conversion of H-2 and CO2 to CH4. A major bottleneck in the process is the gas-liquid mass transfer of H-2.
Results: Fed-batch reactor configuration was tested at mesophilic temperature in laboratory experiments in order to improve the contact time and H-2 mass transfer between the gas and liquid phases. Effluent from an industrial biogas facility served as biocatalyst. The bicarbonate content of the effluent was depleted after some time, but the addition of stoichiometric CO2 sustained H-2 conversion for an extended period of time and prevented a pH shift. The microbial community generated biogas from the added a-cellulose substrate with concomitant H-2 conversion, but the organic substrate did not facilitate H-2 consumption. Fed-batch operational mode allowed a fourfold increase in volumetric H-2 load and a 6.5-fold augmentation of the CH4 formation rate relative to the CSTR reactor configuration. Acetate was the major by-product of the reaction.
Conclusions: Fed-batch reactors significantly improve the efficiency of the biological power-to-gas process. Besides their storage function, biogas fermentation effluent reservoirs can serve as large-scale bio CH4 reactors. On the basis of this recognition, a novel concept is proposed, which merges biogas technology with other means of renewable electricity production for improved efficiency and sustainability.

NATURE COMMUNICATIONS 7: p. 11607. (2016)

Adaptive evolution of complex innovations through stepwise metabolic niche expansion.

Szappanos B, Fritzemeier J, Csorgo B, Lazar V, Lu X, Fekete G, Balint B, Herczeg R, Nagy I, Notebaart RA, Lercher MJ, Pal C, Papp B

A central challenge in evolutionary biology concerns the mechanisms by which complex metabolic innovations requiring multiple mutations arise. Here, we propose that metabolic innovations accessible through the addition of a single reaction serve as stepping stones towards the later establishment of complex metabolic features in another environment. We demonstrate the feasibility of this hypothesis through three complementary analyses. First, using genome-scale metabolic modelling, we show that complex metabolic innovations in Escherichia coli can arise via changing nutrient conditions. Second, using phylogenetic approaches, we demonstrate that the acquisition patterns of complex metabolic pathways during the evolutionary history of bacterial genomes support the hypothesis. Third, we show how adaptation of laboratory populations of E. coli to one carbon source facilitates the later adaptation to another carbon source. Our work demonstrates how complex innovations can evolve through series of adaptive steps without the need to invoke non-adaptive processes.

NUCLEIC ACIDS RESEARCH 44:(7) pp. 3176-3189. (2016)16)

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

Burkovics P, Dome L, Juhasz S, Altmannova V, Sebesta M, Pacesa M, Fugger K, Sorensen CS, Lee MY, Haracska L, Krejci L

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

PLANT PHYSIOLOGY 2016: Paper 15.01679. (2016)

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis L.

Dudits D, Torok K, Cseri A, Paul K, Nagy AV, Nagy B, Sass L, Ferenc G, Vankova R, Dobrev P, Vass I, Ayaydin F

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO2 uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.

MOLECULAR BIOLOGY AND EVOLUTION 33:(5) pp. 1257-1269. (2016)

Indispensability of Horizontally Transferred Genes and Its Impact on Bacterial Genome Streamlining

Karcagi I, Draskovits G, Umenhoffer K, Fekete G, Kovacs K, Mehi O, Baliko G, Szappanos B, Gyorfy Z, Feher T, Bogos B, Blattner FR, Pal C, Posfai G, Papp B

Why are certain bacterial genomes so small and compact? The adaptive genome streamlining hypothesis posits that selection acts to reduce genome size because of themetabolic burden of replicating DNA. To reveal the impact of genome streamlining on cellular traits, we reduced the Escherichia coli genome by up to 20% by deleting regions which have been repeatedly subjects of horizontal transfer in nature. Unexpectedly, horizontally transferred genes not only confer utilization of specific nutrients and elevate tolerance to stresses, but also allow efficient usage of resources to build new cells, and hence influence fitness in routine and stressful environments alike. Genome reduction affected fitness not only by gene loss, but also by induction of a general stress response. Finally, we failed to find evidence that the advantage of smaller genomes would be due to a reduced metabolic burden of replicating DNA or a link with smaller cell size. We conclude that as the potential energetic benefit gained by deletion of short genomic segments is vanishingly small compared with the deleterious side effects of these deletions, selection for reduced DNA synthesis costs is unlikely to shape the evolution of small genomes.

NANOMEDICINE: NANOTECHNOLOGY BIOLOGY AND MEDICINE 2016: p. 1. (2016)

Silver nanoparticles modulate ABC transporter activity and enhance chemotherapy in multidrug resistant cancer

Kovács Dávid, Szőke Krisztina, Igaz Nóra, Spengler Gabriella, Molnár József, Tóth Tímea, Madarász Dániel, Rázga Zsolt, Kónya Zoltán, Boros Imre M, Kiricsi Mónika

The emergence of multidrug resistant (MDR) cancer phenotypes dramatically attenuates the efficiency of antineoplastic drug treatments often leading to the failure of chemotherapy. Therefore there is an urgent need to engineer new therapeutically useful agents and propose innovative approaches able to defeat resistant cancer cells. Although the remarkable anti-cancer features of silver nanoparticles (AgNPs) have already been delineated their impact on MDR cancer has never been investigated. Herein, we report that AgNPs have a notable anti-proliferative effect and induce apoptosis mediated cell death both in drug sensitive and in MDR cancer cells. Furthermore we show evidence that AgNPs exert an inhibitory action on the efflux activity of MDR cancer cells which feature could be exploited to enhance drug accumulation. We verified synergistic interactions of AgNPs with six different antineoplastic agents on drug resistant cells which emphasizes the excellent potential of AgNPs as combinational partners in the chemotherapy of MDR cancer.

AUTOPHAGY 12:(2) pp. 273-286. (2016)

AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects.

Papp D, Kovacs T, Billes V, Varga M, Tarnoci A, Hackler L Jr, Puskas LG, Liliom H, Tarnok K, Schlett K, Borsy A, Padar Z, Kovacs AL, Hegedus K, Juhasz G, Komlos M, Erdos A, Gulyas B, Vellai T

Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases.

ELIFE 5: Paper e12245. 18 p. (2016)

Mutation in Atg5 reduces autophagy and leads to ataxia with developmental delay

Kim M, Sandford E, Gatica D, Qiu Y, Liu X, Zheng Y, Schulman BA, Xu J, Semple I, Ro SH, Kim B, Mavioglu RN, Tolun A, Jipa A, Takats S, Karpati M, Li JZ, Yapici Z, Juhasz G, Lee JH, Klionsky DJ, Burmeister M

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health.

NATURE COMMUNICATIONS 7: Paper 11126. 8 p. (2016)

Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering

Voigt F, Wiedemann L, Zuliani C, Querques I, Sebe A, Mates L, Izsvak Z, Ivics Z, Barabas O

Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases.

JOURNAL OF INVESTIGATIVE DERMATOLOGY 136:(4) pp. 862-865. (2016)

Bacterial sepsis increases survival in metastatic melanoma: Chlamydophila pneumoniae induces macrophage polarization and tumor regression

Buzas K, Marton A, Vizler C, Gyukity-Sebestyen E, Harmati M, Nagy K, Zvara A, Katona RL, Tubak V, Endresz V, Nemeth IB, Olah J, Vigh L, Biro T, Kemeny L

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113:(9) pp. 2502-2507. (2016)

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

Nyerges A, Csorgo B, Nagy I, Balint B, Bihari P, Lazar V, Apjok G, Umenhoffer K, Bogos B, Posfai G, Pal C

Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.

CHEMICAL COMMUNICATIONS Paper 10.1039/C5CC09257D. (2016)

Foldameric probes for membrane interactions by induced β-sheet folding

Zsófia Hegedüs, Ildikó Makra, Norbert Imre, Anasztázia Hetényi, István M Mándity, Éva Monostori, Tamás A Martinek

Design strategies were devised for alpha/beta-peptide foldameric analogues of the antiangiogenic anginex with the goal of mimicking the diverse structural features from the unordered conformation to a folded beta-sheet in response to membrane interactions. Structure-activity relationships were investigated in the light of different beta-sheet folding levels.