Publications

identification and characterization of lfd-2, a predicted fringe protein required for membrane integrity during cell fusion in neurospora crassa.	the molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. in the filamentous fungus neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. in n. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: prm1 and lfd-1. the abse ...	2015	25595444
hypotonic stress-induced calcium signaling in saccharomyces cerevisiae involves trp-like transporters on the endoplasmic reticulum membrane.	saccharomyces cerevisiae cells respond to hypotonic stress (hts) by a cytosolic calcium rise, either generated by an influx of calcium from extracellular medium, when calcium is available, or by a release from intracellular stores in scarcity of extracellular calcium. calcium release from intracellular compartments is peculiarly inhibited by external calcium in a calcineurin-independent and cch1-, but not mid1-, driven manner. hts-induced calcium release is also negatively regulated by the er pr ...	2015	25573187
cellobionic acid utilization: from neurospora crassa to saccharomyces cerevisiae.	economical production of fuels and chemicals from plant biomass requires the efficient use of sugars derived from the plant cell wall. neurospora crassa, a model lignocellulosic degrading fungus, is capable of breaking down the complex structure of the plant cell wall. in addition to cellulases and hemicellulases, n. crassa secretes lytic polysaccharide monooxygenases (lpmos), which cleave cellulose by generating oxidized sugars-particularly aldonic acids. however, the strategies n. crassa emplo ...	2015	26279678
analysis of circadian rhythms in the basal filamentous ascomycete pyronema confluens.	many organisms use circadian clocks to adapt to daily changes in the environment. major insights into the molecular mechanisms of circadian oscillators have been gained through studies of the model organism neurospora crassa; however, little is known about molecular components of circadian clocks in other fungi. an important part of the n. crassa circadian clock is the frequency (frq) gene, homologs of which can be found in sordariomycetes, dothideomycetes, and leotiomycetes, but not eurotiomyce ...	2015	26254031
optimal codons in tremella fuciformis end in c/g, a strong difference with known tremella species.	tremella fuciformis is a popular edible fungus with fruiting bodies that can be produced in large quantities at low costs, while it is easy to transform and cultivate as yeast. this makes it an attractive potential bioreactor. enhanced heterologous gene expression through codon optimization would be useful, but until now codon usage preferences in t. fuciformis remain unknown. to precisely determine the preferred codon usage of t. fuciformis we sequenced the genome of strain tr26 resulting in a ...	2015	26253954
a tool set for the genome-wide analysis of neurospora crassa by rt-pcr.	neurospora crassa is an important model organism for filamentous fungi as well as for circadian biology and photobiology. although the community-accumulated tool set for the molecular analysis of neurospora is extensive, two components are missing: (1) dependable reference genes whose level of expression are relatively constant across light/dark cycles and as a function of time of day and (2) a catalog of primers specifically designed for real-time pcr (rt-pcr). to address the first of these we ...	2015	26248984
two microtubule-plus-end binding proteins lis1-1 and lis1-2, homologues of human lis1 in neurospora crassa.	lis1 is a microtubule (mt) plus-end binding protein that interacts with the dynein/dynactin complex. in humans, lis1 is required for proper nuclear and organelle migration during cell growth. although gene duplication is absent from neurospora crassa, we found two paralogues of human lis1. we named them lis1-1 and lis1-2 and studied their dynamics and function by fluorescent tagging. at the protein level, lis1-1 and lis1-2 were very similar. although, the characteristic coiled-coil motif was not ...	2015	26231681
linear discriminant analysis identifies mitochondrially localized proteins in neurospora crassa.	besides their role as powerhouses, mitochondria play a pivotal role in the spatial organization of numerous enzymatic functions. they are connected to the er, and many pathways are organized through the mitochondrial membranes. thus, the precise definition of mitochondrial proteomes remains a challenging task. here, we have established a proteomic strategy to accurately determine the mitochondrial localization of proteins from the fungal model organism neurospora crassa. this strategy relies on ...	2015	26215788
the phenotype of a phospholipase c (plc-1) mutant in a filamentous fungus, neurospora crassa.	in the filamentous fungus neurospora crassa, phospholipase c may play a role in hyphal extension at the growing tips as part of a growth-sensing mechanism that activates calcium release from internal stores to mediate continued expansion of the hyphal tip. one candidate for a tip-localized phospholipase c is plc-1. we characterized morphology and growth characteristics of a knockout mutant (ko plc-1) and a rip mutated strain (rip plc-1) (missense mutations and a nonsense mutation render the gene ...	2015	26212074
[quantitative assessment of the combined effect of the nitrogen status, light and dehydration of mycelium on conidiation in neurospora crassa].	we performed a quantitative assessment of the conidia yield in neurospora crassa in response to treatment with different conidiation effectors. depending on nitrogen source and intactness of nitrite reductase (nir) and nitrate reductase (nr), light and dehydration affected the number of viable conidia produced by the ascomycete. in most variants of the nitrogen status, the combined action of light and dehydration synergistically increased the conidia yield. conidiation in wild-type cells cultiva ...	2015	26204777
epigenetic and posttranslational modifications in light signal transduction and the circadian clock in neurospora crassa.	blue light, a key abiotic signal, regulates a wide variety of physiological processes in many organisms. one of these phenomena is the circadian rhythm presents in organisms sensitive to the phase-setting effects of blue light and under control of the daily alternation of light and dark. circadian clocks consist of autoregulatory alternating negative and positive feedback loops intimately connected with the cellular metabolism and biochemical processes. neurospora crassa provides an excellent mo ...	2015	26198228
metabolic impacts of using nitrogen and copper-regulated promoters to regulate gene expression in neurospora crassa.	the filamentous fungus neurospora crassa is a long-studied eukaryotic microbial system amenable to heterologous expression of native and foreign proteins. however, relatively few highly tunable promoters have been developed for this species. in this study, we compare the tcu-1 and nit-6 promoters for controlled expression of a gfp reporter gene in n. crassa. although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 ...	2015	26194204
structural and functional characterization of a lytic polysaccharide monooxygenase with broad substrate specificity.	the recently discovered lytic polysaccharide monooxygenases (lpmos) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. nclpmo9c from neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. the crystal structure of the catalytic domain of nclpmo9c revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. the ability o ...	2015	26178376
live imaging of β-1,3-glucan synthase fks-1 in neurospora crassa hyphae.	the subcellular localization and dynamics of fks-1, the putative catalytic subunit of the β-1,3-glucan synthase complex, was analyzed in growing hyphae of neurospora crassa by live confocal microscopy. gfp-tagged fks-1 accumulated at the outer layer of the spitzenkörper (spk), and at the apical plasma membrane (pm). fluorescence recovery after photobleaching analysis revealed arrival of fks-1-containing carriers first at the immediate surroundings of the core region of the spk, and thereafter to ...	2015	26150287
tuning the phase of circadian entrainment.	the circadian clock coordinates daily physiological, metabolic and behavioural rhythms. these endogenous oscillations are synchronized with external cues ('zeitgebers'), such as daily light and temperature cycles. when the circadian clock is entrained by a zeitgeber, the phase difference ψ between the phase of a clock-controlled rhythm and the phase of the zeitgeber is of fundamental importance for the fitness of the organism. the phase of entrainment ψ depends on the mismatch between the intrin ...	2015	26136227
the bzip transcription factor hac-1 is involved in the unfolded protein response and is necessary for growth on cellulose in neurospora crassa.	high protein secretion capacity in filamentous fungi requires an extremely efficient system for protein synthesis, folding and transport. when the folding capacity of the endoplasmic reticulum (er) is exceeded, a pathway known as the unfolded protein response (upr) is triggered, allowing cells to mitigate and cope with this stress. in yeast, this pathway relies on the transcription factor hac1, which mediates the up-regulation of several genes required under these stressful conditions. in this w ...	2015	26132395
endogenous small rna mediates meiotic silencing of a novel dna transposon.	genome defense likely evolved to curtail the spread of transposable elements and invading viruses. a combination of effective defense mechanisms has been shown to limit colonization of the neurospora crassa genome by transposable elements. a novel dna transposon named sly1-1 was discovered in the genome of the most widely used laboratory "wild-type" strain fgsc 2489 (or74a). meiotic silencing by unpaired dna, also simply called meiotic silencing, prevents the expression of regions of the genome ...	2015	26109355
fluorescent markers for the spitzenkörper and exocytosis in zymoseptoria tritici.	fungal hyphae are highly polarized cells that invade their substrate by tip growth. in plant pathogenic fungi, hyphal growth is essential for host invasion. this makes polarity factors and secretion regulators potential new targets for novel fungicides. polarization requires delivery of secretory vesicles to the apical spitzenkörper, followed by polarized exocytosis at the expanding cell tip. here, we introduce fluorescent markers to visualize the apical spitzenkörper and the apical site of exoc ...	2015	26092802
structure of importin-α from a filamentous fungus in complex with a classical nuclear localization signal.	neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. to advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. importin-α (imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (nlss) tha ...	2015	26091498
control of mrna stability in fungi by nmd, ejc and cbc factors through 3'utr introns.	in higher eukaryotes the accelerated degradation of mrnas harboring premature termination codons is controlled by nonsense-mediated mrna decay (nmd), exon junction complex (ejc), and nuclear cap-binding complex (cbc) factors, but the mechanistic basis for this quality-control system and the specific roles of the individual factors remain unclear. using neurospora crassa as a model system, we analyzed the mechanisms by which nmd is induced by spliced 3'-utr introns or upstream open reading frames ...	2015	26048019
suppressing the neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors.	neurospora crassa has been utilized as a model organism for studying biological, regulatory, and circadian rhythms for over 50 years. these circadian cycles are driven at the molecular level by gene transcription events to prepare for environmental changes. n. crassa is typically found on woody biomass and is commonly studied on agar-containing medium which mimics its natural environment. we report a novel method for disrupting circadian gene transcription while maintaining light responsiveness ...	2015	26031221
identification of allorecognition loci in neurospora crassa by genomics and evolutionary approaches.	understanding the genetic and molecular bases of the ability to distinguish self from nonself (allorecognition) and mechanisms underlying evolution of allorecognition systems is an important endeavor for understanding cases where it becomes dysfunctional, such as in autoimmune disorders. in filamentous fungi, allorecognition can result in vegetative or heterokaryon incompatibility, which is a type of programmed cell death that occurs following fusion of genetically different cells. allorecogniti ...	2015	26025978
population genomic analyses reveal possible drivers of population divergence.	recent advances in sequencing technology and efficiency enable new and improved methods to investigate how populations diverge and species evolve. fungi have relatively small and simple genomes and can often be cultured in the laboratory. fungal populations can thus be sequenced for a relatively low cost, which makes them ideal for population genomic analyses. in several recent population genomic studies, wild populations of fungal model organisms and human pathogens have been analysed, for exam ...	2015	26013991
involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in neurospora crassa.	staurosporine-induced cell death in neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular ca(2+) influx and mobilization of ca(2+) from internal stores. here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex i (like the 51kda and 14kda subunits) or the ca(2+)-binding alternative nadph dehydrogenase nde-1 are hypersensitive to staurosporine and incapable of setting ...	2015	26004627
biological significance of photoreceptor photocycle length: vivid photocycle governs the dynamic vivid-white collar complex pool mediating photo-adaptation and response to changes in light intensity.	most organisms on earth sense light through the use of chromophore-bearing photoreceptive proteins with distinct and characteristic photocycle lengths, yet the biological significance of this adduct decay length is neither understood nor has been tested. in the filamentous fungus neurospora crassa vivid (vvd) is a critical player in the process of photoadaptation, the attenuation of light-induced responses and the ability to maintain photosensitivity in response to changing light intensities. de ...	2015	25978382
neurospora heterokaryons with complementary duplications and deficiencies in their constituent nuclei provide an approach to identify nucleus-limited genes.	introgression is the transfer of genes or genomic regions from one species into another via hybridization and back-crosses. we have introgressed four translocations (eb4, ibj5, uk14-1, and b362i) from neurospora crassa into n. tetrasperma. this enabled us to construct two general types of heterokaryons with mat-a and mat-a nuclei of different genotypes: one type is [t + n] (with one translocation nucleus and one normal sequence nucleus), and the other is [dp + df] (with one nucleus carrying a du ...	2015	25897010
genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase production in neurospora crassa.	lignocellulolytic fungal cells suffer endoplasmic reticulum (er) stress during lignocellulase synthesis; however, an understanding of this integrated process on a genome-wide scale remains poor. here, we undertook a systematic investigation of this process in neurospora crassa (n. crassa) using transcriptomic analysis coupled with genetic screens.	2015	25883682
inferring bona fide transfrags in rna-seq derived-transcriptome assemblies of non-model organisms.	de novo transcriptome assembly of short transcribed fragments (transfrags) produced from sequencing-by-synthesis technologies often results in redundant datasets with differing levels of unassembled, partially assembled or mis-assembled transcripts. post-assembly processing intended to reduce redundancy typically involves reassembly or clustering of assembled sequences. however, these approaches are mostly based on common word heuristics and often create clusters of biologically unrelated sequen ...	2015	25880035
mathematical modeling and validation of glucose compensation of the neurospora circadian clock.	autonomous circadian oscillations arise from transcriptional-translational feedback loops of core clock components. the period of a circadian oscillator is relatively insensitive to changes in nutrients (e.g., glucose), which is referred to as "nutrient compensation". recently, a transcription repressor, csp-1, was identified as a component of the circadian system in neurospora crassa. the transcription of csp-1 is under the circadian regulation. intriguingly, csp-1 represses the circadian trans ...	2015	25863073
membrane transporters in self resistance of cercospora nicotianae to the photoactivated toxin cercosporin.	the goal of this work is to characterize membrane transporter genes in cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. previous studies implicated a role for diverse membrane transporters in cercosporin resistance. in this study, transporters identified in a subtractive cdna library between a cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ...	2015	25862648
reconstruction of the carnitine biosynthesis pathway from neurospora crassa in the yeast saccharomyces cerevisiae.	industrial synthesis of l-carnitine is currently performed by whole-cell biotransformation of industrial waste products, mostly d-carnitine and cronobetaine, through specific bacterial species. no comparable system has been established using eukaryotic microorganisms, even though there is a significant and growing international demand for either the pure compound or carnitine-enriched consumables. in eukaryotes, including the fungus neurospora crassa, l-carnitine is biosynthesized through a four ...	2015	25851717
production of cellobionate from cellulose using an engineered neurospora crassa strain with laccase and redox mediator addition.	we report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. a previously engineered strain of neurospora crassa (f5∆ace-1∆cre-1∆ndvb) was shown to produce cellobionate directly from cellulose without the addition of exogenous cellulases. specifically, n. crassa produces cellulases, which hydrolyze cellulose to cellobiose, and cellobiose dehydrogenase (cdh), which oxidizes cellobiose to cel ...	2015	25849253
draft genome sequence of neurospora crassa strain fgsc 73.	we report the elucidation of the complete genome of the neurospora crassa (shear and dodge) strain fgsc 73, a mat-a, trp-3 mutant strain. the genome sequence around the idiotypic mating type locus represents the only publicly available sequence for a mat-a strain. 40.42 megabases are assembled into 358 scaffolds carrying 11,978 gene models.	2015	25838471
combinatorial control of light induced chromatin remodeling and gene activation in neurospora.	light is an important environmental cue that affects physiology and development of neurospora crassa. the light-sensing transcription factor (tf) wcc, which consists of the gata-family tfs wc1 and wc2, is required for light-dependent transcription. sub1, another gata-family tf, is not a photoreceptor but has also been implicated in light-inducible gene expression. to assess regulation and organization of the network of light-inducible genes, we analyzed the roles of wcc and sub1 in light-induced ...	2015	25822411
neurospora importin α is required for normal heterochromatic formation and dna methylation.	heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. in many species, constitutive heterochromatin is decorated with histone h3 tri-methylated at lysine 9 (h3k9me3) and cytosine methylation. in neurospora crassa, a five-protein complex, dcdc, catalyzes h3k9 methylation, which then directs dna methylation. here, we identify and characterize a gene important for dcdc function, dim-3 (defective in methylation-3), which encodes t ...	2015	25793375
dawn- and dusk-phased circadian transcription rhythms coordinate anabolic and catabolic functions in neurospora.	circadian clocks control rhythmic expression of a large number of genes in coordination with the 24 hour day-night cycle. the mechanisms generating circadian rhythms, their amplitude and circadian phase are dependent on a transcriptional network of immense complexity. moreover, the contribution of post-transcriptional mechanisms in generating rhythms in rna abundance is not known.	2015	25762222
deciphering the uniqueness of mucoromycotina cell walls by combining biochemical and phylogenomic approaches.	most fungi from the mucoromycotina lineage occur in ecosystems as saprobes, although some species are phytopathogens or may induce human mycosis. mucoromycotina represent early diverging models that are most valuable for understanding fungal evolution. here we reveal the uniqueness of the cell wall structure of the mucoromycotina rhizopus oryzae and phycomyces blakesleeanus compared with the better characterized cell wall of the ascomycete neurospora crassa. we have analysed the corresponding po ...	2015	25143134
two pentatricopeptide repeat domain proteins are required for the synthesis of respiratory complex i.	in this study pentatricopeptide repeat (ppr) proteins in filamentous ascomycetes are identified and functionally characterized. ppr proteins, which have in common a degenerated 35 amino acid motif often arranged in multiple tandems, are known to be implicated in various steps of rna metabolism in mitochondria and chloroplasts. in filamentous ascomycetes we identified a common set of nine ppr proteins. for seven of these proteins, which were not yet characterized, knockout mutants of neurospora c ...	2015	25108509
viba, a homologue of a transcription factor for fungal heterokaryon incompatibility, is involved in antifungal compound production in the plant-symbiotic fungus epichloë festucae.	symbiotic association of epichloae endophytes (epichloë/neotyphodium species) with cool-season grasses of the subfamily pooideae confers bioprotective benefits to the host plants against abiotic and biotic stresses. while the production of fungal bioprotective metabolites is a well-studied mechanism of host protection from insect herbivory, little is known about the antibiosis mechanism against grass pathogens by the mutualistic endophyte. in this study, an epichloë festucae mutant defective in ...	2015	24906411
functional analysis of two l-arabinose transporters from filamentous fungi reveals promising characteristics for improved pentose utilization in saccharomyces cerevisiae.	limited uptake is one of the bottlenecks for l-arabinose fermentation from lignocellulosic hydrolysates in engineered saccharomyces cerevisiae. this study characterized two novel l-arabinose transporters, lat-1 from neurospora crassa and mtlat-1 from myceliophthora thermophila. although the two proteins share high identity (about 83%), they display different substrate specificities. sugar transport assays using the s. cerevisiae strain eby.vw4000 indicated that lat-1 accepts a broad substrate sp ...	2015	25841015
purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of neurospora crassa mitochondria.	a simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of neurospora crassa mitochondria is described. after fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. covalent chromatography on thiol-activated sepharose cl-4b allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2- ...	2015	2947635
dna-dna and dna-protein crosslinking and repair in neurospora crassa following exposure to nitrogen mustard.	a new method was developed for the analysis of dna-protein crosslinks in neurospora crassa. the formations of dna-protein and dna-dna crosslinks were assayed following exposure of spheroplasts to hn2. both types of crosslink were detected and were found to be repaired during recovery. moreover a mutant sensitive to hn2 was defective in the removal of both types of crosslink.	2015	6239978
ageing of neurospora crassa. iv. induction of senescence in wild type by dietary amino acid analogs and reversal by antioxidants and membrane stabilizers.	the extensional growth rate of wild-type 74a8 n. crassa in the presence of various concentrations of 19 amino acid analogs was measured. seven analogs were not inhibitory at concentrations in the range of one to 10mm. of the remaining 12 analogs, nine inhibited growth in a novel way. the kinetics of growth in the presence of these analogs at 30 degrees were characterized by seven sequential phases: (1)lag; (2) acceleration of growth rate; (3) steady-state growth rate; (4) exponential rate of dec ...	2015	7715
[location of polyphosphatases, hydrolyzing polyphosphates to orthophosphates, in the subcellular structures of neurospora crassa].		2016	4336095
[polyphosphate depolymerase activity in cells of neurospora crassa (28610 as)].		2016	4344204
conversion of d-lysine via l-pepecolic acid in neurospora crassa.	neurospora crassa converts d-lysine to l-pipecolic acid and the latter to l-lysine by a pathway outlined herein and different from mechanisms hitherto known to be involved in interconversions of enantiomers of amino acids.	2016	125972
[isolation and properties of polyphosphatase of neurospora crassa].	polyphosphatase (polyphosphate-phosphohydrolase) has been isolated from mycelium of neurospora crassa and purified to homogenous state. the enzyme is shown to be strictly specific to high molecular weight inorganic polyphosphates. km for phosphate in polymeric form is 6.8-10(-4) m. the molecular weight of this enzyme is 50 000 +/- 3000. to display its activity polyphosphatase requires the presence of bivalent cations of some metals, mg2+ ions being the best activator with co2+, mn2+ and fe2+ ion ...	2016	2861
[detection and some properties of neurospora crassa polyphosphatases hydrolyzing inorganic polyphosphates and orthophosphates].		2016	4333905
[isolation and certain features of a polyphosphate phosphohydrolase deficient mutant of the fungus neurospora crassa].	a polyphosphatase deficient mutant of neurospora crassa has been isolated. the criterion for selecting the mutant was the capacity of the fungus to assimilate polyphosphates as the source of exogenous phosphorus. the mutant like the parent strain ad-6, was an adenine auxotroph but differed from the parent strain by a lower growth rate though, at the stationary stage, its biomass reached the same level as in the strain ad-6. the character of changes in the activity of polyphosphatase in the cours ...	2016	6248740
mutation at the am locus of neurospora crassa.	forty-eight new mutations at the am locus of neurospora crassa have been characterized. nineteen mutations were induced by uv; of these, eight were missense, two were frameshifts, two were nonsense, three were deletions and four were unidentified. twenty-nine mutations were induced with nitrous acid; of these, twenty-one were missense, three were frameshifts, one was nonsense, two were deletions and one was genetically unstable.	2016	6177580
the growth of the isolated hyphal apices of neurospora crassa under carbon starvation.		2016	18841815
fine-scale mapping in neurospora crassa by using genome-wide knockout strains.	fine-scale genetic mapping is often hindered by the lack of adequate markers surrounding the locus of interest. in the filamentous ascomycete neurospora crassa, the genome has been sequenced and an effort has been made to generate genome-wide deletion strains for the entire gene set. accordingly, the hygromycin-resistant marker in each deletion strain can be used as a mapping locus in a classical three-point cross, along with the mapping target and a standard marker. we have demonstrated the fea ...	2016	22067301
melanogenesis, tyrosinase expression, and reproductive differentiation in black and white truffles (ascomycotina).	white and black truffles of the genus tuber are ascomycotina as well as neurospora crassa, which expresses tyrosinase dependently on the reproductive cycle. tyrosinase expression dependent on reproductive differentiation has been also described in black truffles. we present novel and comparative work on melanogenic activities in black and white truffles that both express true tyrosinases. l-tyrosine 3-monooxygenase and l-dopa oxidase activities colocalize as histochemically detected and are simi ...	2016	9170162
potassium distribution in the neurospora crassa hypha.		2016	11029033
kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in neurospora.	kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. they have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions.	2016	10469561
fungal genome sequencing: basic biology to biotechnology.	the genome sequences provide a first glimpse into the genomic basis of the biological diversity of filamentous fungi and yeast. the genome sequence of the budding yeast, saccharomyces cerevisiae, with a small genome size, unicellular growth, and rich history of genetic and molecular analyses was a milestone of early genomics in the 1990s. the subsequent completion of fission yeast, schizosaccharomyces pombe and genetic model, neurospora crassa initiated a revolution in the genomics of the fungal ...	2016	25721271
directed evolution of xylose specific transporters to facilitate glucose-xylose co-utilization.	a highly active xylose specific transporter without glucose inhibition is highly desirable in cost-effective production of biofuels from lignocellulosic biomass. however, currently available xylose specific transporters suffer from low overall activity and most are inhibited by glucose. in this study, we applied a directed evolution strategy to engineer the xylose specific transporter an25 from neurospora crassa with improved xylose transportation capacity. after four rounds of directed evolutio ...	2016	26284760
molecular dissection of so (soft) protein in stress-induced aggregation and cell-to-cell interactive functions in filamentous fungal multicellularity.	filamentous fungi grow by organizing multicellularity through hyphal compartmentalization and cell fusion. so (soft) protein, which was originally identified in neurospora crassa, plays distinct functional roles in cell-to-cell interactions, such as septal plugging and cell fusion. we previously reported that aoso, an aspergillus oryzae so homologue, forms aggregates at the septal pore in response to stress, as well as upon hyphal wounding. however, the functional regions that mediate the multic ...	2016	27109373
identification of a novel l-rhamnose uptake transporter in the filamentous fungus aspergillus niger.	the study of plant biomass utilization by fungi is a research field of great interest due to its many implications in ecology, agriculture and biotechnology. most of the efforts done to increase the understanding of the use of plant cell walls by fungi have been focused on the degradation of cellulose and hemicellulose, and transport and metabolism of their constituent monosaccharides. pectin is another important constituent of plant cell walls, but has received less attention. in relation to th ...	2016	27984587
the introduction of the fungal d-galacturonate pathway enables the consumption of d-galacturonic acid by saccharomyces cerevisiae.	pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. one possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. however, the ethanol-producing organism of choice, saccharomyces cerevisiae, is not able to catabolize d-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, ...	2016	27538689
circadian clocks and the regulation of virulence in fungi: getting up to speed.	you cannot escape time. therefore, it seems wise to learn how to keep track of it and use it to your advantage. circadian clocks are molecular circuits that allow organisms to temporally coordinate a plethora of processes, including gene expression, with a close to 24h rhythm, optimizing cellular function in synchrony with daily environmental cycles. the molecular bases of these clocks have been extensively studied in the fungus neurospora crassa, providing a detailed molecular description. surp ...	2016	27039027
the interaction of fungi with the environment orchestrated by rnai.	the fungal kingdom has been key in the investigation of the biogenesis and function of small rnas (srnas). the discovery of phenomena such as quelling in neurospora crassa represents pioneering work in the identification of the main elements of the rna interference (rnai) machinery. recent discoveries in the regulatory mechanisms in some yeast and filamentous fungi are helping us reach a deeper understanding of the transcriptional and post-transcriptional gene-silencing mechanisms involved in ge ...	2016	26932186
a novel zn2 cys6 transcription factor bcgaar regulates d-galacturonic acid utilization in botrytis cinerea.	d-galacturonic acid (gala) is the most abundant monosaccharide component of pectin. previous transcriptome analysis in the plant pathogenic fungus botrytis cinerea identified eight gala-inducible genes involved in pectin decomposition, gala transport and utilization. co-expression of these genes indicates that a specific regulatory mechanism occurs in b. cinerea. in this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. the bclga1 promoter, cont ...	2016	26691528
the n-acetylglucosamine catabolic gene cluster in trichoderma reesei is controlled by the ndt80-like transcription factor ron1.	chitin is an important structural constituent of fungal cell walls composed of n-acetylglucosamine (glcnac) monosaccharides, but catabolism of glcnac has not been studied in filamentous fungi so far. in the yeast candida albicans, the genes encoding the three enzymes responsible for stepwise conversion of glcnac to fructose-6-phosphate are clustered. in this work, we analysed glcnac catabolism in ascomycete filamentous fungi and found that the respective genes are also clustered in these fungi. ...	2016	26481444
identification and characterization of a novel aspergillus fumigatus rhomboid family putative protease, rbda, involved in hypoxia sensing and virulence.	aspergillus fumigatus is the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. in invasive pulmonary aspergillosis, a. fumigatus spores are inhaled into the lungs, undergoing germination and invasive hyphal growth. the fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. the ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding ...	2016	27068092
structural divergence of the group i intron binding surface in fungal mitochondrial tyrosyl-trna synthetases that function in rna splicing.	the mitochondrial tyrosyl-trna synthetases (mttyrrss) of pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mitochondrial trna(tyr) and act as splicing cofactors for autocatalytic group i introns. previous studies showed that one of these proteins, neurospora crassa cyt-18, binds group i introns by using both its n-terminal catalytic and c-terminal anticodon binding domains and that the catalytic domain uses a newly evolved group i ...	2016	27036943
functional characterization of the woronin body protein wsca of the pathogenic mold aspergillus fumigatus.	woronin bodies are fungal-specific organelles that seal damaged hyphal compartments and thereby contribute to the stress resistance and virulence of filamentous fungi. in this study, we have characterized the aspergillus fumigatus woronin body protein wsca. wsca is homologous to neurospora crassa wsc, a protein that was shown to be important for biogenesis, segregation and positioning of woronin bodies. wsca and wsc both belong to the mpv17/pmp22 family of peroxisomal membrane proteins. an a. fu ...	2016	27016805
de-repression of csp-1 activates adaptive responses to antifungal azoles.	antifungal azoles are the major drugs that are used to treat fungal infections. this study found that in response to antifungal azole stress, neurospora crassa could activate the transcriptional responses of many genes and increase azole resistance by reducing the level of conidial separation 1 (csp-1), a global transcription repressor, at azole-responsive genes. the expression of csp-1 was directly activated by the transcription factors wc-1 and wc-2. upon ketoconazole (ktc) stress, the transcr ...	2016	26781458
small heat shock proteins, phylogeny in filamentous fungi and expression analyses in aspergillus nidulans.	small heat shock proteins (shsps) have been characterized in organisms from all three domains of life and viruses and are involved in a wide range of biological functions. however, the evolution and function of shsp in aspergillus species are largely unknown. in the present work, shsps were identified in 31 filamentous fungi, including species from aspergillus, penicillium, fusarium and magnaporthe, as well as botrytis cinerea and neurospora crassa. phylogenetic analysis revealed high level of d ...	2016	26403724
seeing the world differently: variability in the photosensory mechanisms of two model fungi.	light plays an important role for most organisms on this planet, serving either as a source of energy or information for the adaptation of biological processes to specific times of day. the fungal kingdom is estimated to contain well over a million species, possibly 10-fold more, and it is estimated that a majority of the fungi respond to light, eliciting changes in several physiological characteristics including pathogenesis, development and secondary metabolism. two model organisms for photobi ...	2016	26373782
molecular components of the neurospora crassa ph signaling pathway and their regulation by ph and the pac-3 transcription factor.	environmental ph induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. neurospora crassa shares all six components of the aspergillus nidulans ph signaling pathway, and we investigate here their regulation during an alkaline ph stress response. we show that the n. crassa pal mutant strains, with the exception of δpal-9, which is the a. nidulans pali homolog, exhibit low conidiation and are unable to grow at alkaline ph. ...	2016	27557053
light governs asexual differentiation in the grey mould fungus botrytis cinerea via the putative transcription factor bcltf2.	botrytis cinerea is a plant pathogenic fungus known for its utilization of light as environmental cue to regulate asexual differentiation: conidia are formed in the light, while sclerotia are formed in the dark. as no orthologues of known regulators of conidiation (e.g., aspergillus nidulans brla, neurospora crassa fl) exist in the leotiomycetes, we initiated a de novo approach to identify the functional counterpart in b. cinerea. the search revealed the light-responsive c2h2 transcription facto ...	2016	27347834
gln3 is a main regulator of nitrogen assimilation in candida glabrata.	after candida albicans, the yeast candida glabrata ranks second as an aetiological agent of candidaemia and is the most frequently encountered non-candida albicans species in patients with invasive candidiasis. transcriptome analysis in c. albicans, c. glabrata and cryptoccocus neoformans has revealed that, when engulfed by macrophages, these yeasts upregulate genes involved in nutrient acquisition, including nitrogen transporters such as the general amino acid permease gap1, the dicarboxylic am ...	2016	27222014
fungal sex: the ascomycota.	this article provides an overview of sexual reproduction in the ascomycetes, a phylum of fungi that is named after the specialized sacs or "asci" that hold the sexual spores. they have therefore also been referred to as the sac fungi due to these characteristic structures that typically contain four to eight ascospores. ascomycetes are morphologically diverse and include single-celled yeasts, filamentous fungi, and more complex cup fungi. the sexual cycles of many species, including those of the ...	2016	27763253
selection of pairings reaching evenly across the data (spread): a simple algorithm to design maximally informative fully crossed mating experiments.	we present a novel algorithm for the design of crossing experiments. the algorithm identifies a set of individuals (a 'crossing-set') from a larger pool of potential crossing-sets by maximizing the diversity of traits of interest, for example, maximizing the range of genetic and geographic distances between individuals included in the crossing-set. to calculate diversity, we use the mean nearest neighbor distance of crosses plotted in trait space. we implement our algorithm on a real dataset of ...	2016	26419337
the blue-light receptor cmwc-1 mediates fruit body development and secondary metabolism in cordyceps militaris.	light is an essential factor for pigment formation and fruit body development in cordyceps militaris, a well-known edible and medicinal fungus. cmwc-1, a homolog of the blue-light receptor gene white collar-1 (wc-1) in neurospora crassa, was cloned from the c. militaris genome in our previous study. here, cmwc-1 gene inactivation results in thicker aerial hyphae, disordered fruit body development, a significant reduction in conidial formation, and carotenoid and cordycepin production. these char ...	2016	26476643
loss of hp1 causes depletion of h3k27me3 from facultative heterochromatin and gain of h3k27me2 at constitutive heterochromatin.	methylated lysine 27 on histone h3 (h3k27me) marks repressed "facultative heterochromatin," including developmentally regulated genes in plants and animals. the mechanisms responsible for localization of h3k27me are largely unknown, perhaps in part because of the complexity of epigenetic regulatory networks. we used a relatively simple model organism bearing both facultative and constitutive heterochromatin, neurospora crassa, to explore possible interactions between elements of heterochromatin. ...	2016	26537359
repositioning of drugs using open-access data portal dtome: a test case with probenecid (review).	the one gene-one enzyme hypothesis, first introduced by beadle and tatum in the 1940s and based on their genetic analysis and observation of phenotype changes in neurospora crassa challenged by various experimental conditions, has witnessed significant advances in recent decades. much of our understanding of the association between genes and their phenotype expression has benefited from the completion of the human genome project, and has shown continual transformation guided by the effort direct ...	2016	26572802
fungal cobalamin-independent methionine synthase: insights from the model organism, neurospora crassa.	two families of methionine synthases, distinct in catalytic and structural features, have been encountered: meth, the cobalamin-dependent enzyme and mete, the cobalamin-independent form. the mete family is of mechanistic interest due to the chemically challenging nature of the reaction and is a potential target for antifungal therapeutics since the human genome encodes only meth. here we report the identification, purification, and crystal structure of mete from the filamentous fungus neurospora ...	2016	26657067
heterologous expression of a gh3 β-glucosidase from neurospora crassa in pichia pastoris with high purity and its application in the hydrolysis of soybean isoflavone glycosides.	previous studies have shown isoflavone aglycones to have more biological effects than their counterparts, isoflavone glycones. some β-glucosidases can hydrolyze isoflavone glucosides to release aglycones, and discovery of these has attracted great interest. a glycoside hydrolase (gh) family 3 β-glucosidase (bgl2) gene from neurospora crassa was heterologously expressed in pichia pastoris with high purity. the recombinant bgl2 enzyme displayed its highest activity at ph 5.0 and 60 °c, and had its ...	2016	26596358
antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing.	in eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. we showed previously that antisense transcription triggers dicer-independent sirna (disirna) production and disirna locus dna methylation (dldm) in neurospora crassa here we show that the conserved exonuclease eri-1 (enhanced rnai-1) is a critical component in this process. antisense transcription and eri-1 binding to target rnas are necessary and sufficient to trigger dldm. converge ...	2016	27856616
genome-wide a-to-i rna editing in fungi independent of adar enzymes.	yeasts and filamentous fungi do not have adenosine deaminase acting on rna (adar) orthologs and are believed to lack a-to-i rna editing, which is the most prevalent editing of mrna in animals. however, during this study with the puk1(fgrres_01058) pseudokinase gene important for sexual reproduction in fusarium graminearum, we found that two tandem stop codons, ua(1831)gua(1834)g, in its kinase domain were changed to ug(1831)gug(1834)g by rna editing in perithecia. to confirm a-to-i editing of pu ...	2016	26934920
the tetraspanin tsp3 of neurospora crassa is a vacuolar membrane protein and shares characteristics with idi proteins.	the fungal vacuole is an organelle, which adopts pleiotropic morphologies and functions. in aging and starving hyphae it is the compartment of degradation and recycling of cellular constituents. here we identified tsp3, one of three tetraspanins present in the filamentous ascomycete fungus neurospora crassa, as a vacuolar membrane protein. the protein is detected only in aging and starving cultures and under other conditions, which induce autophagy, such as vegetative incompatibility or the pres ...	2016	26908649
lac-1 and lag-1 with ras-1 affect aging and the biological clock in neurospora crassa.	using an automated cell counting technique developed previously (case et al., ecology and evolution 2014; 4: 3494), we explore the lifespan effects of lac-1, a ceramide synthase gene paralogous to lag-1 in neurospora crassa in conjunction with the band bd (ras-1) gene. we find that the replicative lifespan of a lac-1(ko)bd double mutants is short, about one race tube cycle, and this double mutant lacks a strong ~21-hr clock cycle as shown by race tube and fluorometer analysis of fluorescent stra ...	2016	28031787
hyphal ontogeny in neurospora crassa: a model organism for all seasons.	filamentous fungi have proven to be a better-suited model system than unicellular yeasts in analyses of cellular processes such as polarized growth, exocytosis, endocytosis, and cytoskeleton-based organelle traffic. for example, the filamentous fungus neurospora crassa develops a variety of cellular forms. studying the molecular basis of these forms has led to a better, yet incipient, understanding of polarized growth. polarity factors as well as rho gtpases, septins, and a localized delivery of ...	2016	27990280
cations optimization for protein enrichment in rice straw by mixed cultures of neurospora crassa 14-8 and candida utilis using response surface methodology.	this study investigated the effect of cation optimization by mixed cultures of neurospora crassa and candida utilis on the true protein (tp) content. firstly, to enhance the nutritional contents of rice straw (rs), two fermentation parameters (effect of inoculation ratio and inoculation time) were optimized. it was found that when c. utilis was inoculated 60 h later than n. crassa with the inoculation ratio of 1:5 (n. crassa to c. utilis), the maximum tp yield was obtained. in order to further o ...	2016	27987187
the calmodulin gene in neurospora crassa is required for normal vegetative growth, ultraviolet survival, and sexual development.	we isolated a neurospora crassa mutant of the calmodulin (cmd) gene using repeat-induced point mutation and studied its phenotypes. the cmd (rip) mutant showed a defect in growth, reduced aerial hyphae, decreased carotenoid accumulation, a severe reduction in viability upon ultraviolet (uv) irradiation, and a fertility defect. moreover, meiotic silencing of the cmd gene resulted in a barren phenotype. in addition, we also performed site-directed mutational analysis of the calcium/calmodulin-depe ...	2016	27888323
glucose starvation alters heat shock response, leading to death of wild type cells and survival of map kinase signaling mutant.	a moderate heat shock induces neurospora crassa to synthesize large quantities of heat shock proteins that are protective against higher, otherwise lethal temperatures. however, wild type cells do not survive when carbohydrate deprivation is added to heat shock. in contrast, a mutant strain defective in a stress-activated protein kinase does survive the combined stresses. in order to understand the basis for this difference in survival, we have determined the relative levels of detected proteins ...	2016	27870869
normal chromosome conformation depends on subtelomeric facultative heterochromatin in neurospora crassa.	high-throughput chromosome conformation capture (hi-c) analyses revealed that the 3d structure of the neurospora crassa genome is dominated by intra- and interchromosomal links between regions of heterochromatin, especially constitutive heterochromatin. elimination of trimethylation of lysine 9 on histone h3 (h3k9me3) or its binding partner heterochromatin protein 1 (hp1)-both prominent features of constitutive heterochromatin-have little effect on the hi-c pattern. it remained possible that di- ...	2016	27856763
[influence of oxygenated derivatives of linoleic and linolenic acids on the formation of conidia and protoperithecia in wild-type and photoreceptor complex mutants of neurospora crassa].	the regulatory effect of two oxyderivatives of unsaturated fatty acids (oxylipins), 18-hydroxy-(9z,12z)-octadecadienoic acid (18-hode) and 18-(9z,12z,15z)-octadecatrienoic acid (18-hotre), on the sexual and asexual sporulation of wild-type neurospora crassa strains and wc-1 and wc-1 mutants was studied. in the wild-type strain, 18-hode, unlike 18-hotre, stimulated protoperithecia formation in the dark and in the light. in the same strain, the studied oxylipins influenced conidiagenesis only unde ...	2016	26859959
de novo rna synthesis by rna-dependent rna polymerase activity of telomerase reverse transcriptase.	rna-dependent rna polymerase (rdrp) plays key roles in rna silencing to generate double-stranded rnas. in model organisms, such as caenorhabditis elegans and neurospora crassa, two types of small interfering rnas (sirnas), primary sirnas and secondary sirnas, are expressed; rdrp produces secondary sirnas de novo, without using either dicer or primers, while primary sirnas are processed by dicer. we reported that human telomerase reverse transcriptase (tert) has rdrp activity and produces endogen ...	2016	26830230
meiotic recombination in neurospora crassa proceeds by two pathways with extensive holliday junction migration.	analysis of thousands of δmsh-2 octads using our fluorescent recombination system indicates that, as in other filamentous fungi, symmetric heteroduplex is common in the his-3 region of neurospora crassa. symmetric heteroduplex arises from holliday junction migration, and we suggest this mechanism explains the high frequency of his+ spores in heteroallelic crosses in which recombination is initiated cis to the his-3 allele further from the initiator, cog+. in contrast, when recombination is initi ...	2016	26812651
localization and role of myo-1, an endocytic protein in hyphae of neurospora crassa.	the subapical endocytic collar is a prominent feature of hyphae of neurospora crassa. it comprises a dynamic collection of actin patches associated with a number of proteins required for endocytosis, namely, arp-2/3 complex, fimbrin, coronin, etc. we presently show that myo-1 is another key component of this endocytic collar. a myo-1 sequence was identified in the genome of n. crassa and used it to generate a strain with a myo-1-sgfp allele under the ccg1 promoter. examination of living hyphae b ...	2016	26805950
antifungal mechanisms of a plant defensin mtdef4 are not conserved between the ascomycete fungi neurospora crassa and fusarium graminearum.	defensins play an important role in plant defense against fungal pathogens. the plant defensin, mtdef4, inhibits growth of the ascomycete fungi, neurospora crassa and fusarium graminearum, at micromolar concentrations. we have reported that mtdef4 is transported into the cytoplasm of these fungi and exerts its antifungal activity on intracellular targets. here, we have investigated whether the antifungal mechanisms of mtdef4 are conserved in these fungi. we show that n. crassa and f. graminearum ...	2016	26801962
the lsh/ddm1 homolog mus-30 is required for genome stability, but not for dna methylation in neurospora crassa.	lsh/ddm1 enzymes are required for dna methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. the filamentous fungus neurospora crassa is a tractable system that encodes a single lsh/ddm1 homolog (ncu06306). we report that the neurospora lsh/ddm1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. we show that mus-30-deficient cells have normal dna methylation, but are hypersensi ...	2016	26771905
o-glycan analysis of cellobiohydrolase i from neurospora crassa.	we describe here the composition of the o-linked glycans on the neurospora crassa cellobiohydrolase i (cbhi), which accounts for approximately 40% of the protein secreted by cells growing in the presence of cellulose. cbhi is o-glycosylated with six types of linear, and three types of branched, o-glycans containing approximately equal amounts of mannose and galactose. in addition to the classic fungal o-glycans with reducing end mannoses, we also identified reducing end galactoses which suggest ...	2016	26762173
neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan.	chitosan is a natural polymer with antimicrobial activity. chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ros) in neurospora crassa. we have determined the transcriptional profile of n. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. activation of oxidative metabolism ...	2016	26694141
identification of critical amino acid residues and functional conservation of the neurospora crassa and rattus norvegicus orthologues of neuronal calcium sensor-1.	neuronal calcium sensor-1 (ncs-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (ca(2+)) binding ef-hand domains. we performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the n-terminal myristoylation, a conserved glutamic acid residue responsible for ca(2+) binding in the third ef-hand (ef3), and an unusual non-conserved amino acid argini ...	2016	27796528
accumulation of specific sterol precursors targets a map kinase cascade mediating cell-cell recognition and fusion.	sterols are vital components of eukaryotic cell membranes. defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. however, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. in this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-c ...	2016	27708165
a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus gloeophyllum trabeum and its action on cellulose-xyloglucan complexes.	fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (lpmos) to degrade plant polysaccharides. brown-rot fungi, such as gloeophyllum trabeum, tend to have few lpmos, and information on these enzymes is scarce. the genome of g. trabeum encodes four auxiliary activity 9 (aa9) lpmos (gtlpmo9s), whose coding sequences were amplified from cdna. due to alternative splicing, two variants of gtlpmo9a seem to be produced, a single-domain variant, gtlpmo9a-1, and a longer va ...	2016	27590806