Overexpression of the limk1 Gene in Drosophila melanogaster Can Lead to Suppression of Courtship Memory in Males.

Courtship suppression is a behavioral adaptation of the fruit fly. When majority of the females in a fly population are fertilized and non-receptive for mating, a male, after a series of failed attempts, decreases its courtship activity towards all females, saving its energy and reproductive resources. The time of courtship decrease depends on both duration of unsuccessful courtship and genetically determined features of the male nervous system. Thereby, courtship suppression paradigm can be used for studying molecular mechanisms of learning and memory. p-Cofilin, a component of the actin remodeling signaling cascade and product of LIM-kinase 1 (LIMK1), regulates Drosophila melanogaster forgetting in olfactory learning paradigm. Previously, we have shown that limk1 suppression in the specific types of nervous cells differently affects fly courtship memory. Here, we used Gal4 > UAS system to induce limk1 overexpression in the same types of neurons. limk1 activation in the mushroom body, glia, and fruitless neurons decreased learning index compared to the control strain or the strain with limk1 knockdown. In cholinergic and dopaminergic/serotoninergic neurons, both overexpression and knockdown of limk1 impaired Drosophila short-term memory. Thus, proper balance of the limk1 activity is crucial for normal cognitive activity of the fruit fly.

Positive experience with TNF-α inhibitor in toxic epidermal necrolysis resistant to high-dose systemic corticosteroids.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare, potentially life-threatening syndromes characterized by the development of necrotic epidermal and mucosal lesions. The most common etiologic cause of SJS/TEN is drug-induced mechanisms. The group of drugs with high potential risk includes sulfonamides, anticonvulsants, non-steroidal anti-inflammatory drugs (NSAIDs), allopurinol, phenobarbital, etc. There is no gold standard treatment algorithm for SJS/TEN. In medical practice, systemic glucocorticosteroids (sGCS), intravenous immunoglobulin (IVIG), plasmapheresis, and cyclosporine are used empirically and in various combinations. Recently published studies have demonstrated the efficacy of TNF-α inhibitors as a promising approach in SJS/TEN, including cases resistant to high-dose sGCS, with etanercept and infliximab being the most commonly used drugs. In a large multicenter study by Zhang J et al. (XXXX), 242 patients treated with etanercept, sGCS, or a combination of both had lower mortality compared to the control group. A shorter skin healing time was documented compared to sGCS monotherapy, thus reducing the risk of secondary infections. The published data show a high efficacy with THF-α inhibitor blockade, but the safety of TNF-α inhibitors in patients with SJS/TEN is still questionable due to the paucity of available information. As all clinical research data should be accumulated to provide reliable evidence that the use of TNF-α inhibitors may be beneficial in SJS/TEN, we report a case of etoricoxib-associated SJS with progression to TEN in a 50-year-old woman who was refractory to high-dose sGCS therapy.

Erratum: Aegilops crassa Boiss. repeatome characterized using low-coverage NGS as a source of new FISH markers: application in phylogenetic studies of the Triticeae.

[This corrects the article DOI: 10.3389/fpls.2022.980764.].

Aegilops crassa Boiss. repeatome characterized using low-coverage NGS as a source of new FISH markers: Application in phylogenetic studies of the Triticeae.

Aegilops crassa Boiss. is polyploid grass species that grows in the eastern part of the Fertile Crescent, Afghanistan, and Middle Asia. It consists of tetraploid (4x) and hexaploid (6x) cytotypes (2n = 4x = 28, D1D (Abdolmalaki et al., 2019) XcrXcr and 2n = 6x = 42, D1D (Abdolmalaki et al., 2019) XcrXcrD2D (Adams and Wendel, 2005), respectively) that are similar morphologically. Although many Aegilops species were used in wheat breeding, the genetic potential of Ae. crassa has not yet been exploited due to its uncertain origin and significant genome modifications. Tetraploid Ae. crassa is thought to be the oldest polyploid Aegilops species, the subgenomes of which still retain some features of its ancient diploid progenitors. The D1 and D2 subgenomes of Ae. crassa were contributed by Aegilops tauschii (2n = 2x = 14, DD), while the Xcr subgenome donor is still unknown. Owing to its ancient origin, Ae. crassa can serve as model for studying genome evolution. Despite this, Ae. crassa is poorly studied genetically and no genome sequences were available for this species. We performed low-coverage genome sequencing of 4x and 6x cytotypes of Ae. crassa, and four Ae. tauschii accessions belonging to different subspecies; diploid wheatgrass Thinopyrum bessarabicum (Jb genome), which is phylogenetically close to D (sub)genome species, was taken as an outgroup. Subsequent data analysis using the pipeline RepeatExplorer2 allowed us to characterize the repeatomes of these species and identify several satellite sequences. Some of these sequences are novel, while others are found to be homologous to already known satellite sequences of Triticeae species. The copy number of satellite repeats in genomes of different species and their subgenome (D1 or Xcr) affinity in Ae. crassa were assessed by means of comparative bioinformatic analysis combined with quantitative PCR (qPCR). Fluorescence in situ hybridization (FISH) was performed to map newly identified satellite repeats on chromosomes of common wheat, Triticum aestivum, 4x and 6x Ae. crassa, Ae. tauschii, and Th. bessarabicum. The new FISH markers can be used in phylogenetic analyses of the Triticeae for chromosome identification and the assessment of their subgenome affinities and for evaluation of genome/chromosome constitution of wide hybrids or polyploid species.

The Association of Grain Yield and Agronomical Traits with Genes of Plant Height, Photoperiod Sensitivity and Plastid Glutamine Synthetase in Winter Bread Wheat (Triticum aestivum L.) Collection.

The reduction in plant height caused by mutations in Rht-B1 or Rht-D1 (Reduced height-1) genes in combination with day-length-independent early flowering associated with the Ppd-D1 (Photoperiod-D1) gene were the main factors of the drastic yield increase in bread wheat in the 1960s. Increasing nitrogen use efficiency as well as maintaining high yields under conditions of global climate change are the modern goals of wheat breeding. The glutamine synthetase (GS) enzyme plays a key role in ammonium assimilation in plants. In previous studies, the TaGS2-A1 gene, coding the plastid isoform of GS, was shown to be connected with nitrogen use efficiency in wheat. Using the polymerase chain reaction (PCR) markers, the association of yield and agronomical traits with haplotypes of Rht-B1, Rht-D1, Ppd-D1 and TaGS2-A1 genes was studied in a diverse collection of winter bread wheat cultivars grown in Krasnodar (Russia). In the three-year experiment, semidwarfism and photoperiod insensitivity were confirmed to be highly favorable for the grain yield. The TaGS2-A1b haplotype had a tendency for increased grain yield and lodging resistance, but mainly in plants not possessing the 'green revolution' alleles. Thus, TaGS2-A1b may have potential in breeding wheat cultivars with alternative dwarfing genes or tall cultivars, which may be optimal for growing under certain environments.

cd1 Mutation in Drosophila Affects Phenoxazinone Synthase Catalytic Site and Impairs Long-Term Memory.

Being involved in development of Huntington's, Parkinson's and Alzheimer's diseases, kynurenine pathway (KP) of tryptophan metabolism plays a significant role in modulation of neuropathology. Accumulation of a prooxidant 3-hydroxykynurenine (3-HOK) leads to oxidative stress and neuronal cell apoptosis. Drosophila mutant cardinal (cd1) with 3-HOK excess shows age-dependent neurodegeneration and short-term memory impairments, thereby presenting a model for senile dementia. Although cd gene for phenoxazinone synthase (PHS) catalyzing 3-HOK dimerization has been presumed to harbor the cd1 mutation, its molecular nature remained obscure. Using next generation sequencing, we have shown that the cd gene in cd1 carries a long deletion leading to PHS active site destruction. Contrary to the wild type Canton-S (CS), cd1 males showed defective long-term memory (LTM) in conditioned courtship suppression paradigm (CCSP) at days 5-29 after eclosion. The number of dopaminergic neurons (DAN) regulating fly locomotor activity showed an age-dependent tendency to decrease in cd1 relative to CS. Thus, in accordance with the concept "from the gene to behavior" proclaimed by S. Benzer, we have shown that the aberrant PHS sequence in cd1 provokes drastic LTM impairments and DAN alterations.

Genes Responsible for H2S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster.

The gasotransmitter hydrogen sulfide (H2S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H2S production and metabolism. Here, we investigated the role of H2S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation-reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.

3-Hydroxykynurenine as a Potential Ligand for Hsp70 Proteins and Its Effects on Drosophila Memory After Heat Shock.

Kynurenine products of tryptophan metabolism are modifiers of the nervous activity and oxidative processes in mammals and invertebrates. 3-Hydroxykynurenine (3HOK) in moderate concentrations is a lipid peroxidation inhibitor. However, its accumulation and oxidative auto-dimerization lead to oxidative stress development manifested in age-related neurodegenerative diseases (NDD) and neurological disorders provoked by acute stress. Different forms of stress, the mostly studied being heat shock response, rely on functioning of heat shock proteins of the Hsp70 superfamily. Since kynurenines are called "kids of stress," we performed computational estimation of affinity of 3HOK and other kynurenines binding to predicted ATP site of Drosophila melanogaster Hsp cognate 71 protein (Dhsp71) using AutoDock Vina. The binding energy of 3HOK dimer is - 9.4 kcal/mol; its orientation within the active site is close to that of ATP. This might be a new mechanism of producing a competitive inhibitor of Hsp70 chaperones that decreases organism ability to adapt to heat shock. We also showed that the Drosophila cardinal (cd1) mutant with 3HOK excess, serving as a model for Huntington's disease (HD), manifests severe defects of short-term memory after heat shock applied either in adults or at the prepupal stage.

qPCR as a Selective Tool for Cytogenetics.

qPCR is widely used in quantitative studies of plant genomes and transcriptomes. In this article, this method is considered as an auxiliary step in the preparation and selection of markers for FISH analysis. Several cases from the authors' research on populations of the same species were reviewed, and a comparison of the closely related species, as well as the adaptation of the markers, based on satellite tandem repeats (TRs) using quantitative qPCR data was conducted. In the selected cases, TRs with contrast abundance were identified in the cases of the Dasypyrum, Thinopyrum and Aegilops species, and the transfer of TRs between the wheat and related species was demonstrated. TRs with intraspecific copy number variation were revealed in Thinopyrum ponticum and wheat-wheatgrass partial amphidiploids, and the TR showing predominant hybridization to the sea buckthorn Y chromosome was identified. Additionally, problems such as the absence of a reference gene for qPCR, and low-efficiency and self-complementary primers, were illustrated. In the cases considered here, the qPCR results clearly show high correlation with the subsequent results of the FISH analysis, which confirms the value of this method for cytogenetic studies.

Chromatin Structure and "DNA Sequence View": The Role of Satellite DNA in Ectopic Pairing of the Drosophila X Polytene Chromosome.

Chromatin 3D structure plays a crucial role in regulation of gene activity. Previous studies have envisioned spatial contact formations between chromatin domains with different epigenetic properties, protein compositions and transcription activity. This leaves specific DNA sequences that affect chromosome interactions. The Drosophila melanogaster polytene chromosomes are involved in non-allelic ectopic pairing. The mutant strain agnts3, a Drosophila model for Williams-Beuren syndrome, has an increased frequency of ectopic contacts (FEC) compared to the wild-type strain Canton-S (CS). Ectopic pairing can be mediated by some specific DNA sequences. In this study, using our Homology Segment Analysis software, we estimated the correlation between FEC and frequency of short matching DNA fragments (FMF) for all sections of the X chromosome of Drosophila CS and agnts3 strains. With fragment lengths of 50 nucleotides (nt), CS showed a specific FEC-FMF correlation for 20% of the sections involved in ectopic contacts. The correlation was unspecific in agnts3, which may indicate the alternative epigenetic mechanisms affecting FEC in the mutant strain. Most of the fragments that specifically contributed to FMF were related to 1.688 or 372-bp middle repeats. Thus, middle repetitive DNA may serve as an organizer of ectopic pairing.

Development of new cytogenetic markers for Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang.

Thinopyrum ponticum (Podpera, 1902) Z.-W. Liu & R.-C.Wang, 1993 is an important polyploid wild perennial Triticeae species that is widely used as a source of valuable genes for wheat but its genomic constitution has long been debated. For its chromosome identification, only a limited set of FISH probes has been used. The development of new cytogenetic markers for Th. ponticum chromosomes is of great importance both for cytogenetic characterization of wheat-wheatgrass hybrids and for fundamental comparative studies of phylogenetic relationships between species. Here, we report on the development of five cytogenetic markers for Th. ponticum based on repetitive satellite DNA of which sequences were selected from the whole genome sequence of Aegilops tauschii Cosson, 1849. Using real-time quantitative PCR we estimated the abundance of the found repeats: P720 and P427 had the highest abundance and P132, P332 and P170 had lower quantity in Th. ponticum genome. Using fluorescence in situ hybridization (FISH) we localized five repeats to different regions of the chromosomes of Th. ponticum. Using reprobing multicolor FISH we colocalized the probes between each other. The distribution of these found repeats in the Triticeae genomes and its usability as cytogenetic markers for chromosomes of Th. ponticum are discussed.

Drosophila Model for the Analysis of Genesis of LIM-kinase 1-Dependent Williams-Beuren Syndrome Cognitive Phenotypes: INDELs, Transposable Elements of the Tc1/Mariner Superfamily and MicroRNAs.

Genomic disorders, the syndromes with multiple manifestations, may occur sporadically due to unequal recombination in chromosomal regions with specific architecture. Therefore, each patient may carry an individual structural variant of DNA sequence (SV) with small insertions and deletions (INDELs) sometimes less than 10 bp. The transposable elements of the Tc1/mariner superfamily are often associated with hotspots for homologous recombination involved in human genetic disorders, such as Williams Beuren Syndromes (WBS) with LIM-kinase 1-dependent cognitive defects. The Drosophila melanogaster mutant agnts3 has unusual architecture of the agnostic locus harboring LIMK1: it is a hotspot of chromosome breaks, ectopic contacts, underreplication, and recombination. Here, we present the analysis of LIMK1-containing locus sequencing data in agnts3 and three D. melanogaster wild-type strains-Canton-S, Berlin, and Oregon-R. We found multiple strain-specific SVs, namely, single base changes and small INDEls. The specific feature of agnts3 is 28 bp A/T-rich insertion in intron 1 of LIMK1 and the insertion of mobile S-element from Tc1/mariner superfamily residing ~460 bp downstream LIMK1 3'UTR. Neither of SVs leads to amino acid substitutions in agnts3 LIMK1. However, they apparently affect the nucleosome distribution, non-canonical DNA structure formation and transcriptional factors binding. Interestingly, the overall expression of miRNAs including the biomarkers for human neurological diseases, is drastically reduced in agnts3 relative to the wild-type strains. Thus, LIMK1 DNA structure per se, as well as the pronounced changes in total miRNAs profile, probably lead to LIMK1 dysregulation and complex behavioral dysfunctions observed in agnts3 making this mutant a simple plausible Drosophila model for WBS.

Combination of hypomorphic mutations of the Drosophila homologues of aryl hydrocarbon receptor and nucleosome assembly protein family genes disrupts morphogenesis, memory and detoxification.

Aryl hydrocarbon receptor is essential for biological responses to endogenous and exogenous toxins in mammals. Its Drosophila homolog spineless plays an important role in fly morphogenesis. We have previously shown that during morphogenesis spineless genetically interacts with CG5017 gene, which encodes a nucleosome assembly factor and may affect cognitive function of the fly. We now demonstrate synergistic interactions of spineless and CG5017 in pathways controlling oxidative stress response and long-term memory formation in Drosophila melanogaster. Oxidative stress was induced by low doses of X-ray irradiation of flies carrying hypomorphic mutation of spineless, mutation of CG5017, and their combination. To determine the sensitivity of these mutants to pharmacological modifiers of the irradiation effect, we irradiated flies growing on standard medium supplemented by radiosensitizer furazidin and radioprotector serotonin. The effects of irradiation were investigated by analyzing leg and antenna morphological structures and by using real-time PCR to measure mRNA expression levels for spineless, Cyp6g1 and Gst-theta genes. We also examined long-term memory in these mutants using conditioned courtship suppression paradigm. Our results show that the interaction of spineless and CG5017 is important for regulation of morphogenesis, long-term memory formation, and detoxification during oxidative stress. Since spineless and CG5017 are evolutionary conserved, these results must be considered when evaluating the risk of combining similar mutations in other organisms, including humans.

The 1:1 host-guest complexation between cucurbit[7]uril and styryl dye.

The photophysical properties of aqueous solution of styryl dye, 4-[(E)-2-(3,4-dimethoxyphenyl)ethenyl]-1-ethylpyridinium perchlorate (dye 1), in the presence of cucurbit[7]uril (CB[7]) was studied by means of fluorescence spectroscopy methods. The production of 1:1 host-guest complexes in the range of CB[7] concentrations up to 16 µM with K = 1.0 × 10(6) M(-1) has been observed, which corresponds to appearance of the isosbestic point at 396 nm in the absorption spectra and a 5-fold increase in fluorescence intensity. The decay of fluorescence was found to fit to double-exponential functions in all cases; the calculated average fluorescence lifetime increases from 145 to 352 ps upon the addition of CB[7]. Rotational relaxation times of dye 1 solutions 119 ± 14 ps without CB[7] and 277 ± 35 ps in the presence of CB[7] have been determined by anisotropy fluorescence method. The comparison of the results of quantum-chemical calculations and experimental data confirms that in the host cavity dye 1 rotates as a whole with CB[7].

A tricotage-like failure of nanographene.

The response of a nanographene sheet to external stresses was considered in terms of a mechanochemical reaction. The quantum chemical realization of the approach was based on the coordinate-of-reaction concept for the purpose of introducing a mechanochemical internal coordinate (MIC) that specifies a deformational mode. The related force of response is calculated as the energy gradient along the MIC, while the atomic configuration is optimized over all of the other coordinates under the MIC constant-pitch elongation. The approach is applied to the benzene molecule and (5,5) nanographene. A drastic anisotropy in the microscopic behavior of both objects under elongation along a MIC was observed when the MIC was oriented either along or normally to the C-C bond chain. Both the anisotropy and the high stiffness of the nanographene originate from the response of the benzenoid unit to stress.

Implicit electrostatic solvent model with continuous dielectric permittivity function.

The modification of the electrostatic continuum solvent model considered in the present work is based on the exact solution of the Poisson equation, which can be constructed provided that the dielectric permittivity epsilon of the total solute and solvent system is an isotropic and continuous spatial function. This assumption allows one to formulate a numerically efficient and universal computational scheme that covers the important case of a variable epsilon function inherent to the solvent region. The obtained type of solution is unavailable for conventional dielectric continuum models such as the Onsager and Kirkwood models for spherical cavities and the polarizable continuum model (PCM) for solute cavities of general shape, which imply that epsilon is discontinuous on the boundary confining the excluded volume cavity of the solute particle. Test computations based on the present algorithm are performed for water and several nonaqueous solvents. They illustrate specific features of this approach, called the "smooth boundary continuum model" (SBCM), as compared to the PCM procedure, and suggest primary tentative results of its parametrization for different solvents. The calculation for the case of a binary solvent mixture with variable epsilon in the solvent space region demonstrates the applicability of this approach to a novel application field covered by the SBCM.

Molecular simulation of solvent-induced stokes shift in absorption/emission spectra of organic chromophores.

The values of steady-state solvatochromic Stokes shifts (SS) in absorption/emission electronic spectra of organic chromophores are studied theoretically in the framework of the Hush-Marcus model. Charge distributions for chromophore solutes in their S0 and S1 states are found by means of conventional quantum-chemical methods combined with the continuum PCM approach for treating solvation effects. The solvent reorganization energies, which are expected to correlate with the solvent-induced part of 1/2 SS, are found in a molecular dynamics (MD) simulation which invokes a novel method for separation of the inertial piece of the electrostatic response (Vener, et al. J. Phys. Chem. B 2006, 110, 14950). Computations, performed in two solvents (acetonitrile and benzene), consider three organic dyes: coumarin 153 as a benchmark system and two other chromophores, for which experimental spectra are also reported. The results are found to be in reasonable agreement with the experiment. A consistent treatment of nonlinear effect in the solvent response, promoted by the polarizability of solutes and contributing to the solvent reorganization energies (Ingrosso, et al. J. Phys. Chem. B 2005, 109, 3553), improves the results of computations.

Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour.

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.