The temperature-sensitive Drosophila mutant agnts3 exhibits the restoration of learning defects both after heat shock (HS) and under hypomagnetic conditions (HMC). Previously, agnts3 was shown to have an increased level of LIM kinase 1 (LIMK1). However, its limk1 sequence did not significantly differ from that of the wild-type strain Canton-S (CS). Here, we performed whole-genome and poly(A)-enriched transcriptome sequencing of CS and agnts3 males normally, after HMC, and after HS. Several high-effect agnts3-specific mutations were identified, including MED23 (regulation of HS-dependent transcription) and Spn42De, the human orthologs of which are associated with intellectual disorders. Pronounced interstrain differences between the transcription profiles were revealed. Mainly, they included the genes of defense and stress response, long non-coding RNAs, and transposons. After HS, the differences between the transcriptomes became less pronounced. In agnts3, prosalpha1 was the only gene whose expression changed after both HS and HMC. The normal downregulation of prosalpha1 and Spn42De in agnts3 was confirmed by RT-PCR. Analysis of limk1 expression did not reveal any interstrain differences or changes after stress. Thus, behavioral differences between CS and agnts3 both under normal and stressed conditions are not due to differences in limk1 transcription. Instead, MED23, Spn42De, and prosalpha1 are more likely to contribute to the agnts3 phenotype.

M. E. Lobashev and V. B. Savvateev’s research laid the foundation for studying the relationship between neuroplasticity and reaction to extreme conditions. Common mechanisms underlying adaptive reactions and learning have been proven to exist. Hypoxia is one of the most common damaging factors in various adverse external and internal effects. Severe forms of hypoxia suppress neuroplasticity and cause learning and memory disorders. Chromatin remodelling and expression of genes involved in memory formation and learning may require double-stranded DNA breaks because they accompany intense matrix processes in neurogenesis and serve as indicators of physiological neuronal activity. Stimulation of regulatory cascades involved in learning has an impact on adaptive response formation. For instance, metabolites of the kynurenine pathway of tryptophan metabolism affect the synaptic plasticity processes that regulate memory formation and learning. This article studies the effect of hypoxia on the state of chromosomal apparatus in Drosophila cd mutant (accumulation of 3-hydroxykynurenine). We discover interlinear differences in the frequency of double-stranded DNA breaks following exposure to hypoxia in mutant cd and wild-type CS strain. Obtained data are discussed in terms of the relationship between neuroplasticity processes, circadian rhythm regulation, and mechanisms for adapting to extreme conditions.

Исследования М. Е. Лобашева и В. Б. Савватеева положили начало изучению взаимосвязи между нейропластичностью и развитием реакций на экстремальное воздействие. В настоящее время доказано наличие общих механизмов, лежащих в основе формирования адаптивных реакций и обучения. Гипоксия — один из наиболее распространенных повреждающих факторов при различных неблагоприятных внешних и внутренних воздействиях. Тяжелые формы гипоксии подавляют процессы нейропластичности, вызывают нарушения обучения и памяти. При ремоделировании хроматина и экспрессии генов, вовлеченных в процессы формирования памяти и обучения, возможно, необходимы двухцепочечные разрывы ДНК, поскольку они сопутствуют интенсивным матричным процессам при нейрогенезе и являются показателем физиологической активности нейронов. Стимуляция регуляторных каскадов, участвующих в обучении, влияет на формирование адаптивного ответа. Так, метаболиты кинуренинового пути обмена триптофана влияют на процессы синаптической пластичности, регулирующие формирование памяти и обучение. В настоящей работе изучено влияние гипоксии на состояние хромосомного аппарата у мутанта сd (накопление 3-гидроксикинуренина) дрозофилы. Выявлены межлинейные различия частоты двухцепочечных разрывов ДНК после воздействия гипоксии у мутанта сd и линии дикого типа CS. Данные обсуждаются в свете взаимосвязи процессов нейропластичности, регуляции циркадного ритма и механизмов адаптации к экстремальным воздействиям.

В статье приводится краткий очерк истории учения И. П. Павлова об условных рефлексах, на долгие годы определившего программу исследований института, руководимого академиком Л. А. Орбели, и его значения в развитии новых научных направлений — генетики поведения (высшей нервной деятельности), нейрогенетики, физиологической генетики. Приведены сведения о роли профессора М. Е. Лобашева, выявлении и подчеркивании новых граней в учении И. П. Павлова об условных рефлексах, показавшего значение механизма условного рефлекса в координации многоклеточного организма в процессе тканевой и клеточной дифференциации, в ускорении адаптации организма к экстремальным факторам внешней среды (и таким образом «в какой-то мере выводящего организм из-под контроля элиминирующих факторов внешней среды»), функциональной преемственности в передаче индивидуально приобретаемого опыта между поколениями (и таким образом обеспечивая «сигнальную наследственность», необычайно расширившую приспособительные возможности организма). Описывается вклад профессоров М. Е. Лобашева и В. В. Пономаренко в дальнейшее развитие теории нервизма И. П. Павлова, гипотетически предположивших и экспериментально подтвердивших существенную роль нервной системы в регуляции активности генетического аппарата, в том числе самих нейронов по принципу обратной связи в соответствии с текущими нуждами организма, требованиями внешней среды и индивидуальным опытом. Освещены новые современные направления в исследованиях генетики поведения и нейрогенетики. Подчеркивается мировое признание учения И. П. Павлова об уcловных рефлексах и его роль в анализе теоретических генетических, биологических и физиологических проблем, в практике сельского хозяйства, медицины и педагогики.

M.E. Lobashev and V.В. Savvateev in 1959 obtained unique data on the expansion of the adaptive capabilities of the organism when training the properties of higher nervous activity by the formation of conditioned food reflexes to stimuli that exhaust the nervous system. Apparently, the formation of a conditioned connection help to overcoming stressful effects, adaptation to restrictive conditions, and changes in the functioning of the nervous system. To test this assumption, the influence of stressful influences hypoxia on learning and memory of Drosophila in the paradigm of conditioned reflex suppression of courtship was studied. The results were obtained on the enhancement of the ability to learn under hypoxic exposure. These experimental conditions did not affect memory formation. The effect of hypoxia on chromosomes through the formation of double-stranded breaks was revealed. The data are discussed in light of the relationship between neuroplasticity processes and mechanisms of adaptation to stressors.

В настоящее время нейродегенеративные заболевания (НДЗ) имеют весьма широкое распространение. По данным Всемирной организации здравоохранения (ВОЗ) в 2015 году деменция затронула 47 млн человек во всем мире, и, по прогнозам, эта цифра к 2030 году достигнет 75 млн, а к 2050 году — 132 млн. Нейрофизиологи во всем мире стремятся познать этиологию и патогенез НДЗ. Известно, что одной из причин возникновения нейрокогнитивных патологий является нарушение экспрессии гена limk1. Кроме того, согласно современным представлениям, основу интеллектуальных проблем при нейрологических повреждениях мозга составляет активное забывание, регулируемое сигнальным каскадом ремоделирования актина, ключевым звеном которого является фермент LIMK1. В работе проведен анализ формирования и динамики изменения краткосрочной и среднесрочной памяти у линий Drosophila melanogaster, полиморфных по гену limk1 (Canton-S, Oregon-R и agnts3). Полиморфизм по гену limk1 дрозофилы сказывается на содержании его продукта (мутант agnts3 характеризуется 2,5-кратным повышением содержания LIMK1 по сравнению с CS) и приводит к нарушениям поведения ухаживания и обучения. Результаты настоящего исследования двух линий дикого типа и мутанта agnts3 с измененной структурой гена limk1 показывают, что нарушения структуры данного гена могут являться причиной нарушения процессов обучения и забывания.

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.

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.

According to present knowledge, systemic realization of genetic activity in the dynamic spatial organization of the genome in the nucleus provides such a level of plasticity of complex biological systems that allows them to adequately respond to environmental stimuli or signals during the development, modulate and shift the balance of contacting chromatin components and dimensions of their interactions, resulting in structural rearrangements. The chromosome positions within the nucleus determine both normal development and progression of genomic diseases, i.e., changes according to the environmental requirements, current needs of the organism, and its individual experience. At the same time, the striking output of the evolution of higher organisms, largely ignored to date, is that only 1.2% of the mammalian genome encodes proteins and the vast majority of the expressed information is in RNA. There are hundreds of thousands of non-coding (nc) RNAs, as well as many other yet-to-be-discovered small regulatory RNAs. A new paradigm envisions the interactions between these two worlds, the one of protein and the other of RNA, as providing a dynamic link between the transcriptome and the environment and, therefore, the progressive maturation and functional plasticity of the nervous system in health and disease. Also, a wide repertoire of ncRNAs plays an important role in chromatin organization, gene expression, and disease etiology via a signal cascade of actin remodeling (LIMK1, cofilin, actin). The activity of the protein kinase LIMK1 that controls spine development, local dendritic translation at postsynaptic sites and ionotropic glutamate receptor trafficking is regulated by a brain-specific miRNA miR-134. This miRNA is localized to the synapto-dendritic compartment of rat hippocampal neurons and negatively regulates the size of dendritic spines - postsynaptic sites of excitatory synaptic transmission. Moreover, LIMK1 hemizygosity is considered to cause cognitive defects in a genome disorder Williams syndrome. Drosophila is a helpful model organism to determine the sequence of events in this system of hierarchical relationships. Drosophila LIMK1 gene (agnostic) with a specific chromosome architecture around the gene capable of generating miRNAs, recapitulates many features both of Williams syndrome and of neurodegenerative disorders. Mutants in the gene have increased expression of LIMK1 and cofilin, modified chromosome packaging and homologous and nonhomologous pairing, implemented in different rates of unequal recombination. Also, they display congofilic inclusions both in the adult brain and larval tissues presumably leading to severe defects in learning and memory during courtship conditioning

At most, many protein-misfolding diseases develop as environmentally induced sporadic disorders. Recent studies indicate that the dynamic interplay between a wide repertoire of noncoding RNAs and the environment play an important role in brain development and pathogenesis of brain disorders. To elucidate this new issue, novel animal models which reproduce the most prominent disease manifestations are required. For this, transgenic Drosophila strains were constructed to express small highly structured, non-coding RNA under control of a heat shock promoter. Expression of the RNA induced formation of intracellular aggregates revealed by Thioflafin T in embryonic cell culture and Congo Red in the brain of transgenic flies. Also, this strongly perturbed the brain control of locomotion monitored by the parameters of sound production and memory retention of young 5-day-old males. This novel model demonstrates that expression of non-coding RNA alone is sufficient to trigger neuropathology.

The inherent limitations of genetic analysis in humans and other mammals as well as striking conservation of most genes controlling nervous system functioning in flies and mammals made Drosophila an attractive model to investigate various aspects of brain diseases. Since RNA research has made great progress in recent years here we present an overview of studies demonstrating the role of various non-coding RNAs in neurodegeneration and stress response in Drosophila as a model organism. We put special emphasis on the role of non-coding micro RNAs, hsr-omega transcripts, and artificial small highly structured RNAs as triggers of neuropathology including aggregates formation, cognitive abnormalities and other symptoms. Cellular stress is a conspicuous feature of many neurodegenerative diseases and the production of specialized proteins protects the nerve cells against aggregates formation. Therefore, herein we describe some data implicating various classes of non-coding RNAs in stress response in Drosophila. All these findings highlight Drosophila as an important model system to investigate various brain diseases potentially mediated by some non-coding RNAs including polyglutamine diseases, Alzheimer's disease, Huntigton's disease, and many others.

Protein aggregation is a hallmark of many neurodegenerative diseases. RNA chaperones have been suggested to play a role in protein misfolding and aggregation. Noncoding, highly structured RNA recently has been demonstrated to facilitate transformation of recombinant and cellular prion protein into proteinase K-resistant, congophilic, insoluble aggregates and to generate cytotoxic oligomers in vitro. Transgenic Drosophila melanogaster strains were developed to express highly structured RNA under control of a heat shock promoter. Expression of a specific construct strongly perturbed fly behavior, caused significant decline in learning and memory retention of adult males, and was coincident with the formation of intracellular congophilic aggregates in the brain and other tissues of adult and larval stages. Additionally, neuronal cell pathology of adult flies was similar to that observed in human Parkinson's and Alzheimer's disease. This novel model demonstrates that expression of a specific highly structured RNA alone is sufficient to trigger neurodegeneration, possibly through chaperone-like facilitation of protein misfolding and aggregation.

In this paper, we discuss an efficient and robust implementation of a linear time algorithm due to [1] for computing the minimum perimeter triangle that circumscribes a convex n-gon. Our implementation is in C++, and utilizes the OpenGL graphics library for visualization and animation. The proposed implementation is efficient in the sense that it complies with the algorithm’s linear time complexity while achieving a small constant factor. The implementation is robust in the sense that it will work for all input instances.