Morphometric analysis of supraoptic neurons of the rat hypothalamic nuclei under conditions of prolonged illumination

The article reviews the results of studies of the morphofunctional state of neurons of the supraoptic nuclei of the rat hypothalamus under conditions of different duration of light regime. Under standard light regime in rats, a diurnal rhythm of morphofunctional activity of supraoptic nucleus neurons with maximum activity during daytime (before 2 p.m.) is recorded. In animals subjected to prolonged light exposure, more pronounced changes in the morphofunctional state of the supraoptic neurons of the hypothalamus at 2 a.m. than at 2 p.m. were established. Thus, the neuronal nucleus area was 94.08 ± 9.55 μm2 and was significantly greater than that in intact animals. The nucleo-cytoplasmic ratio of supraoptic hypothalamic neuron at 2 a.m. was lower than that in intact animals due to a decrease in specific nucleus volume. In comparison with the day period (2 p.m.), before 2 a.m. there was revealed a decrease of the neuron body area of supraoptic nuclei of hypothalamus due to possible decrease of the area of nucleus and nucleolus of cells. This was the reason for the increase in the nucleo-cytoplasmic ratio in the neurons under observation at night, which was 2.51 ± 0.023 units. Constant light regime did not cause inversion of the rhythm of morphofunctional activity of the neurons under study, the maximum values, as in intact animals, occurred in the daytime observation period

СТРЕС-ІНДУКОВАНІ ЗМІНИ МОРФОФУНКЦІОНАЛЬНОГО СТАНУ НЕЙРОНІВ НАДЗОРОВОГО ЯДРА ГІПОТАЛАМУСА ЩУРІВ

SUMMARY. Motor activity is an important feature of animals and humans, this is one of the conditions for their normal existence and development, which depends on the level of illumination. Restriction of motor activity (hypokinesia, or immobilization) is a powerful stress factor that can cause various pathological processes. The hypothalamus, as the highest subcortical center of the autonomic nervous system, exerts a powerful regulating effect on all vital functions of the body, including maintaining the homeostatic balance of the living system, which is disturbed as a result of stressors, in particular immobilization. In connection with the important role of the large neurosecretory cells of the hypothalamic supraoptic nucleus (SON) in the realization of the adaptive capabilities of the organism, it is urgent to study the nature of their response when the organism of experimental animals is exposed to immobilization stress. The aim – to learn the effect of immobilization stress on the morpho-functional state of the SON of the rat hypothalamus. Material and Methods. The experiments were carried out on nonlinear male white rats weighing 200–220 g; immobilization stress was modeled by keeping the rats in special plastic cage-cases for 6 hours daily during 7 days.  The animals were divided into 2 series of studies, in each of which the sampling of biomaterial was carried out at 2 p.m. and 2 a.m. using morpho-functional and statistical research methods. Results and Discussion. The study of the morphometric characteristics of the neurons of the SON of the hypothalamus revealed the diurnal dynamics of the indices. Under the standard light regime in rats, the circadian rhythm of the morpho-functional activity of the neurons of the SON was recorded with a maximum activity at night (2 a.m.). When the animals were kept under immobilization stress at 2 p.m., the area of the neuron of the SON of the hypothalamus reached (305.67±7.939) μm2 and was significantly higher (by 9.97 %) relative to the same value in intact rats. At the same time, there was an increase in the size of its cytoplasm by 17.0 %, and the nuclear-cytoplasmic ratio, which reached (3.69±0.019) units. Immobilization stress led at 2 p.m. to a significant decrease in the RNA concentration in the nucleus by 33.1 %, in the nucleolus and cytoplasm of neurons of the SON of the hypothalamus relative to these values ​in intact animals. Keeping animals under conditions of immobilization stress caused more pronounced changes in the morpho-functional state of the neurons of the SON of the hypothalamus at 2 a.m. rather than at 2 p.m. Thus, the area of the neuron nucleus was (261.94±6.240) μm2 and was significantly less (by 14.3 %) than that in intact animals. These changes were accompanied by a decrease in the area of the nucleolus and cytoplasm of the neuron, which was (165.33±4.642) μm2 and was significantly less (by 24.2 %) than that in animals kept under standard illumination. It was noted that the stay of animals under immobilization stress disturbed the circadian rhythm of the morpho-functional activity of the neurons of the SON of the hypothalamus. Their greater activity, in contrast to the rats under normal lighting conditions, was recorded during the daytime observation period. Conclusions. 1. Under the standard light regime, the daily rhythm of the morpho-functional activity of the neurons of the SON with the maximum activity at night was recorded in rats. 2. Immobilization stress causes a disturbance in the rhythm of the morpho-functional activity of the studied neurons. 3. Keeping animals under conditions of immobilization stress caused more pronounced changes in the morpho-functional state of neurons in the SON of the hypothalamus at 2 a.m. than at 2 p.m., which was accompanied by a decrease in the area of ​​the nucleus, nucleolus and cytoplasm of the neuron, which were significantly less than those in intact animals.РЕЗЮМЕ. Двигательная активность является важным свойством животных и человека, одним из условий их нормального существования и развития, зависящим от уровня освещенности. Ограничение двигательной активности (гипокинезия, или иммобилизация) – мощный стресорный фактор, который вызывает разнообразные патологические процессы. Гипоталамус, как высший подкорковый центр автономной (вегетативной) нервной системы, оказывает мощное регулирующее влияние на все жизненно важные функции организма, в том числе и на поддержание гомеостатического равновесия живой системы, которая нарушается в результате деятельности стрессоров, в частности иммобилизации. В связи с важной ролью крупноклеточных нейронов супраоптического ядра (СОЯ) гипоталамуса в реализации адаптационных возможностей организма актуальным является изучение характера их реагирования при воздействии на организм экспериментальных животных иммобилизационного стресса (ИС). Цель – изучить влияние иммобилизационного стресса на морфофункциональное состояние СОЯ гипоталамуса крыс. Материал и методы. Эксперименты проведены на нелинейных самцах белых крыс массой 200–220 г. Длительный ИС моделировали путем содержания крыс в специальных пластиковых клетках-пеналах в течение 6 ч ежедневно на протяжении 7 суток. Животные были поделены на 2 серии исследований, в каждой из которых забор биоматериала осуществляли в 14.00 и 02.00 ч с применением морфофункциональных и статистических методов исследования. Результаты. Изучение морфометрических характеристик нейронов СОЯ гипоталамуса выявило суточную динамику показателей. При стандартном световом режиме у крыс регистрируется суточный ритм морфофункциональной активности нейронов СОЯ гипоталамуса с максимумом активности в ночное время (02.00 ч). При содержании животных в условиях ИС в 14.00 ч. площадь нейрона СОЯ гипоталамуса достигала (305,67±7,939) мкм2 и была достоверно выше (на 9,97 %) относительно аналогичной величины в контрольной группе крыс. В то же время отмечено увеличение площади его цитоплазмы на 17,0 %. ИС привел в 14.00 ч к достоверному уменьшению концентрации РНК в ядре на 33,1 %, ядрышке и цитоплазме нейронов СОЯ гипоталамуса относительно величин контрольной группы животных. Содержание животных в условиях ИС вызвало более выраженные изменения морфофункционального состояния нейронов СОЯ гипоталамуса в 02.00 ч, чем в 14.00. Так, площадь ядра нейрона составила (261,94±6,240) мкм2 и была достоверно меньше (на 14,3 %) аналогичной в контрольной группе животных. Указанные изменения сопровождались уменьшением площади ядрышка и цитоплазмы нейрона, которая составляла (165,33±4,642) мкм2 и была достоверно ниже (на 24,2 %) величины у животных, содержавшихся при стандартном режиме освещения. Пребывание животных в условиях ИС нарушало суточный ритм морфофункциональной активности нейронов СОЯ гипоталамуса. Большую их активность, в отличие от крыс, находившихся при стандартном освещении, регистрировали в дневной период наблюдения. Выводы. 1. При стандартном световом режиме у крыс регистрируется суточный ритм морфофункциональной активности нейронов СОЯ гипоталамуса с максимумом активности в ночное время. 2. ИС вызывает нарушение ритма морфофункциональной активности исследуемых нейронов. 3. Содержание животных в условиях ИС вызвало более выраженные изменения морфофункционального состояния нейронов СОЯ гипоталамуса в 02.00 ч, чем в 14.00 ч, сопровождающиеся достоверным уменьшением площадей ядра, ядрышка и цитоплазмы нейрона относительно аналогичных величин в контрольной группе животных.РЕЗЮМЕ. Рухова активність є важливою властивістю тварин і людини, це одна з умов їх нормального існування та розвитку, яка залежить від рівня освітленості. Обмеження рухової активності (гіпокінезія, або іммобілізація) – потужний стресорний фактор, який викликає різноманітні патологічні процеси. Гіпоталамус, як вищий підкірковий центр автономної (вегетативної) нервової системи, має потужний регулювальний вплив на всі життєво важливі функції організму, у тому числі й на підтримання гомеостатичної рівноваги живої системи, яка порушується в результаті діяльності стресорів, зокрема іммобілізації. У зв’язку з важливою роллю великоклітинних надзорових ядер гіпоталамуса в реалізації адаптаційних можливостей організму актуальним є вивчення характеру їх реагування при впливі на організм експериментальних тварин іммобілізаційного стресу. Мета – вивчення впливу іммобілізаційного стресу на морфофункціональний стан надзорових ядер гіпоталамуса щурів. Матеріал і методи. Експерименти проведені на нелінійних самцях білих щурів масою 200–220 г. Тривалий іммобілізаційний стрес моделювали шляхом утримання щурів у спеціальних пластикових клітках-пеналах впродовж 6 год щоденно 7 діб поспіль. Тварин було поділено на 2 серії досліджень, у кожній з яких забір біоматеріалу здійснювали о 14.00 і о 02.00 год із застосуванням морфофункціональних та статистичних методів дослідження. Результати. Вивчення морфометричних характеристик нейронів надзорових ядер гіпоталамуса виявило добову динаміку показників. За стандартного світлового режиму у щурів реєстрували добовий ритм морфофункціональної активності нейронів надзорових ядер гіпоталамуса з максимумом активності в нічний час (02.00 год). При утримуванні тварин в умовах іммобілізаційного стресу о 14.00 год площа нейрона надзорових ядер гіпоталамуса сягала (305,67±7,939) мкм2 і була вірогідно більшою (на 9,97 %) від аналогічної величини в контрольній групі щурів. Водночас, виявлено зростання площі його цитоплазми на 17,0 %. Іммобілізаційний стрес призвів о 14.00 год до вірогідного зменшення концентрації РНК в ядрі на 33,1 %, в ядерці та цитоплазмі нейронів надзорових ядер гіпоталамуса щодо величин контрольної групи тварин. Утримування тварин в умовах іммобілізаційного стресу викликало більш виражені зміни морфофункціонального стану нейронів надзорових ядер гіпоталамуса о 02.00 год, ніж о 14.00 год. Так, площа ядра нейрона становила (261,94±6,240) мкм2 і була вірогідно меншою (на 14,3 %) за аналогічну в контрольній групі тварин. Вказані зміни супроводжувалися зменшенням площі ядерця та цитоплазми нейрона, яка становила (165,33±4,642) мкм2 і була вірогідно меншою (на 24,2 %) від такої в тварин, яких утримували за стандартного режиму освітлення. Перебування тварин за умов іммобілізаційного стресу порушувало добовий ритм морфофункціональної активності нейронів надзорових ядер гіпоталамуса. Більшу їх активність, на відміну від щурів, які перебували за звичайного освітлення, реєстрували у денний період спостереження. Висновки. 1. За стандартного світлового режиму у щурів реєструється добовий ритм морфофункціональної активності нейронів надзорових ядер гіпоталамуса з максимумом активності в нічний час. 2. Іммобілізаційний стрес спричиняє порушення ритму морфофункціональної активності досліджуваних нейронів. 3. Утримування тварин в умовах іммобілізаційного стресу викликало більш виражені зміни морфофункціонального стану нейронів надзорових ядер гіпоталамуса о 02.00 год, ніж о 14.00 год, що супроводжувалося вірогідним зменшенням площ ядра, ядерця та цитоплазми нейрона щодо аналогічних величин у контрольній групі тварин

An intuitionistic approach to scoring DNA sequences against transcription factor binding site motifs

Background: Transcription factors (TFs) control transcription by binding to specific regions of DNA called transcription factor binding sites (TFBSs). The identification of TFBSs is a crucial problem in computational biology and includes the subtask of predicting the location of known TFBS motifs in a given DNA sequence. It has previously been shown that, when scoring matches to known TFBS motifs, interdependencies between positions within a motif should be taken into account. However, this remains a challenging task owing to the fact that sequences similar to those of known TFBSs can occur by chance with a relatively high frequency. Here we present a new method for matching sequences to TFBS motifs based on intuitionistic fuzzy sets (IFS) theory, an approach that has been shown to be particularly appropriate for tackling problems that embody a high degree of uncertainty. Results: We propose SCintuit, a new scoring method for measuring sequence-motif affinity based on IFS theory. Unlike existing methods that consider dependencies between positions, SCintuit is designed to prevent overestimation of less conserved positions of TFBSs. For a given pair of bases, SCintuit is computed not only as a function of their combined probability of occurrence, but also taking into account the individual importance of each single base at its corresponding position. We used SCintuit to identify known TFBSs in DNA sequences. Our method provides excellent results when dealing with both synthetic and real data, outperforming the sensitivity and the specificity of two existing methods in all the experiments we performed. Conclusions: The results show that SCintuit improves the prediction quality for TFs of the existing approaches without compromising sensitivity. In addition, we show how SCintuit can be successfully applied to real research problems. In this study the reliability of the IFS theory for motif discovery tasks is proven

Markov Chain-based Promoter Structure Modeling for Tissue-specific Expression Pattern Prediction

Transcriptional regulation is the first level of regulation of gene expression and is therefore a major topic in computational biology. Genes with similar expression patterns can be assumed to be co-regulated at the transcriptional level by promoter sequences with a similar structure. Current approaches for modeling shared regulatory features tend to focus mainly on clustering of cis-regulatory sites. Here we introduce a Markov chain-based promoter structure model that uses both shared motifs and shared features from an input set of promoter sequences to predict candidate genes with similar expression. The model uses positional preference, order, and orientation of motifs. The trained model is used to score a genomic set of promoter sequences: high-scoring promoters are assumed to have a structure similar to the input sequences and are thus expected to drive similar expression patterns. We applied our model on two datasets in Caenorhabditis elegans and in Ciona intestinalis. Both computational and experimental verifications indicate that this model is capable of predicting candidate promoters driving similar expression patterns as the input-regulatory sequences. This model can be useful for finding promising candidate genes for wet-lab experiments and for increasing our understanding of transcriptional regulation

Notch and MAML-1 Complexation Do Not Detectably Alter the DNA Binding Specificity of the Transcription Factor CSL

Canonical Notch signaling is initiated when ligand binding induces proteolytic release of the intracellular part of Notch (ICN) from the cell membrane. ICN then travels into the nucleus where it drives the assembly of a transcriptional activation complex containing the DNA-binding transcription factor CSL, ICN, and a specialized co-activator of the Mastermind family. A consensus DNA binding site motif for the CSL protein was previously defined using selection-based methods, but whether subsequent association of Notch and Mastermind-like proteins affects the DNA binding preferences of CSL has not previously been examined.Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors. Measurements were taken both in the absence and in the presence of Mastermind-like-1 (MAML1). Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins.These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes

Predicting the binding preference of transcription factors to individual DNA k-mers

Motivation: Recognition of specific DNA sequences is a central mechanism by which transcription factors (TFs) control gene expression. Many TF-binding preferences, however, are unknown or poorly characterized, in part due to the difficulty associated with determining their specificity experimentally, and an incomplete understanding of the mechanisms governing sequence specificity. New techniques that estimate the affinity of TFs to all possible k-mers provide a new opportunity to study DNA–protein interaction mechanisms, and may facilitate inference of binding preferences for members of a given TF family when such information is available for other family members. Results: We employed a new dataset consisting of the relative preferences of mouse homeodomains for all eight-base DNA sequences in order to ask how well we can predict the binding profiles of homeodomains when only their protein sequences are given. We evaluated a panel of standard statistical inference techniques, as well as variations of the protein features considered. Nearest neighbour among functionally important residues emerged among the most effective methods. Our results underscore the complexity of TF–DNA recognition, and suggest a rational approach for future analyses of TF families. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.Canadian Institutes of Health ResearchOntario Research FundNational Institutes of Health (U.S.)National Human Genome Research Institute (U.S.

Applying whole-genome studies of epigenetic regulation to study human disease

Epigenetics may be broadly defined as the study of processes that produce a heritable phenotype that is not strictly dependent on DNA sequence. The definition has traditionally been restricted to processes that occur in the cell’s nucleus, with the term ‘heritable’ having a loose meaning that can be applied to either the entire organism or single cells. For example, a process that produces a phenotype only in a specific cell type (for instance, chromatin-mediated maintenance of a differentiated state) is usually considered epigenetic even if it is not directly inherited, but instead must be re-established or actively maintained at each cell division. Given this definition, the field of epigenetics has long focused on proteins that affect DNA packaging, and thereby affect the utilization of the genetic information encoded in the DNA template. This focus extends to the enzymatic modification of those proteins, and to the enzymatic modification of the DNA template itself, primarily DNA methylation

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays

A graph-based motif detection algorithm models complex nucleotide dependencies in transcription factor binding sites

Given a set of known binding sites for a specific transcription factor, it is possible to build a model of the transcription factor binding site, usually called a motif model, and use this model to search for other sites that bind the same transcription factor. Typically, this search is performed using a position-specific scoring matrix (PSSM), also known as a position weight matrix. In this paper we analyze a set of eukaryotic transcription factor binding sites and show that there is extensive clustering of similar k-mers in eukaryotic motifs, owing to both functional and evolutionary constraints. The apparent limitations of probabilistic models in representing complex nucleotide dependencies lead us to a graph-based representation of motifs. When deciding whether a candidate k-mer is part of a motif or not, we base our decision not on how well the k-mer conforms to a model of the motif as a whole, but how similar it is to specific, known k-mers in the motif. We elucidate the reasons why we expect graph-based methods to perform well on motif data. Our MotifScan algorithm shows greatly improved performance over the prevalent PSSM-based method for the detection of eukaryotic motifs