Exome sequencing in rare neurological disorders

PhD ThesisNeurological disorders are complex traits, manifesting as a range of diverse phenotypes. The current diagnostic approach involves either stepwise testing, which is expensive and time consuming, or targeted next generation sequencing with a limited portfolio of genes. Both of these approaches have a lower diagnostic yield. Whole exome sequencing may be a more advantageous and faster method to discover disease causing gene mutations. This study evaluates the use of whole exome sequencing for diagnostic purposes in neurological disorders. Whole exome sequencing was performed in a heterogeneous cohort of patients with suspected inherited ataxia as an example of a neurological disorder, with the aim to identify candidate gene mutations. The study cohort consisted of 35 affected individuals from 22 randomly selected families of white European descent with no known consanguinity. All common sporadic, inherited and metabolic causes were excluded on routine clinical investigations prior to inclusion in this study. Whole exome sequencing was performed on 30 affected individuals. In-house bioinformatic analysis was based on previously published tools. A variant filtering algorithm excluded synonymous variants and focused on protein altering variants, nonsense mutations, exonic insertions/deletions and splice site variants. Minor allele frequency (MAF) was set at 1% in dbSNP137, 1000 genomes (April 2012 data release) and NHLBI-ESP6500 databases as well as in 286 unrelated in-house controls. Selection of the remaining variants was based on mode of inheritance. The variants were prioritized for brain and nerve cell expression and defined using carefully selected criteria. Genetic analysis was supported further by molecular genetic approaches (Sanger sequencing, reverse transcription PCR, quantitative pyrosequencing, cloning for allelic cis-trans study) and proteomics (Western blotting, immunohistochemistry). Confirmed pathogenic variants were found in 9/22 probands (41%) implicating 6 genes (KCNC3, SPG7, TUBB4A, SLC1A3, SACS and NPC1). Likely de novo dominant TUBB4A mutations were found in two families. In one family quantitative pyrosequencing revealed varying degrees of mosaicism in the mildly affected mother and heterozygosity in the severely affected offspring. In silico analysis further supported pathogenicity of the mutation and revealed that it could potentially disrupt ! ! ii! polymerizations of αβ-tubulin heterodimers. Possible pathogenic variants were identified in 5/22 probands (23%) implicating 5 genes (ZFYVE26, ZFYVE27, WFS1, WNK1 and FASTKD2). A predicted splice site mutation was detected in three members of an autosomal dominant pedigree in the previously described gene ZFYVE27. The ZFYVE27 protein (protrudin) levels were increased approximately 2.5 fold in the cerebellum but not in the frontal cortex of the affected individual. Protrudin is an endoplasmic reticulum (ER) protein and its anomalies have previously been shown to cause ER stress. In this study levels of the master regulator of ER stress, BiP/GRP78, were significantly increased in the patient’s cerebellum, which may indicate the ER pathology. In one family with possible pathogenic compound heterozygous FASTKD2 mutations, the in silico splice-site prediction was validated by sequencing analysis of cDNA clones. Likely de novo compound heterozygous mutations in ZFYVE26 (SPG15) in one family was validated with sequencing of cloned alleles and the result confirmed occurrence of the mutations in trans, therefore supporting their autosomal recessive inheritance. In conclusion, the likely molecular diagnosis in 14 out of 22 families (64%) was defined; a total of 11 genes were implicated. Disease genes previously described in isolated families were validated and the clinical phenotypes of known disease genes was broadened. This study has also demonstrated genetic heterogeneity of hereditary ataxias but shows the impact of exome sequencing in a group of patients difficult to diagnose genetically

Piezoelectric Response of Cadmium Sulfide Nanorod Crystals Grown from Gas Phase by Using Ag and Au Catalyst

The paper describes macroscopic and AFM measurements of piezoelectric response of a macroscopic array of nanorod crystals of CdS and single nanorod crystal of CdS, respectively. The CdS nanorod crystals grown using Ag catalyst are presented for the first time. It is shown that these crystals have an irregular shape of six-sided polyhedron and demonstrate piezoelectric response similar to their counterpart crystals grown from Au catalyst which have a perfect hexagonal shape and wurtzite structure. The irregular shape of Ag catalyzed CdS nanocrystals is discussed based on the assumption of their sphalerite crystal symmetry

Синтез та біологічна активність нових четвертинних солей адамантановмісних діалкіламінопропанолів

The emergence and spread of multidrug-resistant pathogens leads to a decrease in efficacy of antibiotic therapy, prolongation of the length of patient’s hospital stay and an increase in treatment costs. The screening of potential antimicrobial agents among the new classes of chemical compounds is one of the promising methods to overcome the problem of resistance.Objectives. The synthesis and screening studies of antimicrobial activity of quaternary salts of adamantane derivatives (3a – 3l) with the aim to find of new prospective compound with good activityMaterials and methods. The synthesis and investigation of physicochemical properties of new adamantan-based dialkylaminopropanol quaternary salts were carried out. The evaluation of antimicrobial action against S. aureus, E. coli and C. albicans strains were performed.Results and discussion. The results showed that the inhibitory activities of quaternary salts with 1-adamantylethyl radical in their alkoxy group were significantly higher than those of the compounds with 1-adamantyl and 1-adamantyloxyethyl radicals in their alkoxy group.Conclusions. 3c was the most active compound tested against all strains, with MIC between 1.56 and 3.12 µg/mL, and its antimicrobial activity was similar to that of myramistin.Актуальность. Появление и распространение устойчивых штаммов возбудителей инфекционных заболеваний приводит к снижению эффективности антибиотикотерапии, способствует увеличению срока госпитализации пациентов и расходов на их лечение. Одним из путей борьбы с резистентностью является поиск активных соединений среди новых химических классов и разработка на их основе эффективных препаратов.Цель работы. Синтез и скрининговые исследования антимикробного действия четвертичных солей адамантансодержащих диалкиламинопропанолов с целью поиска новых перспективных активных соединений.Материалы и методы. Осуществлен синтез и изучены физико-химические свойства четвертичных солей адамантансодержащих диалкиламинопропанолов. Была проведена оценка их антимикробного действия в отношении S. aureus, E. coli и C. albicans.Результаты и их обсуждение. Показано, что ингибирующая активность четвертичных солей с 1-адамантилэтильным радикалом в алкоксигруппе была значительно выше, чем у соединений с 1-адамантильным и 1-адамантилоксиэтильным радикалами.Выводы. Наиболее выраженная активность в отношении всех штаммов была установлена у соединения 3с, которое по показателю антимикробной активности не уступает препарату сравнения мирамистину (МПК был в диапазоне 1,56-3,12 мкг/мл).Актуальність. Поява та розповсюдження стійких штамів збудників інфекційних хвороб призводить до зниження ефективності антибіотикотерапії, спричиняє збільшення терміну госпіталізації пацієнтів та витрат на їх лікування. Одним із шляхів протидії резистентності є пошук активних сполук серед нових хімічних класів та розробка на їх основі ефективних препаратів. Мета роботи. Синтез та скринінгові дослідження антимікробної дії четвертинних солей адамантановмісних діалкіламінопропанолів (3a-3l) з метою пошуку нових перспективних активних сполук.Матеріали та методи. Здійснено синтез та вивчені фізико-хімічні властивості четвертинних солей адамантановмісних діалкіламінопропанолів. Було оцінено їх антимікробну дію відносно S. aureus, E. coli та C. albicans.Результати та їх обговорення. Було встановлено, що інгібуюча активність четвертинних солей з 1-адамантилетиловим радикалом у алкоксигрупі була значно вищою, ніж у сполук з 1-адамантиловим та 1-адаман-тилоксіетильним радикалами.Висновки. Найбільш виражену інгібуючу активність по відношенню до всіх штамів показала сполука 3с, яка за антимікробною активністю не поступалась препарату порівняння мірамістину (МІК був в інтервалі 1,56-3,12 мкг/мл)

Formation of self-organized organic-inorganic hybrids

The morphology features and peculiarities of current-voltage characteristics of selforganized organic–silicon hybrids were investigated. The organic layers were formed by chemical bath deposition at room temperatures of phosphorus doped n-type FZ Si-patterned substrate. The pattern was formed by etching in anisotropic etch on the base of aqueous solution of potassium hydrate KOH and isopropyl alcohol. The following aqueous solutions of organic heterocyclic aromatic compounds were used for hybrids formation: sulfacyl sodium, procainamide hydrochloride (novocain) and lamotridgine. These hybrids have shown different types of morphology. This depends on substrate properties, time deposition and organic concentration in water solution. The photovoltaic effect of organic-pattern silicon is the result of chemisorptions of functional amine, amide, carboxyl, thiols and halogen groups on silicon pattern-type surface. At the same time these results have proven that the substrate of start and classic morphology in pyramid form is favored for formation of organic-silicon hybrids for photovoltaic application.Досліджено морфологічні властивості та особливості характеристик струм–напруга для самоорганізованих кремнійорганічних гібридів. Органічні шари було одержано хімічним осадженням за кімнатної температури легованих фосфором візерункових кремнієвих субстратів FZ n-типу. Візерунок формували витравлюванням в анізотропних травниках на основі водного розчину гідрату калію КОН та ізопропилового спирту. В подальшому для отримання гібридів використовували водні розчини органічних гетероциклічних сполук: сульфосаліцилового натрію, гідро хлориду прокаінаміду (новокаїну) і ламотріджину. Ці гібриди показали різну морфологію. Вона залежить від властивостей субстрату, часу осадження та концентрації органічної складової у водних розчинах. Фотогальванічний ефект кремнійорганічного рисунка є результатом хемосорбції функціональних груп амінів, амідів, карбоксилу, тріолів та галогену на поверхні кремнію. Водночас, ці результати підтверджують,що субстрат початкової і класичної морфології у вигляді піраміди кращий для утворення кремнійорганічних гібридів фотогальванічного застосування.Исследованы морфологические свойства и особенности характеристик ток–напряжение для самоорганизующихся кремнийорганических гибридов. Органические слои были получены химическим осаждением при комнатной температуре легированных фосфором узорчатых кремниевых субстратов FZ n-типа. Узор формировали вытравливанием в анизотропных травителях на основе водного раствора гидрата калия KOH и изопропилового спирта. В дальнейшем для получения гибридов использовали водные растворы органических гетероциклических соединений: сульфосалицилового натрия, гидрохлорида прокаинамида (новокаина) и ламотритриджина. Эти гибриды показали различную морфологию. Она зависит от свойств субстрата, времени осаждения и концентрации органической составляющей в водных растворах. Фотогальванический эффект кремнийорганического рисунка является результатом хемосорбции функциональных групп аминов, амидов, карбоксила, триолов и галогена на поверхности кремния. В то же самое время, эти результаты подтверждают, что субстрат начальной и классической морфологии в виде пирамиды является предпочтительным для образования кремнийорганических гибридов фотогальванического применения

Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death

Background: Animals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant "apoptosis-like programmed cell death" (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies.Results: Here, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40-55 degrees C and 85 degrees C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 degrees C, the only striking morphological difference between cell deaths induced by 40-55 degrees C or 85 degrees C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents.Conclusions: We conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways

Frequency of rare recessive mutations in unexplained late onset cerebellar ataxia.

Sporadic late onset cerebellar ataxia is a well-described clinical presentation with a broad differential diagnosis that adult neurologists should be familiar with. However, despite extensive clinical investigations, an acquired cause is identified in only a minority of cases. Thereafter, an underlying genetic basis is often considered, even in those without a family history. Here we apply whole exome sequencing to a cohort of 12 patients with late onset cerebellar ataxia. We show that 33% of 'idiopathic' cases harbor compound heterozygous mutations in known ataxia genes, including genes not included on multi-gene panels, or primarily associated with an ataxic presentation

Heat and drought induced transcriptomic changes in barley varieties with contrasting stress response phenotypes

Drought and heat stress substantially impact plant growth and productivity. When subjected to drought or heat stress, plants exhibit reduction in growth resulting in yield losses. The occurrence of these two stresses together intensifies their negative effects. Unraveling the molecular changes in response to combined abiotic stress is essential to breed climate-resilient crops. In this study, transcriptome profiles were compared between stress-tolerant (Otis), and stress-sensitive (Golden Promise) barley genotypes subjected to drought, heat, and combined heat and drought stress for five days during heading stage. The major differences that emerged from the transcriptome analysis were the overall number of differentially expressed genes was relatively higher in Golden Promise (GP) compared to Otis. The differential expression of more than 900 transcription factors in GP and Otis may aid this transcriptional reprogramming in response to abiotic stress. Secondly, combined heat and water deficit stress results in a unique and massive transcriptomic response that cannot be predicted from individual stress responses. Enrichment analyses of gene ontology terms revealed unique and stress type-specific adjustments of gene expression. Weighted Gene Co-expression Network Analysis identified genes associated with RNA metabolism and Hsp70 chaperone components as hub genes that can be useful for engineering tolerance to multiple abiotic stresses. Comparison of the transcriptomes of unstressed Otis and GP plants identified several genes associated with biosynthesis of antioxidants and osmolytes were higher in the former that maybe providing innate tolerance capabilities to effectively combat hostile conditions. Lines with different repertoire of innate tolerance mechanisms can be effectively leveraged in breeding programs for developing climate-resilient barley varieties with superior end-use traits

EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression

The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific

A plant virus protein, NIa-pro, interacts with Indole-3-acetic acid-amido synthetase, whose levels positively correlate with disease severity

Potato virus Y (PVY) is an economically important plant pathogen that reduces the productivity of several host plants. To develop PVY-resistant cultivars, it is essential to identify the plant-PVY interactome and decipher the biological significance of those molecular interactions. We performed a yeast two-hybrid (Y2H) screen of Nicotiana benthamiana cDNA library using PVY-encoded NIa-pro as the bait. The N. benthamiana Indole-3-acetic acid-amido synthetase (IAAS) was identified as an interactor of NIa-pro protein. The interaction was confirmed via targeted Y2H and bimolecular fluorescence complementation (BiFC) assays. NIa-pro interacts with IAAS protein and consequently increasing the stability of IAAS protein. Also, the subcellular localization of both NIa-pro and IAAS protein in the nucleus and cytosol was demonstrated. By converting free IAA (active form) to conjugated IAA (inactive form), IAAS plays a crucial regulatory role in auxin signaling. Transient silencing of IAAS in N. benthamiana plants reduced the PVY-mediated symptom induction and virus accumulation. Conversely, overexpression of IAAS enhanced symptom induction and virus accumulation in infected plants. In addition, the expression of auxin-responsive genes was found to be downregulated during PVY infection. Our findings demonstrate that PVY NIa-pro protein potentially promotes disease development via modulating auxin homeostasis