Probing Retroviral and Retrotransposon Genome Structures: The “SHAPE” of Things to Come

Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100–500 nt) RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots) that may play a critical regulatory role. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide “connectivity map,” conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges

Expression of alternatively spliced human T-cell leukemia virus type 1 mRNAs is influenced by mitosis and by a novel cis-acting regulatory sequence

Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system

The effect of naringin on early spheno-occipital growth as measured by the width of the hypertrophic zone during endochondral ossification

Poster Session - Theme: General Dentistry and Oral HealthThis journal suppl. entitled: Special Issue: Abstracts of the 2012 FDI Annual World Dental CongressOBJECTIVE: To assess the effect of the flavonoid naringin on the early growth of spheno-occipital synchondrosis by measuring the width of the hypertrophic zone in an in vitro mouse model. MATERIALS AND METHODS: Experimental protocol was approved by the University of Hong Kong’s Committee on the Use of Live Animals in Teaching and Research. Fifty 1-day old BALB/c mice were randomly assign...postprin

Individualized Housing Modifies the Immune–Endocrine System in CD1 Adult Male Mice

In the last years, different research groups have made considerable efforts to improve the care and use of animals in research. Mice (Mus musculus) are the most widely used animal species in research in the European Union and are sociable and hierarchical creatures. During experiments, researchers tend to individualize males, but no consideration is given to whether this social isolation causes them stress. The aim of this study was, therefore, to explore whether 4 weeks of social isolation could induce changes in different physiological parameters in adult Crl:CD1(ICR) (CD1) males, which may interfere with experimental results. Body weight, blood cells, and fecal corticosterone metabolites levels were the analyzed parameters. Blood and fecal samples were collected at weeks 1 and 4 of the experimental procedure. Four weeks of single housing produced a significant time-dependent decrease in monocytes and granulocytes. Fecal corticosterone metabolite levels were higher in single-housed mice after 1 week and then normalized after 4 weeks of isolation. Body weight, red blood cells, and platelets remained unchanged in both groups during this period. We can, therefore, conclude that social isolation affects some immune and endocrine parameters, and that this should be taken into account in the interpretation of research data.This study was supported by the University of the Basque Country (UPV/EHU) GIU18/103 grant

Chronic social instability stress down-regulates IL-10 and up-regulates CX3CR1 in tumor-bearing and non-tumor-bearing female mice

Extensive literature has reported a link between stress and tumor progression, and between both of these factors and mental health. Despite the higher incidence of affective disorders in females and the neurochemical dif-ferences according to sex, female populations have been understudied. The aim of this study was therefore to analyze the effect of stress on tumor development in female OF1 mice. For this purpose, subjects were inoculated with B16F10 melanoma cells and exposed to the Chronic Social Instability Stress (CSIS) model. Behavioral, neurochemical and neuroendocrine parameters were analyzed. Female mice exposed to CSIS exhibited reduced body weight and increased arousal, but there was no evidence of depressive behavior or anxiety. Exposure to CSIS did not affect either corticosterone levels or tumor development, although it did provoke an imbalance in cerebral inflammatory cytokines, decreasing IL-10 expression (IL-6/IL-10 and TNF-alpha/IL-10); chemokines, increasing CX3CR1 expression (CX3CL1/CX3CR1); and glucocorticoid receptors, decreasing GR expression (MR/ GR). In contrast, tumor development did not alter body weight and, although it did alter behavior, it did so to a much lesser extent. Tumor inoculation did not affect corticosterone levels, but increased the MR/GR ratio in the hippocampus and provoked an imbalance in cerebral inflammatory cytokines and chemokines, although differently from stress. These results underscore the need for experimental approaches that allow us to take sex differences into account when exploring this issue, since these results appear to indicate that the female response to stress is mediated by mechanisms different from those often proposed in relation to male mice.This study was supported by the Spanish Ministry of Science, Innovation RTI2018–098264-B-I00 (MCIU/AEI/FEDER, UE), the UPV/EHU GIU18/103 and the PIBA 2019–22 Project Grants

BIOMASS AS A SOURCE OF RENEWABLE ENERGY IN POLAND

Розглянуто особливості використання біомаси для збільшення обсягів виробництва відновлюваної енергії в Польщі, що є однією з основних передумов виконання вимог кліматичного пакету Європейського Союзу (до 2020 р.). Серед різноманітних видів біомаси найбільшу питому вагу у виробництві відновлюваної енергії має сектор твердого біопалива з рослинної чи тваринної сировини, з продуктів життєдіяльності організмів чи промислових відходів органічного походження, де у 2013 р. вироблено 46,4 % електроенергії та 97 % теплової енергії від загального обсягу виробництва "зеленої" енергії. Оцінено можливості збільшення обсягів виробництва енергії з біомаси у сучасній польській економіці.Рассмотрены особенности использования биомассы для увеличения выработки возобновляемой энергии в Польше, что является одним из главных условий выполнения требований климатического пакета Европейского Союза (до 2020 г.). Среди разнообразных видов биомассы наибольший удельный вес в производстве возобновляемой энергии имеет сектор твердого биотoплива из растительного или животного сырья, из продуктов жизнедеятельности организмов или органических промышленных отходов, в котором в 2013 г. произведено 46,4 % электроэнергии и 97 % тепловой энергии из общего объема производства "зеленой" энергии. Оценены возможности увеличения выработки энергии из биомассы в современной польской экономике.Preconditions for using biomass in order to increase Poland's energy production out of renewable sources as it is required by the European Union's climate package (by 2020) are considered. Among various types of biomass, the highest share in renewable energy production is kept by the sector of biofuel production out of products of either plant or animal origin, and organic industrial residues, where 46,4 % of electrical energy and 97 % of thermic energy in the total amount of "green" energy production is supplied as of 2013. Potential directions of an increase in the energy production out of biomass in the contemporary Poland's economy are discussed

Alder (Alnus glutinosa) carr in Poland

The paper aims at the analysis of ecological and regional variation of forest communities from the class Alnetea glutinosae in Poland. A previous syntaxonomical position of the association Carici elongatae-Alnetum has been revised. Instead, two new alder-carr associations have been described. They differ in character and differential species, with dissimilar trophic requirements. The group of species with Sphagnum squarrosum is differential for acidophilous bog moss alder carrs (Sphagno squarrosi-Alnetum), while the group of species with Ribes nigrum differentiates mesotrophic black-currant alder carr (Ribo nigri-Alnetum). Both alder-carr associations occur throughout lowland Poland. Their floristic compositions differ regionally. Suboceanic and mid-European species grow in alder carr of western Poland, while boreal and boreocontinental species enter from the northern-east. The studies on the regional variation are based on the analysis of distribution of differential species groups. These have resulted in the delimitation of geographical races of alder carr. Both alder-carr associations differ from one another in their internal variation. Sphagno squarrosi-Alnetum varies greatly from region to region. For this association four geographical races have been distinguished. In the Ribo nigri-Alnetum internal trophic variation plays a more important role. Therefore, it has been divided into four subassociations and two geographical races. In central Poland black-currant alder carr occurs in a form with both geographical races but with different subassociations.In der vorliegenden Arbeit wird die ökologische und regionale Variabilität der Waldgesellschaften aus der Klasse Alnetea glutinosae in Polen analysiert. Die bisherige syntaxonomische Auffassung des Carici elongatae-Alnetum wird überprüft. Es ergeben sich neue Erlenbruchwald-Gesellschaften mit eigenen Kenn- und Trennarten, welche verschiedene trophische Ansprüche haben. Die Artengruppe mit Sphagnum squarrosum kennzeichnet die azidophytischen Torfmoos-Erlenwälder (Sphagno squarrosi-Alnetum), die Gruppe mit Ribes nigrum die mesophytischen Schwarzjohannisbeeren-Erlenwälder (Ribo nigri-Alnetum). Beide Pflanzengesellschaften kommen im ganzen polnischen Tiefland vor. Die regionalen Unterschiede in ihrer Artenzusammensetzung werden betont. In den Erlenbrüchern West-Polens sind subatlantische und mitteleuropäische Arten zu finden; vom Nordosten dringen boreale und boreokontinentale Arten ein. Die Untersuchung der regionalen Variabilität beruhte auf der Analyse der Verbreitung von Differentialartengruppen. Die Ergebnisse bilden die Grundlage für die Abgrenzung regionaler Ausbildungen. Die innere Variabilität der zwei Bruchwald-Assoziationen ist unterschiedlich. Für das Sphagno squarrosi-Alnetum ist eine deutliche regionale Variabilität [mit vier Regionalausbildungen] charakteristisch. Im Ribo nigri-Alnetum spielt die trophische Variabilität eine größere Rolle; dieser entspricht einer Gliederung in vier Subassoziationen. Jede Subassoziation kommt in zwei Regionalausbildungen vor, die im Übergangsgebiet von Zentralpolen zusammentreffen. Sie vertreten dort jedoch verschiedene Subassoziationen

Assessment of compliance of products serving the purposes of the national security

The paper presents principles of assessment of compliance to be observed by the suppliers of products serving the purposes of the national security and ordered by organisational units subject to or supervised by the competent minister of interior, arising from the act of November 17th, 2006. The authors also discussed the certification procedures and reference documents applied in the certification process carried out by the Product Certification Department (ZCW) of ITB MORATEX, a body accredited by the Ministry of Interior and Administration (MSWiA) in respect of OIB

Probing the Structures of Viral RNA Regulatory Elements with SHAPE and Related Methodologies

Viral RNAs were selected by evolution to possess maximum functionality in a minimal sequence. Depending on the classification of the virus and the type of RNA in question, viral RNAs must alternately be replicated, spliced, transcribed, transported from the nucleus into the cytoplasm, translated and/or packaged into nascent virions, and in most cases, provide the sequence and structural determinants to facilitate these processes. One consequence of this compact multifunctionality is that viral RNA structures can be exquisitely complex, often involving intermolecular interactions with RNA or protein, intramolecular interactions between sequence segments separated by several thousands of nucleotides, or specialized motifs such as pseudoknots or kissing loops. The fluidity of viral RNA structure can also present a challenge when attempting to characterize it, as genomic RNAs especially are likely to sample numerous conformations at various stages of the virus life cycle. Here we review advances in chemoenzymatic structure probing that have made it possible to address such challenges with respect to cis-acting elements, full-length viral genomes and long non-coding RNAs that play a major role in regulating viral gene expression

Label-Free Detection of Human Coronaviruses in Infected Cells Using Enhanced Darkfield Hyperspectral Microscopy (EDHM)

Human coronaviruses (HCoV) are causative agents of mild to severe intestinal and respiratory infections in humans. In the last 15 years, we have witnessed the emergence of three zoonotic, highly pathogenic HCoVs. Thus, early and accurate detection of these viral pathogens is essential for preventing transmission and providing timely treatment and monitoring of drug resistance. Herein, we applied enhanced darkfield hyperspectral microscopy (EDHM), a novel non-invasive, label-free diagnostic tool, to rapidly and accurately identify two strains of HCoVs, i.e., OC43 and 229E. The EDHM technology allows collecting the optical image with spectral and spatial details in a single measurement without direct contact between the specimen and the sensor. Thus, it can directly map spectral signatures specific for a given viral strain in a complex biological milieu. Our study demonstrated distinct spectral patterns for HCoV-OC43 and HCoV-229E virions in the solution, serving as distinguishable parameters for their differentiation. Furthermore, spectral signatures obtained for both HCoV strains in the infected cells displayed a considerable peak wavelength shift compared to the uninfected cell, indicating that the EDHM is applicable to detect HCoV infection in mammalian cells