De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment

PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences—not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs

Mesenchymal Stem Cell Therapy for Spinal Cord Contusion: A Comparative Study on Small and Large Animal Models

Here, we provide a first comparative study of the therapeutic potential of allogeneic mesenchymal stem cells derived from bone marrow (BM-MSCs), adipose tissue (AD-MSCs), and dental pulp (DP-MSCs) embedded in fibrin matrix, in small (rat) and large (pig) spinal cord injury (SCI) models during subacute period of spinal contusion. Results of behavioral, electrophysiological, and histological assessment as well as immunohistochemistry and real-time polymerase chain reaction analysis suggest that application of AD-MSCs combined with a fibrin matrix within the subacute period in rats (2 weeks after injury), provides significantly higher post-traumatic regeneration compared to a similar application of BM-MSCs or DP-MSCs. Within the rat model, use of AD-MSCs resulted in a marked change in: (1) restoration of locomotor activity and conduction along spinal axons; (2) reduction of post-traumatic cavitation and enhancing tissue retention; and (3) modulation of microglial and astroglial activation. The effect of an autologous application of AD-MSCs during the subacute period after spinal contusion was also confirmed in pigs (6 weeks after injury). Effects included: (1) partial restoration of the somatosensory spinal pathways; (2) reduction of post-traumatic cavitation and enhancing tissue retention; and (3) modulation of astroglial activation in dorsal root entry zone. However, pigs only partially replicated the findings observed in rats. Together, these results indicate application of AD-MSCs embedded in fibrin matrix at the site of SCI during the subacute period can facilitate regeneration of nervous tissue in rats and pigs. These results, for the first time, provide robust support for the use of AD-MSC to treat subacute SCI

Key role of piRNAs in telomeric chromatin maintenance and telomere nuclear positioning in Drosophila germline

Abstract Background Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) have been described in various eukaryotes; however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we performed an in-depth study of the biogenesis of telomeric piRNAs and their function in telomere homeostasis in the germline. Results To fully characterize telomeric piRNA clusters, we integrated the data obtained from analysis of endogenous telomeric repeats, as well as transgenes inserted into different telomeric and subtelomeric regions. The small RNA-seq data from strains carrying telomeric transgenes demonstrated that all transgenes belong to a class of dual-strand piRNA clusters; however, their capacity to produce piRNAs varies significantly. Rhino, a paralog of heterochromatic protein 1 (HP1) expressed exclusively in the germline, is associated with all telomeric transgenes, but its enrichment correlates with the abundance of transgenic piRNAs. It is likely that this heterogeneity is determined by the sequence peculiarities of telomeric retrotransposons. In contrast to the heterochromatic non-telomeric germline piRNA clusters, piRNA loss leads to a dramatic decrease in HP1, Rhino, and trimethylated histone H3 lysine 9 in telomeric regions. Therefore, the presence of piRNAs is required for the maintenance of telomere chromatin in the germline. Moreover, piRNA loss causes telomere translocation from the nuclear periphery toward the nuclear interior but does not affect telomere end capping. Analysis of the telomere-associated sequences (TASs) chromatin revealed strong tissue specificity. In the germline, TASs are enriched with HP1 and Rhino, in contrast to somatic tissues, where they are repressed by Polycomb group proteins. Conclusions piRNAs play an essential role in the assembly of telomeric chromatin, as well as in nuclear telomere positioning in the germline. Telomeric arrays and TASs belong to a unique type of Rhino-dependent piRNA clusters with transcripts that serve simultaneously as piRNA precursors and as their only targets. Telomeric chromatin is highly sensitive to piRNA loss, implying the existence of a novel developmental checkpoint that depends on telomere integrity in the germline

Eu-heterochromatic Rearrangements Induce Replication of Heterochromatic Sequences Normally Underreplicated in Polytene Chromosomes of Drosophila melanogaster

In polytene chromosomes of D. melanogaster the heterochromatic pericentric regions are underreplicated (underrepresented). In this report, we analyze the effects of eu-heterochromatic rearrangements involving a cluster of the X-linked heterochromatic (Xh) Stellate repeats on the representation of these sequences in salivary gland polytene chromosomes. The discontinuous heterochromatic Stellate cluster contains specific restriction fragments that were mapped along the distal region of Xh. We found that transposition of a fragment of the Stellate cluster into euchromatin resulted in its replication in polytene chromosomes. Interestingly, only the Stellate repeats that remain within the pericentric Xh and are close to a new eu-heterochromatic boundary were replicated, strongly suggesting the existence of a spreading effect exerted by the adjacent euchromatin. Internal rearrangements of the distal Xh did not affect Stellate polytenization. We also demonstrated trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin; replication of transposed Stellate sequences was suppressed by a deletion of Xh and restored by addition of Y heterochromatin. This phenomenon is discussed in light of a possible role of heterochromatic proteins in the process of heterochromatin underrepresentation in polytene chromosomes

Determination of Main Spectral and Luminescent Characteristics of Winter Wheat Seeds Infected with Pathogenic Microflora

In connection with the constant growth of demand for high-quality food products, there is a need to develop effective methods for storing agricultural products, and the registration and predicting infection in the early stages. The studying of the physical properties of infected plants and seeds has fundamental importance for determining crop losses, conducting a survey of diseases, and assessing the effectiveness of their control (assessment of the resistance of crops and varieties, the effect of fungicides, etc.). Presently, photoluminescent methods for diagnosing seeds in the ultraviolet and visible ranges have not been studied. For research, seeds of winter wheat were selected, and were infected with one of the most common and dangerous diseases for plants—fusarium. The research of luminescence was carried out based on a hardware–software complex consisting of a multifunctional spectrofluorometer “Fluorat-02-Panorama”, a computer with software “Panorama Pro” installed, and an external camera for the samples under study. Spectra were obtained with a diagnostic range of winter wheat seeds of 220–400 nm. Based on the results obtained for winter wheat seeds, it is possible to further develop a method for determining the degree of fusarium infection

Stellate Repeats: Targets of Silencing and Modules Causing cis-Inactivation and trans-Activation

The mechanism of silencing of testis expressed X-linked Stellate repeats by homologous Y-linked Suppressor of Stellate [Su(Ste)] repeats localized in the crystal locus was studied. The double stranded RNA as a product of symmetrical transcription of Su(Ste) repeat and small iinterfaceSu(Ste) siRNA were revealed suggesting the mechanism of RNA interference (RNAi) for Stellate silencing. The relief of Stellate silencing as a result of impaired complementarity between the sequences of putative target Stellate transcripts and Su(Ste) repeats was shown. The role of RNAi mechanism in the silencing of heterochromatic retrotransposon GATE inserted in Stellate cluster was revealed. The studies of cis-effects of Stellate tandem repeats causing variegated expression of juxtaposed reporter genes were extended and the lacZ variegation in imaginal disc was shown. The exceptional case of a non-variegated expression of mini-white gene juxtaposed to Stellate repeats in a construct inserted into the 39C region was shown to be accompanied by trans-activation in homozygous state. Trans-activation effect was retained after transposition of this construct into heterochromatic environment in spite of strong variegation of a mini-white gene