The pulmonary-proteoliposome as a new therapeutic approach for Coronaviruses

We are proposing the use of pulmonary-proteoliposome as a new therapeutic approach for Coronaviruses. The designed strategy represents a potential treatment to reduce the overall viral load in the lungs and to help the immune system to successfully stave off the infection

Notch pathway connections in primary leukaemia samples of limited size

Abstract Background: The Notch pathway combined with other signalling molecules acts specifically for the development of each blood cell type and differentiation stage. A causative role of Notch dysfunction in leukaemia development has been found in many studies so, initially only for T- acute lymphoblastic leukaemia (T-ALL) but more recently also for B cell and myeloid leukaemia. The aim of our study is to introduce a method for multiple direct analysis of the Notch pathway partners in a population of only 500 or fewer cells. The notion of this method consists in gaining insight into gene expression at the level of the malignant clone population. A small number of cells is a significant limitation when working on primary cells either when freshly isolated or when analysed after several days in cocultures. Methods: The primers were designed to avoid genomic amplification through the selection of 3′ and 5′ primers that hybridise with different exons. Cell lines and primary cells were collected and multiplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) performed on a descending number of cells, ranging from 2,500 cells up to 50 cells per sample, for the Notch pathway genes and other transcription factors important for cell differentiation. ImageJ program, STATISTICA 13.1 software package and Student’s t-test were used for statistical evaluation. We checked protein expression by western blot. Results: We characterised the gene expression levels of Notch, Ikaros and Parp genes in leukaemia cell lines of B and T origin and in primary leukaemia samples of limited size. We further compared our results to the cDNA analysis obtained by total RNA isolation from a large number of cells as routinely performed in clinical laboratories, and finally tested the method described on primary cells from leukaemia patients. Conclusions: This rapid multiple gene expression analysis of a small population of cells provides efficient cell classification determining malignant changes as an important additional information for clinical leukaemia diagnostics as well as for in vitro studies of primary cells

Distinct Regulation of the Expression of Satellite DNAs in the Beetle Tribolium castaneum

In the flour beetle, Tribolium castaneum (peri)centromeric heterochromatin is mainly composed of a major satellite DNA TCAST1 interspersed with minor satellites. With the exception of heterochromatin, clustered satellite repeats are found dispersed within euchromatin. In order to uncover a possible satellite DNA function within the beetle genome, we analysed the expression of the major TCAST1 and a minor TCAST2 satellite during the development and upon heat stress. The results reveal that TCAST1 transcription was strongly induced at specific embryonic stages and upon heat stress, while TCAST2 transcription is stable during both processes. TCAST1 transcripts are processed preferentially into piRNAs during embryogenesis and into siRNAs during later development, contrary to TCAST2 transcripts, which are processed exclusively into piRNAs. In addition, increased TCAST1 expression upon heat stress is accompanied by the enrichment of the silent histone mark H3K9me3 on the major satellite, while the H3K9me3 level at TCAST2 remains unchanged. The transcription of the two satellites is proposed to be affected by the chromatin state: heterochromatin and euchromatin, which are assumed to be the prevalent sources of TCAST1 and TCAST2 transcripts, respectively. In addition, distinct regulation of the expression might be related to diverse roles that major and minor satellite RNAs play during the development and stress response

Transcription of satellite DNAs in insects

Chromatin condensation is an important regulatory mechanism of gene silencing as well as gene activation for the hundreds of functional protein genes harbored in heterochromatic regions of different insect species. Being the major heterochromatin constituents, satellite DNAs serve important roles in heterochromatin regulation in insect in general. Their expression occurs in all developmental stages, being the highest during embryogenesis. Satellite DNA transcrips range from small RNAs, corresponding in size to siRNA, and piwiRNAs, to large, a few Kb long RNAs. The long transcripts are preferentially nonpolyadenylated and remain in the nucleus. The actively regulated expression of satDNAs by cis or trans elements as well as by environmental stress, rather than constitutive transcription, speaks in favour of their involvement in differentiation, development, and environmental response

Intra-specific variability and unusual organization of the repetitive units in a satellite DNA from Rana dalmatina: molecular evidence of a new mechanism of DNA repair acting on satellite DNA.

We have characterized the S1 satellite from eight European populations of Rana dalmatina by Southern blot, cloning and a new method that determines the sequence variability of repetitive units in the genome. This report completes our previous studies on this satellite DNA family, thus providing the first characterization of the overall variability of the structure and genomic organization of a satellite DNA within a species and among related species. The S1 satellite from R. dalmatina has a pericentromeric location on ten chromosome pairs and presents two homologous repeats S1a (494 bp) and S1b (332 bp), mostly organized as composite S1a-S1b repetitive units. In other brown frog species, both repeats have different sequences and locations, and are usually organized as separate arrays, although composite S1a-S1b repeats represent a minor, widely variable component in Rana italica. The average genomic sequences indicate that the species contains an enormous number of variants of each repeat derived from a unique, species-specific common sequence. The repeat variability is restricted to specific base changes in specific sequence positions in all population samples. Our data show that the structure and evolution of S1 satellite family is not due to crossing-over and gene conversion, but to a mechanism that maintains the ability of the satellite DNA to assemble in constitutive heterochromatin by replacing altered satellite segments with new arrays generated by rolling circle amplification. The mode of action of this repair process not only directly explains the intra- and inter-specific variability of the structure and organization of the S1 satellite repeats from European brown frogs, but also accounts for all general features of satellite DNA in eukaryotes, including its discontinuous evolution. This repair mechanism can maintain the satellite structure in a species indefinitely, but also promote a rapid generation of new variants or types of satellite DNA when environmental conditions favor the formation of new species

SATELLITE DNA - UKE ELEMENTS DISPERSED WlTHlN EUCHROMATIN OF BEETTE Tribolium castoneum

Satellite DNA - like elements dispersed in the within euchromatin of Tribolium castaneu

Epigenetic regulation of genes mediated by satellite DNA

Satellite DNA is a highly repetitive DNA, organized in long tandem arrays, located in the heterochromatic regions of chromosomes. Repeats of satellite DNAs can be excised from their heterochromatin loci and integrate into euchromatin. This scenario was proposed to describe euchromatic dispersion of TCAST1, the species-specific major satellite DNA of beetle Triboliumcastaneum, which is associated with numerous protein-coding genes. Dispersed repeats of TCAST1 (dTcast1) were present as monomeric or multimeric repeat units in intragenic (introns), as well as, intergenic regions. Our assumption was that dTcast1can influence the expression of associated genes. Our results show that dTcast1 affects the adjacent genes under physiological condition by inducing a slight downregulation of gene expression. The effect is more pronounced after heat stress when transient increase of satellite DNA transcripts processed into TCAST1-siRNAs is induced. We showed that a temporary formation of heterochromatic state characterized by increased level of H3K9met2/3 at dTcast insertion sites and their spreading to the proximal regions is responsible for downregulation of nearby genes. In conclusion, dTcast1 satellite elements influence the level of expression of their associated genes through RNA interference-based „heterochromatinization“and the level of suppression is positively correlated with the amount of transcripts of TCAST1 satellite DNA. Insertion of satellite DNA repeats within euchromatin provides genes with regulatory elements that modulate their activity in particular in response to environmental stress. Variation in satellite repeats insertion among individuals can in some cases provide phenotypic variation that could be acted upon by selection enabling satellite DNA to contribute to the evolution of gene regulatory networks

The first characterisation of the overall variability of repetitive units in a species reveals unexpected features of satellite DNA.

We investigated the overall variability of the S1a satellite DNA repeats in ten European populations of Rana temporaria by a new procedure that determines the average sequence of the repeats in a genome. The average genomic sequences show that only 17% of the S1a repeat sequence (494 bp) is variable. The variable positions contain the same major and minor bases in all or many of the population samples tested, but the percentages of these bases can greatly vary among populations. This indicates the presence in the species of an enormous number of repeats having a different distribution of bases in these variable positions. Individual genomes contain thousands of repeat variants, but these mixtures have very similar characteristics in all populations because they present the same type of restricted and species-specific variability. Southern blots analyses and sequences of cloned S1a repeats fully support this conclusion. The S1 satellite DNA of other European brown frog species also presents properties indicating the same type of variability. This first characterisation of the overall repeat variability of a satellite DNA in a species has revealed features that cannot be determined by gene conversion and crossing over. Our results suggest that a specific directional process based on rolling circle amplification should play a relevant role in the evolution of satellite DNA

DNA demethylation and heterochromatin remodelling upon heat stress

Conversion of environmental signals into epigenetic information is thought to occur widely but has been poorly studied as yet. It is proposed that changes in the expression of molecules involved in chromatin remodelling or their modification might play a role in this process. Here we study remodelling of heterochromatin upon heat stress in the red flour beetle Tribolium castaneum (Tenebrionidae, Coleoptera), one of the most important stored products pest. We show that the expression of abundant satellite DNA TCAST which is located within the constitutive pericentromeric heterochromatin, is strongly induced following heat shock and is accompanied by increase in repressive epigenetic modifications of histones at TCAST regions. Upon recovery from heat stress, the expression of satellite DNA-associated siRNAs as well as histone modifications is quickly restored. Bisulphite sequencing of TCAST satellite DNA reveals cytosine methylation not only restricted to CpG sites but also found at CpA, CpT and CpC sites, and preserved within heterochromatin during all developmental phases. The level of methylation decreases after heat shock depending on the duration and results in a complete demethylation after prolonged treatment. Our results indicate that heat shock induced satellite DNA demethylation and expression affect epigenetic state of constitutive heterochromatin in insects. It can be hypothesized that transient remodelling of heterochromatin is part of a physiological gene expression program activated under stress conditions. We propose the existence of a negative-feedback mechanism in which satellite DNA-associated siRNAs induced by heat stress, repress their own transcription by providing additional H3K9me3 anchorage sites for the chromodomain protein such as HP1, and in this way restore heterochromatic state