Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models.

BACKGROUND: The majority of genes in the human genome is present in two copies but the expression levels of both alleles is not equal. Allelic imbalance is an aspect of gene expression relevant not only in the context of genetic variation, but also to understand the pathophysiology of genes implicated in genetic disorders, in particular, dominant genetic diseases where patients possess one normal and one mutant allele. Polyglutamine (polyQ) diseases are caused by the expansion of CAG trinucleotide tracts within specific genes. Spinocerebellar ataxia type 3 (SCA3) and Huntington's disease (HD) patients harbor one normal and one mutant allele that differ in the length of CAG tracts. However, assessing the expression level of individual alleles is challenging due to the presence of abundant CAG repeats in the human transcriptome, which make difficult the design of allele-specific methods, as well as of therapeutic strategies to selectively engage CAG sequences in mutant transcripts. RESULTS: To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice. CONCLUSIONS: Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.

Genetic tests based on the RT-PCR reaction in the diagnostics of SARS-CoV-2 infection.

INTRODUCTION: Since the SARS-CoV-2 emergence in 2019/2020, at least 158 million infections with this pathogen have been recorded, of which 3.29 million infected people have died. Due to the non-specific symptoms of SARS-CoV-2 infection, laboratory tests based on RT-PCR (reverse transcription and polymerase chain reaction) are mainly used in the diagnosis of COVID-19 disease. AIM: The aim of this study is to compare the molecular tests available on the Polish market for the diagnosis of SARS-CoV2 infection. RESULTS: Based on the data provided by the manufacturers and the performed laboratory analyses, we have shown that the available diagnostic kits differ mainly in the sensitivity and duration of the reaction. CONCLUSION: Due to the ongoing COVID-19 pandemic, the indicated parameters are key to effective control of the spread of SARS-CoV2, and therefore should be mainly taken into account when choosing and purchasing by diagnostic centres.

Nuclear speckles are detention centers for transcripts containing expanded CAG repeats.

The human genetic disorders caused by CAG repeat expansions in the translated sequences of various genes are called polyglutamine (polyQ) diseases because of the cellular "toxicity" of the mutant proteins. The contribution of mutant transcripts to the pathogenesis of these diseases is supported by several observations obtained from cellular models of these disorders. Here, we show that the common feature of cell lines modeling polyQ diseases is the formation of nuclear CAG RNA foci. We performed qualitative and quantitative analyses of these foci in numerous cellular models endogenously and exogenously expressing mutant transcripts by fluorescence in situ hybridization (FISH). We compared the CAG RNA foci of polyQ diseases with the CUG foci of myotonic dystrophy type 1 and found substantial differences in their number and morphology. Smaller differences within the polyQ disease group were also revealed and included a positive correlation between the foci number and the CAG repeat length. We show that expanded CAA repeats, also encoding glutamine, did not trigger RNA foci formation and foci formation is independent of the presence of mutant polyglutamine protein. Using FISH combined with immunofluorescence, we demonstrated partial co-localization of CAG repeat foci with MBNL1 alternative splicing factor, which explains the mild deregulation of MBNL1-dependent genes. We also showed that foci reside within nuclear speckles in diverse cell types: fibroblasts, lymphoblasts, iPS cells and neuronal progenitors and remain dependent on integrity of these nuclear structures.

In Vitro Expansion of CAG, CAA, and Mixed CAG/CAA Repeats.

Polyglutamine diseases, including Huntington's disease and a number of spinocerebellar ataxias, are caused by expanded CAG repeats that are located in translated sequences of individual, functionally-unrelated genes. Only mutant proteins containing polyglutamine expansions have long been thought to be pathogenic, but recent evidence has implicated mutant transcripts containing long CAG repeats in pathogenic processes. The presence of two pathogenic factors prompted us to attempt to distinguish the effects triggered by mutant protein from those caused by mutant RNA in cellular models of polyglutamine diseases. We used the SLIP (Synthesis of Long Iterative Polynucleotide) method to generate plasmids expressing long CAG repeats (forming a hairpin structure), CAA-interrupted CAG repeats (forming multiple unstable hairpins) or pure CAA repeats (not forming any secondary structure). We successfully modified the original SLIP protocol to generate repeats of desired length starting from constructs containing short repeat tracts. We demonstrated that the SLIP method is a time- and cost-effective approach to manipulate the lengths of expanded repeat sequences.

Implications of Poly(A) Tail Processing in Repeat Expansion Diseases.

Repeat expansion diseases are a group of more than 40 disorders that affect mainly the nervous and/or muscular system and include myotonic dystrophies, Huntington's disease, and fragile X syndrome. The mutation-driven expanded repeat tract occurs in specific genes and is composed of tri- to dodeca-nucleotide-long units. Mutant mRNA is a pathogenic factor or important contributor to the disease and has great potential as a therapeutic target. Although repeat expansion diseases are quite well known, there are limited studies concerning polyadenylation events for implicated transcripts that could have profound effects on transcript stability, localization, and translation efficiency. In this review, we briefly present polyadenylation and alternative polyadenylation (APA) mechanisms and discuss their role in the pathogenesis of selected diseases. We also discuss several methods for poly(A) tail measurement (both transcript-specific and transcriptome-wide analyses) and APA site identification-the further development and use of which may contribute to a better understanding of the correlation between APA events and repeat expansion diseases. Finally, we point out some future perspectives on the research into repeat expansion diseases, as well as APA studies.

Purification of phage display-modified bacteriophage T4 by affinity chromatography.

BACKGROUND: Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. RESULTS: Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. CONCLUSIONS: Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.

[Bacteriophage T4: molecular aspects of bacterial cell infection and the role of capsid proteins]. / Bakteriofag T4: molekularne aspekty infekcji komórki bakteryjnej, rola bialek kapsydowych.

Bacteriophage T4 of the Myoviridae family is ubiquitous in the environment and living organisms. In microbiology it has become a universal research model for the mechanisms of many biological processes, including bacteriophage infection. T4 phage is a tailed phage, the most frequent bacteriophage group. It is made up of a head, a contractile tail, and dsDNA. Its tail is a complex structure composed of a collar and its whiskers, a tail tube, a base plate, short fibers, and tail fibers. All these elements cooperate in effective infection. The main host of bacteriophage T4 is Escherichia coli. Adsorption on the bacterial surface is crucial for infection. It depends on specific receptors: lipopolysaccharides and OmpC protein. The high bacteriophage specificity requires specific structures (compositions) of both bacterial and bacteriophage (gp12, gp37) elements. The introduction of phage DNA into the bacterium engages a group of proteins, for example those essential for effective tail contraction and membrane fusion and those with enzymatic activity. In the infected bacterial cell, T4 starts to control cell metabolism with phage replication and expression factors. The final stage of infection is assemblage and lysis. Here the role of bacterial and bacteriophage elements in the above processes is presented and their cooperation with regard to currently identified molecular regions of activity.

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.