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RFC1 disease, caused by biallelic repeat expansion in RFC1 , is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I–V β = −1.06, P < 0.001; lobules VI–VII β = −0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion. [ABSTRACT FROM AUTHOR]

Abstract Background Current solutions for the analysis of Western Blot images lack either transparency and reproducibility or can be tedious to use if one has to ensure the reproducibility of the analysis. Results Here, we present an open-source gel image analysis program, IOCBIO Gel. It is designed to simplify image analysis and link the analysis results with the metadata describing the measurements. The software runs on all major desktop operating systems. It allows one to use it in either a single-researcher environment with local storage of the data or in a multiple-researcher environment using a central database to facilitate data sharing within the research team and beyond. By recording the original image and all operations performed on it, such as image cropping, subtraction of background, sample lane selection, and integration boundaries, the software ensures the reproducibility of the analysis and simplifies making corrections at any stage of the research. The analysis results are available either through direct access to the database used to store it or through the export of the relevant data. Conclusions The software is not only limited to Western Blot image analysis and can be used to analyze images obtained as a part of many other widely used biochemical techniques such as isoelectric focusing. By recording the original data and all the analysis steps, the program improves reproducibility in the analysis and contributes to the implementation of FAIR principles in the related fields.

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions (SEIs) and double-Holliday junctions (dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication. [ABSTRACT FROM AUTHOR]

Background: Current solutions for the analysis of Western Blot images lack either transparency and reproducibility or can be tedious to use if one has to ensure the reproducibility of the analysis. Results: Here, we present an open-source gel image analysis program, IOCBIO Gel. It is designed to simplify image analysis and link the analysis results with the metadata describing the measurements. The software runs on all major desktop operating systems. It allows one to use it in either a single-researcher environment with local storage of the data or in a multiple-researcher environment using a central database to facilitate data sharing within the research team and beyond. By recording the original image and all operations performed on it, such as image cropping, subtraction of background, sample lane selection, and integration boundaries, the software ensures the reproducibility of the analysis and simplifies making corrections at any stage of the research. The analysis results are available either through direct access to the database used to store it or through the export of the relevant data. Conclusions: The software is not only limited to Western Blot image analysis and can be used to analyze images obtained as a part of many other widely used biochemical techniques such as isoelectric focusing. By recording the original data and all the analysis steps, the program improves reproducibility in the analysis and contributes to the implementation of FAIR principles in the related fields. [ABSTRACT FROM AUTHOR]

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients' blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method. [ABSTRACT FROM AUTHOR]

Telomere length (TL) limits somatic cell replication. However, the shortest among the telomeres in each nucleus, not mean TL, is thought to induce replicative senescence. Researchers have relied on Southern blotting (SB), and techniques calibrated by SB, for precise measurements of TL in epidemiological studies. However, SB provides little information on the shortest telomeres among the 92 telomeres in the nucleus of human somatic cells. Therefore, little is known about the accumulation of short telomeres with age, or whether it limits the human lifespan. To fill this knowledge void, we used the Telomere‐Shortest‐Length‐Assay (TeSLA), a method that tallies and measures single telomeres of all chromosomes. We charted the age‐dependent buildup of short telomeres (<3 kb) in human hematopoietic cells from 334 individuals (birth‐89 years) from the general population, and 18 patients with dyskeratosis congenita‐telomere biology disorders (DC/TBDs), whose hematopoietic cells have presumably reached or are close to their replicative limit. For comparison, we also measured TL with SB. We found that in hematopoietic cells, the buildup of short telomeres occurs in parallel with the shortening with age of mean TL. However, the proportion of short telomeres was lower in octogenarians from the general population than in patients with DC/TBDs. At any age, mean TL was longer and the proportion of short telomeres lower in females than in males. We conclude that though converging to the TL‐mediated replicative limit, hematopoietic cell telomeres are unlikely to reach this limit during the lifespan of most contemporary humans. [ABSTRACT FROM AUTHOR]

Rheum australe (Himalayan Rhubarb, Polygonaceae), an endangered medicinal and vegetable herb owes its age-old remedying properties to the bio-active phyto-constituents viz. anthraquinones, stilbenoids, chromones and dietary flavonoids. Polyketide pathway primarily involving the intricate Type III polyketide synthases (PKSs) contributes to the biosynthesis of these phyto-constituents. In the present study, we perform a homology-based approach to isolate an 1176 bp full-length cds sequence of the RaALS gene showing an equitable level of sequence similarity to related Type III PKSs at both nucleic acid and amino acid levels. In silico characterization revealed the presence of highly conserved amino acid residues found in nearly all Type III PKSs including the conserved active-site residues, signature motif and cyclization pocket residues with an exception of Ile256 and Gly258. Docking studies established major interactions between the starter acetyl-CoA and RaALS. Copy number analysis suggested slender evolution in Type III PKS in R. australe having a single copy of RaALS gene. qRT-PCR analyses revealed corroboration between the higher expression of RaALS in leaves followed by stem and root with that of the metabolite concentration. Expression studies further showed a direct increase of RaALS transcripts with the growing metabolite accretion in relation to altitude suggesting a probable involvement of specific Type III PKS in biosynthesis of the major phyto-constituents. Furthermore, abiotic stressors viz. methyl jasmonate, salicylic acid and UV light enhanced RaALS transcription hinting towards its role in defense mechanism in R. australe and highlighting the significance of RaALS as a prospective target for metabolic engineering. [ABSTRACT FROM AUTHOR]

Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus that is exerting a major restraint on global tomato production. In this transgenic approach, an RNA interference (RNAi)-based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction, and stimulus to biotic and abiotic disease was engineered targeting the AC1/Rep gene of the Oman strain of TYLCV-OM. The Rep-amiRNA constructs presented an effective approach in regulating the expression of the Rep gene against TYLCV as a silencing target to create transgenic Solanum lycopersicum L. plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a Southern hybridization analysis were performed to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1-transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with the TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the current study proposes that genetically transformed tomato plants expressing amiRNA could be a potential approach for engineering tolerance in plants against TYLCV infection and conceivably for the inhibition of viral diseases against different strains of whitefly-transmitted begomoviruses in Oman. [ABSTRACT FROM AUTHOR]

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients’ blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method.

Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus that is exerting a major restraint on global tomato production. In this transgenic approach, an RNA interference (RNAi)-based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction, and stimulus to biotic and abiotic disease was engineered targeting the AC1/Rep gene of the Oman strain of TYLCV-OM. The Rep-amiRNA constructs presented an effective approach in regulating the expression of the Rep gene against TYLCV as a silencing target to create transgenic Solanum lycopersicum L. plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a Southern hybridization analysis were performed to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1-transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with the TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the current study proposes that genetically transformed tomato plants expressing amiRNA could be a potential approach for engineering tolerance in plants against TYLCV infection and conceivably for the inhibition of viral diseases against different strains of whitefly-transmitted begomoviruses in Oman.

Telomeres, repeating TTAGGG sequences at the ends of eukaryotic chromosomes, increase genomic stability. Telomere shortening occurs not only during DNA replication associated with cell division but also under oxidative stress, where reactive oxygen species damage DNA. Therefore, changes in telomere length can be used to evaluate chronic cost or stress responses incurred by individuals. The phylogenetically unique Chondrichthyes are among the least-studied groups of marine vertebrates. Telomere data are limited and have only been reported in a few Chondrichthyes species. In this study, we measured telomere length and quantified oxidative stress and antioxidant power in 17 Chondrichthyes species whose telomere length has not been measured before. The presence of telomere sequences > 30 bp and lower values of oxidative stress were confirmed in most species. Average telomere length was not correlated with oxidative stress and antioxidant power in 15 species for which both measurements were available. It would be desirable in the future to elucidate the nature of telomeres in Chondrichthyes, and their direct relationship with reactive oxygen species. [ABSTRACT FROM AUTHOR]

DNA methylation of three cultivars, each of the fruit tree species pear, plum and apple, was analyzed by the methylation-sensitive amplified polymorphism (MSAP) marker. All three fruit tree cultivars were found to contain apparently lower levels of methylation at the 5′-CCGG sites than all other plant species, such as rice and wheat, studied by the same method. Sequencing of the representative loci isolated from the MSAP profiles indicated that both protein-coding genes and transposable elements (TEs) were involved in low methylation. Gel blotting using isolated MSAP fragments and fragment mixtures representing two major types of TEs (copia- and gypsy-like) as hybridization probes confirmed the unexpected low DNA methylation levels at the 5′-CCGG sites in these three fruit tree genomes. Our results suggest that the three asexually propagated perennial fruit trees may indeed contain unusual lower levels of DNA methylation, especially in TEs at the 5′-CCGG sites. Additionally, our results may also suggest that the often used MSAP marker, which targets only one kind of specific methylation-sensitive sites recognized by a pair of isoschizomers (e.g., 5′-CCGG by HpaII/MspI), is not always representative of other cytosine sites (e.g., CHH) or CG sites other than those of 5′-CCGGs in some plant species. [ABSTRACT FROM AUTHOR]

Cultivated potato (Solanum tuberosum) is susceptible to many pests and pathogens, but the most important threat to potato production is, so far, the late blight disease, caused by the oomycete Phytophthora infestans. Resistance genes from the wild Solanum sp. have been used by breeders to develop late-blight-resistant cultivars. Two sets of Black differentials potato genotypes (R1, R2, ..., R11) were used to identify a new marker for resistance gene R11 of potato to late blight. RAPD polymorphic bands were isolated, cloned, and converted into SCAR primers. By amplification of genomic DNA with SCAR primers followed by enzymatic digestion with HinfI restriction enzyme, and verified by Southern blotting, a marker of R11 resistance gene of potato to Phytophthora infestans was identified. [ABSTRACT FROM AUTHOR]

Gel image analyses are often difficult to reproduce, as the most commonly used software, the ImageJ Gels plugin, does not automatically record any steps in the analysis process. This protocol provides detailed steps for image analysis using IOCBIO Gel software with western blot as an example; however, the protocol is applicable to all images obtained by electrophoresis, such as Southern blotting, northern blotting, and isoelectric focusing. IOCBIO Gel allows multiple sample analyses, linking the original image to all the operations performed on it, which can be stored in a central database or on a PC, ensuring ease of access and the possibility to perform corrections at each analysis stage. In addition, IOCBIO Gel is lightweight, with only minimal computer requirements. Key features • Free and open-source software for analyzing gel images. • Reproducibility. • Can be used with images obtained by electrophoresis, such as western blotting, Southern blotting, isoelectric focusing, and more., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

DNA methylation of three cultivars, each of the fruit tree species pear, plum and apple, was analyzed by the methylation-sensitive amplified polymorphism (MSAP) marker. All three fruit tree cultivars were found to contain apparently lower levels of methylation at the 5′-CCGG sites than all other plant species, such as rice and wheat, studied by the same method. Sequencing of the representative loci isolated from the MSAP profiles indicated that both protein-coding genes and transposable elements (TEs) were involved in low methylation. Gel blotting using isolated MSAP fragments and fragment mixtures representing two major types of TEs (copia- and gypsy-like) as hybridization probes confirmed the unexpected low DNA methylation levels at the 5′-CCGG sites in these three fruit tree genomes. Our results suggest that the three asexually propagated perennial fruit trees may indeed contain unusual lower levels of DNA methylation, especially in TEs at the 5′-CCGG sites. Additionally, our results may also suggest that the often used MSAP marker, which targets only one kind of specific methylation-sensitive sites recognized by a pair of isoschizomers (e.g., 5′-CCGG by HpaII/MspI), is not always representative of other cytosine sites (e.g., CHH) or CG sites other than those of 5′-CCGGs in some plant species.

Telomeres are located at the ends of chromosomes and have specific sequences with a distinctive structure that safeguards genes. They possess capping structures that protect chromosome ends from fusion events and ensure chromosome stability. Telomeres shorten in length during each cycle of cell division. When this length reaches a certain threshold, it can lead to genomic instability, thus being implicated in various diseases, including cancer and neurodegenerative disorders. The possibility of telomeres serving as a biomarker for aging and age-related disease is being explored, and their significance is still under study. This is because post-mitotic cells, which are mature cells that do not undergo mitosis, do not experience telomere shortening due to age. Instead, other causes, for example, exposure to oxidative stress, can directly damage the telomeres, causing genomic instability. Nonetheless, a general agreement has been established that measuring telomere length offers valuable insights and forms a crucial foundation for analyzing gene expression and epigenetic data. Numerous approaches have been developed to accurately measure telomere lengths. In this review, we summarize various methods and their advantages and limitations for assessing telomere length., Competing Interests: The authors have no potential conflicts of interest to disclose. The funder played no role in the study’s design, data collection, analysis, interpretation of the data, writing of the report, or the decision to submit the report for publication., (© 2024 The Authors.)

Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus exerting a major restraint on world tomato production. In this transgenic approach, an RNA interference (RNAi) based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction and stimulus to biotic and a biotic disease was engineered targeting AC1/Rep gene of Oman strain of TYLCV-OM. The Rep-amiRNA constructs presentan effective approach in regulating the expression of Rep gene against TYLCV as a silencing target to create transgenic tomato plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a southern hybridization analysis was achieved to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1 transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the outcomes of this study advised that transformed plants expressing amiRNA could be an essential approach for engineering tolerant plants against TYLCV infection and conceivably for the inhibition of viral disease against different strains of whitefly transmitted begomoviruses in Oman. [ABSTRACT FROM AUTHOR]

• The Trichomonas vaginalis genome contains (TTTTAGGG) n tandem repeats. • The repeat units are located at the end of T. vaginalis chromosomes. • T. vaginalis telomere length varies from 1.0–1.5 kb. Trichomoniasis is the most common nonviral sexually transmitted disease; it is caused by Trichomonas vaginalis and seriously threatens human reproductive health. Telomeres are specialised DNA–protein complexes at the ends of chromosomes that have a protective function. The aim of the present study was to identify and characterise the telomeric DNA of T. vaginalis —which has not been previously reported—by multiple molecular methods including sequencing, the Bal nuclease (BAL) 31 nuclease assay, fluorescence in situ hybridisation (FISH), and Southern blotting. We found numerous repeated units of TTTTAGGG in T. vaginalis genomic DNA digested with S1 nuclease in combination with Xba I restriction enzyme. The (TTTTAGGG) n tandem repeats were also highly sensitive to BAL 31 exonuclease digestion. We confirmed that the (TTTTAGGG) n repeats were located at the end of T. vaginalis chromosomes by FISH. Restriction enzyme digestion combined with Southern blotting using a digoxigenin-labelled (TTTTAGGG) 5 probe showed that the T. vaginalis telomeric DNA length varied from 1.0 to 1.5 kb. This is the first report on the telomeric DNA sequence of T. vaginalis which includes the length and distribution on chromosomes; our findings lay a foundation for further study on telomere maintenance mechanisms in T. vaginalis. [ABSTRACT FROM AUTHOR]

Repeat expansion diseases, including fragile X syndrome, Huntington's disease, and C9orf72 -related motor neuron disease and frontotemporal dementia, are a group of disorders associated with polymorphic expansions of tandem repeat nucleotide sequences. These expansions are highly repetitive and often hundreds to thousands of repeats in length, making accurate identification and determination of repeat length via PCR or sequencing challenging. Here we describe a protocol for monitoring repeat length in Drosophila models carrying 1,000 repeat C9orf72 -related dipeptide repeat transgenes using Southern blotting. This protocol has been used regularly to check the length of these lines for over 100 generations with robust and repeatable results and can be implemented for monitoring any repeat expansion in Drosophila ., Competing Interests: Competing interestsThe authors declare no competing interests, (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)