Repeat Contraction Research Articles

CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair

BackgroundThe expansion of CAG/CTG repeats in functionally unrelated genes is a causative factor in many inherited neurodegenerative disorders, including Huntington’s disease (HD), spinocerebellar ataxias (SCAs), and myotonic dystrophy type 1 (DM1). Despite many years of research, the mechanism responsible for repeat instability is unknown, and recent findings indicate the key role of DNA repair in this process. The repair of DSBs induced by genome editing tools results in the shortening of long CAG/CTG repeats in yeast models. Understanding this mechanism is the first step in developing a therapeutic strategy based on the controlled shortening of repeats. The aim of this study was to characterize Cas9-induced DSB repair products at the endogenous HTT locus in human cells and to identify factors affecting the formation of specific types of sequences.ResultsThe location of the cleavage site and the surrounding sequence influence the outcome of DNA repair. DSBs within CAG repeats result in shortening of the repeats in frame in ~ 90% of products. The mechanism of this contraction involves MRE11-CTIP and RAD51 activity and DNA end resection. We demonstrated that a DSB located upstream of CAG repeats induces polymerase theta-mediated end joining, resulting in deletion of the entire CAG tract. Furthermore, using proteomic analysis, we identified novel factors that may be involved in CAG sequence repair.ConclusionsOur study provides new insights into the complex mechanisms of CRISPR/Cas9-induced shortening of CAG repeats in human cells.

Utility of Optical Genome Mapping in Repeat Disorders.

Genomic repeat sequences are patterns of nucleic acids that exist in multiple copies throughout the genome. More than 60 Mendelian disorders are caused by the expansion or contraction of these repeats. Various specific methods for determining tandem repeat variations have been developed. However, these methods are highly specific to the genomic region being studied and sometimes require specialized tools. In this study, we have investigated the use of Optical Genome Mapping (OGM) as a diagnostic tool for detecting repeat disorders. We evaluated 19 patients with a prediagnosis of repeat disorders and explained the molecular etiology of 9 of them with OGM (5 patients with Facioscapulohumeral Muscular Dystrophy (FSHD), 2 patients with Friedreich's Ataxia (FA), 1 patient with Fragile X Syndrome (FXS), and 1 patient with Progressive Myoclonic Epilepsy 1A (EPM1A)). We confirmed OGM results with more widely used fragment analysis techniques. This study highlights the utility of OGM as a diagnostic tool for repeat expansion and contraction diseases such as FA, FXS, EPM1A, and FSHD.

Chloroplast genome evolution of Berberis (Berberidaceae): Implications for phylogeny and metabarcoding

Berberidis Radix (Sankezhen), a typical multi-origin Chinese medicinal material, originates from the dried roots of plants of the Berberis genus and is used to treat various ailments. These species have similar morphologies, potentially leading to misidentifications that can impact medicine efficacy. Therefore, developing suitable molecular markers to identify medicinal species is imperative. Furthermore, discrepancies exist in the taxonomy of the Berberis genus. In the present study, we de novo assembled the chloroplast genomes of six Berberis species (Berberis woomungensis C. Y. Wu, Berberis pruinosa Franch., Berberis thunbergii DC., Berberis chinensis Poir., Berberis wilsoniae Hemsl., and Berberis sp.) that commonly constitute Berberidis Radix and compared them with previously reported genomes. Our comparative analysis revealed similarities in genome structure, relative synonymous codon usage, amino acid frequency, repeats, and substitutions. Higher synonymous substitutions, indicative of predominant purifying selection on protein-coding genes, were observed compared to non-synonymous substitutions. However, positive selection was identified in six genes across 29 Berberis species—accD, matK, ndhD, rbcL, ycf1, and ycf2—highlighting their potential roles in adaptive responses to specific environmental conditions within the genus. Inverted repeats expansion and contraction affected the rate of mutations and were associated with the phylogenetic classification of Berberis. Our phylogenetic analysis supported the division of the Berberis complex into four genera, which corroborates previous studies involving extensive sampling. We identified the ndhD-ccsA region as the most polymorphic region and applied this region to Chinese patent medicines containing Berberidis Radix through metabarcoding. The metabarcoding analysis confirmed that five Berberis species commonly constitute Berberidis Radix in Chinese patent medicines. In conclusion, this study provides insight into the molecular evolution of the chloroplast genome and the phylogeny of the Berberis genus. In addition, metabarcoding provides insight into the species composition of Berberidis Radix in Chinese patent medicines.

Polyphyly of Boehmeria (Urticaceae) congruent with plastome structural variation.

Boehmeria is a taxonomically challenging group within the nettle family (Urticaceae). The polyphyly of the genus has been proposed by previous studies with respect to five genera (Debregeasia, Cypholophus, Sarcochlamys, Archiboehmeria, and Astrothalamus). Extensive homoplasy of morphological characters has made generic delimitation problematic. Previous studies in other plant groups suggest that plastome structural variations have the potential to provide characters useful in reconstructing evolutionary relationships. We aimed to test this across Boehmeria and its allied genera by mapping plastome structural variations onto a resolved strongly supported phylogeny. In doing so, we expanded the sampling of the plastome to include Cypholophus, Sarcochlamys, Archiboehmeria, and Astrothalamus for the first time. The results of our phylogenomic analyses provide strong support for Sarcochlamys as being more closely related to Leucosyke puya than to Boehmeria and for the clustering of Boehmeria s.l. into four subclades. The sizes of the plastomes in Boehmeria s.l. ranged from 142,627 bp to 170,958 bp. The plastomes recovered a typical quadripartite structure comprising 127~146 genes. We observe several obvious structural variations across the taxa such as gene loss and multiple gene duplication, inverted repeat (IR) contraction and wide expansions, and inversions. Moreover, we recover a trend for these variations that the early clades were relatively conserved in evolution, whereas the later diverging clades were variable. We propose that the structural variations documented may be linked to the adaptation of Boehmeria s.l. to a wide range of habitats, from moist broadleaf forests in Asia to xeric shrublands and deserts in Africa. This study confirms that variation in plastome gene loss/duplication, IR contraction/expansion, and inversions can provide evidence useful for the reconstruction of evolutionary relationships.

DNA Base Damage Repair Crosstalks with Chromatin Structures to Contract Expanded GAA Repeats in Friedreich's Ataxia.

Trinucleotide repeat (TNR) expansion is the cause of over 40 neurodegenerative diseases, including Huntington's disease and Friedreich's ataxia (FRDA). There are no effective treatments for these diseases due to the poor understanding of molecular mechanisms underlying somatic TNR expansion and contraction in neural systems. We and others have found that DNA base excision repair (BER) actively modulates TNR instability, shedding light on the development of effective treatments for the diseases by contracting expanded repeats through DNA repair. In this study, temozolomide (TMZ) was employed as a model DNA base damaging agent to reveal the mechanisms of the BER pathway in modulating GAA repeat instability at the frataxin (FXN) gene in FRDA neural cells and transgenic mouse mice. We found that TMZ induced large GAA repeat contraction in FRDA mouse brain tissue, neurons, and FRDA iPSC-differentiated neural cells, increasing frataxin protein levels in FRDA mouse brain and neural cells. Surprisingly, we found that TMZ could also inhibit H3K9 methyltransferases, leading to open chromatin and increasing ssDNA breaks and recruitment of the key BER enzyme, pol β, on the repeats in FRDA neural cells. We further demonstrated that the H3K9 methyltransferase inhibitor BIX01294 also induced the contraction of the expanded repeats and increased frataxin protein in FRDA neural cells by opening the chromatin and increasing the endogenous ssDNA breaks and recruitment of pol β on the repeats. Our study provides new mechanistic insight illustrating that inhibition of H3K9 methylation can crosstalk with BER to induce GAA repeat contraction in FRDA. Our results will open a new avenue for developing novel gene therapy by targeting histone methylation and the BER pathway for repeat expansion diseases.

Complete chloroplast genome and comparison of herbicides toxicity on Aeschynomene indica (Leguminosae) in upland direct-seeding paddy field

BackgroundIndian jointvetch (Aeschynomene indica) is a common and pernicious weed found in the upland direct-seeding rice fields in the lower reaches of the Yangtze River in China. However, there are few reports on the degree of harm, genetic characteristics, and management methods of this weed. The purpose of this study is to clarify the harm of Indian jointvetch to upland direct-seeding rice, analyze the genetic characteristics of this weed based on chloroplast genomics and identify its related species, and screen herbicides that are effective in managing this weed in upland direct-seeding rice fields.ResultsIn a field investigation in upland direct-seeding rice paddies in Shanghai and Jiangsu, we determined that the plant height and maximum lateral distance of Indian jointvetch reached approximately 134.2 cm and 57.9 cm, respectively. With Indian jointvetch present at a density of 4/m2 and 8/m2, the yield of rice decreased by approximately 50% and 70%, respectively. We further obtained the first assembly of the complete chloroplast (cp.) genome sequence of Indian jointvetch (163,613 bp). There were 161 simple sequence repeats, 166 long repeats, and 83 protein-encoding genes. The phylogenetic tree and inverted repeat region expansion and contraction analysis based on cp. genomes demonstrated that species with closer affinity to A. indica included Glycine soja, Glycine max, and Sesbania cannabina. Moreover, a total of 3281, 3840, and 3838 single nucleotide polymorphisms were detected in the coding sequence regions of the cp. genomes of S. cannabina voucher IBSC, G. soja, and G. max compared with the A. indica sequence, respectively. A greenhouse pot experiment indicated that two pre-emergence herbicides, saflufenacil and oxyfluorfen, and two post-emergence herbicides, florpyrauxifen-benzyl and penoxsulam, can more effectively manage Indian jointvetch than other common herbicides in paddy fields. The combination of these two types of herbicides is recommended for managing Indian jointvetch throughout the entire growth period of upland direct-seeding rice.ConclusionsThis study provides molecular resources for future research focusing on the identification of the infrageneric taxa, phylogenetic resolution, and biodiversity of Leguminosae plants, along with recommendations for reliable management methods to control Indian jointvetch.

WITHDRAWN: Chloroplast genome evolution of Berberis (Berberidaceae): Implications for phylogeny and metabarcoding

Berberidis Radix (Sankezhen), a typical multi-origin Chinese medicinal material, originates from the dried roots of the Berberis genus plants and is used to treat a range of ailments. These species have similar morphology potentially leading to misidentification that can impact the medicine's efficacy. Therefore, it is imperative to develop suitable molecular markers to identify medicinal species. Furthermore, discrepancies exist in the taxonomy of the Berberis genus. In the current study, we de novo assembled the chloroplast genomes of six Berberis species (Berberis julianae, Berberis tsienii, Berberis pruinosa, Berberis thunbergii, Berberis poiretii, and Berberis wilsoniae) that commonly constitute Berberidis Radix and compared them with previously reported genomes. Our comparative analysis revealed similarities in genome structure, relative synonymous codon usage, amino acid frequency, repeats, and substitutions. Higher synonymous substitutions, indicative of predominant purifying selection on protein-coding genes, were observed compared to non-synonymous substitutions. However, positive selection was identified for six genes across 29 Berberis species—accD, matK, ndhD, rbcL, ycf1 and ycf2—highlighting their potential roles in adaptive responses to specific environmental conditions within the genus. Inverted repeats expansion and contraction affected the rate of mutations and is associated with the phylogenetic classification of Berberis. Our phylogenetic analysis supported the division of the Berberis complex into four genera, which corroborates the findings of previous studies involving extensive sampling. We identified the ndhD-ccsA region as the most polymorphic region and applied this region to Chinese patent medicines containing Berberidis Radix through metabarcoding. Metabarcoding analysis confirmed that five Berberis species commonly constitute Berberidis Radix in Chinese patent medicines. In conclusion, this study provides insight into the molecular evolution of the chloroplast genome and the phylogeny of the Berberis genus. In addition, through metabarcoding, provides insight into the species composition of Berberidis Radix in Chinese patent medicines.

Comparative and phylogenetic analyses of nine complete chloroplast genomes of Orchidaceae

The orchid family has 200,000 species and 700 genera, and it is found worldwide in the tropics and subtropics. In China, there are 1247 species and subspecies of orchids belonging to the Orchidaceae family. Orchidaceae is one of the most diverse plant families in the world, known for their lush look, remarkable ecological tolerance, and capability for reproduction. It has significant decorative and therapeutic value. In terms of evolution, the orchid family is one of the more complicated groups, but up until now, little has been known about its affinities. This study examined the properties of 19 chloroplast (cp) genomes, of which 11 had previously been published and nine had only recently been revealed. Following that, topics such as analysis of selection pressure, codon usage, amino acid frequencies, repeated sequences, and reverse repeat contraction and expansion are covered. The Orchidaceae share similar cp chromosomal characteristics, and we have conducted a preliminary analysis of their evolutionary connections. The cp genome of this family has a typical tepartite structure and a high degree of consistency across species. Platanthera urceolata with more tandem repeats of the cp genome. Similar cp chromosomal traits can be seen in the orchidaceae. Galearis roborowskyi, Neottianthe cucullata, Neottianthe monophylla, Platanthera urceolata and Ponerorchis compacta are the closest cousins, according to phylogenetic study.

Massive contractions of myotonic dystrophy type 2-associated CCTG tetranucleotide repeats occur via double-strand break repair with distinct requirements for DNA helicases.

Myotonic dystrophy type 2 (DM2) is a genetic disease caused by expanded CCTG DNA repeats in the first intron of CNBP. The number of CCTG repeats in DM2 patients ranges from 75 to 11,000, yet little is known about the molecular mechanisms responsible for repeat expansions or contractions. We developed an experimental system in Saccharomyces cerevisiae that enables the selection of large-scale contractions of (CCTG)100 within the intron of a reporter gene and subsequent genetic analysis. Contractions exceeded 80 repeat units, causing the final repetitive tract to be well below the threshold for disease. We found that Rad51 and Rad52 are involved in these massive contractions, indicating a mechanism that uses homologous recombination. Srs2 helicase was shown previously to stabilize CTG, CAG, and CGG repeats. Loss of Srs2 did not significantly affect CCTG contraction rates in unperturbed conditions. In contrast, loss of the RecQ helicase Sgs1 resulted in a 6-fold decrease in contraction rate with specific evidence that helicase activity is required for large-scale contractions. Using a genetic assay to evaluate chromosome arm loss, we determined that CCTG and reverse complementary CAGG repeats elevate the rate of chromosomal fragility compared to a short-track control. Overall, our results demonstrate that the genetic control of CCTG repeat contractions is notably distinct among disease-causing microsatellite repeat sequences.

Genome instability footprint under rapamycin and hydroxyurea treatments.

The mutational processes dictating the accumulation of mutations in genomes are shaped by genetic background, environment and their interactions. Accurate quantification of mutation rates and spectra under drugs has important implications in disease treatment. Here, we used whole-genome sequencing and time-resolved growth phenotyping of yeast mutation accumulation lines to give a detailed view of the mutagenic effects of rapamycin and hydroxyurea on the genome and cell growth. Mutation rates depended on the genetic backgrounds but were only marginally affected by rapamycin. As a remarkable exception, rapamycin treatment was associated with frequent chromosome XII amplifications, which compensated for rapamycin induced rDNA repeat contraction on this chromosome and served to maintain rDNA content homeostasis and fitness. In hydroxyurea, a wide range of mutation rates were elevated regardless of the genetic backgrounds, with a particularly high occurrence of aneuploidy that associated with dramatic fitness loss. Hydroxyurea also induced a high T-to-G and low C-to-A transversion rate that reversed the common G/C-to-A/T bias in yeast and gave rise to a broad range of structural variants, including mtDNA deletions. The hydroxyurea mutation footprint was consistent with the activation of error-prone DNA polymerase activities and non-homologues end joining repair pathways. Taken together, our study provides an in-depth view of mutation rates and signatures in rapamycin and hydroxyurea and their impact on cell fitness, which brings insights for assessing their chronic effects on genome integrity.

Comparative Chloroplast Genome Analyses of Six Hemlock Trees in East Asia: Insights into Their Genomic Characterization and Phylogenetic Relationship

Hemlocks (Pinaceae: Tsuga) are widely distributed in North America and East Asia, forming a reticulate evolutionary structure in East Asia with significant ecological importance. To clarify the chloroplast genome characteristics and phylogenetic relationships among Tsuga species, we analyzed the chloroplast genomes of T. chinensis var. tchekiangensis, T. chinensis, T. diversifolia, T. dumosa, T. forrestii, and T. sieboldii, performing associated phylogenetic analyses. The results reveal that the chloroplast genome lengths among the six Tsuga species vary from 120,520 to 121,010 bp, encompassing about 108 to 112 genes, including 35/32 tRNA genes and 4 rRNA genes. A codon usage analysis highlighted a preference for A/U-ending codons, and all six nucleotide types have A/T bases and a prevalence of mononucleotides. Notably, all Tsuga species exhibit inverted repeat (IR) contractions and possess unique hexanucleotides absent in the other species of Pinaceae, potentially making them more susceptible to gene recombination or rearrangement during evolution. While most variations are observed in non-coding regions, particularly in intergenic fragments, substantial variation sites are also present within the genes. The phylogenetic tree, constructed using chloroplast genomes, substantiates the sister taxa relationship between Tsuga and Nothotsuga. Furthermore, it confirms that T. chinensis var. tchekiangensis exhibits a closer relationship with T. forrestii than with T. chinensis. These findings not only provide partial evidence that T. chinensis may not constitute a monophyletic species but also underscore the necessity of reevaluating the taxonomic status of T. chinensis var. tchekiangensis. In addition, while the RSCU cluster analysis is basically consistent with the phylogenetic analysis, it also highlights a distinct differentiation between Nothotsuga and Tsuga. This study not only provides molecular-level phylogenetic classification evidence of Pinaceous genera via chloroplast genome analyses but also offers compelling evidence for further exploring the relationships and species delimitation among the hemlocks of East Asia.

Comparative Analysis of Plastomes in Elsholtzieae: Phylogenetic Relationships and Potential Molecular Markers.

The Elsholtzieae, comprising ca. 7 genera and 70 species, is a small tribe of Lamiaceae (mint family). Members of Elsholtzieae are of high medicinal, aromatic, culinary, and ornamentals value. Despite the rich diversity and value of Elsholtzieae, few molecular markers or plastomes are available for phylogenetics. In the present study, we employed high-throughput sequencing to assemble two Mosla plastomes, M. dianthera and M. scabra, for the first time, and compared with other plastomes of Elsholtzieae. The plastomes of Elsholtzieae exhibited a quadripartite structure, ranging in size from 148,288 bp to 152,602 bp. Excepting the absence of the pseudogene rps19 in Elsholtzia densa, the exhaustive tally revealed the presence of 132 genes (113 unique genes). Among these, 85 protein-coding genes (CDS), 37 tRNA genes, 8 rRNA genes, and 2 pseudogenes (rps19 and ycf1) were annotated. Comparative analyses showed that the plastomes of these species have minor variations at the gene level. Notably, the E. eriostchya plastid genome exhibited increased GC content regions in the LSC and SSC, resulting in an increased overall GC content of the entire plastid genome. The E. densa plastid genome displayed modified boundaries due to inverted repeat (IR) contraction. The sequences of CDS and intergenic regions (IGS) with elevated variability were identified as potential molecular markers for taxonomic inquiries within Elsholtzieae. Phylogenetic analysis indicated that four genera formed monophyletic entities, with Mosla and Perilla forming a sister clade. This clade was, in turn, sister to Collinsonia, collectively forming a sister group to Elsholtzia. Both CDS, and CDS + IGS could construct a phylogenetic tree with stronger support. These findings facilitate species identification and DNA barcoding investigations in Elsholtzieae and provide a foundation for further exploration and resource utilization within this tribe.

Comparison of Biological and Genetic Characteristics between Two Most Common Broad-Leaved Weeds in Paddy Fields: Ammannia arenaria and A. multiflora (Lythraceae).

Ammannia arenaria and A. multifloras, morphologically similar at the seedling stage, are the most common broad-leaved weeds in paddy fields. Our study showed that A. arenaria occupied more space than A. multifloras when competing with rice. However, A. multifloras germination has lower temperature adaptability. No difference in sensitivity to common herbicides between two Ammannia species was observed. Chloroplast (cp) genomes could be conducive to clarify their genetic relationship. The complete cp genome sequences of A. arenaria (158,401 bp) and A. multiflora (157,900 bp) were assembled for the first time. In A. arenaria, there were 91 simple sequence repeats, 115 long repeats, and 86 protein-encoding genes, one, sixteen, and thirty more than those in A. multiflora. Inverted repeats regions expansion and contraction and the phylogenetic tree based on cp genomes demonstrated the closely relationship between the two species. However, in A. arenaria, 20 single nucleotide polymorphisms in the CDS region were detected compared to A. multiflora, which can be used to distinguish the two species. Moreover, there was one unique gene, infA, only in A. arenaria. This study provides reliable molecular resources for future research focusing on the infrageneric taxa identification, phylogenetic resolution, population structure, and biodiversity of Ammannia species.

Site-specific R-loops induce CGG repeat contraction and fragile X gene reactivation

Here, we describe an approach to correct the genetic defect in fragile X syndrome (FXS) via recruitment of endogenous repair mechanisms. A leading cause of autism spectrum disorders, FXS results from epigenetic silencing of FMR1 due to a congenital trinucleotide (CGG) repeat expansion. By investigating conditions favorable to FMR1 reactivation, we find MEK and BRAF inhibitors that induce a strong repeat contraction and full FMR1 reactivation in cellular models. We trace the mechanism to DNA demethylation and site-specific R-loops, which are necessary and sufficient for repeat contraction. A positive feedback cycle comprising demethylation, de novo FMR1 transcription, and R-loop formation results in the recruitment of endogenous DNA repair mechanisms that then drive excision of the long CGG repeat. Repeat contraction is specific to FMR1 and restores the production of FMRP protein. Our study therefore identifies a potential method of treating FXS in the future.

Comparison of the chloroplast genomes and phylogenomic analysis of Elaeocarpaceae.

Elaeocarpaceae is a vital family in tropical and subtropical forests. Compared with the important position of Elaeocarpaceae species in forest ecosystem and the concern of medicinal value, the most research on Elaeocarpaceae are classification and taxonomy. Molecular systematics has corrected the morphological misjudgment, and it belongs to Oxalidales. Phylogenetic and divergence time estimates of Elaeocarpaceae is mostly constructed by using chloroplast gene fragments. At present, although there are reports on the chloroplast structure of Elaeocarpaceae, a comprehensive analysis of the chloroplast structure of Elaeocarpaceae is lacking. To understand the variation in chloroplast sequence size and structure in Elaeocarpaceae, the chloroplast genomes of nine species were sequenced using the Illumina HiSeq 2500 platform and further assembled and annotated with Elaeocarpus japonicus and Sloanea sinensis (family Elaeocarpaceae) as references. A phylogenomic tree was constructed based on the complete chloroplast genomes of the 11 species representing five genera of Elaeocarpaceae. Chloroplast genome characteristics were examined by using Circoletto and IRscope software. The results revealed the following: (a) The 11 sequenced chloroplast genomes ranged in size from 157,546 to 159,400 bp. (b) The chloroplast genomes of Elaeocarpus, Sloanea, Crinodendron and Vallea lacked the rpl32 gene in the small single-copy (SSC) region. The large single-copy (LSC) region of the chloroplast genomes lacked the ndhK gene in Elaeocarpus, Vallea stipularis, and Aristotelia fruticosa. The LSC region of the chloroplast genomes lacked the infA gene in genus Elaeocarpus and Crinodendron patagua. (c) Through inverted repeat (IR) expansion and contraction analysis, a significant difference was found between the LSC/IRB and IRA/LSC boundaries among these species. Rps3 was detected in the neighboring regions of the LSC and IRb regions in Elaeocarpus. (d) Phylogenomic analysis revealed that the genus Elaeocarpus is closely related to Crinodendron patagua on an independent branch and Aristotelia fruticosa is closely related to Vallea stipularis, forming a clade with the genus Sloanea. Structural comparisons showed that Elaeocarpaceae diverged at 60 Mya, the genus Elaeocarpus diverged 53 Mya and that the genus Sloanea diverged 0.44 Mya. These results provide new insight into the evolution of the Elaeocarpaceae.

Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks

Expanded CAG/CTG repeats are sites of DNA damage, leading to repeat length changes. Homologous recombination (HR) is one cause of repeat instability and we hypothesized that gap filling was a driver of repeat instability during HR. To test this, we developed an assay such that resection and ssDNA gap fill-in would occur across a (CAG)70 or (CTG)70 repeat tract. When the ssDNA template was a CTG sequence, there were increased repeat contractions and a fragile site was created leading to large-scale deletions. When the CTG sequence was on the resected strand, resection was inhibited, resulting in repeat expansions. Increased nucleolytic processing by deletion of Rad9, the ortholog of 53BP1, rescued repeat instability and chromosome breakage. Loss of Rad51 increased contractions implicating a protective role for Rad51 on ssDNA. Together, our work implicates structure-forming repeats as an impediment to resection and gap-filling which can lead to mutations and large-scale deletions.

Structural variation among assembled genomes facilitates development of rapid and low-cost NOR-linked markers and NOR-telomere junction mapping in Arabidopsis.

Genome-wide structural variants we identified and new NOR-linked markers we developed would be useful for future genome-wide association studies (GWAS), and for new gene/trait mapping purposes. Bioinformatic alignment of the assembled genomes of Col-0 and Sha ecotypes of Arabidopsis thaliana revealed ~ 13,000 genome-wide structural variants involving simple insertions or deletions and repeat contractions or expansions. Using some of these structural variants, we developed new, rapid, and low-cost PCR-based molecular markers that are genetically linked to the nucleolus organizer regions (NORs). A. thaliana has two NORs, one each on chromosome 2 (NOR2) and chromosome 4 (NOR4). Both NORs are ~ 4Mb each, and hundreds of 45S ribosomal RNA (rRNA) genes are tandemly arrayed at these loci. Using previously characterized recombinant inbred lines (RILs) derived from Sha xCol-0 crosses, we validated the utility of the newly developed NOR-linked markers in genetically mapping rRNA genes and the associated telomeres to either NOR2 or NOR4. Lastly, we sequenced Sha genome using Oxford Nanopore Technology (ONT) and used the data to obtain sequences of NOR-telomere junctions, and with the help of RILs, we mapped them as new genetic markers to their respective NORs (NOR2-TEL2N and NOR4-TEL4N). The structural variants obtained from this study would serve as valuable data for genome-wide association studies (GWAS), and to rapidly design more genome-wide genetic (molecular) markers for new gene/trait mapping purposes.

Effect of focused ultrasound peripheral nerve stimulation on muscle mechanical properties: An in vivo murine model

Electrical muscle stimulation is limited to surface muscle activation; however, focused ultrasound (fUS) has the potential to stimulate deep skeletal muscles. This study aims to evaluate the effects of fUS peripheral nerve stimulation on muscle mechanical properties in an in vivo murine model. The sciatic nerve of 19 Sprague-Dawley rats was insonified with 3.68-MHz fUS (p+ = 6.4 MPa, p− = 5 MPa) using 10-ms pulses (0.9-ms on/0.1-ms off, 10 times) repeated at 0.5-Hz for 30, 90, or 180 severy second day for two weeks. Stimulation was monitored using electromyography (EMG) with indwelling electrodes inserted in the gastrocnemius muscle belly. Bilateral muscles were harvested for tensile mechanical testing (n = 15) and histology (n = 4). Muscle contraction was confirmed via EMG in 16/19 rats. Elastic moduli of stimulated muscles (133.2 ± 42.1 kPa) and controls (101.4 ± 39.2 kPa) were not different (ANOVA, p = 0.053). A trend toward increased elastic modulus with number of muscle stimulations was observed until 450 stimulations (not significant: ANOVA, p = 0.437), after which elastic modulus began to decrease. No tissue damage was observed histologically. The results of this pilot study demonstrate that fUS can induce repeatable muscle contraction that may improve muscle strength and mechanical properties over time. [Work supported by PSU College of Engineering Seed Grant 2021.]

Gene-Environment Interactions in Repeat Expansion Diseases: Mechanisms of Environmentally Induced Repeat Instability.

Short tandem repeats (STRs) are units of 1-6 base pairs that occur in tandem repetition to form a repeat tract. STRs exhibit repeat instability, which generates expansions or contractions of the repeat tract. Over 50 diseases, primarily affecting the central nervous system and muscles, are characterized by repeat instability. Longer repeat tracts are typically associated with earlier age of onset and increased disease severity. Environmental exposures are suspected to play a role in the pathogenesis of repeat expansion diseases. Here, we review the current knowledge of mechanisms of environmentally induced repeat instability in repeat expansion diseases. The current evidence demonstrates that environmental factors modulate repeat instability via DNA damage and induction of DNA repair pathways, with distinct mechanisms for repeat expansion and contraction. Of particular note, oxidative stress is a key mediator of environmentally induced repeat instability. The preliminary evidence suggests epigenetic modifications as potential mediators of environmentally induced repeat instability. Future research incorporating an array of environmental exposures, new human cohorts, and improved model systems, with a continued focus on cell-types, tissues, and critical windows, will aid in identifying mechanisms of environmentally induced repeat instability. Identifying environmental modulators of repeat instability and their mechanisms of action will inform preventions, therapies, and public health measures.

Online Classification of Transient EMG Patterns for the Control of the Wrist and Hand in a Transradial Prosthesis

Decoding human motor intentions by processing electrophysiological signals is a crucial, yet unsolved, challenge for the development of effective upper limb prostheses. Pattern recognition of continuous myoelectric (EMG) signals represents the state-of-art for multi-DoF prosthesis control. However, this approach relies on the unreliable assumption that repeatable muscular contractions produce repeatable patterns of steady-state EMGs. Here, we propose an approach for decoding wrist and hand movements by processing the signals associated with the onset of contraction (transient EMG). Specifically, we extend the concept of a transient EMG controller for the control of both wrist and hand, and tested it online. We assessed it with one transradial amputee and 15 non-amputees via the Target Achievement Control test. Non-amputees successfully completed 95% of the trials with a median completion time of 17 seconds, showing a significant learning trend ( <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">p</i> < 0.001). The transradial amputee completed about the 80% of the trials with a median completion time of 26 seconds. Although the performance proved comparable with earlier studies, the long completion times suggest that the current controller is not yet clinically viable. However, taken collectively, our outcomes reinforce earlier hypothesis that the transient EMG could represent a viable alternative to steady-state pattern recognition approaches.