Low Copy Research Articles

Development of a LAMP assay for the rapid visual detection of the emerging tick-borne Songling virus

BackgroundSongling virus (SGLV) within the genus Orthonairovirus, family Nairoviridae, is an emerging tick-borne virus associated with human febrile illness. However, no rapid detection method for SGLV has been established.MethodsIn this study, four primer sets targeting the nucleocapsid protein gene of SGLV were designed for use in the LAMP assay and evaluated to identify the optimal primer set. Recombinant plasmids were constructed and utilized for assessing the sensitivity of the assay. Tacheng tick virus 1 (TcTV-1)-, Beiji nairovirus (BJNV)-, Yezo virus (YEZV)-, severe fever with thrombocytopenia syndrome virus (SFTSV)-, and tick-borne encephalitis virus (TBEV)-positive tick samples were utilized to assess the specificity. Field-collected ticks were also evaluated as biological specimens to validate the assay.ResultsA SGLV-specific LAMP assay was established with a detection limit of 1 × 10–2 copies/μl and could be visually confirmed by a color change from purple to blue in SGLV-positive samples. No cross-reactivity was observed in the detection of TcTV-1, BJNV, YEZV, SFTSV, and TBEV using the LAMP assay. In addition to the detection of the same seven high-copy numbers of SGLV as the SYBR Green quantitative RT-PCR assay within a reduced timeframe, the developed LAMP method also effectively identified an additional sample with a low copy number in the field-collected tick samples.ConclusionsWe successfully developed a sensitive, specific, and cost-effective visual method for the rapid detection of SGLV using the LAMP assay, which can be applied in pathogenesis and epidemiological surveillance studies of SGLV.Graphical

Increased Susceptibility of Cardiac Tissue to PM2.5-Induced Toxicity in Uremic Cardiomyopathic Rats Is Linked to Elevated Levels of Mitochondrial Dysfunction.

Patients with chronic kidney disease (CKD) frequently develop uremic cardiomyopathy, characterized by mitochondrial dysfunction as one of its pathologically significant mediators. Given that PM2.5 specifically targets cardiac mitochondria, exacerbating toxicity, this study addresses the potential alterations in the severity of PM2.5 toxicity in the context of CKD conditions. Female Wistar rats were exposed to PM2.5 at a concentration of 250 μg/m3 daily for 3 h for 21 days after which an adenine-induced CKD model was developed. While both PM2.5 exposure and the induction of CKD in rats lead to cardiomyopathy, the CKD animals exposed to PM2.5 exhibited a notably severe extent of myocardial hypertrophy and fibrosis. ECG recordings in CKD+ PM2.5 animals revealed a depressed ST segment and prolonged QRS interval, with both PM2.5 and CKD animals displaying an elevated ST segment. Subcellular level analysis confirmed a significantly low mitochondrial copy number and a severe decline in mitochondrial bioenergetic function in the CKD+ PM2.5 group. The prominent decline in PGC1-α further affirmed the severe mitochondrial functional deterioration in CKD+ PM2.5 animals compared to other experimental groups. Additionally, myocardial calcification was enhanced in CKD+ PM2.5 animals, heightening the susceptibility of CKD animals to PM2.5 toxicity. In summary, our findings suggest that the increased vulnerability of CKD myocardium to PM2.5-induced toxicity may be attributed to severe mitochondrial damage and increased calcification in the myocardium.

Mitochondrial DNA copy number and neurocognitive outcomes in children.

Low mitochondria DNA copy number (mtDNAcn) has been linked to cognitive decline. However, the role of mtDNAcn in healthy cognitive development is unclear. We hypothesized early-life mtDNAcn would be associated with children's learning and memory. We quantified mtDNAcn in umbilical cord blood and child blood at ages 5-7 from participants in a prospective birth cohort. We administered the Children's Memory Scale (CMS) at ages 9-14 (N = 342) and the Wechsler Intelligence Scale for Children (WISC-IV) at ages 7 and 9 (N = 457). Associations between mtDNAcn tertiles and CMS and WISC were evaluated with linear regression and linear mixed-effects models, respectively. We examined non-linear associations using generalized additive mixed models. Relative to the middle tertile of mtDNAcn, lower childhood mtDNAcn was associated with lower WISC Working Memory (β = -2.65, 95% CI [-5.24, -0.06]) and Full-Scale IQ (β = -3.71 [-6.42, -1.00]), and higher CMS Visual Memory (β = 4.70 [0.47, 8.93]). Higher childhood mtDNAcn was linked to higher CMS Verbal Memory (β = 7.75 [2.50, 13.01]). In non-linear models, higher childhood mtDNAcn was associated with lower WISC Verbal Comprehension. Our study provides novel evidence that mtDNAcn measured in childhood is associated with children's neurocognitive performance. mtDNAcn may be a marker of healthy child development. Mitochondrial DNA copy number (mtDNAcn) may serve as a biomarker for early-life neurocognitive performances in the children's population. Both low and high mtDNAcn may contribute to poorer neurocognition, reflected through learning and memory abilities. This research elucidated the importance of investigating mitochondrial biomarkers in healthy populations and facilitated advancements of future studies to better understand the associations between mitochondrial markers and adverse children's health outcomes.

Direct genome sequencing of respiratory viruses from low viral load clinical specimens using the target capture sequencing technology.

The use of metagenomic next-generation sequencing technology to obtain complete viral genome sequences directly from clinical samples with low viral load remains challenging-especially in the case of respiratory viruses-due to the low copy number of viral versus host genomes. To overcome this limitation, target capture sequencing for the enrichment of specific genomes has been developed and applied for direct genome sequencing of viruses. However, as the efficiency of enrichment varies depending on the probes, the type of clinical sample, etc., validation is essential before target capture sequencing can be applied to clinical diagnostics. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A. We focused on clinical specimens containing low copy numbers of viral genomes. Target capture sequencing yielded approximately 180- and 2,000-fold higher read counts of SARS-CoV-2 and influenza A virus, respectively, than metagenomic sequencing when the RNA extracted from specimens contained 59.3 copies/µL of SARS-CoV-2 or 625.1 copies/µL of influenza A virus. In addition, the target capture sequencing identified sequence reads in all SARS-CoV-2- or influenza type A-positive specimens with <26 RNA copies/µL, some of which also yielded >70% of the full-length genomes of SARS-CoV-2 or influenza A virus. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with viruses other than SARS-CoV-2, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.IMPORTANCETarget capture sequencing has been developed and applied for direct genome sequencing of viruses in clinical specimens to overcome the low detection sensitivity of metagenomic next-generation sequencing. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A, focusing on clinical specimens containing low copy numbers of viral genomes. Our results showed that the target capture sequencing yielded dramatically higher read counts than metagenomic sequencing for both viruses. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with other viruses, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.

ExoSloNano: Multi-Modal Nanogold Tags for identification of Macromolecules in Live Cells & Cryo-Electron Tomograms.

In situ cryo-Electron Microscopy (cryo-EM) enables the direct interrogation of structure-function relationships by resolving macromolecular structures in their native cellular environment. Tremendous progress in sample preparation, imaging and data processing over the past decade has contributed to the identification and determination of large biomolecular complexes. However, the majority of proteins are of a size that still eludes identification in cellular cryo-EM data, and most proteins exist in low copy numbers. Therefore, novel tools are needed for cryo-EM to identify the vast majority of macromolecules across multiple size scales (from microns to nanometers). Here, we introduce and validate novel nanogold probes that enable the detection of specific proteins using cryo-ET (cryo-Electron Tomography) and resin-embedded correlated light and electron microscopy (CLEM). We demonstrate that these nanogold probes can be introduced into live cells, in a manner that preserves intact molecular networks and cell viability. We use this system to identify both cytoplasmic and nuclear proteins by room temperature EM, and resolve associated structures by cryo-ET. We further employ gold particles of different sizes to enable future multiplexed labeling and structural analysis. By providing high efficiency protein labeling in live cells and molecular specificity within cryo-ET tomograms, we establish a broadly enabling tool that significantly expands the proteome available to electron microscopy.

An automated commercial open access assay for detection of Mycoplasma genitalium macrolide resistance.

Azithromycin, a macrolide antibioticum, is the first-line treatment for Mycoplasma genitalium (MG), but resistant MG is an increasing problem. Macrolide resistance-mediated mutations (MRM) has been linked to point mutations in region V of the MG 23S rRNA gene. We have evaluated an open access analyzer (Panther Fusion, Hologic Inc) for detectability of MRM (mutations A2071G and A2072G) and MG wild type (WT) in clinical samples. Also, the agreement of the Panther Fusion assay results with a corresponding established In-house MRM-WT PCR (ABI 7500) was calculated. Left over material from 55 clinical samples positive for MG by the Aptima test (Hologic) based on transcription-mediated amplification (TMA), collected from January to February 2023 in Region Skåne, Sweden, was analyzed. Specific amplification curves were generated for positive controls of MG mutations (A2071G and A2072G) and WT by the Panther Fusion assay. The limit of detection (LOD) was 5.3 copies/mL for WT, 8.1 copies/mL for mutation A2071G, and 81 copies/mL for mutation A2072G. The overall concordance was 91% between the Panther Fusion and the In-house PCR (Kappa 0.621, 95% CI; 0.327-0.914) for detection of WT or MRM in MG-positive clinical samples. The Panther Fusion detected MRM in 20% (11/55) and WT in 62% (34/55) of the samples. The corresponding In-house PCR results were 25% (14/55) and 65% (36/55). In summary, the Panther Fusion assay demonstrated detection of low copy number of MRM and WT of MG. Among clinical samples substantial agreement between the Panther Fusion and In-house PCR results was observed. Integrating MG-analysis (TMA) and MRM-WT assay on the Panther platform could make MRM testing more readily available. However, the Panther Fusion had a lower success rate (82% vs 90%) for macrolide susceptibility testing, hence testing with a complementary method should be considered for samples where neither WT nor MRM MG are detectable.

Cumulative effects of mutation accumulation on mitochondrial function and fitness

The impact of mutations on the mitochondria deserves specific interest due to the crucial role played by these organelles on numerous cellular functions. This study examines the effects of repeated bottlenecks on mitochondrial function and fitness. Daphnia pulex mutation accumulation lines (MA) lines were maintained for over 120 generations under copper and no copper conditions. Following the MA propagation, Daphnia from MA lines were raised under optimal and high temperatures for two generations before assessing mitochondrial and phenotypic traits. Spontaneous mutation accumulation under copper led to a later age at maturity and lowered fecundity in the MA lines. Mitochondrial respiration was found to be 10% lower in all mutation accumulation (MA) lines as compared to the non-MA control. MtDNA copy number was elevated in MA lines compared to the control under optimal temperature suggesting a compensatory mechanism. Three MA lines propagated under low copper had very low mtDNA copy number and fitness, suggesting mutations might have affected genes involved in mtDNA replication or mitochondrial biogenesis. Overall, our study suggests that mutation accumulation had an impact on life history traits, mtDNA copy number, and mitochondrial respiration. Some phenotypic effects were magnified under high temperatures. MtDNA copy number appears to be an important mitigation factor to allow mitochondria to cope with mutation accumulation up to a certain level beyond which it can no longer compensate.

Clinicopathologic Features and Digital Imaging Analysis of HER2 Protein in Breast Carcinomas With Different HER2 Fluorescence in Situ Hybridization Patterns

BackgroundHER2-targeted therapies have significantly improved outcomes for patients with HER2-positive breast cancer (BC), which represents 15% to 20% of all BC cases. HER2 status is assessed via immunohistochemistry (IHC) and/or in situ hybridization (ISH), dividing BCs into five groups (G1-G5). Patients and methodsIn a study of 2,702 primary BC cases, comprising 12.7% G1, 0.2% G2, 2.8% G3, 8.5% G4, and 75.9% G5, we analyzed clinicopathologic features and HER2 protein expression digitally for each ISH group. ResultsNotably, G5 cases had a higher proportion of lobular carcinoma (13.9%) compared to other groups. G3 cases showed the highest percentage of grade 3 tumors (56.9%), while G5 cases had the lowest (21.4%). Additionally, G5 cases had the highest rate of estrogen receptor (ER) positivity (84.6%), while G1-HC (high copy number) cases had the lowest (70.4%). Most G1-HC cases were HER2 IHC 3+ (76.1%), while most G5 cases were IHC 0/1+ (75.7%). IHC 2+ was most common in G1-LC (low copy number) and G3 cases (83.8% and 90.7%, respectively), with G4 cases predominantly IHC 2+ (56.3%) and IHC 1+ (30.1%). Discordant HER2 IHC and ISH results were observed in 12 cases (0.4%), including 7 G1-HC (2.3%), 4 G1-LC (10.8%), and 1 G5 case (0.1%). Digital quantification of HER2 IHC levels in all groups except G5 revealed that G1-HC tumors had the highest HER2 protein expression, followed by G3, with G4 showing the lowest. ConclusionThese findings offer valuable insights into the clinicopathologic characteristics and future management for different HER2 ISH groups.

Human hypofunctional NCF1 variants promote pulmonary fibrosis in the bleomycin-induced mouse model and patients with systemic sclerosis via expansion of SPP1+ monocytes-derived macrophages

ObjectiveWe assessed the role of a systemic lupus erythematosus causal hypofunctional variant, neutrophil cytosolic factor 1 (NCF1)-p.Arg90His (p.R90H) substitution, in systemic sclerosis (SSc).MethodsAssociation of NCF1-H90 with SSc was performed in...

CHAMP: A Centrifugal Microfluidics-Based CRISPR/Cas12b-Combined Real-Time LAMP One-Pot Method for Mycoplasma pneumoniae Infection Diagnosis.

The Mycoplasma pneumoniae outbreak poses health risks to community residents. However, it still has limitations for current clinical diagnostic methods (qPCR nucleic acid assay or IgM immunoassay), including specialized handling, expensive equipment, prolonged turnaround time, and false positives and negatives, highlighting the need to improve clinical diagnostic methods. Herein, we present a novel centrifugal microfluidics-based method for rapidly diagnosing M. pneumoniae infections (CHAMP system). This user-friendly method combines CRISPR/Cas12b and real-time loop-mediated isothermal amplification (LAMP) in a one-pot reaction, offering high sensitivity, specificity, and simplicity for methodology. By adding fully automated nucleic acid magnetic bead-extracted samples to a prepackaged centrifugal microfluidics chip, 48 samples can be automated tested simultaneously within 15 to 60 min at 60 °C. 427 clinical nasopharyngeal swab specimens were used for validation, demonstrating good positive and negative predictive values and good diagnostic sensitivity, specificity, and significant time savings. This method is particularly suitable for detecting low nucleic acid copies of M. pneumoniae samples.

Dendritically localized RNAs are packaged as diversely composed ribonucleoprotein particles with heterogeneous copy number states.

Localization of mRNAs to dendrites is a fundamental mechanism by which neurons achieve spatiotemporal control of gene expression. Translationally repressed neuronal mRNA transport granules, also referred to as ribonucleoprotein particles (RNPs), have been shown to be trafficked as single or low copy number RNPs and as larger complexes with multiple copies and/or species of mRNAs. However, there is little evidence of either population in intact neuronal circuits. Using single molecule fluorescence in situ hybridization studies in the dendrites of adult rat and mouse hippocampus, we provide evidence that supports the existence of multi-transcript RNPs with the constituents varying in amounts for each RNA species. By competing-off fluorescently labeled probe with serial increases of unlabeled probe, we detected stepwise decreases in Arc RNP number and fluorescence intensity, suggesting Arc RNAs localize to dendrites in both low- and multiple-copy number RNPs. When probing for multiple mRNAs, we find that localized RNPs are heterogeneous in size and colocalization patterns that vary per RNA. Further, localized RNAs that are targeted by the same trans-acting element (FMRP) display greater levels of colocalization compared to an RNA not targeted by FMRP. Simultaneous visualization of a dozen FMRP-targeted mRNA species using highly multiplexed imaging demonstrates that dendritic RNAs are mostly trafficked as heteromeric cargoes of multiple types of RNAs (at least one or more RNAs). Moreover, the composition of these RNA cargoes, as assessed by colocalization, correlates with the abundance of the transcripts even after accounting for the expected differences in colocalization based on expression. Collectively, these results suggest that dendritic RNPs are packaged as heterogeneous co-assemblies of different mRNAs and that RNP contents may be driven, at least partially, by highly abundant dendritic RNAs; a model that favors efficiency over fine-tuned control for sustaining long-distance trafficking of thousands of messenger molecules.

MAPT haplotype-associated transcriptomic changes in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a neurodegenerative movement and cognitive disorder characterized by abnormal accumulation of the microtubule-associated protein tau in the brain. Biochemically, inclusions in PSP are enriched for tau proteoforms with four microtubule-binding domain repeats (4R), an isoform that arises from alternative tau pre-mRNA splicing. While preferential aggregation and reduced degradation of 4R tau protein is thought to play a role in inclusion formation and toxicity, an alternative hypothesis is that altered expression of tau mRNA isoforms plays a causal role. This stems from the observation that PSP is associated with common variation in the tau gene (MAPT) at the 17q21.31 locus which contains low copy number repeats flanking a large recurrent genomic inversion. The complex genomic structural changes at the locus give rise to two dominant haplotypes, termed H1 and H2, that have the potential to markedly influence gene expression. Here, we explored haplotype-dependent differences in gene expression using a bulk RNA-seq dataset derived from human post-mortem brain tissue from PSP (n = 84) and controls (n = 77) using a rigorous computational pipeline, including alternative pre-mRNA splicing. We found 3579 differentially expressed genes in the temporal cortex and 10,011 in the cerebellum. We also found 7214 differential splicing events in the temporal cortex and 18,802 in the cerebellum. In the cerebellum, total tau mRNA levels and the proportion of transcripts encoding 4R tau were significantly increased in PSP compared to controls. In the temporal cortex, the proportion of reads that expressed 4R tau was increased in cases compared to controls. 4R tau mRNA levels were significantly associated with the H1 haplotype in the temporal cortex. Further, we observed a marked haplotype-dependent difference in KANSL1 expression that was strongly associated with H1 in both brain regions. These findings support the hypothesis that sporadic PSP is associated with haplotype-dependent increases in 4R tau mRNA that might play a causal role in this disorder.

7p22.3 microdeletion: a case study of a patient with congenital heart defect, neurodevelopmental delay and epilepsy

BackgroundChromosome 7 has regions enriched with low copy repeats (LCRs), which increase the likelihood of chromosomal microdeletion disorders. Documented microdeletion disorders on chromosome 7 include both well-known Williams syndrome and more rare cases. It is noteworthy that most cases of various microdeletions are characterized by phenotypic signs of neuropsychological developmental disorders, which, however, have a different genetic origin. The localization of the microdeletions, the genes included in the region, as well as the structural features of the sequences of these genes have a cumulative influence on the phenotypic characteristics of the individuals for each specific case and the severity of the manifestations of disorders. The consideration of these features and their detailed analysis is important for a correct and comprehensive assessment of the disease.ResultsThe article describes a clinical case of 7p22.3 microdeletion in a patient with congenital heart defect and neurological abnormalities - epilepsy, combined with moderate mental and motor developmental delay.ConclusionsThrough detailed genetic analyses, we are improving the clinical description of the rare 7p22.3 microdeletion and thus creating a basis for future genetic counseling and research into targeted therapies.

Dried Blood Matrix as a New Material for the Detection of DNA Viruses.

The gold standard for diagnosing viruses such as the Hepatitis B Virus has remained largely unchanged, relying on conventional methods involving extraction, purification, and polymerase chain reaction (PCR). This approach is hindered by limited availability, as it is time-consuming and requires highly trained personnel. Moreover, it suffers from low recovery rates of the nucleic acid molecules for samples with low copy numbers. To address the challenges of complex instrumentation and low recovery rate of DNA, a drying process coupled with thermal treatment of whole blood is employed, resulting in the creation of a dried blood matrix characterized by a porous structure with a high surface-to-volume ratio where it also inactivates the amplification inhibitors present in whole blood. Drawing on insights from Brunauer-Emmett-Teller (BET)- Barrett-Joyner-Halenda (BJH) analysis, scanning electron microscopy (SEM), and fluorescence recovery after photobleaching (FRAP), detection assay is devised for HBV, as a demonstration, from whole blood with high recovery of DNA and simplified instrumentation achieving a limit of detection (LOD) of 10 IU mL-1. This assay can be completed in <1.5 h using a simple heater, can be applied to other DNA viruses, and is expected to be suitable for point-of-care, especially in low-resource settings.

The mutagenic effect of cold plasma on Medicago sativa L.

To explore the feasibility of using cold plasma as a mutagenesis breeding technology for forage crops, in this study we used the Medicago sativa L. cultivar, Zhongcao No. 3, as the experimental material. The effects of plasma treatments on Medicago sativa L. were analyzed through the use of plasma and activated water. Treatments with plasma and activated water inhibited plant height but promoted root growth. By creating a closed environment, adding a dielectric barrier plate, and combining these two treatment methods, the greatest impact can be had on the growth of Medicago sativa L. seeds. After treatment, the plant heights were approximately half those of the control group, and the root lengths were approximately 1.6 times those of the control group. Through emission spectroscopy, it was found that active particles such as O, NO2, and N2* were present and could be considered to have produced plasma-activated water through contact with the water surface, thus affecting the survival and growth of the seeds. Whole-genome resequencing (WGRS) was performed on the wild-type and selected mutants after treatment, with an average sequencing depth of 115.93×, an average genome alignment rate of 91.72 %, and an average genome coverage rate of 91.85 %. Various types of mutations were detected and annotated. After filtering, 7,822,324 SNP (single nucleotide polymorphisms) sites, 2,161,917 indel sites, 200,544 SV sites, 238 CNV (copy number variation) sites. The SNPs, indels (insertions/deletions), and SVs (structural variations) were mainly heterozygous, with heterozygosity rates of 87.13 %, 92.16 %, and 83.49 %, respectively. The CNVs were dominated by low copy numbers, accounting for 53.77 %. These results indicate that plasma treatment has significant effects on plant growth and genome of Medicago sativa L.

Evaluation of the sensitivity of a measles diagnostic real-time RT-PCR assay incorporating recently observed priming mismatch variants, 2024.

We investigated a variant of measles virus that encodes three mismatches to the reverse priming site for a widely used diagnostic real-time RT-PCR assay; reduction of sensitivity was hypothesised. We examined performance of the assay in context of the variant using in silico data, synthetic RNA templates and clinical specimens. Sensitivity was reduced observed at low copy numbers for templates encoding the variant sequence. We designed and tested an alternate priming strategy, rescuing the sensitivity of the assay.

Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells.

To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We experimentally tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.

Identification of transposable element families from pangenome polymorphisms

BackgroundTransposable Elements (TEs) are segments of DNA, typically a few hundred base pairs up to several tens of thousands bases long, that have the ability to generate new copies of themselves in the genome. Most existing methods used to identify TEs in a newly sequenced genome are based on their repetitive character, together with detection based on homology and structural features. As new high quality assemblies become more common, including the availability of multiple independent assemblies from the same species, an alternative strategy for identification of TE families becomes possible in which we focus on the polymorphism at insertion sites caused by TE mobility.ResultsWe develop the idea of using the structural polymorphisms found in pangenomes to create a library of the TE families recently active in a species, or in a closely related group of species. We present a tool, pantera, that achieves this task, and illustrate its use both on species with well-curated libraries, and on new assemblies.ConclusionsOur results show that pantera is sensitive and accurate, tending to correctly identify complete elements with precise boundaries, and is particularly well suited to detect larger, low copy number TEs that are often undetected with existing de novo methods.

High heteroplasmy is associated with low mitochondrial copy number and selection against non-synonymous mutations in the snail Cepaea nemoralis

Molluscan mitochondrial genomes are unusual because they show wide variation in size, radical genome rearrangements and frequently show high variation (> 10%) within species. As progress in understanding this variation has been limited, we used whole genome sequencing of a six-generation matriline of the terrestrial snail Cepaea nemoralis, as well as whole genome sequences from wild-collected C. nemoralis, the sister species C. hortensis, and multiple other snail species to explore the origins of mitochondrial DNA (mtDNA) variation. The main finding is that a high rate of SNP heteroplasmy in somatic tissue was negatively correlated with mtDNA copy number in both Cepaea species. In individuals with under ten mtDNA copies per nuclear genome, more than 10% of all positions were heteroplasmic, with evidence for transmission of this heteroplasmy through the germline. Further analyses showed evidence for purifying selection acting on non-synonymous mutations, even at low frequency of the rare allele, especially in cytochrome oxidase subunit 1 and cytochrome b. The mtDNA of some individuals of Cepaea nemoralis contained a length heteroplasmy, including up to 12 direct repeat copies of tRNA-Val, with 24 copies in another snail, Candidula rugosiuscula, and repeats of tRNA-Thr in C. hortensis. These repeats likely arise due to error prone replication but are not correlated with mitochondrial copy number in C. nemoralis. Overall, the findings provide key insights into mechanisms of replication, mutation and evolution in molluscan mtDNA, and so will inform wider studies on the biology and evolution of mtDNA across animal phyla.

Copy number variations of CCND1 gene and chromosome 11 centromere in acral melanoma

Objective: To study the correlation between the copy number variations of CCND1 gene and chromosome 11 and their associations with clinicopathologic features in acral melanoma. Methods: Thirty-three acral melanoma cases diagnosed at the Department of Pathology of Peking University Third Hospital, Beijing, China from January 2018 to August 2021 were collected. Fluorescence in situ hybridization (FISH) was used to detect the copy number of CCND1 gene and centromere of chromosome 11. The relationship between the copy numbers of CCND1 and chromosome 11 centromere, and the correlation between CCND1 copy number and clinicopathologic characteristics were analyzed. Results: There were 15 male and 18 female patients, with an age ranging from 22-86 years. 63.6% (21/33) of the patients had an increased CCND1 gene copy number. 21.2% (7/33) of patients with increased CCND1 copy number had an accompanying chromosome 11 centromere copy number increase. 27.3% (9/33) of the cases had a low copy number of CCND1 gene, and 4 of them (4/33, 12.1%) were accompanied by chromosome 11 centromere copy number increase. 36.4% (12/33) of the cases had a high copy number of CCND1 gene, and 3 (3/33, 9.1%) of them were accompanied by chromosome 11 centromere copy number increase. No cases with CCND1 low copy number increase showed CCND1/CEP11 ratio greater than 2.00. The 11 cases with CCND1 high copy number increase showed CCND1/CEP11 ratio greater than or equal to 2.00. However, there was no significant correlation between CCND1 copy number increase and any of the examined clinicopathologic features such as age, sex, histological type, Breslow thickness, ulcer and Clark level. Conclusions: CCND1 copy number increase is a significant molecular alteration in acral melanoma. In some cases, CCND1 copy number increase may be accompanied by the copy number increase of chromosome 11. For these cases the copy number increase in CCND1 gene may be a result of the copy number change of chromosome 11.