Clinical Utility of Circulating Cell-Free DNA as a Liquid Biopsy in Cats With Various Tumours.

Only a limited number of tumour biomarkers are currently available in veterinary medicine, particularly in cats. Cell-free DNA (cfDNA) is an extracellular DNA fragment released upon cell death and is considered a minimally invasive biomarker for the diagnosis and monitoring of various human malignancies. This study aimed to clarify the utility of circulating cfDNA as a liquid biopsy for various feline tumours. Plasma samples were collected from 44 cats with various tumours, 24 cats with other diseases and 10 healthy controls. A follow-up study was conducted in three tumour-bearing patients. All cfDNA concentrations were quantified via real-time polymerase chain reaction (PCR), which provided short and long fragments of a newly identified feline LINE-1 gene. We found that cfDNA levels were significantly higher in cats with various tumours than in those with other diseases or healthy controls. The cfDNA concentration was not correlated with serum amyloid A (SAA) levels. Cats with tumours exhibited elevated cfDNA levels that predicted tumour-bearing with a sensitivity and specificity of 50.5% and 91.2%, respectively (AUC 0.736; p < 0.001). In lymphoma cases, cats with high cfDNA levels had significantly shorter survival times than those with low cfDNA levels (median: 33 days vs. 178 days; p = 0.003). In addition, the cfDNA levels of the three patients correlated with clinical status during follow-up. Collectively, these findings indicate the potential of cfDNA as a useful biomarker for the diagnosis, therapeutic monitoring and prognostic assessment of tumours in cats.

Cytogenetic bands and sharp peaks of Alu underlie large-scale segmental regulation of nuclear genome architecture.

Cytogenetic bands reflect genomic organization in large blocks of DNA with similar properties. Because banding patterns are invariant, this organization may often be assumed unimportant for genome regulation. Results here challenge that view. Findings here suggest cytogenetic bands reflect a visible framework upon which regulated genome architecture is built. Given Alu and L1 densities differ in cytogenetic bands, we examined their distribution after X-chromosome inactivation or formation of senescent-associated heterochromatin foci (SAHFs). Alu-rich regions remain outside both SAHFs and the Barr Body (BB), affirming that the BB is not the whole chromosome but a condensed, L1-rich core. Hi-C analysis of senescent cells demonstrates large (~10 Mb) G-bands remodel as a contiguous unit, gaining distal intrachromosomal interactions as syntenic G-bands coalesce into SAHFs. Striking peaks of Alu within R-bands strongly resist condensation. Thus, large-scale segmental genome architectur relates to dark versus light cytogenetic bands and Alu-peaks, implicating both in chromatin regulation.

Altered Methylation Levels in LINE-1 in Dental Pulp Stem Cell-Derived Osteoblasts.

OBJECTIVES: Long interspersed nuclear element-1 (LINE-1) and Alu elements are major targets of methylation, an epigenetic mechanism that is associated with several biological processes. Alterations of methylation of LINE-1 and Alu have been reported in cancers, diseases, and ageing. However, these alterations have not been studied in osteogenic differentiation of dental pulp stem cells (DPSCs), which are a promising source of tissue regeneration. METHOD: This study was performed to investigate the methylation level of LINE-1 and Alu in dental pulp stem cell-derived osteoblasts (DPSC-DOs). By using the combined bisulfite restriction analysis, the levels of total methylation and 4 patterns of methylated cytosine-phosphate-guanine (CpG) dinucleotides of LINE-1 and Alu were compared between DPSC-DOs and DPSCs. RESULT: The levels of total methylation and hypermethylated CpG dinucleotides of LINE-1 were significantly lower (P = .015 and .021, respectively), whilst levels of one pattern of partial methylated CpG dinucleotides were significantly higher in DPSC-DOs than DPSCs (P = .021). The methylation of Alu was not significantly different between DPSCs and DPSC-DOs. CONCLUSIONS: Methylation alterations of LINE-1 but not Alu were found in osteogenic differentiation of DPSCs. The results of this study offer foundational insights into osteoblast differentiation from an epigenetic perspective and may contribute to advancements in bone regeneration therapy in the future.

Impact of prematurity on LINE-1 promoter methylation.

Aim: Promoter methylation of LINE-1 may be affected by prematurity, but there is little evidence in the literature.Materials & methods: Blood from premature and full-term neonates on days 0, 5, 30 and 90 was analyzed for DNA methylation percentage in a promoter region of the LINE-1, after bisulfite conversion and pyrosequencing.Results: Premature infants, as a whole, showed significantly lower methylation percentage at birth, but this difference diminished over time. However, the subgroup of extremely premature (<28 weeks gestational age) had higher methylation percentages, similar to full-term newborns.Conclusion: This research underscores the critical role of prematurity on the methylation pattern of LINE-1. These findings underline the complexity of epigenetic regulation in prematurity and emphasize the need for further studies.

Premature birth can have significant effects on a baby's development and long-term health. This study investigates how being born prematurely affects a process called DNA methylation, which can influence how genes are turned on or off. Specifically, we examined the LINE-1 promoter, a frequently occurring region of DNA known for its role in regulating gene activity.We collected blood samples from both premature and full-term newborns at birth and at several points in the early months of life. Our findings showed that premature babies have lower levels of LINE-1 promoter methylation at birth compared with full-term babies. These differences in methylation could possibly affect the babies' development and health as they grow.Our research highlights the need for continued study in this area to explore how these epigenetic changes impact long-term health and to develop strategies to mitigate these effects.

Retrotransposons and Diabetes Mellitus.

Retrotransposons are invasive genetic elements, which replicate by copying and pasting themselves throughout the genome in a process called retrotransposition. The most abundant retrotransposons by number in the human genome are Alu and LINE-1 elements, which comprise approximately 40% of the human genome. The ability of retrotransposons to expand and colonize eukaryotic genomes has rendered them evolutionarily successful and is responsible for creating genetic alterations leading to significant impacts on their hosts. Previous research suggested that hypomethylation of Alu and LINE-1 elements is associated with global hypomethylation and genomic instability in several types of cancer and diseases, such as neurodegenerative diseases, obesity, osteoporosis, and diabetes mellitus (DM). With the advancement of sequencing technologies and computational tools, the study of the retrotransposon's association with physiology and diseases is becoming a hot topic among researchers. Quantifying Alu and LINE-1 methylation is thought to serve as a surrogate measurement of global DNA methylation level. Although Alu and LINE-1 hypomethylation appears to serve as a cellular senescence biomarker promoting genomic instability, there is sparse information available regarding their potential functional and biological significance in DM. This review article summarizes the current knowledge on the involvement of the main epigenetic alterations in the methylation status of Alu and LINE-1 retrotransposons and their potential role as epigenetic markers of global DNA methylation in the pathogenesis of DM.

Multi-ancestry GWAS reveals loci linked to human variation in LINE-1- and Alu-copy numbers.

Long INterspersed Element-1 (LINE-1; L1) and Alu are two families of transposable elements (TEs) occupying ~17% and ~11% of the human genome, respectively. Though only a small fraction of L1 copies is able to produce the machinery to mobilize autonomously, Alu elements and degenerate L1 copies can hijack their functional machinery and mobilize in trans. The expression and subsequent copy number expansion of L1 and Alu can exert pathological effects on their hosts, promoting genome instability, inflammation, and cell cycle alterations. These features have made L1 and Alu promising focus subjects in studies of aging and aging diseases where they can become active. However, the mechanisms regulating variation in their expression and copy number remain incompletely characterized. Moreover, the relevance of known mechanisms to diverse human populations remains unclear, as mechanisms are often characterized in isogenic cell culture models. To address these gaps, we leveraged genomic data from the 1000 Genomes Project to carry out a trans-ethnic GWAS of L1 and Alu insertion global singletons. These singletons are rare insertions observed only once in a population, potentially reflecting recently acquired L1 and Alu integrants or structural variants, and which we used as proxies for L1/Alu-associated copy number variation. Our computational approach identified single nucleotide variants in genomic regions containing genes with potential and known TE regulatory properties, and it enriched for single nucleotide variants in regions containing known regulators of L1 expression. Moreover, we identified many reference TE copies and polymorphic structural variants that were associated with L1/Alu singletons, suggesting their potential contribution to TE copy number variation through transposition-dependent or transposition-independent mechanisms. Finally, a transcriptional analysis of lymphoblastoid cells highlighted potential cell cycle alterations in a subset of samples harboring L1/Alu singletons. Collectively, our results (i) suggest that known TE regulatory mechanisms may also play regulatory roles in diverse human populations, (ii) expand the list of genic and repetitive genomic loci implicated in TE copy number variation, and (iii) reinforce the links between TEs and disease.

Comprehensive analysis of the expression patterns and function of the FTO-LINE1 axis in yak tissues and muscle satellite cells.

Background: The long interspersed nuclear element 1 (LINE1) retrotransposon has been identified as a specific substrate for fat mass and obesity-related gene (FTO), which facilitates the removal of N6-methyladenosine modifications from its targeted RNAs. Methods: This study examined the dynamic interaction between FTO and LINE1 in yak tissues and muscle satellite cells, utilizing RT-qPCR, RNA immunoprecipitation (RIP), immunofluorescence staining, and techniques involving overexpression and interference of FTO and LINE1 to elucidate the relationship between FTO and LINE1 in yak tissues and muscle satellite cells. Results: Cloning and analysis of the FTO coding sequence in Jiulong yak revealed a conserved protein structure across various Bos breeds, with notable homology observed with domestic yak, domestic cattle, and Java bison. Comprehensive examination of FTO and LINE1 gene expression patterns in Jiulong yaks revealed consistent trends across tissues in both sexes. FTO mRNA levels were markedly elevated in the heart and kidney, while LINE1 RNA was predominantly expressed in the heart. Immunoprecipitation confirmed the direct interaction between the FTO protein and LINE1 RNA in yak tissues and muscle satellite cells. The FTO-LINE1 axis was confirmed by a significant decrease in LINE1 RNA enrichment following its expression interference in yak muscle satellite cells. Overexpression of FTO substantially reduced the expression of recombinant myogenic factor 5 (MYF5). However, FTO interference had no discernible effect on MYF5 and myoblast determination protein 1 (MYOD1) mRNA levels. Immunofluorescence analysis revealed no alterations in Ki-67 protein expression following FTO interference or overexpression. However, phalloidin staining demonstrated enhancement in the myotube fusion rate of yak muscle satellite cells upon LINE1 interference. Conclusion: This comprehensive mapping of the FTO and LINE1 mRNA expression patterns establishes a direct interaction between the FTO protein and LINE1 RNA in yak. The findings suggest that FTO overexpression promotes muscle satellite cells differentiation, whereas LINE1 negatively regulates myotube fusion. The study provides fundamental insights into the role of the FTO-LINE1 axis in determining the fate of muscle satellite cells in yak, laying a solid theoretical foundation for future investigations.

Investigation of chimeric transcripts derived from LINE-1 and Alu retrotransposons in cerebellar tissues of individuals with autism spectrum disorder (ASD).

LINE-1 and Alu retrotransposons are components of the human genome and have been implicated in many human diseases. These elements can influence human transcriptome plasticity in various mechanisms. Chimeric transcripts derived from LINE-1 and Alu can also impact the human transcriptome, such as exonization and post-transcriptional modification. However, its specific role in ASD neuropathology remains unclear, particularly in the cerebellum tissues. We performed RNA-sequencing of post-mortem cerebellum tissues from ASD and unaffected individuals for transposable elements profiling and chimeric transcript identification. The majority of free transcripts of transposable elements were not changed in the cerebellum tissues of ASD compared with unaffected individuals. Nevertheless, we observed that chimeric transcripts derived from LINE-1 and Alu were embedded in the transcripts of differentially expressed genes in the cerebellum of ASD, and these genes were related to developments and abnormalities of the cerebellum. In addition, the expression levels of these genes were correlated with the significantly decreased thickness of the molecular layer in the cerebellum of ASD. We also found that global methylation and expression of LINE-1 and Alu elements were not changed in ASD, but observed in the ASD sub-phenotypes. Our findings showed associations between transposable elements and cerebellar abnormalities in ASD, particularly in distinct phenotypic subgroups. Further investigations using appropriate models are warranted to elucidate the structural and functional implications of LINE-1 and Alu elements in ASD neuropathology.

Lamivudine modulates the expression of neurological impairment-related genes and LINE-1 retrotransposons in brain tissues of a Down syndrome mouse model.

Elevated activity of retrotransposons is increasingly recognized to be implicated in a wide range of neurodegenerative and neurodevelopmental diseases, including Down syndrome (DS), which is the most common chromosomal condition causing intellectual disability globally. Previous research by our group has revealed that treatment with lamivudine, a reverse transcriptase inhibitor, improved neurobehavioral phenotypes and completely rescued hippocampal-dependent recognition memory in a DS mouse model, Ts65Dn. We hypothesized that retrotransposition rates would increase in the Ts65Dn mouse model, and lamivudine could block retrotransposons. We analyzed the differentially expressed long interspersed element-1 (LINE-1 or L1) mapping on MMU16 and 17, and showed for the first time that retrotransposition could be associated with Ts65Dn's pathology, as misregulation of L1 was found in brain tissues associated with trisomy. In the cerebral cortex, 6 out of 26 upregulated L1s in trisomic treated mice were located in the telomeric region of MMU16 near Ttc3, Kcnj6, and Dscam genes. In the hippocampus, one upregulated L1 element in trisomic treated mice was located near the Fgd4 gene on MMU16. Moreover, two downregulated L1s rescued after treatment with lamivudine were located in the intronic region of Nrxn1 (MMU17) and Snhg14 (MMU7), implicated in a variety of neurodegenerative disorders. To gain further insight into the mechanism of this improvement, we here analyzed the gene expression profile in the hippocampus and cerebral cortex of trisomic mice treated and no-treated with lamivudine compared to their wild-type littermates. We found that treatment with lamivudine rescued the expression of 24% of trisomic genes in the cortex (located on mouse chromosome (MMU) 16 and 17) and 15% in the hippocampus (located in the human chromosome 21 orthologous regions), with important DS candidate genes such as App and Ets2, rescued in both regions.

Mapping of long interspersed element-1 (L1) insertions by TIPseq provides information about sub chromosomal genetic variation in human embryos.

PURPOSE: Retrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos. METHODS: Sixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls. RESULTS: Twenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos. CONCLUSION: Several studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.

LINE-1 transposable element renaissance in aging and age-related diseases.

Transposable elements (TEs) are essential components of eukaryotic genomes and subject to stringent regulatory mechanisms to avoid their potentially deleterious effects. However, numerous studies have verified the resurrection of TEs, particularly long interspersed nuclear element-1 (LINE-1), during preimplantation development, aging, cancer, and other age-related diseases. The LINE-1 family has also been implicated in several aging-related processes, including genomic instability, loss of heterochromatin, DNA methylation, and the senescence-associated secretory phenotype (SASP). Additionally, the role of the LINE-1 family in cancer development has also been substantiated. Research in this field has offered valuable insights into the functional mechanisms underlying LINE-1 activity, enhancing our understanding of aging regulation. This review provides a comprehensive summary of current findings on LINE-1 and their roles in aging and age-related diseases.

Competition between two HUSH complexes orchestrates the immune response to retroelement invasion.

The human silencing hub (HUSH) preserves genome integrity through the epigenetic repression of invasive genetic elements. However, despite our understanding of HUSH as an obligate complex of three subunits, only loss of MPP8 or Periphilin, but not TASOR, triggers interferon signaling following derepression of endogenous retroelements. Here, we resolve this paradox by characterizing a second HUSH complex that shares MPP8 and Periphilin but assembles around TASOR2, an uncharacterized paralog of TASOR. Whereas HUSH represses LINE-1 retroelements marked by the repressive histone modification H3K9me3, HUSH2 is recruited by the transcription factor IRF2 to repress interferon-stimulated genes. Mechanistically, HUSH-mediated retroelement silencing sequesters the limited pool of the shared subunits MPP8 and Periphilin, preventing TASOR2 from forming HUSH2 complexes and hence relieving the HUSH2-mediated repression of interferon-stimulated genes. Thus, competition between two HUSH complexes intertwines retroelement silencing with the induction of an immune response, coupling epigenetic and immune aspects of genome defense.

m6A-Mediated IRS1 Regulates the Development of Oral Squamous Cell Carcinoma through p53/Line-1 Signaling.

BACKGROUND: The importance of N6-methyladenosine (m6A) modification in tumorigenesis and progression have been highlighted. This study aimed to investigate the modification of insulin receptor substrate 1 (IRS1) by m6A and its role in oral squamous cell carcinoma (OSCC). METHODS: Bioinformatics was employed to predict differential genes related to epithelial-mesenchymal transition (EMT) in OSCC. Seventeen pairs of OSCC and paracancerous tissue samples were collected. The impact of IRS1 on OSCC cell growth and EMT was evaluated. The fluctuations in IRS1 enrichment and the involvement of p53/Line-1 were investigated. RESULTS: IRS1 was highly expressed in OSCC. IRS1 silencing decreased OSCC cell proliferation and increased apoptosis. IRS1 silencing hindered EMT by regulating related markers. IRS1 silencing upregulated p53 and downregulated Line-1 ORF1p. The p53 inhibition reversed the effects of IRS1 silencing and induced EMT in OSCC cells. Furthermore, the m6A modification of IRS1 was increased in OSCC cells. IRS1 were positively regulated by the m6A regulators methyltransferase-like 14 (METTL14) and YTH domain-containing protein 1 (YTHDC1). IRS1 bound to YTHDC1, and YTHDC1 knockdown inhibited the IRS1 nuclear export. The obesity-associated protein (FTO) negatively regulated IRS1, and FTO overexpression reversed the IRS1-induced OSCC tumor growth. CONCLUSIONS: m6A methylation-mediated IRS1 regulated EMT in OSCC through p53/Line-1. These findings provide potential therapeutic strategies for managing OSCC.

An Evaluation of DNA Methylation Levels and Sleep in Relation to Hot Flashes: A Cross-Sectional Study.

Objectives: We aimed to evaluate the DNA methylation levels in perimenopausal and postmenopausal women, measured through Long Interspersed Element-1 (LINE-1) and Alu, and the sleep parameters in relation to the presence of hot flashes (HFs). Methods: This cross-sectional study included 30 peri- or postmenopausal women aged between 45 and 55. The menopausal status was determined according to STRAW + 10 criteria and all participants had a low cardiovascular disease (CVD) risk profile determined by Framingham risk score. The sample was divided into two groups based on the presence or absence of HFs documented in their medical history during their initial visit: Group 1 (n = 15) with HFs present and Group 2 (n = 15) with HFs absent. The patients had polysomnography test and HFs were recorded both by sternal skin conductance and self-report overnight. Genomic DNA was extracted from the women's blood and methylation status was analyzed by fluorescence-based real-time quantitative PCR. The quantified value of DNA methylation of a target gene was normalized by ß-actin. The primary outcome was the variation in methylation levels of LINE-1 and Alu and sleep parameters according to the presence of HFs. Results: LINE-1 and Alu methylation levels were higher in Group 1 (HFs present), although statistically non-significant. LINE-1 methylation levels were negatively correlated with age. Sleep efficiency was statistically significantly lower for women in Group 1 (HFs present) (74.66% ± 11.16% vs. 82.63% ± 7.31%; p = 0.03). The ratio of duration of awakening to total sleep time was statistically significantly higher in Group 1 (HFs present) (22.38% ± 9.99% vs. 15.07% ± 6.93, p = 0.03). Objectively recorded hot flashes were significantly higher in Group 1 (4.00 ± 3.21 vs. 1.47 ± 1.46, p = 0.03). None of the cases in Group 2 self-reported HF despite objectively recorded HFs during the polysomnography. The rate of hot flash associated with awakening was 41.4% in the whole sample. Conclusions: Women with a history of hot flashes exhibited lower sleep efficiency and higher awakening rates. Although a history of experiencing hot flashes was associated with higher LINE-1 and Alu methylation levels, no statistical significance was found. Further studies are needed to clarify this association. This study was funded by the Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa. Project number: TTU-2021-35629.

The transcription factor PAX5 activates human LINE1 retrotransposons to induce cellular senescence.

As a hallmark of senescent cells, the derepression of Long Interspersed Elements 1 (LINE1) transcription results in accumulated LINE1 cDNA, which triggers the secretion of the senescence-associated secretory phenotype (SASP) and paracrine senescence in a cGAS-STING pathway-dependent manner. However, transcription factors that govern senescence-associated LINE1 reactivation remain ill-defined. Here, we predict several transcription factors that bind to human LINE1 elements to regulate their transcription by analyzing the conserved binding motifs in the 5'-untranslated regions (UTR) of the commonly upregulated LINE1 elements in different types of senescent cells. Further analysis reveals that PAX5 directly binds to LINE1 5'-UTR and the binding is enhanced in senescent cells. The enrichment of PAX5 at the 5'-UTR promotes cellular senescence and SASP by activating LINE1. We also demonstrate that the longevity gene SIRT6 suppresses PAX5 transcription by directly binding to the PAX5 promoter, and overexpressing PAX5 abrogates the suppressive effect of SIRT6 on stress-dependent cellular senescence. Our work suggests that PAX5 could serve as a potential target for drug development aiming to suppress LINE1 activation and treat senescence-associated diseases.

Insertion of short L1 sequences generates inter-strain histone acetylation differences in the mouse.

BACKGROUND: Gene expression divergence between populations and between individuals can emerge from genetic variations within the genes and/or in the cis regulatory elements. Since epigenetic modifications regulate gene expression, it is conceivable that epigenetic variations in cis regulatory elements can also be a source of gene expression divergence. RESULTS: In this study, we compared histone acetylation (namely, H3K9ac) profiles in two mouse strains of different subspecies origin, C57BL/6 J (B6) and MSM/Ms (MSM), as well as their F1 hybrids. This identified 319 regions of strain-specific acetylation, about half of which were observed between the alleles of F1 hybrids. While the allele-specific presence of the interferon regulatory factor 3 (IRF3) binding sequence was associated with allele-specific histone acetylation, we also revealed that B6-specific insertions of a short 3' fragment of LINE-1 (L1) retrotransposon occur within or proximal to MSM-specific acetylated regions. Furthermore, even in hyperacetylated domains, flanking regions of non-polymorphic 3' L1 fragments were hypoacetylated, suggesting a general activity of the 3' L1 fragment to induce hypoacetylation. Indeed, we confirmed the binding of the 3' region of L1 by three Krüppel-associated box domain-containing zinc finger proteins (KZFPs), which interact with histone deacetylases. These results suggest that even a short insertion of L1 would be excluded from gene- and acetylation-rich regions by natural selection. Finally, mRNA-seq analysis for F1 hybrids was carried out, which disclosed a link between allele-specific promoter/enhancer acetylation and gene expression. CONCLUSIONS: This study disclosed a number of genetic changes that have changed the histone acetylation levels during the evolution of mouse subspecies, a part of which is associated with gene expression changes. Insertions of even a very short L1 fragment can decrease the acetylation level in their neighboring regions and thereby have been counter-selected in gene-rich regions, which may explain a long-standing mystery of discrete genomic distribution of LINEs and SINEs.

Diagnostic value of combined detection of plasma cfDNA concentration and integrity in NSCLC.

Aim: To evaluate the value of combined detection of plasma cfDNA concentration and integrity in the early diagnosis of NSCLC. Methods: Real-time fluorescence quantitative PCR was used to determine the concentration and integrity of plasma cfDNA in 71 NSCLC patients and 53 healthy people. Results: Combined detection of plasma cfDNA concentration and integrity had higher diagnostic power in differentiating NSCLC patients with stage I/II from healthy people than detection of plasma cfDNA concentration alone or integrity alone. The AUC, sensitivity and specificity of the combined detection of plasma cfDNA concentration and integrity were 0.781, 0.62 and 0.85. Conclusion: Combined detection of plasma cfDNA concentration and integrity could improve the diagnostic value in NSCLC detection.

The discovery of cfDNA has opened up a wide range of new possibilities for the diagnosis of cancer. CfDNA provides a noninvasive diagnostic approach for early screening, early detection and monitoring of patients with cancer. Currently, the application of cfDNA in clinical practice for NSCLC patients has been widely reported, which mainly focused on DNA methylation detection, oncogenic driver gene mutation detection. However, few studies have evaluated the diagnostic value of combined detection of plasma cfDNA concentration and integrity for NSCLC patients. Our study suggests that the combination of plasma cfDNA concentration and integrity has higher AUC value in differentiating NSCLC patients from healthy individuals than plasma cfDNA concentration alone or integrity alone.

Accurate detection of copy number aberrations in FFPE samples using the mFAST-SeqS approach.

BACKGROUND: Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions. METHODS: Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach. RESULTS: Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0-2] in normal tissues (n = 4), 3[1-7] in premalignant lesions (n = 9) and 21[13-48] in cancers (n = 10). In anal samples, median [IQR] were 0[0-1] in normal tissues (n = 4), 14[6-38] in premalignant lesions (n = 4) and 18[9-31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq. CONCLUSION: mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples.

Retrotransposons in embryogenesis and neurodevelopment.

Retrotransposable elements (RTEs) are genetic elements that can replicate and insert new copies into different genomic locations. RTEs have long been identified as 'parasitic genes', as their mobilization can cause mutations, DNA damage, and inflammation. Interestingly, high levels of retrotransposon activation are observed in early embryogenesis and neurodevelopment, suggesting that RTEs may possess functional roles during these stages of development. Recent studies demonstrate that RTEs can function as transcriptional regulatory elements through mechanisms such as chromatin organization and noncoding RNAs. It is clear, however, that RTE expression and activity must be restrained at some level during development, since overactivation of RTEs during neurodevelopment is associated with several developmental disorders. Further investigation is needed to understand the importance of RTE expression and activity during neurodevelopment and the balance between RTE-regulated development and RTE-mediated pathogenesis.

Improved LINE-1 Detection through Pattern Matching by Increasing Probe Length.

Long Interspersed Element-1 (LINE-1 or L1) is an autonomous transposable element that accounts for 17% of the human genome. Strong correlations between abnormal L1 expression and diseases, particularly cancer, have been documented by numerous studies. L1PD (LINE-1 Pattern Detection) had been previously created to detect L1s by using a fixed pre-determined set of 50-mer probes and a pattern-matching algorithm. L1PD uses a novel seed-and-pattern-match strategy as opposed to the well-known seed-and-extend strategy employed by other tools. This study discusses an improved version of L1PD that shows how increasing the size of the k-mer probes from 50 to 75 or to 100 yields better results, as evidenced by experiments showing higher precision and recall when compared to the 50-mers. The probe-generation process was updated and the corresponding software is now shared so that users may generate probes for other reference genomes (with certain limitations). Additionally, L1PD was applied to other non-human genomes, such as dogs, horses, and cows, to further validate the pattern-matching strategy. The improved version of L1PD proves to be an efficient and promising approach for L1 detection.