Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of similar to 150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as "hot" LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3 ' transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3 ' transduction events stemming from a particular hot LINE-1.Peer reviewe

EGFR exon 19-deletion aberrantly regulate ERCC1 expression that may partly impaired DNA damage repair ability in non-small cell lung cancer

Background Epidermal growth factor receptor (EGFR) activating mutations are usually associated with DNA damage repair (DDR) deficiency. However, the precise mechanism has remained elusive. In this study, we aimed to investigate whether EGFR exon 19 deletion mutation downstream signals contributed to DDR deficiency by downregulation of excision repair cross-complementation group-1 (ERCC1), a key factor in DDR, expression and function. Methods We first measured cell survival, DNA damage (gamma-H2AX foci formation) and damage repair (ERCC1 and RAD51 foci formation) ability in response to DNA cross-linking drug in EGFR exon 19 deletion and EGFR wild-type cells separately. We then investigated the involvement of EGFR downstream signals in regulating ERCC1 expression and function in EGFR exon 19 deletion cells as compared with EGFR wild-type ones. Results We observed increased gamma-H2AX, but impaired ERCC1 and RAD51 nuclear foci formation in EGFR exon 19 deletion cells as compared with EGFR wild-type ones treated with DNA cross-linker. In addition, we identified that inhibition of EGFR exon 19 deletion signals increased ERCC1 expression, whereas blocked wild-type EGFR signals decreased ERCC1 expression, on both mRNA and protein levels. Furthermore, EGFR exon 19 deletion downstream signals not only inhibited ERCC1 expression but also influenced ERCC1 foci formation in response to DNA cross-linker. Conclusion Our findings indicated that the aberrant EGFR exon 19 deletion signals were not only associated with decreased expression of ERCC1 but were also involved in impaired ERCC1 recruitment in response to DNA cross-link damage, thereby providing us with more evidence for exploring the mechanism of DDR deficiency in EGFR mutant NSCLC.Peer reviewe

The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wildtype lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis

The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wildtype lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis.Peer reviewe

Detection of subclonal L1 transductions in colorectal cancer by long-distance inverse-PCR and Nanopore sequencing

Long interspersed nuclear elements-1 (L1s) are a large family of retrotransposons. Retrotransposons are repetitive sequences that are capable of autonomous mobility via a copy-and-paste mechanism. In most copy events, only the L1 sequence is inserted, however, they can also mobilize the flanking non-repetitive region by a process known as 3' transduction. L1 insertions can contribute to genome plasticity and cause potentially tumorigenic genomic instability. However, detecting the activity of a particular source L1 and identifying new insertions stemming from it is a challenging task with current methodological approaches. We developed a long-distance inverse PCR (LDI-PCR) based approach to monitor the mobility of active L1 elements based on their 3' transduction activity. LDI-PCR requires no prior knowledge of the insertion target region. By applying LDI-PCR in conjunction with Nanopore sequencing (Oxford Nanopore Technologies) on one L1 reported to be particularly active in human cancer genomes, we detected 14 out of 15 3' transductions previously identified by whole genome sequencing in two different colorectal tumour samples. In addition we discovered 25 novel highly subclonal insertions. Furthermore, the long sequencing reads produced by LDI-PCR/Nanopore sequencing enabled the identification of both the 5' and 3' junctions and revealed detailed insertion sequence information.Peer reviewe

Observation of multiple van Hove singularities and correlated electronic states in a new topological ferromagnetic kagome metal NdTi3Bi4

Kagome materials have attracted enormous research interest recently owing to its diverse topological phases and manifestation of electronic correlation due to its inherent geometric frustration. Here, we report the electronic structure of a new distorted kagome metal NdTi3Bi4 using a combination of angle resolved photoemission spectroscopy (ARPES) measurements and density functional theory (DFT) calculations. We discover the presence of two at bands which are found to originate from the kagome structure formed by Ti atoms with major contribution from Ti dxy and Ti dx2-y2 orbitals. We also observed multiple van Hove singularities (VHSs) in its electronic structure, with one VHS lying near the Fermi level EF. In addition, the presence of a surface Dirac cone at the G point and a linear Dirac-like state at the K point with its Dirac node lying very close to the EF indicates its topological nature. Our findings reveal NdTi3Bi4 as a potential material to understand the interplay of topology, magnetism, and electron correlation.Comment: 7 pages, 4 figure

Observation of multiple flat bands and topological Dirac states in a new titanium based slightly distorted kagome metal YbTi3Bi4

Kagome lattices have emerged as an ideal platform for exploring various exotic quantum phenomena such as correlated topological phases, frustrated lattice geometry, unconventional charge density wave orders, Chern quantum phases, superconductivity, etc. In particular, the vanadium based nonmagnetic kagome metals AV3Sb5 (A= K, Rb, and Cs) have seen a flurry of research interest due to the discovery of multiple competing orders. Here, we report the discovery of a new Ti based kagome metal YbTi3Bi4 and employ angle-resolved photoemission spectroscopy (ARPES), magnetotransport in combination with density functional theory calculations to investigate its electronic structure. We reveal spectroscopic evidence of multiple flat bands arising from the kagome lattice of Ti with predominant Ti 3d character. Through our calculations of the Z2 indices, we have identified that the system exhibits topological nontriviality with surface Dirac cones at the Gamma point and a quasi two-dimensional Dirac state at the K point which is further confirmed by our ARPES measured band dispersion. These results establish YbTi3Bi4 as a novel platform for exploring the intersection of nontrivial topology, and electron correlation effects in this newly discovered Ti based kagome lattice.Comment: 8 pages, 5 figure

Utilizing Reduced Graphene Oxide-Iron Nanoparticles Composite to Enhance and Accelerate the Removal of Methyl Blue Organic Dye in Wastewater

In this work, a nano-composite is used to remove dye from wastewater of different industries. For this purpose, thesynthesis of a magnetic 1:1 composite made of iron nanoparticles (NPs) using reduced graphene oxide is a novel techniqueand tested for Methyl Blue (MB) dye adsorption from aqueous solution. In this study Fe nanoparticles in reduced Graphenecomposite (FGOC) has been prepared using Graphene Oxide (GO). X-ray diffraction, FTIR spectroscopy and Ramanspectroscopy, are used to identify the structures. Many methods have been developed for MB removal in wastewater. One ofthe most popular methods is adsorption because it is simple and high-efficiency, and the adsorbent is crucial. It reached amaximum MB adsorption at pH 7. The kinetic study indicated that the adsorption of MB process was fitted well to thequasi-first-order and quasi-second-order kinetic models. The isotherm study revealed that the MB adsorption process obeyedthe Langmuir and Freundlich adsorption Isotherms models. The GO adding content and absorption conditions on the methylblue removal efficiencies were investigated. This adsorbent is easily recovered by an external magnetic field from thetreated wastewater and has high reusability

Locus-specific LINE-1 expression in clinical ovarian cancer specimens at the single-cell level

Long interspersed nuclear elements (LINE-1s/L1s) are a group of retrotransposons that can copy themselves within a genome. In humans, it is the most successful transposon in nucleotide content. L1 expression is generally mild in normal human tissues, but the activity has been shown to increase significantly in many cancers. Few studies have examined L1 expression at single-cell resolution, thus it is undetermined whether L1 reactivation occurs solely in malignant cells within tumors. One of the cancer types with frequent L1 activity is high-grade serous ovarian carcinoma (HGSOC). Here, we identified locus-specific L1 expression with 3′ single-cell RNA sequencing in pre- and post-chemotherapy HGSOC sample pairs from 11 patients, and in fallopian tube samples from five healthy women. Although L1 expression quantification with the chosen technique was challenging due to the repetitive nature of the element, we found evidence of L1 expression primarily in cancer cells, but also in other cell types, e.g. cancer-associated fibroblasts. The expression levels were similar in samples taken before and after neoadjuvant chemotherapy, indicating that L1 transcriptional activity was unaffected by clinical platinum-taxane treatment. Furthermore, L1 activity was negatively associated with the expression of MYC target genes, a finding that supports earlier literature of MYC being an L1 suppressor.Peer reviewe

Multiple Shocks, Coping and Welfare Consequences: Natural Disasters and Health Shocks in the Indian Sundarbans

Background: Based on a household survey in Indian Sundarbans hit by tropical cyclone Aila in May 2009, this study tests for evidence and argues that health and climatic shocks are essentially linked forming a continuum and with exposure to a marginal one, coping mechanisms and welfare outcomes triggered in the response is significantly affected. Data & Methods: The data for this study is based on a cross-sectional household survey carried out during June 2010. The survey was aimed to assess the impact of cyclone Aila on households and consequent coping mechanisms in three of the worst-affected blocks (a sub-district administrative unit), viz. Hingalganj, Gosaba and Patharpratima. The survey covered 809 individuals from 179 households, cross cutting age and gender. A separate module on health-seeking behaviour serves as the information source of health shocks defined as illness episodes (ambulatory or hospitalized) experienced by household members. Key findings: Finding reveals that over half of the households (54%) consider that Aila has dealt a high, damaging impact on their household assets. Result further shows deterioration of health status in the period following the incidence of Aila. Finding suggests having suffered multiple shocks increases the number of adverse welfare outcomes by 55%. Whereas, suffering either from the climatic shock (33%) or the health shock (25%) alone increases such risks by a much lesser extent. The multiple-shock households face a significantly higher degree of difficulty to finance expenses arising out of health shocks, as opposed to their counterparts facing only the health shock. Further, these households are more likely to finance the expenses through informal loans and credit from acquaintances or moneylenders. Conclusion: This paper presented empirical evidence on how natural and health shocks mutually reinforce their resultant impact, making coping increasingly difficult and present significant risks of welfare loss, having short as well as long-run development manifestations.DFI