Somatic nuclear mitochondrial DNA insertions are prevalent in the human brain and accumulate over time in fibroblasts.

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in nonhuman species and recently demonstrated to occur in rare instances from one human generation to the next. Here, we investigated numtogenesis dynamics in humans in 2 ways. First, we quantified Numts in 1,187 postmortem brain and blood samples from different individuals. Compared to circulating immune cells (n = 389), postmitotic brain tissue (n = 798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples, we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex (DLPFC) compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, an increase in the number of brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment (NCI) who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures whole-genome sequencing design in a human fibroblast model that recapitulates several molecular hallmarks of aging. These longitudinal experiments revealed a gradual accumulation of 1 Numt every ~13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven by cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derived SURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7-fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human postmitotic tissues produces functionally relevant human Numts over timescales shorter than previously assumed.

linc00958/miR-185-5p/RSF-1 modulates cisplatin resistance and angiogenesis through AKT1/GSK3ß/VEGFA pathway in cervical cancer.

BACKGROUND: Chemoresistance is one of the major obstacles that lead to poor prognosis in cervical cancer. linc00958 was reported to be an oncogene in cervical cancer. However, its role in mediating chemoresistance remains to be revealed. PURPOSE: To explore the regulatory mechanisms of linc00958 in cisplatin-resistant cervical cancer cells and further validate in xenograft mice. METHODS: Online bioinformatic tools were used to conduct the pre-investigation of linc00958/miR-185-5p/RSF-1 and predict the associations between RSF-1 and AKT1/GSK3ß/VEGFA in cervical cancer. RT-qPCR measured the RNA expression levels of linc00958/miR-185-5p/RSF-1 in SiHa and SiHa/DDP. Cell survival rates were evaluated by CCK8 methods after cells were exposed to differential concentrations of DDP. Dual-luciferase reporter methods were used to measure luciferase activity. Western blot measured RSF-1 protein and phosphorylated changes of AKT1/GSK3ß. Immunofluorescence was employed to observe VEGFA secretion in vitro. Tube formation was applied to evaluate the in-vitro changes of angiogenesis. The SiHa/DDP cells stably transfected with pLKO-sh-NC or pLKO-sh-linc00958 plasmids, were injected into mice, establishing xenograft models. The changes in mice weight and tumor volumes were recorded. H&E staining and Immunohistochemistry (IHC) method was further performed. RESULTS: linc00958 expression was higher in SiHa/DDP cells. High linc00958 expression was associated with low overall survival. In SiHa/DDP cells linc00958/miR-185-5p/RSF-1 axis inhibited the cellular resistance to cisplatin and suppressed VEGFA and the tube formation through AKT1/GSK3ß/VEGFA pathway. The knockdown of linc00958 inhibited RSF-1 and Ki67, curbing tumor growth; it also inhibited VEGFA and CD34, decreasing angiogenesis in mice. CONCLUSION: linc00958/miR-185-5p/RSF-1 modulates cisplatin resistance and angiogenesis through AKT1/GSK3ß/VEGFA pathway in cervical cancer.

SearcHPV: A novel approach to identify and assemble human papillomavirus-host genomic integration events in cancer.

BACKGROUND: Human papillomavirus (HPV) is a well-established driver of malignant transformation at a number of sites, including head and neck, cervical, vulvar, anorectal, and penile squamous cell carcinomas; however, the impact of HPV integration into the host human genome on this process remains largely unresolved. This is due to the technical challenge of identifying HPV integration sites, which includes limitations of existing informatics approaches to discovering viral-host breakpoints from low-read-coverage sequencing data. METHODS: To overcome this limitation, the authors developed SearcHPV, a new HPV detection pipeline based on targeted capture technology, and applied the algorithm to targeted capture data. They performed an integrated analysis of SearcHPV-defined breakpoints with genome-wide linked-read sequencing to identify potential HPV-related structural variations. RESULTS: Through an analysis of HPV+ models, the authors showed that SearcHPV detected HPV-host integration sites with a higher sensitivity and specificity than 2 other commonly used HPV detection callers. SearcHPV uncovered HPV integration sites adjacent to known cancer-related genes, including TP63, MYC, and TRAF2, and near regions of large structural variation. The authors further validated the junction contig assembly feature of SearcHPV, which helped to accurately identify viral-host junction breakpoint sequences. They found that viral integration occurred through a variety of DNA repair mechanisms, including nonhomologous end joining, alternative end joining, and microhomology-mediated repair. CONCLUSIONS: In summary, SearcHPV is a new optimized tool for the accurate detection of HPV-human integration sites from targeted capture DNA sequencing data.

The role of miR-29c/B7-H3 axis in children with allergic asthma.

BACKGROUND: MicroRNAs play roles in the pathogenesis of bronchial asthma. However, the mechanism of miR-29c in allergic asthma remains unclear. This study is to elucidate the regulation of Th cell differentiation by miR-29c in mononuclear macrophages. METHODS: A total of 52 children with asthma exacerbation and 26 children as controls were enrolled in the study. CD14+ monocytes were isolated from the peripheral blood. Differential expressions of microRNAs were evaluated using microarray analysis and miR-29c expression in monocytes was determined by qRT-PCR. The plasma B7-H3 was determined by ELISA. Transfection studies and luciferase reporter assay were performed to confirm target gene of miR-29c and its function. RESULTS: Compared to controls, 88 miRNAs in blood monocytes were up-regulated and 41 miRNAs down-regulated including miR-29c in asthma children. Children with asthma exacerbation had significantly lower level of miR-29c and higher level of plasma B7-H3 compared to controls (both P < 0.05). Functional studies based on luciferase reporter assay and immunofluorescence staining suggest that B7-H3 is the direct target of miR-29c and transfection anti-miR-29c into macrophages could enhance ROR-γt and GATA-3 expression in co-cultured CD4+ T cells and increase levels of IL-4 and IL-17 in supernatants. CONCLUSION: The axis of miR-29c/B7-H3 plays an important role in children with asthma through regulating Th2/Th17 cell differentiation and may provide new targets for treatment of asthma.

Somatic nuclear mitochondrial DNA insertions are prevalent in the human brain and accumulate over time in fibroblasts.

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in non-human species 1-3 and recently demonstrated to occur in rare instances from one human generation to the next 4. Here we investigated numtogenesis dynamics in humans in two ways. First, we quantified Numts in 1,187 post-mortem brain and blood samples from different individuals. Compared to circulating immune cells (n=389), post-mitotic brain tissue (n=798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, more brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures WGS design in a human fibroblast model that recapitulates several molecular hallmarks of aging 5. These longitudinal experiments revealed a gradual accumulation of one Numt every ~13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derived SURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7-fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human post-mitotic tissues produce functionally-relevant human Numts over timescales shorter than previously assumed.

Experimental Research on a Capsule Robot with Spring-Connected Legs.

Based on a previous study of a novel capsule robot (CR) with spring-connected legs that could collect intestinal juice for biopsy, in this research, an experiment system is designed, and two experiments are carried out. One of the experiments measures the torque and cutting force of this CR, and the other experiment tests and evaluates the biopsy function of this CR. In the measuring experiment, we analyze how the magnetic torque exerted on this CR changes. In the experiment with a biopsy, we decompose the biopsy actions and select the most effective biopsy action. The result of the experiments shows that this CR can collect and store biopsy samples ideally, and the most effective biopsy action is the rotation with legs extended.

Downregulation of SOX21-AS1 Alleviated Cisplatin Resistance in Cervical Cancer Through Epithelial-Mesenchymal Transition Inhibition.

Cisplatin is widely used in chemotherapies in cervical cancer (CC). Nevertheless, drug resistance in cancer patients poses a major threat to efficacy of treatment. To explore the underlying modulatory mechanism of SOX21-AS1 in cisplatin resistance in CC cell and mice models, Gepia database was referred for SOX21-AS1 expression in cancer tissues and normal ones. Reverse transcription quantitative real-time polymerase chain reaction was used to measure the differential expression of SOX21-AS1 in parental Siha cells and cisplatin-resistant Siha/DDP cells. Luciferase reporter gene assays were conducted to verify putative bindings between SOX21-AS1 and miR-9-3p. Western blot method was employed to evaluate the changes in cleaved-caspase 7 protein expression. Cisplatin resistance was evaluated in each transfected group using cell counting kit 8 method after cells were exposed to cisplatin (0, 7.5, 15, 30, 60, 120, and 240 µg/mL) for 24 hours. Flow cytometry method was used to measure the apoptosis rates. Cell migration and invasion were measured using Transwell assays. Immunofluorescence method was applied to observe epithelial to mesenchymal transition (EMT) markers, including E-cadherin, Snail, matrix metalloproteinase (MMP)3, and MMP9. Siha/DDP cell groups stably transfected with sh-NC and sh-SOX21-AS1 were injected through tail vein of Balb/C mice. Lung tissue sections were used for hematoxylin and eosin staining and immunohistochemistry analysis. SOX1-AS1 expression was higher in cancer tissues than normal ones and was also higher in Siha/DDP rather than Siha cells. SOX21-AS1 was targeted by miR-9-3p in CC cells. Downregulation of SOX21-AS1 or overexpression of miR-9-3p inhibited cisplatin resistance in Siha/DDP cells and reduced cell invasion and migration and attenuated EMT progression. In vivo, the SOX21-AS1 knockdown led to less severe lung metastasis. Downregulation of SOX21-AS1 alleviated cisplatin resistance in CC through EMT inhibition.

Analysis of Human Papilloma Virus Content and Integration in Mucoepidermoid Carcinoma.

Mucoepidermoid Carcinomas (MEC) represent the most common malignancies of salivary glands. Approximately 50% of all MEC cases are known to harbor CRTC1/3-MAML2 gene fusions, but the additional molecular drivers remain largely uncharacterized. Here, we sought to resolve controversy around the role of human papillomavirus (HPV) as a potential driver of mucoepidermoid carcinoma. Bioinformatics analysis was performed on 48 MEC transcriptomes. Subsequent targeted capture DNA sequencing was used to annotate HPV content and integration status in the host genome. HPV of any type was only identified in 1/48 (2%) of the MEC transcriptomes analyzed. Importantly, the one HPV16+ tumor expressed high levels of p16, had high expression of HPV16 oncogenes E6 and E7, and displayed a complex integration pattern that included breakpoints into 13 host genes including PIK3AP1, HIPI, OLFM4,SIRT1, ARAP2, TMEM161B-AS1, and EPS15L1 as well as 9 non-genic regions. In this cohort, HPV is a rare driver of MEC but may have a substantial etiologic role in cases that harbor the virus. Genetic mechanisms of host genome integration are similar to those observed in other head and neck cancers.

Early HPV ctDNA Kinetics and Imaging Biomarkers Predict Therapeutic Response in p16+ Oropharyngeal Squamous Cell Carcinoma.

PURPOSE: In locally advanced p16+ oropharyngeal squamous cell carcinoma (OPSCC), (i) to investigate kinetics of human papillomavirus (HPV) circulating tumor DNA (ctDNA) and association with tumor progression after chemoradiation, and (ii) to compare the predictive value of ctDNA to imaging biomarkers of MRI and FDG-PET. EXPERIMENTAL DESIGN: Serial blood samples were collected from patients with AJCC8 stage III OPSCC (n = 34) enrolled on a randomized trial: pretreatment; during chemoradiation at weeks 2, 4, and 7; and posttreatment. All patients also had dynamic-contrast-enhanced and diffusion-weighted MRI, as well as FDG-PET scans pre-chemoradiation and week 2 during chemoradiation. ctDNA values were analyzed for prediction of freedom from progression (FFP), and correlations with aggressive tumor subvolumes with low blood volume (TVLBV) and low apparent diffusion coefficient (TVLADC), and metabolic tumor volume (MTV) using Cox proportional hazards model and Spearman rank correlation. RESULTS: Low pretreatment ctDNA and an early increase in ctDNA at week 2 compared with baseline were significantly associated with superior FFP (P < 0.02 and P < 0.05, respectively). At week 4 or 7, neither ctDNA counts nor clearance were significantly predictive of progression (P = 0.8). Pretreatment ctDNA values were significantly correlated with nodal TVLBV, TVLADC, and MTV pre-chemoradiation (P < 0.03), while the ctDNA values at week 2 were correlated with these imaging metrics in primary tumor. Multivariate analysis showed that ctDNA and the imaging metrics performed comparably to predict FFP. CONCLUSIONS: Early ctDNA kinetics during definitive chemoradiation may predict therapy response in stage III OPSCC.

Improving read alignment through the generation of alternative reference via iterative strategy.

There is generally one standard reference sequence for each species. When extensive variations exist in other breeds of the species, it can lead to ambiguous alignment and inaccurate variant calling and, in turn, compromise the accuracy of downstream analysis. Here, with the help of the FPGA hardware platform, we present a method that generates an alternative reference via an iterative strategy to improve the read alignment for breeds that are genetically distant to the reference breed. Compared to the published reference genomes, by using the alternative reference sequences we built, the mapping rates of Chinese indigenous pigs and chickens were improved by 0.61-1.68% and 0.09-0.45%, respectively. These sequences also enable researchers to recover highly variable regions that could be missed using public reference sequences. We also determined that the optimal number of iterations needed to generate alternative reference sequences were seven and five for pigs and chickens, respectively. Our results show that, for genetically distant breeds, generating an alternative reference sequence can facilitate read alignment and variant calling and improve the accuracy of downstream analyses.