Deletion Rate Research Articles

GLaMST: grow lineages along minimum spanning tree for b cell receptor sequencing data

BackgroundB cell affinity maturation enables B cells to generate high-affinity antibodies. This process involves somatic hypermutation of B cell immunoglobulin receptor (BCR) genes and selection by their ability to bind antigens. Lineage trees are used to describe this microevolution of B cell immunoglobulin genes. In a lineage tree, each node is one BCR sequence that mutated from the germinal center and each directed edge represents a single base mutation, insertion or deletion. In BCR sequencing data, the observed data only contains a subset of BCR sequences in this microevolution process. Therefore, reconstructing the lineage tree from experimental data requires algorithms to build the tree based on partially observed tree nodes.ResultsWe developed a new algorithm named Grow Lineages along Minimum Spanning Tree (GLaMST), which efficiently reconstruct the lineage tree given observed BCR sequences that correspond to a subset of the tree nodes. Through comparison using simulated and real data, GLaMST outperforms existing algorithms in simulations with high rates of mutation, insertion and deletion, and generates lineage trees with smaller size and closer to ground truth according to tree features that highly correlated with selection pressure.ConclusionsGLaMST outperforms state-of-art in reconstruction of the BCR lineage tree in both efficiency and accuracy. Integrating it into existing BCR sequencing analysis frameworks can significant improve lineage tree reconstruction aspect of the analysis.

Evaluation of UroVysion for Urachal Carcinoma Detection.

Background: Patients with hematuria who are positive for urinary fluorescence in situ hybridization (FISH) are generally considered to have urothelial carcinoma. We determined whether UroVysion FISH could be used for the diagnosis of urachal carcinoma.Methods: Seven cases of urachal carcinoma with haematuria subjected to FISH analysis were retrospectively analyzed in our hospital from May 2012 to November 2019. Paraffin-embedded tissue sections from one FISH-positive and one FISH-negative urachal carcinoma were processed in strict accordance with the instructions of the UroVysion kit. Meanwhile, FISH data from the other 414 hematuria patients were collected as controls.Results: All 7 patients with urachal carcinoma were diagnosed with adenocarcinoma. According to Sheldon stage, six patients had stage IIIa and one patient had stage IVb. The sensitivity and specificity of urinary FISH for the diagnosis of urachal carcinoma were 71.43% (5/7) and 94.61% (281/297), respectively. The rates of polysomy for chromosomes 3 and 7 in positive patients were both 100% (5/5), whereas the rate of polysomy for chromosome 17 was 40% (2/5), and the chromosome 9p21 region (p16) gene deletion rate was 20% (1/5). Histological assessment and cytological FISH were consistent for urachal carcinoma. No significant difference was observed in the diagnostic efficacy between urachal carcinoma and urothelial carcinoma (71.43 vs. 87.18%, P = 0.245).Conclusions: Taken together, UroVysion FISH was found to be positive in a high proportion of pathologically confirmed urachal carcinoma of late stage with hematuria. Its chromosomal aberrations may be different from those of urothelial carcinoma, but more studies are needed to clarify their genetic background. Not all tumors showing abnormalities by FISH are urothelial carcinomas.

Clinical implications of mismatch repair deficiency screening in patients with mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN)

IntroductionMixed neuroendocrine non-neuroendocrine neoplasms (MiNEN) are rare tumors, mainly encountered in the gastroenteropancreatic tract. Based on the limited available data, MiNEN is usually a highly aggressive neoplasm combining a high-grade neuroendocrine and a non-neuroendocrine component, associated with a poor prognostic outlook. Deficient DNA mismatch repair (MMR) results in microsatellite instability, which is a useful screening marker for identifying patients with Lynch syndrome and a prognostic factor for chemotherapeutic interventions. Little information on MMR status in MiNEN is available in published studies. Therefore, the purpose of this study was to explore the status and putative role of MMR on MiNEN. MethodsWe investigated the MMR status in 44 cases and characterized their clinicopathological features and prognoses. Immunohistochemistry was performed for four mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2). ResultsMean age at diagnosis was 61 years, and 75% of the patients were male. Lymph node metastases were observed in 14 (35.9%) patients. The most common tumor localizations were gastric (28 patients, 63.6%). Lack of immunohistochemical expression of MMR proteins was shown in 38.6% of cases. The common deletion rates of one or more proteins were 29.4% (5/17) for MLH1/PMS2 and 23.5% (4/17) for MLH1. Correlation between clinicopathological parameters showed that MMR deficiency was significantly associated with early TNM stage and better prognoses in patients with MiNEN. ConclusionMiNENs showed frequent losses of MMR protein expression, which contributes to the knowledge of the pathological and clinical aspects of MiNEN tumors.

A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.

Renal Pathology and Cytogenetic Examination in Multiple Myeloma Patients with Renal Impairment--Retrospective Analysis

To explore the renal pathology and cytogenetic features in the multiple myeloma (MM) patients with renal impairment. The clinical data of newly diagnosed MM patients with renal impairment in our hospital from January 2009 to January 2019 were analyzed retrospectively, and the relationship between FISH results and results of renal pathological exanimation was analyzed statistically by using SPSS 20.0. A total of 20 patients underwent renal biopsy, included 12 males and 8 females. FISH result showed that out of 20 patients, 7 cases presented interstitial nephritis, among which 3 cases were negative for FISH, and in the remaining cases the rate of IgH rearrangement, 1q21 amplification, RB1 deletion, D13S319 deletion, and P53 deletion detection was 42.86%, 28.57%, 28.57%, 28.57% and 14.29% respectively, the detection positive rate was statistically significantly lower as compared with total probe positive rate (P＜0.01). There were 6 cases of cast nephropathy, among which IgH rearrangement, the rate of 1q21 amplification, RB1 deletion, D13S319 deletion, and P53 deletion detection was 66.67%, 50%, 66.67%, 50% and 0% respectively. Compared with the total probe positive rate, there was no statistical significance (P＞0.05). There were 4 cases of acute tubular necrosis, among which the detection rates of IgH rearrangement, 1q21 amplification, RB1 deletion, D13S319 deletion, and P53 deletion was 100%, 50%, 50%, 25% and 25%, respectively. Compared with the total probe positive rate, there was no statistical significance (P＞0.05). There were one case of amyloidosis, and one case of tubular nephropathy with amyloidosis, the detection with 5 probes were all positive. One case of light chain deposition disease was positive for RB1 gene deletion + D13S319 gene deletion. FISH in the MM patients with different renal pathological changes is characterized by heterogeneity, which can be used to predict the risk of renal damage and speculate possible renal pathological types to guide prognosis.

Associations of mitochondrial DNA copy number and deletion rate with early pregnancy loss

Early pregnancy loss (EPL) is a common event worldwide. Previous studies show that mitochondrial DNA (mtDNA) copy number (CN) is associated with semen parameters and preimplantation embryo viability, indicating the predictive potential of mtDNA CN for ongoing pregnancy outcomes. However, no relevant study has assessed the relationship between mtDNA CN and EPL. Thus, we aimed to determine whether mtDNA CN and mtDNA 4977-bp deletion rate (DR) in chorionic villous tissue are associated with EPL. Chorionic villous tissue total DNA was extracted from 75 EPL cases and 75 healthy controls. Chromosomal analysis was conducted using copy number variation (CNV) sequencing. The mtDNA CN and DR were measured in samples without pathogenic CNVs. The association between mtDNA CN or DR and EPL risk were estimated using logistic regression. The EPL group had a significantly different mtDNA CN (P < 0.001) and DR (P = 0.005) compared to the control group. Both biomarkers were independent risk factors for EPL (CN odds ratio 1.71, 95% confidence interval 1.17 to 2.49, P = 0.005; DR odds ratio 1.07, 95% confidence interval 1.02 to 1.12, P = 0.006). These results suggest that higher mtDNA CN and DR levels are strongly associated with EPL and represent independent risk factors for EPL. Further studies validating these findings and exploring the underlying biological mechanisms are warranted.

Correlation of extracellular polymeric substances and microbial community structure in denitrification biofilm exposed to adverse conditions.

Microbial community may respond to different adverse conditions and result in the variation of extracellular polymeric substances (EPS) in denitrification biofilm; this study discovered the role of EPS in accordance with the analysis of cyclic diguanylate (c-di-GMP) and electron equilibrium (EE) under low organic loading rate, shock organic loading rate and low temperature conditions. Good nitrate removal performance could be achieved under shock organic loading rate and low temperature conditions; however, owing to the low organic loading rate, the carbon source was preferentially utilized for biomass growth. Tightly bound EPS (TB-EPS) contents progressively increased and facilitated cell adhesion and biofilm formation. The stable TB protein (TB-PN) content in TB-EPS built a cross-linked network to maintain internal biofilm structure and led to the rapid biosynthesis of polysaccharides, which could further enhance microbial adhesion and improve nitrate removal. C-di-GMP played an important role in biomass retention and biofilm formation, based on the correlation analysis of c-di-GMP and EPS. TB polysaccharide (TB-PS) contents presented a significant positive correlation with c-di-GMP content, microbial adhesion and biofilm stabilization was further enhanced through c-di-GMP regulation. In addition, a remarkable negative correlation between electron deletion rate (EDR) and TB-PN and TB-PS was discovered, and TB-PS was required to serve as energy source to enhance denitrification according to EE analysis. Surprisingly, dynamic microbial community was observed due to the drastic community succession under low temperature conditions, and the discrepancy between the dominant species for denitrification was found under shock organic loading rate and low temperature conditions. The notable increase in bacterial strains Simlicispira, Pseudomonas and Chryseobacterium was conducive to biofilm formation and denitrification under shock organic loading rate, while Dechloromonas and Zoogloea dramatically enriched for nitrate removal under low temperature conditions. The high abundance of Dechloromonas improved the secretion of EPS through the downstream signal transduction, and the c-di-GMP conserved in Pseudomonas concurrently facilitated to enhance exopolysaccharide production to shock organic loading rate and low temperature conditions.

Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region.

Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5 kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with DNA double-strand breaks (DSBs) generated by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at single-guide RNA target sites. Our results indicated that the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammalian cells, and also caused frequent mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.

Genome-wide copy number variation-, validation- and screening study implicates a new copy number polymorphism associated with suicide attempts in major depressive disorder.

The genetic basis of suicide attempts (SA) remains unclear. Especially the role of copy number variations (CNVs) remains to be elucidated. The present study aimed to identify susceptibility variants associated with SA among Chinese with major depressive disorder (MDD), covering both CNVs and single-nucleotide polymorphisms (SNPs). We conducted a genome-wide association study (GWAS) on MDD patients with and without SA and top results were tested in a replication study. A genome-wide CNV study was also performed. Subsequently, a validation assay using qRT-PCR technology was performed to confirm any associated CNVs and then applied to the entire cohort to examine the association. Although GWAS did not identify any SNPs reaching genome-wide significance, we identified TPH2 as the top susceptibility gene (p-value=2.75e-05) in gene-based analysis, which is a strong biological candidate for its role in the serotonergic system. As for CNV analysis, we found that the global rate of CNV was higher in SA than that in non-SA subjects (p-value=0.023). Genome-wide CNV study revealed an SA-associated CNV region that achieved genome-wide significance (corrected p-value=0.014). The associated CNV was successfully validated with a more rigorous qRT-PCR assay and identified to be a common variant in this cohort. Its deletion rate was higher in SA subjects [OR=2.05 (1.02-4.12), adjusted p-value=0.045]. Based on the GTEx database, genetic variants that probed this CNV were significantly associated with the expression level of ZNF33B in two brain regions (p-value<4.2e-05). In stratified analysis, the CNV showed a significant effect [OR=2.58 (1.06-6.27), p-value=0.039] in those with high neuroticism but not in those with average or low neuroticism. We identified a new common CNV likely involved in the etiology of SA. This finding sheds light on an important role of common CNVs in the pathophysiology of SA, suggesting a new promising avenue for investigating its genetic architecture.

Progress in the correlation between PTPN12 gene expression and human tumors.

Background:The global morbidity of cancer is rising rapidly. Despite advances in molecular biology, immunology, and cytotoxic and immune-anticancer therapies, cancer remains a major cause of death worldwide. Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a new member of the cytoplasmic protein tyrosine phosphatase family, isolated from a cDNA library of adult colon tissue. Thus far, no studies have reviewed the correlation between PTPN12 gene expression and human tumors.Methods:This article summarizes the latest domestic and international research developments on how the expression of PTPN12 relates to human tumors. The extensive search in Web of Science and PubMed with the keywords including PTPN12, tumor, renal cell carcinoma, proto-oncogenes, tumor suppressor genes was undertaken.Results:More and more studies have shown that a tumor is essentially a genetic disease, arising from a broken antagonistic function between proto-oncogenes and tumor suppressor genes. When their antagonistic effect is out of balance, it may cause uncontrolled growth of cells and lead to the occurrence of tumors. PTPN12 is a tumor suppressor gene, so inhibiting its activity will lead directly or indirectly to the occurrence of tumors.Conclusion:The etiology, prevention, and treatment of tumors have become the focus of research around the world. PTPN12 is a tumor suppressor gene. In the future, PTPN12 might serve as a novel molecular marker to benefit patients, and even the development of tumor suppressor gene activation agents can form a practical research direction.

Association between TP53 gene deletion and protein expression in esophageal squamous cell carcinoma and its prognostic significance.

The aim of the present study was to investigate the association between tumor protein 53 (TP53) gene deletion and protein expression and clinical features in esophageal squamous cell carcinoma (ESCC), and to evaluate the predictive value of these two characteristics in the prognosis of ESCC. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were performed to detect the expression of p53 protein and gene deletion in ESCC tissue samples from different ethnic groups in Xinjiang, in order to analyze their association with clinicopathological characteristics and patient prognosis, as well as the sensitivity and specificity of the two methods. In addition, the results were further validated by tissue microarray from a different region. The positive rate of p53 protein expression was 54.5% (201/369) in the multi-ethnic group, and was significantly different between sex (P=0.026) and between tumor differentiation groups (P=0.032). FISH demonstrated that the TP53 gene deletion rate was 31.8% (68/214), which was significantly different between different tumor differentiation (P=0.002), lymph node metastasis (P=0.005) and vascular invasion (P<0.001) groups. The survival rate of patients with TP53 gene deletion was significantly lower than those without TP53 gene deletion (P<0.05). The positive rate of p53 protein expression in the tissue microarray was 58.1% (68/117), which was significantly different between the depth of invasion groups (P=0.011). The TP53 gene deletion rate was 47.9% (56/117), which significantly differed according to lymph node metastasis (P=0.003) and TNM stage (P=0.01). In addition, the total concordance rates of the two methods were 60.3 and 64.1%, respectively. There were also significant differences in the positive rate of TP53 gene deletion and protein expression in different stages of ESCC (P<0.05), which increased gradually with the progression of ESCC. The deletion of the TP53 gene in esophageal cancer was associated with poor prognosis and may be an important biomarker for evaluating the prognosis of patients with ESCC. The combination of FISH and IHC methods could significantly improve the detection rate of TP53 gene abnormalities and the accuracy of prognostic assessment of ESCC.

Abstract B081: Common copy number aberrations in breast cancer in Latinas

Abstract Background: Latinos represent the largest and fastest-growing minority population in the US; breast cancer is the most common cancer and causes the most cancer deaths in this population. Copy number amplifications and deletions are an important determinant of the biology of breast tumors and, in some cases, may help to guide treatment. However, little is known about the copy number profile of breast tumors from Latinas. For example, in The Cancer Genome Atlas (TCGA), one of the largest publicly available tumor mutation datasets, there are data on only 31 Latina (out of a total of &amp;gt;1000) breast cancer cases. Therefore, we used whole exome sequencing on matched tumor/normal pairs in 146 tumors from 142 women, to investigate copy number aberrations in breast tumors from Latinas. Methods: We enrolled 142 Latinas with invasive breast cancer and obtained blood and tumor tissue (formalin-fixed paraffin embedded). We performed whole exome sequencing of 146 pairs of tumor (somatic) and normal (germline) samples resulting in 142 tumor-normal pairs for analysis. Target coverage for normal was ~30X and for tumor was ~100X. Sequence data were aligned using Burrows-Wheeler Aligner, and copy number aberrations were called using CNVkit. We estimated genetic ancestry from the germline genotype data using ADMIXTURE. Results: Age at diagnosis ranged from 31 to 75 years with a median age of 48 years. Histologically, 83% were estrogen-receptor positive, 71 % were progesterone-receptor positive, and 17% were human epidermal growth factor receptor positive; 87% were Stage 1 or 2 at diagnosis. The mean genetic ancestry in our sample was ~52.3% European, ~41.6% Indigenous American, and ~6.2% African. Large copy number amplifications were detected mostly commonly at 1q and 8q, and large deletions were detected at 11q, 16q and 17p. Genes commonly gained/amplified included MYC (43%), FGFR1 (19.2%), CCND1 (18.0%), and ERRB2 (14.4%). Genes commonly deleted in at least one chromosome include TP53, MAP2K4, BRCA1, PP2R2A, CHEK2, and PALB2. Summary: Breast tumors from Latinas display similar rates of large chromosomal copy number gains and losses at common positions and similar rates of gene-specific copy number amplifications and deletions compared non-Latina European ancestry women. Citation Format: Elad Ziv, Daniel Schmolze, Donglei Hu, Aaron Adamson, Shu Tao, Charleen Adams, Linda Steele, Scott Huntsman, Jeffrey Weitzel, Jung Byun, Kevin Gardner, Eliseo Perez-Stable, Anna Napoles-Springer, Susan Neuhausen. Common copy number aberrations in breast cancer in Latinas [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B081.

Racial diversity of genomic alterations in lung adenocarcinomas.

9543 Background: Lung cancer is the leading cause of cancer related death in the United States in all racial groups. However, the overall death rate from lung cancer is different among white, black and Asian. Although differences in socioeconomic status and treatments received might be the contributing factors, the biology, particularly genomic alteration of cancer might also have an important impact. To date, the differences of genomic alterations among all racial patients are poorly understood. Methods: The American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE, version 7.0) database was analyzed. A total of 8908 patients with lung adenocarcinoma (LUAD) were identified. Among them, 5652 patients who had comprehensive sequencing results (gene panel ≥ 275) were selected for further analysis. Patients with unknown race, undefined and Pacific Island (only 3 patients) were excluded. Finally, 5360 patients were included in the study. The prevalence and distribution of genomic alteration cross all racial groups were analyzed. Results: Overall 5951 samples from 5360 patients were collected (85.7% white, 7.9% Asian, 4.7% black, 0.2% Native American and 1.4% other). Most patients have only one sample. The median mutation counts in white, black, Asian, Native American and others are 7, 6, 5, 5, 7 respectively (Kruskal–Wallis test, P&lt; 0.001). Asian have significantly higher rates of insertion and deletions than other races (14.8% of Asian versus 9.8% of white, 10.7% of black, 10.4% of Native American, 11.1% of other; Pearson’s chi-square test, &lt; 0.001). TP53, EGFR, KRAS and STK11 are the most frequent alterations in white, black and other. EGFR, TP53, KRAS and APC are the most frequent alterations in Asian. STK11 mutations are rare in both Asian and Native American. Native Americans have more alterations of LRP1B, ARID2 and ATM, although the patients’ number remains small. ATM and KEAP1 mutations are also common in white and black. EGFR is the highest discrepancy gene in racial distribution. 78.9% Asian, 47.7% black, 36.4% of native American had EGFR alteration in comparison to 29.6% in white (Fisher's exact test, P &lt; 0.001). KRAS is the second highest discrepancy gene in racial distribution. 11.6% Asian, 26.3% black, 27.3% of Native American had KRAS alteration in comparison to 36.1% in white (Fisher's exact test, P&lt; 0.001). Conclusions: Our study demonstrated the potential diversity of genomic alterations across all racial groups with potential impact on therapeutic decisions.

Impact of transposable elements on genome size variation between two closely related crustacean species

Identifying and quantifying genome size variation among species and understanding the underlying causes is a long-standing objective in evolutionary biology. Here, we investigated the basis of genome size variation between two closely related species of terrestrial isopods: Armadillidium vulgare and Armadillidium nasatum. The two species diverged 25 million years ago and the A. vulgare genome is ~500 megabases larger than the A. nasatum genome (1.7 vs. 1.2 gigabases, respectively). Our analyses indicated that genome size difference is essentially attributed to transposable elements (TEs). We found that the deletion rate may be slightly higher in A. nasatum than in A. vulgare, but it is unlikely to explain the observed genome size difference. As the two genomes largely share the same TE families, differential transpositional activity also contributes to the observed variation. Analyses of TE expression suggested that the cumulative expression level of all expressed TEs was higher in A. nasatum than in A. vulgare. Assuming TE expression level is a good proxy for TE transpositional activity, our results suggest that the two species may have recently been experiencing different TE transposition dynamics. Overall, our results illustrate the important impact TEs can have on genome structure and evolution between closely related species.

Numerical study of hard rock breakage under indenter impact by the hybrid FDEM

Achieving efficient crushing of hard rocks has always been an urgent problem in mining and tunneling. A novel numerical model of rock fragmentation was developed by the hybrid FDEM (finite and discrete element method) to investigate the mechanism of hard rock breakage produced by indenter impact. Zero-thickness discrete elements embedded into the finite model were used to describe the micromechanics of the rock microscopic cementation via the mixed-mode cohesive traction response. The breakage patterns could be well reproduced by the developed numerical model, which was consistent with the experimental results. The rock breakage features, such as impact crater, crushing zone, small debris, large fragments, symmetrical cracks and lateral cracks, were closely related to the variation in the contact force. The element deletion rate at the crushing stage was larger than that of the relatively stable stage and unloading stage, with the lowest rate of element deletion. The formation mechanisms of the crushing zone, small debris, large fragments, and symmetrical cracks were different. The crushing zone formation related to the cohesive element failure in shear action; the cohesive element failure for small debris formation was caused by the combination of tensile and shear action; the symmetrical and lateral crack formations were both caused by the tensile action on the failed cohesive element, but the tensile stress that produced the cohesive element failure in the lateral crack path was from the compression stress reflected in the boundary.

A Synthetic Genetic Circuit Enables Precise Quantification of Direct Repeat Deletion in Bacteria.

Quantification of the rate of direct repeat deletion (DRD) is an important aspect in the research of DNA rearrangement. The widely used tetracycline selection method usually introduces antibiotic pressure to the tested organism, which may interfere with the DRD process. Also the length of repeat arm (LRA) is limited by the length of the TetR coding sequence. On the basis of the fluorescent microscopy and high-throughput imaging processing, here we developed a two-module genetic circuit, termed TFDEC (which stands for three-color fluorescence-based deletion event counter), to quantify the DRD rate under neutral conditions. DRD events were determined from the state of a three-state fluorescent logic gate constructed through coupling of an OR gate and an AND gate. TFDEC was applied in Pseudomonas aeruginosa, and we found that the DRD rate was RecA-dependent for long repeat arms (>500 bp) and RecA-independent for short repeat arms (<500 bp), which was consistent with the case in Escherichia coli. In addition, the increase of DRD rate followed an S-shaped curve with the increase of LRA, while treating cells with ciprofloxacin did not change the LRA-dependence of DRD. We also detected a significant increased DRD rate for long repeat arms in the uvrD (8-fold) and radA (4-fold) mutants. Our results show that the TFDEC method could be used as a complement tool for quantification of the DRD rate in the future.

Sequencing barcode construction and identification methods based on block error-correction codes.

Multiplexed sequencing relies on specific sample labels, the barcodes, to tag DNA fragments belonging to different samples and to separate the output of the sequencers. However, the barcodes are often corrupted by insertion, deletion and substitution errors introduced during sequencing, which may lead to sample misassignment. In this paper, we propose a barcode construction method, which combines a block error-correction code with a predetermined pseudorandom sequence to generate a base sequence for labeling different samples. Furthermore, to identify the corrupted barcodes for assigning reads to their respective samples, we present a soft decision identification method that consists of inner decoding and outer decoding. The inner decoder establishes the hidden Markov model (HMM) for base insertion/deletion estimation with the pseudorandom sequence, and adapts the forward-backward (FB) algorithm to output the soft information of each bit in the block code. The outer decoder performs soft decision decoding using the soft information to effectively correct multiple errors in the barcodes. Simulation results show that the proposed methods are highly robust to high error rates of insertions, deletions and substitutions in the barcodes. In addition, compared with the inner decoding algorithm of the barcodes based on watermarks, the proposed inner decoding algorithm can greatly reduce the decoding complexity.

Study on the Expression and Significance of P16 Gene Methylation Based on Information Technology Statistics in Female Cervical Cancer in Hunan Povince

To explore the significance of p16 gene methylation and abnormal expression in cervical cancer, Methylation specific PCR (MSP) was used to detect 5 ′ CpG island methylation of p16 gene promoter in 60 cervical cancer tissues of different pathological types and clinical stages, who was chosen in Human province. Detection of homozygous deletion of exon 1 (E1) and exon 2 (E2) of p16 gene by PCR. Immunohistochemical analysis of p16 protein expression. The results showed that there was no methylation of p16 gene in normal control tissues and paracancerous tissues and no E1 and E2 deletions and abnormal p16 protein expression were found. The methylation rate of 60 cervical cancer samples was 21.67% (13 / 60), the deletion rate of p16 gene was 15.00% (9 / 60), and the expression rate of p16 protein was 51.67% (31 / 60). It can be seen that the positive expression rate of p16 gene protein decreased with the increase of clinical stage. The results suggest that p16 gene inactivation is common in cervical cancer and closely related to the pathological grade. p16 gene methylation plays a role in the development of cervical cancer.

영어 어말 설단폐쇄음의 탈락 양상: 고빈도 단어를 중심으로

Word frequency exerts a strong influence on coronal stop deletion. In general, high frequency words undergo word-final coronal stop deletion at a higher rate than low frequency words. This deletion process is undergone in consonant cluster simplification for economy of articulation. This study aims to investigate aspects of English word-final coronal stop deletion across a word boundary in high frequency words. Data were extracted from the Buckeye Corpus of Spontaneous American English Speech depending on morphological, phonological, phonetic and sociolinguistic variations. The results showed that different phonological environments favored one type of coronal stop variations such as [t], flapping, glottaling and deletion. Deletion was most prevalent for coronal stops in C/t,d/#C environment while coronal stops frequently underwent flapping in V/t,d/#V environment. In C/t,d/#C environment adjacent identical features were prohibited (Obligatory Contour Principle). More specifically, when the preceding consonants were more similar to coronal stops, the rate of coronal stop deletion was higher. The following consonants also influenced the coronal stop deletion. The rate of deletion was higher before sonorants than before obstruents. Therefore, actual education of coronal stop deletion should be done depending on various environments and factors.

CRISPR/Cas9 increases mitotic gene conversion in human cells.

Gene conversion is a process of transferring genetic material from one homologous sequence to another. Most reported gene conversions are meiotic although mitotic gene conversion is also described. When using CRISPR/Cas9 to target the human hemoglobin subunit beta (HBB) gene, hemoglobin subunit delta (HBD) gene footprints were observed in HBB gene. However, it is unclear whether these were the results of gene conversion or PCR-mediated sequence shuffling between highly homologous sequences. Here we provide evidence that the HBD footprints in HBB were indeed results of gene conversion. We demonstrated that the CRISPR/Cas9 facilitated unidirectional sequence transfer from the homologous gene without double-strand breaks (DSB) to the one with DSBs, and showed that the rates of HBD footprint in HBB were positively correlated to the HBB insertion and deletion rates. We further showed that when targeting HBD gene, HBB footprints could also be observed in HBD gene. The mitotic gene conversion was observed not only in immortalized HEK293T cells, but also in human primary cells. Our work reveals mitotic gene conversion as an often overlooked effect of CRISPR/Cas9-mediated genome editing.