Bp Mutation Research Articles

Oxidized LDL Regulates Endothelin-1 and Oxidative Stress in Vascular Endothelial Cells: Role of Extracellular Regulated Kinase1/2 (ERK1/2)

Article Oxidized LDL Regulates Endothelin-1 and Oxidative Stress in Vascular Endothelial Cells: Role of Extracellular Regulated Kinase1/2 (ERK1/2) Haishan Xu 1,#, Jinhong Duan 1,#, Jun Tao 2, Wen Wang 3, Yunqing Wu 1,^, Shunling Dai 1,*, and Jun Ren 4,5, 1 Faculty of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005 China 2 Department of Cardiovascular Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510000 China 3 Department of Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China 4 Department of Cardiology, Zhongshan Hospital Fudan University, Shanghai 200032, China 5 National Clinical Research Center for Interventional Medicine, Shanghai 200032, China # These two authors contributed equally to this work ^ Deceased * Correspondence: daishunling@aliyun.com (Shunling Dai); corresponding author:jren_aldh2@outlook.com (Jun Ren) Abstract: It is perceived that oxidized low density lipoprotein (oxLDL) perturbs endothelial function and fosters endothelin-1 (ET-1) secretion although the underlying mechanism remains elusive. This study was designed to decipher potential mechanisms underscoring oxLDL-evoked regulation of ET-1 and signaling pathways involved in endothelial cells. ET-1 mRNA expression, secretion and promoter function were determined using RT-PCR, enzyme immunometric and luciferase assays, respectively. GO and GSEA bioinformatics analyses depicted differentially expressed genes (DEGs) mainly associated with cell proliferation, cell division, cellular structure, energy supply, and apoptosis in oxLDL-challenged endothelial cells. Incubation of oxLDL overtly increased ROS production, apoptosis, mRNA level, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVECs), the effects were mitigated by N-Acetyl Cysteine (NAC). Moreover, oxLDL challenge evoked phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) in HUVECs, the effect was reversed by NAC and MEK inhibitor PD98059. NAC and PD98059 nullified oxLDL- induced rises in mRNA expression, secretion and promoter activity of ET-1. Truncation of 5’-flanking sequence of ET-1 (–566 bpLuc to –250 bpLuc) displayed elevated luciferase activity with 24-h oxLDL incubation. Fusion plasmid from –233 and –185 bp Luc drastically dampened luciferase activity in basal and oxLDL-challenged HUVECs. Transfection of reporter construct –250 bp Luc with a 2 bp mutation at AP-1 locus, removed basal and oxLDL- evoked rises in ET-1 promoter activity. Collectively, our findings support that oxLDL evoked activation of ERK1/2 signaling likely through ROS production, en route to upregulation of endothelial transcriptional factor AP-1, resulting in expression and secretion of ET-1.

Identification and characterisation of de novo germline structural variants in two commercial pig lines using trio-based whole genome sequencing

BackgroundDe novo mutations arising in the germline are a source of genetic variation and their discovery broadens our understanding of genetic disorders and evolutionary patterns. Although the number of de novo single nucleotide variants (dnSNVs) has been studied in a number of species, relatively little is known about the occurrence of de novo structural variants (dnSVs). In this study, we investigated 37 deeply sequenced pig trios from two commercial lines to identify dnSVs present in the offspring. The identified dnSVs were characterised by identifying their parent of origin, their functional annotations and characterizing sequence homology at the breakpoints.ResultsWe identified four swine germline dnSVs, all located in intronic regions of protein-coding genes. Our conservative, first estimate of the swine germline dnSV rate is 0.108 (95% CI 0.038–0.255) per generation (one dnSV per nine offspring), detected using short-read sequencing. Two detected dnSVs are clusters of mutations. Mutation cluster 1 contains a de novo duplication, a dnSNV and a de novo deletion. Mutation cluster 2 contains a de novo deletion and three de novo duplications, of which one is inverted. Mutation cluster 2 is 25 kb in size, whereas mutation cluster 1 (197 bp) and the other two individual dnSVs (64 and 573 bp) are smaller. Only mutation cluster 2 could be phased and is located on the paternal haplotype. Mutation cluster 2 originates from both micro-homology as well as non-homology mutation mechanisms, where mutation cluster 1 and the other two dnSVs are caused by mutation mechanisms lacking sequence homology. The 64 bp deletion and mutation cluster 1 were validated through PCR. Lastly, the 64 bp deletion and the 573 bp duplication were validated in sequenced offspring of probands with three generations of sequence data.ConclusionsOur estimate of 0.108 dnSVs per generation in the swine germline is conservative, due to our small sample size and restricted possibilities of dnSV detection from short-read sequencing. The current study highlights the complexity of dnSVs and shows the potential of breeding programs for pigs and livestock species in general, to provide a suitable population structure for identification and characterisation of dnSVs.

The role of chronic infection with toxoplasmosis in mitochondrial DNA 4,977 bp common deletion in sperm of men infertility.

Toxoplasma has the ability to infect a wide range of living organisms, including humans, and the infection found all over the world. The current study included 200 blood and semen samples collected from married men from which 100 samples of infertile men and 100 non-infertile men as control. The infertile men were divided into two groups: group A, which includes 35 (35%) infertile patients have IgG antibody sero-positive with toxoplasmosis, and group B, which includes 65 (65%) infertile people without toxoplasmosis were detected by rapid test for Toxo IgG/IgM antibody and then ensure by enzyme linked sorbent immunoassay (ELISA). The aging of men involved in the study extended between 20-57 years. The study revealed group A Toxoplasma IgG positive have mean concentration 65.10±13.84, was measured by ELISA in the serum of patients. Studies have shown mean age of men in group A and group B patients were 32.77±7.83 and 30.31±6.14, respectively. The age from 20-29 years as well as from 30 to 39 years high number of infertility in both A and B group patients. There is no significant distribution between group A and group B patients according to age of men infertility. The common deletion 4,977 bp mutation in mitochondrial sperm DNA(mt-DNA) was detected using the Gap-polymerase chain reaction (PCR) technique. Although the results showed that group A have high rate of common deletion mutation compare to group B but non-significant, while there is no deletion detection in the control group. The current results showed that reducing the mean total number of sperms in group A patient common deletion high significant compared to group B. The mean percentage of slow movement high significant than group B patient but non-significant mean percentage of death sperm although have high mean than to group B patient and abnormal sperm shape percentage have significant is higher in group A patient (common deletion) compared to group B.

Abstract 3535: Whole genome sequencing of single circulating tumor cells from localized high-risk prostate cancer patients: Laser microdissection and 3Dtelomere profile

Abstract Advances in the development of precision medicine for prostate cancer patients rely on accurate identification of the genomic features underlying a patient's tumor. Genomic analysis of circulating tumor cells (CTCs) could provide a unique and accessible representation of tumor diversity, however for the use of CTCs as a less-invasive sampling of prostate cancer and as biomarkers for patient stratification and selection of targeted therapy, complex platforms are necessary to ensure an efficient enrichment (isolation), detection (identification-imaging) and characterization strategies (molecular profiles). In this study, we combined a filtration-based CTC isolation technology with prostate cancer cell-specific antibodies to identify the prostate cancer CTCs. After identification, we performed the 3D telomere profiling prior to laser microdissection for whole genome sequencing of singles CTCs from 11 localized high-risk prostate cancer patient samples (36 CTCs in total). We identified unique and common somatic single nucleotide variants (SSNVs) and copy number alterations (CNAs) that can be used to predict high-risk lethal prostate cancer and treatment response in patients with clinically localized high- risk prostate cancers. The distribution of telomeres intensities show a range between 0 to 190.000 (a.u) and telomeres parameters were similar as described previously for high risk patients by our group. We analyzed 36 CTCs from 11 different patient samples and compared with normal lymphocytes from the same patient using whole genome sequencing (WGS). We identified 184 common mutated genes in all CTCs and two mutated genes with the same base pair (bp) mutation (PMS2 and EIF2AK4). We also identified 503 consensus mutations found in all patient but not in all CTCs and 1728 unique mutations found in all or any CTCs of only one patient, showing the heterogeneity that arises in single cells. The list of genes affected are all involved in potential pathways for tumor development such as immune system, signal transduction, homeostasis, gene expression, chromatin organization, DNA repair, cell cycle, programmed cell death, cell-cell communication and others. MCPH1, also known as BRIT1, is a key regulatory gene of the DNA damage response pathway and was mutated in all CTCs from prostate patients. MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. The PMS2 gene is a member of a set of genes known as the mismatch repair (MMR) genes which promotes also genomic Instability. Those mutations can be closely linked with the genomic instability we found assessing the telomere profiles. The CDH17 gene is a member of the cadherin superfamily and in contrast to classic cadherins, such as E- and N-cadherin, LI-cadherin possess biological functions distinct from classic cadherins and reduced expression of LI-cadherin is closely associated with tumor progression and lymph node metastasis of human colorectal carcinoma. These mutations were also found in all CTCs. All this information is needed for individualized therapy and predict drug resistance, phenotypic transition which is heterogeneous from cell to cell from patient to patient. Citation Format: Aline Rangel Pozzo. Whole genome sequencing of single circulating tumor cells from localized high-risk prostate cancer patients: Laser microdissection and 3Dtelomere profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3535.

Detection of a 4 bp Mutation in the 3'UTR Region of Goat Sox9 Gene and Its Effect on the Growth Traits.

Simple SummaryThe sex determining region Y (SRY)-type high mobility group (HMG) box 9 (Sox9) gene is critically important in the formation and development of cartilage and is considered the “main regulator” of chondrogenesis. Additionally, a large number of studies have shown that mutations in a single allele of human Sox9 can lead to campomelic dysplasia syndrome. Therefore, the mutations of Sox9 have been the subject of increasing interest among researchers. However, no studies to date have examined the association between Sox9 gene variants and growth traits in goats. Here, we detected a 4 bp indel in the 3′Untranslated Regions (3′UTR) region of Sox9 in Shaanbei white cashmere (SBWC) goats (n = 1109) and studied the association between this indel and growth traits. The 4 bp indel of Sox9 was significantly associated with body length, heart girth, hip width, and all body measurement indexes (p < 0.05) in SBWC goats. Thus, this deletion could be used as an effective molecular marker for maximizing the growth traits of goats in breeding programs.The SRY-type HMG box 9 (Sox9) gene plays an important role in chondrocyte development as well as changes in hypertrophic chondrocytes, indicating that Sox9 can regulate growth in animals. However, no studies to date have examined the correlation between variations in Sox9 and growth traits in goats. Here, we found a 4 bp indel in the 3′UTR of Sox9 and verified its association with growth traits in Shaanbei white cashmere goats (n = 1109). The frequencies of two genotypes (ID and II) were 0.397 and 0.603, respectively, and polymorphic information content (PIC) values showed that the indel had a medium PIC (PIC > 0.25). The 4 bp indel was significantly correlated with body length (p = 0.006), heart girth (p = 0.001), and hip width (p = 4.37 × 10 −4). Notably, individuals with the ID genotype had significantly superior phenotypic traits compared with individuals bearing the II genotype. Hence, we speculated that the 4 bp indel is an important mutation affecting growth traits in goat, and may serve as an effective DNA molecular marker for marker-assisted selection in goat breeding programs.

Screening of Deletion Variants within the Goat PRDM6 Gene and Its Effects on Growth Traits.

Simple SummaryGenome-wide association studies found that the PR/SET Domain 6 (PRDM6) gene mutation was associated with bone development, bone density, and body mass index. This study found a 12 bp deletion variation within the PRDM6 gene in Shaanbei white cashmere goats in a large sample size (n = 1044). This variation polymorphism was associated with multiple goat growth traits in the yearling period, including heart girth (p = 0.027), cannon circumference (p = 0.008), chest depth (p = 2.10 × 10−5), chest width (p = 0.004), body height (p = 0.032), body length (p = 0.044), and hip-width (p = 0.014). The effects of the 12 bp variation were found to make no difference on adult goat growth traits. Taken together, these results demonstrate that the 12 bp deletion variant plays an important role in the early growth and development of goats and could be considered as a useful and effective molecular marker for goat breeding selection in the growth stage.By genome-wide association studies, the PRDM6 gene has been shown to affect multiple, apparently unrelated inherited traits, including bone density and body mass index. Therefore, it is considered a potentially pleiotropic gene. In this study, we identified a 12 bp deletion variant (NC_030814.1:rs651603667, g: 79985625-79985636delTTGACTGATCCA) within the PRDM6 gene in a large sample (SBWC goats; n = 1044). All goat samples were collected in Shaanxi province in July 2018. The frequency of the wt allele was higher than the frequency of the del allele, and this mutation polymorphism confirmed to be consistent with the Hardy–Weinberg equilibrium (p > 0.05). Further results showed that in a group of goats in the yearling period (18 months old, n = 567), this deletion variant of the PRDM6 gene was associated with heart girth (p = 0.027), cannon circumference (p = 0.008), chest depth (p = 2.10 × 10−5), chest width (p = 0.004), body height (p = 0.032), body length (p = 0.044) and hip-width (p = 0.014). For adult SBWC goats (36 months old, n = 477), the effects of the 12 bp variation on growth-related traits were found to make no difference. These findings show that the 12 bp deletion within the goat PRDM6 gene plays an important role in the early growth and development of goats. Using the 12 bp mutation, breeders can quickly and effectively select excellent individual goats at an early stage.

Frequency of somatic mutations in TERT promoter, TP53 and CTNNB1 genes in patients with hepatocellular carcinoma from Southern Italy.

Somatic mutations in the TERT promoter and in the TP53 and CTNNB1 genes are considered drivers for hepatocellular carcinoma (HCC) development. They show variable frequencies in different geographic areas, possibly depending on liver disease etiology and environmental factors. TP53, CTNNB1 and TERT genetic mutations were investigated in tumor and non-tumor liver tissues from 67 patients with HCC and liver tissue specimens from 41 control obese subjects from Southern Italy. Furthermore, TERT expression was assessed by reverse transcription-quantitative PCR. Neither CTNNB1 mutations or TP53 R249S substitution were detected in any case. The TP53 R72P polymorphism was found in 10/67 (14.9%) tumors, but was not found in either non-tumor tissues (P=0.001) or controls (P=0.009). TERT gene promoter mutations were found in 29/67 (43.3%) tumor tissues but were not found in either non-tumor (P<0.0001) or control liver specimens (P<0.0001). The most frequent mutation in the tumors was the known hot spot at −124 bp from the TERT ATG start site (−124G>A, 28 cases, 41.8%; P<0.0001). A new previously never reported TERT promoter mutation (at −297 bp from the ATG, −297C>T) was found in 5/67 (7.5%) tumors, in 0/67 (0%) non-tumor (P<0.0001), and in 0/41 (0%) controls (P=0.07). This mutation creates an AP2 consensus sequence, and was found alone (1 case) or in combination (4 cases) with the −124 bp mutation. The mutation at −124 and −297 bp induced a 33-fold (P<0.0001) and 40-fold increase of TERT expression levels, respectively. When both mutations were present, TERT expression levels were increased >300-fold (P=0.001). A new TERT promoter mutation was identified, which generates a de novo binding motif for AP2 transcription factors, and which significantly increases TERT promoter transcriptional activity.

Genetic variations in the sheep SIRT7 gene and their correlation with body size traits.

As a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase and ADP ribosyl transferase, the silent information regulator 7 (Sirtuin 7, SIRT7) plays a crucial role in regulating the differentiation of adipocytes and myoblasts, lipid metabolism, glucose metabolism, and cellular growth in mammals. It has been hypothesized that SIRT7 affects growth traits in animals; therefore, in this study, the potential insertion/deletion (indel) of genetic variations within the ovine SIRT7 gene and their correlation with sheep growth traits were explored. A total of 709 individuals from five Chinese and Mongolian sheep breeds were analyzed. Two novel indel loci of the sheep SIRT7 gene were detected and were named 5 promoter region-insertion-7 bp (5 promoter region-7 bp) and 3 UTR-insertion-17 bp (3 UTR-17 bp), respectively. In all of the sheep breeds, frequencies of the 5 promoter region-7 bp mutation were low, whereas mutations of 3 UTR-17 bp were high in Tong sheep and Lanzhou fat-tail sheep (LFTS). Furthermore, both indel polymorphisms had significant associations with different growth characteristics (). Among these associations, the 3 UTR-17 bp was highly correlated with rump width in small-tail Han sheep (STHS, rams; ), and individuals with the ID genotype had better chest depth values than those with the II genotype. In this paper, two novel indels within the sheep SIRT7 gene were identified, and genetic diversity and its effects on body size traits were explored. These findings will potentially provide useful DNA markers for the improvement of economic traits in sheep genetic breeding.

Tần suất và đột biến gen LMP1 của virus Epstein-Barr ở mẫu sinh thiết vòm của bệnh nhân ung thư vòm mũi họng điều trị tại Bệnh viện Ung bướu Cần Thơ

Latent Membrane Protein 1 (LMP1) gen of Epstein-Barr virus (EBV) (LMP1 EBV) and the mutation in LMP1 EBV were important for the formation of malignant tumors in nasopharyngeal carcinoma (NPC) in patients with EBV infection. The objective of the study was to determine the frequency and loss 30 bp mutation of the LMP1 gene in the biopsy specimen of the NPC patient. A cross sectional descriptive study was carried out of 65 biopsy specimes of patients, who was confirmed as nasopharynreal carcinoma at the Can Tho Oncology Hospital. Polymerase Chain Reaction (PCR) with LMP1 primer (168373-168174) was used to determine the frequency of LMP1 EBV gene and sequening technique was applied to identify LMP1 gene mutation. As a result, the rate of LMP1 EBV in nasopharyx biopsy specimens was 61.5% (40/65). Besides, there was 57.5% (23/40) of 30 bp loss mutant on LMP1 EBV gene was indentified among of LMP1 EBV in nasopharyx biopsy specimens. In conclusions, the rate of LMP1 EBV on biopsy specimens of NPC in Can Tho was 61.5% with the common mutation pattern of 30bp loss.

Comprehensive behavioral and biochemical outcomes of novel murine models of GM1-gangliosidosis and Morquio syndrome type B

Deficiencies in the lysosomal hydrolase β-galactosidase (β-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse β-gal encoding gene, Glb1, was targeted to generate both models of β-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1W274L), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of β-gal (β-gal−/−). Glb1W274L mice showed a significant reduction in β-gal enzyme activity (8.4–13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, β-gal−/− mice were devoid of β-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). β-gal−/− mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of β-galactosidase deficiency with residual enzyme activity has been developed.

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

Generation, Characterization and Genetic Modification of Human iPSCs Containing Calr, MPL and JAK2 Mutations Found in MPN Patients

Introduction: Myeloproliferative neoplasms (MPNs) that include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are associated with multiple somatic and myeloid-specific mutations. A major challenge to the study of these diseases has been the lack of model systems that can accurately represent their complexity; the clonal heterogeneity within the hematopoietic stem/progenitor cells (HSPC) population is confounding and it is technically challenging to isolate and expand patient HSPC clones in vitro. To overcome this hurdle, we have utilized induced pluripotent stem cell (iPSC) technology to generate disease and clone-specific iPSC lines that preserve the genetic identity of HSPC clones with the JAK2-V617F mutation (Ye et al., 2009 in Blood). Recent advances in MPN genetics have identified CALR and MPL mutations in the majority of JAK2-V617F negative MPN. We therefore created additional panels of iPSCs from patients carrying CALR and MPL mutations in addition to our JAK2-mutated iPSCs. In addition, we used CRISPR/Cas9 technology to study genetic lesions and their contribution the phenotypes by using iPSC in vitro hematopoietic differentiation assays (Ye, et al., 2015 in Molecular Therapy).Methods: We generated iPSC lines from MPN patients with JAK2V617F, CALR or MPL mutations using peripheral blood CD34+ cells by transient expression of reprogramming factors in plasmid vectors. We characterized the pluripotency and karyotype of the derived iPSC lines, and determined if they contained the same somatic mutation as found in blood samples of each MPN patient. Then the derived iPSC cell lines were differentiated to CD34+ HSPCs, erythroid or megakaryocytic cell lineages using defined in vitro culture systems. Erythroid differentiation was determined by flow cytometric analysis of CD235a (Glycophrin A)-positive and CD45-negative cells. Megakaryocytic differentiation was determined by the levels of CD41+CD42+ cells.Results: In vitro differentiation assays have shown increased erythroid differentiation of HSPCs derived from patient-specific iPSCs that carry the JAK2V617F mutation, recapitulating a major PV disease feature. Editing a PV-derived JAK2V617F/V617F iPSC line to JAK2V617F/WT abrogated endogenous erythroid colony (EEC) formation, erythroid expansion and altered erythropoietin dose-response curve characteristic of the parent line. Conversely, gene editing a JAK2V617F/WT to a JAK2V617F/V617F iPSC line from a different PV patient conferred EEC formation and expanded erythropoiesis. In addition, data generated from these isogenic iPSCs suggested the contribution of additional genetic lesions to MPN disease phenotypes in JAK2-V617F positive patients. We also generated iPSC lines from MPN patients with the CALR type II (+5 bp) mutation and the rare MPL-V501L mutation. We found that the iPSC line containing a heterozygous MPL-V501L mutation (derived from an ET patient) appeared normal with iPSC typical morphology and phenotypes (expressing OCT4 and TRA-1-60 pluripotency markers) as the wildtype and JAK2-V617F iPSCs. We also generated four iPSC lines from a MF patient: all the four clones contained the homozygous CALR type II (+5 bp) mutation as found in the blood cells of the MF patient. Two iPSC clones we examined had a normal karyoptype. However, the iPSC lines showed signs of increased spontaneous differentiation when being cultured under the standard culture condition, based on cell morphology and reduction of OCT4 & TRA-1-60 expression. We are currently testing the potential of these iPSC lines containing the CALR and MPLmutations in hematopoietic differentiation, focusing on erythrocytic and megakaryocytic differentiation activities.Conclusions: we believe that by comparative analysis of isogenic iPSCs containing the common and rare MPN driver mutations, both common and specific pathways essential for MPNs can be determined. Precise genome editing or surgery of these mutations by engineered targeting may also provide a novel approach to eliminate the malignant MPN clones.This project is supported in part by the MPN Research Foundation and LLS. DisclosuresNo relevant conflicts of interest to declare.

Characterization and fine mapping of osh15(t), a novel dwarf mutant gene in rice (Oryza sativa L.)

Plant height is one of the most important agronomic traits of plant architecture, and also affects grain yield in rice. In this study, we obtained a novel dwarf rice mutant of japonica variety Shennong9816, designated Shennong9816d. Compared with wild-type, the Shennong9816d plant height was significantly reduced, and the tiller number significantly increased. Additionally, the mutant yield component, and the number of large and small vascular bundles were significantly decreased compared with wild-type. Genetic analysis indicated that the Shennong9816d dwarf phenotype was controlled by a recessive nuclear gene, while the plant was shown to be sensitive to gibberellic acid. Using a large F2 population derived from a cross between Shennong9816d and the indica rice variety Habataki, the osh15(t) gene was fine mapped between RM20891 and RM20898, within a physical distance of 73.78 kb. Sequencing analysis showed that Shennong9816d carries a 1 bp mutation and a 30 bp insertion in the OSH15 region. These results suggest that osh15(t) is a novel allelic mutant originally derived from japonica variety Shennong9816, which may be useful for introducing the semi-dwarf phenotype to improve plant architecture in rice breeding practice.

Mitochondrial injury and cognitive function in HIV infection and methamphetamine use.

In this work, we evaluated the association of human immunodeficiency virus (HIV) infection and methamphetamine (METH) use with mitochondrial injury in the brain and its implication on neurocognitive impairment. Mitochondria carry their genome (mtDNA) and play a critical role in cellular processes in the central nervous system. METH is commonly used in HIV-infected populations. HIV infection and METH use can cause damage to mtDNA and lead to neurocognitive morbidity. We evaluated HIV infection and METH use with mitochondrial injury in the brain. We obtained white and gray matter from Brodmann areas 7, 8, 9, 46 of the following: HIV-infected individuals with history of past METH use (HIV+METH+, n = 16), HIV-infected individuals with no history of past METH use (HIV+METH-, n = 11), and HIV-negative controls (HIV-METH-, n = 30). We used the 'common deletion', a 4977 bp mutation, as a measurement of mitochondrial injury, and quantified levels of mtDNA and 'common deletion' by droplet digital PCR, and evaluated in relation to neurocognitive functioning [Global Deficit Score (GDS)]. Levels of mtDNA and mitochondrial injury were highest in white matter of Brodmann area 46. A higher relative proportion of mtDNA carrying the 'common deletion' was associated with lower GDS (P < 0.01) in HIV+METH+ but higher GDS (P < 0.01) in HIV+METH-. Increased mitochondrial injury was associated with worse neurocognitive function in HIV+METH- individuals. Among HIV+METH+ individuals, an opposite effect was seen.

Super-resolution mapping of wetland inundation from remote sensing imagery based on integration of back-propagation neural network and genetic algorithm

Mapping the spatio-temporal characteristics of wetland inundation has an important significance to the study of wetland environment and associated flora and fauna. High temporal remote sensing imagery is widely used for this purpose with the limitations of relatively low spatial resolutions. In this study, a novel method based on integration of back-propagation neural network (BP) and genetic algorithm (GA), so-called IBPGA, is proposed for super-resolution mapping of wetland inundation (SMWI) from multispectral remote sensing imagery. The IBPGA-SMWI algorithm is developed, including the fitness function and integration search strategy. IBPGA-SMWI was evaluated using Landsat TM/ETM+ imagery from the Poyanghu wetland in China and the Macquarie Marshes in Australia. Compared with traditional SMWI methods, IBPGA-SMWI consistently achieved more accurate super-resolution mapping results in terms of visual and quantitative evaluations. In comparison with GA-SMWI, IBPGA-SMWI not only improved the accuracy of SMWI, but also accelerated the convergence speed of the algorithm. The sensitivity analysis of IBPGA-SMWI in relation to standard crossover rate, BP crossover rate and mutation rate was also carried out to discuss the algorithm performance. It is hoped that the results of this study will enhance the application of median-low resolution remote sensing imagery in wetland inundation mapping and monitoring, and ultimately support the studies of wetland environment.

Comparative Analysis of Context-Dependent Mutagenesis Using Human and Mouse Models

Substitution rates strongly depend on their nucleotide context. One of the most studied examples is the excess of C > T mutations in the CG context in various groups of organisms, including vertebrates. Studies on the molecular mechanisms underlying this mutation regularity have provided insights into evolution, mutagenesis, and cancer development. Recently several other hypermutable motifs were identified in the human genome. There is an increased frequency of T > C mutations in the second position of the words ATTG and ATAG and an increased frequency of A > C mutations in the first position of the word ACAA. For a better understanding of evolution, it is of interest whether these mutation regularities are human specific or present in other vertebrates, as their presence might affect the validity of currently used substitution models and molecular clocks. A comprehensive analysis of mutagenesis in 4 bp mutation contexts requires a vast amount of mutation data. Such data may be derived from the comparisons of individual genomes or from single nucleotide polymorphism (SNP) databases. Using this approach, we performed a systematical comparison of mutation regularities within 2–4 bp contexts in Mus musculus and Homo sapiens and uncovered that even closely related organisms may have notable differences in context-dependent mutation regularities.

Characterization and expression of the gyrA gene from quinolone resistant Yersinia ruckeri strains isolated from Atlantic salmon (Salmo salar L.) in Norway

Yersinia ruckeri, the causative agent of yersiniosis has been reported in a number of fish species but the most vulnerable are the salmonids including Atlantic salmon (Salmo salar L.). In the present study Y. ruckeri isolates were collected from diseased Atlantic salmon juveniles and characterized for resistance against quinolones. Isolates were screened using disk diffusion assays and MIC determination. The QRDR regions of the gyrA, gyrB, parC and parE genes were sequenced. Quinolone resistant isolates revealed a single bp mutation which replaced serine by arginine at position 83 in the GyrA protein while no mutations were found in gyrB, parC and parE genes. Isolates were also screened for plasmid encoded qnrA, qnrB and qnrS genes but they were found absent. Cloning of gyrA from susceptible and resistant isolates into heterologous Y. ruckeri was not successful. The different gyrA alleles from susceptible and resistant isolates of Y. ruckeri were cloned into Escherichia coli TOPO 10 and E. coli DH5α. While cloning of the resistant allele into the sensitive host had no effect, cloning of the quinolone susceptible gyrA allele into quinolone resistant E. coli DH5α increased the inhibition zone diameter from 25mm to 38mm and decreased the MIC from 4μg/ml to 2μg/ml, suggesting dominance of wild type gyrA over the mutant allele. It is assumed that the wild type GyrA protein has more affinity to form the gyrase–DNA complex than mutant GyrA even in the presence of high levels of the mutated enzyme.

New words in human mutagenesis

BackgroundThe substitution rates within different nucleotide contexts are subject to varying levels of bias. The most well known example of such bias is the excess of C to T (C > T) mutations in CpG (CG) dinucleotides. The molecular mechanisms underlying this bias are important factors in human genome evolution and cancer development. The discovery of other nucleotide contexts that have profound effects on substitution rates can improve our understanding of how mutations are acquired, and why mutation hotspots exist.ResultsWe compared rates of inherited mutations in 1-4 bp nucleotide contexts using reconstructed ancestral states of human single nucleotide polymorphisms (SNPs) from intergenic regions. Chimp and orangutan genomic sequences were used as outgroups. We uncovered 3.5 and 3.3-fold excesses of T > C mutations in the second position of ATTG and ATAG words, respectively, and a 3.4-fold excess of A > C mutations in the first position of the ACAA word.ConclusionsAlthough all the observed biases are less pronounced than the 5.1-fold excess of C > T mutations in CG dinucleotides, the three 4 bp mutation contexts mentioned above (and their complementary contexts) are well distinguished from all other mutation contexts. This provides a challenge to discover the underlying mechanisms responsible for the observed excesses of mutations.

Speeding Cis-Trans Regulation Discovery by Phylogenomic Analyses Coupled with Screenings of an Arrayed Library of Arabidopsis Transcription Factors

Transcriptional regulation is an important mechanism underlying gene expression and has played a crucial role in evolution. The number, position and interactions between cis-elements and transcription factors (TFs) determine the expression pattern of a gene. To identify functionally relevant cis-elements in gene promoters, a phylogenetic shadowing approach with a lipase gene (LIP1) was used. As a proof of concept, in silico analyses of several Brassicaceae LIP1 promoters identified a highly conserved sequence (LIP1 element) that is sufficient to drive strong expression of a reporter gene in planta. A collection of ca. 1,200 Arabidopsis thaliana TF open reading frames (ORFs) was arrayed in a 96-well format (RR library) and a convenient mating based yeast one hybrid (Y1H) screening procedure was established. We constructed an episomal plasmid (pTUY1H) to clone the LIP1 element and used it as bait for Y1H screenings. A novel interaction with an HD-ZIP (AtML1) TF was identified and abolished by a 2 bp mutation in the LIP1 element. A role of this interaction in transcriptional regulation was confirmed in planta. In addition, we validated our strategy by reproducing the previously reported interaction between a MYB-CC (PHR1) TF, a central regulator of phosphate starvation responses, with a conserved promoter fragment (IPS1 element) containing its cognate binding sequence. Finally, we established that the LIP1 and IPS1 elements were differentially bound by HD-ZIP and MYB-CC family members in agreement with their genetic redundancy in planta. In conclusion, combining in silico analyses of orthologous gene promoters with Y1H screening of the RR library represents a powerful approach to decipher cis- and trans-regulatory codes.

Targeted Knockdown of EGR-1 Inhibits IL-8 Production and IL-8-mediated Invasion of Prostate Cancer Cells through Suppressing EGR-1/NF-κB Synergy

IL-8 produced by prostate cancer cells may be responsible for the androgen-independent growth of advanced prostate cancers. Accumulating evidence from microarray analyses and animal genetic models highlights the central involvement of the transcription factor early growth response-1 (EGR-1) in prostate carcinoma progression. It is unknown, however, whether knockdown of EGR-1 inhibits IL-8 production and IL-8-mediated tumor metastasis. Here we show that EGR-1 knockdown by a specific shRNA-Egr1 inhibited gene transcription and production of IL-8 by the human prostate cancer cell line DU145. Conversely, enforced expression of EGR-1 in EGR-1-lacking PC3 prostate cancer cells markedly enhanced IL-8 transcription and secretion. By using wild type and a series of mutant IL-8 promoter luciferase constructs, we found that the NF-kappaB binding site is important for EGR-1 regulation of IL-8. Furthermore, silencing EGR-1 suppressed a synergistically functional interaction between EGR-1 and NF-kappaB. Consequently, knockdown of EGR-1 inhibited IL-8-mediated tumor colony formation and invasion. Thus, targeted knockdown of EGR-1 could be an effective therapeutic approach against prostate cancer.