10-base Pair Research Articles

Validation of a Chromosome 14 Risk Haplotype for Idiopathic Epilepsy in the Belgian Shepherd Dog Found to Be Associated with an Insertion in the RAPGEF5 Gene.

An idiopathic epilepsy (IE) risk haplotype on canine chromosome (CFA) 14 has been reported to interact with the CFA37 common risk haplotype in the Belgian shepherd (BS). Additional IE cases and control dogs were genotyped for the risk haplotypes to validate these previous findings. In the new cohort, the interaction between the two regions significantly elevated IE risk. When the haplotypes were analyzed individually, particular haplotypes on both CFA14 (ACTG) and 37 (GG) were associated with elevated IE risk, though only the CFA37 AA was significantly associated (p < 0.003) with reduced risk in the new cohort. However, the CFA14 ACTG risk was statistically significant when the new and previous cohort data were combined. The frequency of the ACTG haplotype was four-fold higher in BS dogs than in other breeds. Whole genome sequence analysis revealed that a 3-base pair predicted disruptive insertion in the RAPGEF5 gene, which is adjacent to the CFA14 risk haplotype. RAPGEF5 is involved in the Wnt-β-catenin signaling pathway that is crucial for normal brain function. Although this risk variant does not fully predict the likelihood of a BS developing IE, the association with a variant in a candidate gene may provide insight into the genetic control of canine IE.

Abstract 3996: A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice

Abstract Introduction: Glioblastoma (GBM) is the most frequent treatment resistant and poor prognosis primary brain tumor. Age-adjusted incidence among Caucasians is approx. twice that of Africans. Genetic changes in ethnicity may represent druggable targets for development of new treatments for GBM. Chondroitin sulfate proteoglycan 4 (CSPG4) with sequence homology to neuron-glial-2 (NG2), henceforth CSPG4/NG2, is a transmembrane proteoglycan that is upregulated in GBM, resulting in leaky neovasculature and high cell proliferation, and is independently prognostic for poor survival. Mechanisms regulating CSPG4/NG2 expression remain to be elucidated. Experiments: Tumor-derived DNA whole exome sequences (WES) from mixed American GBM patients (n=300) collected into the TCGA database and blood-derived DNA WES from controls (n = 2504) in the 1000 Genome database were analyzed for mutations in the CSPG4 gene by GATK-3.5. We used prime editing (PE) to introduce a 13-base pair (bp) deletion through design of PE guide RNA (n=27), screening GBM cells (n=14) for CSPG4/NG2 expression, transfecting and expanding PE cells, confirming editing using Sanger sequencing. We functionally characterized the mutation in PE cells and transplanted in orthotopic PDX mouse models compared to wild type (WT) and Cas9 control cells. Western blotting, BrdU proliferation assays, clonogenic- and wound healing assays were used to investigate function. Gelatin zymography assessed CSPG4/NG2 matrix metalloproteinase (MMP) cross-talk during invasion on mixed extracellular matrix- or collagen IV- coated transwells. Results: A novel 13bp frameshift deletion in exon 3 of the CSPG4 gene was identified from WES of GBM samples deposited in TCGA. The mutation was cancer specific (p&amp;lt;0.001). Comparison of frequency in various ethnicities revealed the mutation to be more frequent among African American GBM patients (p=0.019). We generated 3 clones heterozygous for the CSPG4/NG2 deletion in patient derived GBMs. A homozygous deletion was lethal to the cells. The 13bp deletion reduced CSPG4/NG2 expression by approx. 60%, slowed tumor cell proliferation compared to both WT (p&amp;lt;0.001) and Cas9 cells (p&amp;lt;0.0001). PE cells had significantly reduced colony formation (p&amp;lt;0.001), scratch wound healing (p&amp;lt;0.001), had a weaker invasive phenotype when challenged with collagen IV (p&amp;lt;0.001), and reduced cleavage of collagen I by MMP2 that was corroborated by RNAseq gene expression. Reduced growth of PE cells in vivo was verified by multiplex immunohistochemistry on ex vivo brain sections and identified biomarkers for differential tumor development. Tumor growth was reduced in vivo in mice. Conclusions: We identified a novel 13bp deletion in CSPG4/NG2 with phenotypic effect that could be used as a future molecular target in GBM treatment, and present the first successful application of PE in human GBM cells. Citation Format: Victoria Smith Arnesen, Susina Suntharalingam, Żaneta Matuszek, Shahin Sarowar, Stian Knappskog, Stein Atle Lie, David R. Liu, Mohummad Aminur Rahman, Martha Chekenya. A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3996.

Deciphering the mechanism of p73 recognition of p53 response elements using the crystal structure of p73-DNA complexes and computational studies

p73 belongs to p53 family transcription factor activating more than 50% of cell fate p53 target genes involved in cell cycle, apoptosis, DNA damage response alongside neuronal system development and differentiation by binding to 20-bp response elements (REs) having sequence motif (PPPC-A/T-T/A-GYYY) where P-purines and Y-pyrimidines with each 10-bp separated by minimum 0 to 13-bp spacer. The promiscuous nature of recognizing both cell fate and development genes and the underlying RE selectivity mechanism by p73 is not well understood. Here, we report the molecular details of p73 recognizing the REs using the crystal structure of p73 DNA binding domain (DBD) in complex with 12 base pair DNA sequence 5'-cAGGCATGCCTg-3′ and molecular dynamics simulations with six different p53 natural promoter sequences. Each 20-base pair natural promoter forms a different major/minor groove due to the presence of nucleotides A/T, A/C, G/G, T/T and G/T at positions 3, 8, 13, 18 uniquely recognized by p73 key residues Lys138 and Arg268. The loops L1 and L3 bearing these residues influence inter-and intra-dimer interfaces interactions and hence p73 forms a unique tetramer with each natural promoter sequence. Structural features of the DNA and the spacing between half-sites influence p73 tetramerization and its transactivation function.

Recurrent mutations in topoisomerase IIα cause a previously undescribed mutator phenotype in human cancers

Topoisomerases nick and reseal DNA to relieve torsional stress associated with transcription and replication and to resolve structures such as knots and catenanes. Stabilization of the yeast Top2 cleavage intermediates is mutagenic in yeast, but whether this extends to higher eukaryotes is less clear. Chemotherapeutic topoisomerase poisons also elevate cleavage, resulting in mutagenesis. Here, we describe p.K743N mutations in human topoisomerase hTOP2α and link them to a previously undescribed mutator phenotype in cancer. Overexpression of the orthologous mutant protein in yeast generated a characteristic pattern of 2- to 4-base pair (bp) duplications resembling those in tumors with p.K743N. Using mutant strains and biochemical analysis, we determined the genetic requirements of this mutagenic process and showed that it results from trapping of the mutant yeast yTop2 cleavage complex. In addition to 2- to 4-bp duplications, hTOP2α p.K743N is also associated with deletions that are absent in yeast. We call the combined pattern of duplications and deletions ID_TOP2α. All seven tumors carrying the hTOP2α p.K743N mutation showed ID_TOP2α, while it was absent from all other tumors examined (n = 12,269). Each tumor with the ID_TOP2α signature had indels in several known cancer genes, which included frameshift mutations in tumor suppressors PTEN and TP53 and an activating insertion in BRAF. Sequence motifs found at ID_TOP2α mutations were present at 80% of indels in cancer-driver genes, suggesting that ID_TOP2α mutagenesis may contribute to tumorigenesis. The results reported here shed further light on the role of topoisomerase II in genome instability.

Functional evaluation of immunoregulatory molecules HLA-G, galectin-1, and IL-10 in people living with HIV.

Objective(s):Investigate polymorphisms and expressions of human leukocyte antigen-G (HLA-G), galectin-1 (Gal-1), and interleukin-10 (IL-10) in people living with HIV (PLHIV) with and without comorbidities to help understanding the mechanisms involved in triggering these disorders in PLHIV and in their prognosis.Design:Here we evaluated the potential correlation between the genetic polymorphism and/or protein levels of HLA-G, Gal-1, and IL-10 with and without comorbidities of PLHIV.Methods:Two hundred HIV patients under antiretroviral treatment (83 with comorbidities and 117 without comorbidities) and 200 healthy individuals (controls) were genotyped, using PCR, for HLA-G 14-base pair polymorphism located at the 3’ untranslated region in exon 8 insertion/insertion (Ins/Ins: low HLA-G expression) or deletion/deletion (Del/Del: high HLA-G expression). Soluble levels of HLA-G (sHLA-G), Gal-1, and IL-10 were quantified by enzyme-linked immunosorbet assay.Results:HIV patients without comorbidities exhibited higher frequency of 14-base pair Del/Del genotype than HIV patients with comorbidities. As expected, HIV patients Ins/Ins with and without comorbidities produced less sHLA-G than controls. However, HIV patients Del/Del with comorbidities expressed sHLA-G more than controls and HIV patients Del/Del without comorbidities. Interestingly, patients that showed low levels sHLA-G, and presence of comorbidities, exhibited high Gal-1 serum levels. However, an increase in soluble levels of IL-10 in PLHIV was observed when compared to controls, especially in the PLHIV group without comorbidities suggesting, a protective role of IL-10 in the development of comorbidities.Conclusions:These data suggested that the high expression of sHLA-G and IL-10 or Gal-1 could be associated and could be associated with the development or not of comorbidities in PLHIV.

South Asian-Specific MYBPC3 Δ25bp Deletion Carriers Display Hypercontraction and Impaired Diastolic Function Under Exercise Stress.

Background: A 25-base pair (25bp) intronic deletion in the MYBPC3 gene enriched in South Asians (SAs) is a risk allele for late-onset left ventricular (LV) dysfunction, hypertrophy, and heart failure (HF) with several forms of cardiomyopathy. However, the effect of this variant on exercise parameters has not been evaluated.Methods: As a pilot study, 10 asymptomatic SA carriers of the MYBPC3Δ25bp variant (52.9 ± 2.14 years) and 10 age- and gender-matched non-carriers (NCs) (50.1 ± 2.7 years) were evaluated at baseline and under exercise stress conditions using bicycle exercise echocardiography and continuous cardiac monitoring.Results: Baseline echocardiography parameters were not different between the two groups. However, in response to exercise stress, the carriers of Δ25bp had significantly higher LV ejection fraction (%) (CI: 4.57 ± 1.93; p < 0.0001), LV outflow tract peak velocity (m/s) (CI: 0.19 ± 0.07; p < 0.0001), and higher aortic valve (AV) peak velocity (m/s) (CI: 0.103 ± 0.08; p = 0.01) in comparison to NCs, and E/A ratio, a marker of diastolic compliance, was significantly lower in Δ25bp carriers (CI: 0.107 ± 0.102; p = 0.038). Interestingly, LV end-diastolic diameter (LVIDdia) was augmented in NCs in response to stress, while it did not increase in Δ25bp carriers (CI: 0.239 ± 0.125; p = 0.0002). Further, stress-induced right ventricular systolic excursion velocity s' (m/s), as a marker of right ventricle function, increased similarly in both groups, but tricuspid annular plane systolic excursion increased more in carriers (slope: 0.008; p = 0.0001), suggesting right ventricle functional differences between the two groups.Conclusions: These data support that MYBPC3Δ25bp is associated with LV hypercontraction under stress conditions with evidence of diastolic impairment.

Structural specificity of groove binding mechanism between imidazolium-based ionic liquids and DNA revealed by synchrotron-UV Resonance Raman spectroscopy and molecular dynamics simulations

The predicted capability of Ionic Liquids (ILs) in stabilizing the native structure of nucleic acids is relevant in biotechnology, especially for DNA storage and handling. In the present work, we implement a joint combination of advanced spectroscopic techniques such as synchrotron radiation-UV Resonance Raman spectroscopy (SR-UVRR) and molecular dynamics (MD) simulations for deepening insight into the sequence and structural specificity of the binding interactions between imidazolium-based ILs and both the phosphate groups and nucleobases in the minor and major grooves of double-stranded DNA. A 30-base pair double-stranded DNA structure has been chosen as a model of natural DNA. The experimental and simulation results give evidence of the predominance of a groove binding mechanism between ILs cations and DNA, with preferential interactions among guanine residues and the shorter alkyl-chain length on imidazolium cations. Raman experiments allowed us to detect both cooperative transition and reversible pre-melting structural transformations that involve specific tracts in the structure of DNA and are turned on at lower temperatures for guanine residues than for adenine ones. The more marked effect on the pre-melting states of adenine operated by the imidazolium-based ILs with chloride as anion suggests a selective strong interaction of this anion with the DNA’s adenine-rich tracts. MD simulation results reveal the influence of ILs on the structural properties of DNA and provide more details about the solvation, interaction, stability and flexibility of DNA in the hydrated ILs. According to MD analyses, simultaneous electrostatic and hydrophobic interactions drive the shorter alkyl-chain length of imidazolium cations to have greater interplays with the DNA major groove.

Multiplexed DNA-Directed Patterning of Antibodies for Applications in Cell Subpopulation Analysis

Antibodies provide the functional biospecificity that has enabled the development of sensors, diagnostic tools, and assays in both laboratory and clinical settings. However, as multimarker screening becomes increasingly necessary due to the heterogeneity and complexity of human pathology, new methods must be developed that are capable of coordinating the precise assembly of multiple, distinct antibodies. To address this technological challenge, we engineered a bottom-up, high-throughput method in which DNA patterns, comprising unique 20-base pair oligonucleotides, are patterned onto a substrate using photolithography. These microfabricated surface patterns are programmed to hybridize with, and instruct the multiplexed assembly of, antibodies conjugated with the complementary DNA strands. We demonstrate that this simple, yet robust, approach preserves the antibody-binding functionality in two common applications: antibody-based cell capture and label-free surface marker screening. Using a simple proof-of-concept capture device, we achieved high purity separation of a breast cancer cell line, MCF-7, from a blood cell line, Jurkat, with capture purities of 77.4% and 96.6% when using antibodies specific for the respective cell types. We also show that antigen-antibody interactions slow cell trajectories in flow in the next-generation microfluidic node-pore sensing (NPS) device, enabling the differentiation of MCF-7 and Jurkat cells based on EpCAM surface-marker expression. Finally, we use a next-generation NPS device patterned with antibodies against E-cadherin, N-cadherin, and β-integrin-three markers that are associated with epithelial-mesenchymal transitions-to perform label-free surface marker screening of MCF10A, MCF-7, and Hs 578T breast epithelial cells. Our high-throughput, highly versatile technique enables rapid development of customized, antibody-based assays across a host of diverse diseases and research thrusts.

Detection of 15-bp Deletion Mutation within PLAG1 Gene and Its Effects on Growth Traits in Goats.

Simple SummaryGoats have always served as an important domesticated livestock. PLAG1 is a major gene that affects the stature and growth of animals. Body size traits are very important for goats as it directly affects the economic characteristics of meat and cashmere production. This study showed that the 15-base pair (bp) InDel (rs637141549) can significantly affect growth traits such as body weight, height, height at hip cross, chest circumference, hip width and body index of goats through the detection of large samples (n = 1581) in four indigenous breeds. Accordingly, it is suggested that the deletion mutation can be used as a potential molecular marker that significantly affects goat growth traits. Moreover, the 15bp deletion mutation can be used as a potential molecular marker, which significantly affects the growth traits of goats and plays an important role in animal husbandry production. Stature and weight are important growth and development traits for animals, which also significantly affect the productivity of livestock. Polymorphic adenoma gene 1 (PLAG1) is located in the growth-related quantitative trait nucleotides (QTN), and its variation has been determined to significantly affect the body stature of bovines. This study found that novel 15-bp InDel could significantly influence important growth traits in goats. The frequencies of genotypes of the 15-bp mutation and relationship with core growth traits such as body weight, body height, height at hip cross, chest circumference, hip width and body index were explored in 1581 individuals among 4 Chinese native goat breeds. The most frequent genotypes of Shaanbei white Cashmere goat (SWCG), Inner Mongolia White Cashmere goat (IMCG) and Guanzhong Dairy goat (GZDG) were II genotypes (insertion/insertion), and the frequency of ID genotype (insertion/deletion) was found to be slightly higher than that of II genotype in Hainan Black goat (HNBG), showing that the frequency of the I allele was higher than that of the D allele. In adult goats, there were significant differences between 15-bp variation and body weight, chest circumference and body height traits in SWCG (p < 0.05). Furthermore, the locus was also found to be significantly correlated with the body index of HNBG (p = 0.044) and hip width in GZDG (p = 0.002). In regard to lambs, there were significant differences in height at the hip cross of SWCG (p = 0.036) and hip width in IMWC (p = 0.005). The corresponding results suggest that the 15-bp InDel mutation of PLAG1 is associated with the regulation of important growth characteristics of both adult and lamb of goats, which may serve as efficient molecular markers for goat breeding.

Evaluating toxicity of Varroa mite (Varroa destructor)-active dsRNA to monarch butterfly (Danaus plexippus) larvae.

Varroa mites (Varroa destructor) are parasitic mites that, combined with other factors, are contributing to high levels of honey bee (Apis mellifera) colony losses. A Varroa-active dsRNA was recently developed to control Varroa mites within honey bee brood cells. This dsRNA has 372 base pairs that are homologous to a sequence region within the Varroa mite calmodulin gene (cam). The Varroa-active dsRNA also shares a 21-base pair match with monarch butterfly (Danaus plexippus) calmodulin mRNA, raising the possibility of non-target effects if there is environmental exposure. We chronically exposed the entire monarch larval stage to common (Asclepias syriaca) and tropical (Asclepias curassavica) milkweed leaves treated with concentrations of Varroa-active dsRNA that are one- and ten-fold higher than those used to treat honey bee hives. This corresponded to concentrations of 0.025–0.041 and 0.211–0.282 mg/g leaf, respectively. Potassium arsenate and a previously designed monarch-active dsRNA with a 100% base pair match to the monarch v-ATPase A mRNA (leaf concentration was 0.020–0.034 mg/g) were used as positive controls. The Varroa mite and monarch-active dsRNA’s did not cause significant differences in larval mortality, larval or pupal development, pupal weights, or adult eclosion rates when compared to negative controls. Irrespective of control or dsRNA treatment, larvae that consumed approximately 7500 to 10,500-mg milkweed leaf within 10 to 12 days had the highest pupal weights. The lack of mortality and sublethal effects following dietary exposure to dsRNA with 21-base pair and 100% base pair match to mRNAs that correspond to regulatory genes suggest monarch mRNA may be refractory to silencing by dsRNA or monarch dsRNase may degrade dsRNA to a concentration that is insufficient to silence mRNA signaling.

Genetic polymorphism of dopamine receptor D4 (DRD4) gene in ten Indian rhesus macaques (Macaca mulatta mulatta)

Advances in molecular genetical research have revealed a new platform for studying the configuration and activity correlations of dopamine receptor D4 (DRD4) gene in the non-human primates and human. Among the other five dopamine receptor genes, dopamine receptor D4 (DRD4) gene has variable number of tandem repeats which are polymorphic and located in the third cytoplasmic loop of the receptor. These polymorphism are expressed in the repeat numbers of 48-base pair (bp) sequence coding for 16 amino acids. This study is designed to observe the genetic polymorphism of dopamine receptor D4 gene within the ten Indian rhesus macaques (Macaca mulatta mulatta). This research is also planned to compare the genetic sequences among the Indian rhesus macaque DRD4 genetic sequences of our recent study, rhesus macaque DRD4 gene reference sequence provided by ENSEMBL genome browser and human DRD4 gene reference sequence provided by National Center for Biotechnology Information (NCBI), USA. In our study, only DRD4 5-repeat allele variant is observed in all the ten selected Indian rhesus macaques. Sequencing data of DRD4 gene of these rhesus macaques indicate that rhesus macaques exhibit 48-bp tandem repeats in the DRD4 receptor gene which is similar to humans and there is intraspecies variability within the rhesus macaque DRD4 genetic sequences.

Mono- and bilayer smectic liquid crystal ordering in dense solutions of “gapped” DNA duplexes

Although its mesomorphic properties have been studied for many years, only recently has the molecule of life begun to reveal the true range of its rich liquid crystalline behavior. End-to-end interactions between concentrated, ultrashort DNA duplexes-driving the self-assembly of aggregates that organize into liquid crystal phases-and the incorporation of flexible single-stranded "gaps" in otherwise fully paired duplexes-producing clear evidence of an elementary lamellar (smectic-A) phase in DNA solutions-are two exciting developments that have opened avenues for discovery. Here, we report on a wider investigation of the nature and temperature dependence of smectic ordering in concentrated solutions of various "gapped" DNA (GDNA) constructs. We examine symmetric GDNA constructs consisting of two 48-base pair duplex segments bridged by a single-stranded sequence of 2 to 20 thymine bases. Two distinct smectic layer structures are observed for DNA concentration in the range [Formula: see text] mg/mL. One exhibits an interlayer periodicity comparable with two-duplex lengths ("bilayer" structure), and the other has a period similar to a single-duplex length ("monolayer" structure). The bilayer structure is observed for gap length ≳10 bases and melts into the cholesteric phase at a temperature between 30 °C and 35 °C. The monolayer structure predominates for gap length ≲10 bases and persists to [Formula: see text]C. We discuss models for the two layer structures and mechanisms for their stability. We also report results for asymmetric gapped constructs and for constructs with terminal overhangs, which further support the model layer structures.

Lack of association between 4-base pair insertion/deletion (rs3783553) polymorphism within the 3′UTR of IL1A and breast cancer: A preliminary report

As a complex disease, various factors are implicated in breast cancer (BC) etiopathogenesis. Mounting evidence indicating that cytokines are one of the crucial elements of carcinogenesis. Interleukin (IL)-1a is a key regulatory cytokine involved in inflammation, immune system activities, and several human diseases, including BC. Eventually, polymorphisms of the IL1A gene can alter IL-1a expression and function. The present study explored the possible relationship between rs3783553as a functional polymorphism in the 3′ untranslated region of IL1A and BC risk. This case-control study was completed on 190 BC patients and 200 age-matched healthy female subjects. Genotyping for rs3783553 was done by mismatched polymerase chain reaction-restriction fragment length polymorphism. The results showed that rs3783553 was not associated with BC risk in any hereditary models (OR = 0.75, 95%CI = 0.50–1.13, p = 0.18, ins/del vs ins/ins and OR = 0.92, 95%CI = 0.29–2.95, p = 0.99, del/del vs ins/ins for codominant; OR = 0.77, 95%CI = 0.51–1.14, p = 0.22, ins/del + del/del vs ins/ins for dominant; OR = 1.05, 95%CI = 0.33–3.32, p = 0.99, del/del vs ins/ins + ins/del for recessive) as well as allele frequency (OR = 0.85, 95%CI = 0.62–1.16, p = 0.33, del vs ins). Our finding indicated the lack of association between rs3783553 and susceptibility to BC in a sample from southeast of Iran. Further studies in other ethnic groups with a larger study population should be done to elucidate the exact association of rs3783553 and BC susceptibility.

A CRISPR-Cas9-integrase complex generates precise DNA fragments for genome integration.

CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9′s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy

AbstractPoint mutations of D835/I836 of the FLT3 gene have been reported in adult acute myeloid leukemia (AML), but not in pediatric AML or acute lymphoblastic leukemia (ALL). FLT3-D835/I836 mutations were found in 6 (5.4%) of 112 children with ALL older than 1 year and in 8 (16.0%) of 50 infants with ALL. Missense mutations were found in 11 patients, 3-base pair deletions in 2 patients, and a deletion/insertion in 1 patient. Remarkably, FLT3-D835/I836 mutations were found in 8 (18.2%) of 44 infants with ALL with MLL rearrangements and in 4 (21.5%) of 19 patients with hyperdiploid ALL, but they were not found in any patients older than 1 year who had TEL-AML1 (n = 11), E2APBX1 (n = 4), or BCR-ABL (n = 6) fusion genes. Although infant ALL patients with mutations had poorer prognoses than did those without mutations, pediatric ALL patients with mutations who were older than 1 year had good prognoses. We also found FLT3-D835 mutations in 2 of 11 leukemic cell lines with MLL rearrangements. FLT3 was highly phosphorylated in these cell lines with FLT3-D835 mutations, leading to constitutive activation of downstream targets such as signal transducer and activator of transcription 5 (STAT5) without FLT3 ligand stimulation. These results suggested that FLT3-D835/I836 mutations are one of the second genetic events in infant ALL with MLL rearrangements or pediatric ALL with hyperdiploidy. (Blood. 2004;103:1085-1088)

Interaction of fumigaclavine C with High Mobility Group Box 1 protein (HMGB1) and its DNA complex: A computational approach

The fumigaclavines represent a small group of clavine-type alkaloids produced by the pathogenic fungus Aspergillus fumigatus. The leading compound in the family is fumigaclavine C (Fm-C) endowed with potent anti-inflammatory properties. Fm-C represses the production of several inflammatory cytokines in cells via a mechanism implicating a reduced nucleo-cytoplasmic transport and extracellular export of the alarmin protein HMGB1, through a direct drug-protein interaction, and a down-regulation of HMGB1 expression. We have investigated the interaction of Fm-C with HMGB1 using two complementary forms of the HMG-box protein, in its free and DNA-bound configurations, using molecular modeling. We identified up to six potential binding sites for Fm-C in the vicinity of the B-box of HMGB1, with the site designated Lys-103 being the most favored and maintained when the protein is bound to a 16-base pair DNA oligonucleotide. Structure-binding relationships have been explored through the comparison of the HMGB1-binding properties of fumigaclavines A, B and C, and the related alkaloid lysergic acid diethylamide (LSD). Both the C-9 acetyl group and C-2 dimethylallyl side chain of Fm-C contribute importantly to the protein interaction. LSD appears also to form stable complexes with free HMGB1. According to the calculated empirical energies of interaction (ΔE), the compounds rank in the order: Fm-C ∼ LSD < Fm-A < Fm-B, for binding to HMGB1. The study helps to better comprehend the mechanism of action of Fm-C, and its anti-inflammatory and anticancer properties.

Rational Design of Aptamer-Tagged tRNAs.

Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.

Analysis of clinicopathological and molecular features of crawling-type gastric adenocarcinoma

BackgroundCrawling-type adenocarcinoma (CRA) is an important gastric cancer (GC) subtype that exhibits a specific histological pattern and has characteristic clinicopathological findings. Despite its characteristic histology, little is known about the molecular characteristics of CRA.MethodsWe examined 177 GC cases, including 51 cases of CRA and 126 cases having conventional differentiated adenocarcinomas (CDAs). Results for immunohistochemistry (mucin phenotype; Muc5AC, Muc6, Muc2 and CD10, CDX-2, MLH-1, p53 and β-catenin), mutation analysis (TP53, KRAS and BRAF), microsatellite instability (BAT25, BAT26, D2S123, D5S346 and D17S250), DNA methylation status by a two-panel method (RUNX3, MINT31, LOX, NEUROG1, ELMO1 and THBD), MLH-1 promoter methylation, and allelic imbalance (AI; 1p, 3p, 4p, 5q, 8p, 9p, 13q, TP53, 18q and 22q) were examined.ResultsCRAs were more likely to occur in the middle third of the stomach, in younger patients and to be macroscopically depressed. Nuclear accumulation of β-catenin and loss of MLH-1 expression were less frequent among CRA cases compared to CDA cases. At a molecular level, CRA is often characterized by the deletion mutation c.529_546 (18-base pair deletion at codon 177–182 in exon 5) in the TP53 gene (10 cases). Although the low methylation epigenotype was significantly more frequent for CRAs compared to CDAs, multiple AIs were more often seen in CRAs relative to CDAs.ConclusionsThe results demonstrated that TP53 mutations, particularly c.529_546del, and multiple AIs are closely associated with CRA carcinogenesis. Our results suggest that CRA is an independent entity of GC in terms of clinicopathologic and molecular findings.

CRISPR Nickase-Mediated Base Editing in Yeast.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled efficient, markerless genome editing in a wide range of organisms. However, there is an off-target effect and a limit to the area of precise editing. Bases that can be precisely edited are limited to within the 20-base pair gRNA-targeting site and protospacer adjacent motif (PAM) sequence. We have developed a CRISPR nickase system that can perform a precise genome-wide base editing in Saccharomyces cerevisiae using a single Cas9 nickase. This system can precisely edit a broader genomic region by the avoidance of double-strand break (DSB) and subsequent non-homologous end joining (NHEJ). Furthermore, unintended mutations were not found at off-target sites in this system. In combination with yeast gap repair cloning, precise genome editing of yeast cells can be performed in 5days. Here, we describe the methods for precise and convenient genome editing using this novel CRISPR nickase system.

Genetic, clinical, molecular, and pathogenic aspects of the South Asian-specific polymorphic MYBPC3Δ25bp variant.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by ventricular enlargement, diastolic dysfunction, and increased risk for sudden cardiac death. Sarcomeric genetic defects are the predominant known cause of HCM. In particular, mutations in the myosin-binding protein C gene (MYBPC3) are associated with ~ 40% of all HCM cases in which a genetic basis has been established. A decade ago, our group reported a 25-base pair deletion in intron 32 of MYBPC3 (MYBPC3Δ25bp) that is uniquely prevalent in South Asians and is associated with autosomal dominant cardiomyopathy. Although our studies suggest that this deletion results in left ventricular dysfunction, cardiomyopathies, and heart failure, the precise mechanism by which this variant predisposes to heart disease remains unclear. Increasingly appreciated, however, is the contribution of secondary risk factors, additional mutations, and lifestyle choices in augmenting or modifying the HCM phenotype in MYBPC3Δ25bp carriers. Therefore, the goal of this review article is to summarize the current research dedicated to understanding the molecular pathophysiology of HCM in South Asians with the MYBPC3Δ25bp variant. An emphasis is to review the latest techniques currently applied to explore the MYBPC3Δ25bp pathogenesis and to provide a foundation for developing new diagnostic strategies and advances in therapeutics.