scholarly journals Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands

2021 ◽  
Vol 22 (23) ◽  
pp. 13159
Author(s):  
Hijiri Hasegawa ◽  
Ikkei Sasaki ◽  
Kaori Tsukakoshi ◽  
Yue Ma ◽  
Kazuo Nagasawa ◽  
...  
Keyword(s):  
Structural Changes ◽  
Cytosine Methylation ◽  
Disease Diagnosis ◽  
Microtiter Plate ◽  
Cpg Methylation ◽  
Microtiter Plate Assay ◽  
Significant Difference ◽  
G Quadruplex ◽  
Structural Differences ◽  
Symptomatic Diagnosis

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.

scholarly journals Comparison of biofilm formation and efflux pumps in ESBL and carbapenemase producing Klebsiella pneumoniae

2018 ◽  
Vol 12 (03) ◽  
pp. 156-163 ◽  
Author(s):  
Burak Yazgan ◽  
Ibrahim Türkel ◽  
Rıdvan Güçkan ◽  
Kılınç Kılınç ◽  
Tuba Yıldırım
Keyword(s):  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Cell Communication ◽  
Opportunistic Pathogen ◽  
Efflux Pumps ◽  
Microtiter Plate ◽  
Virulence Determinants ◽  
Clinical Environment ◽  
Microtiter Plate Assay ◽  
Significant Difference

Introduction: Klebsiella pneumoniae is an opportunistic pathogen that causes a range of diseases. The appearance of extended-spectrum β-lactamase -and carbapenemase-producing strains, in addition to the biofilm-forming phenotype, is a major problem in the clinical environment. Methodology: A total of 33 clinical K. pneumoniae isolates were used in this study. Antimicrobial susceptibilities were assessed by a disc diffusion assay. Biofilm formation was determined by a microtiter plate assay, staining with 1% crystal violet and measuring  absorbance after destaining. Moreover, expression of acrA, kdeA, ketM, kpnEF, and kexD efflux associated genes  was measured by qRT-PCR. Results: Isolates displayed high resistance to β-lactams such as cefazolin, cefuroxime, ceftriaxone, cefepime, piperacillin-tazobactam, imipenem, and meropenem and decreased resistance to gentamicin, amikacin, ciprofloxacin, and levofloxacin. ESBL-producing isolates formed more biofilm than carbapenemase-producing isolates. The mRNA expression levels in KPC isolates for acrA (2-fold), kdeA (2.7-fold), ketM (2.2-fold), and kpnEF (3.4-fold) were significantly increased compared to ESBL-producing isolates. There was no significant difference in kexD expression level. Conclusions: Under the conditions used here ESBL-producing isolates formed more biofilm than KPC postive isolates; this was associated with virulence determinants which were also transferred by plasmids together with ESBLs enzymes. Moreover, the upregulation of acrA, kdeA, ketM, and kpnEF efflux pumps was seen in carbapenemase-producing isolates demonstrating that high expression of efflux pumps alone could not confer resistance but may act as a physiological determinant such as bacterial pathogenicity and virulence, and cell-to-cell communication for bacteria.

Promotor Demethylation as a Mechanism of HOX11 Activation in Adult T-ALL?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2288-2288
Author(s):  
Ulrike Baak ◽  
Helmut Orawa ◽  
Nicola Goekbuget ◽  
Olaf Hopfer ◽  
Thomas Burmeister ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Cytosine Methylation ◽  
Methylation Status ◽  
Cpg Methylation ◽  
Methylation Array ◽  
Aberrant Expression ◽  
Methylation Specific Pcr ◽  
Specific Pcr ◽  
Significant Difference

Abstract Promotor demethylation of oncogenes has been associated with transcriptional activation in cancer cells. The proto-oncogene HOX11/TLX1 has been found to be aberrantly expressed in up to 30% of adult T-ALL patients. In few, a translocation between the HOX11 locus at 10q24 and the T-cell receptor locus has been identified. In the majority of cases the mechanism leading to HOX11 reactivation remains unclear. It had been proposed that an epigenetical modification by demethylation of the proximal HOX11 promotor could be responsible for an aberrant expression of HOX11. To test this hypothesis we have correlated the methylation status of CpG residues in the proximal HOX11 promotor with the gene expression status of HOX11 in adult T-ALL samples from the German Multicenter ALL Study (GMALL) 5/93 and 6/99. HOX11 expression was measured in 286 pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR as described previously. The methylation status was then randomly analyzed after bisulphite treatment by methylation-specific PCR (MSP) in 53 T-ALL samples with HOX11 expression (HOX11 positive) and 102 samples without HOX11 expression (HOX11 negative). Of the 150 analyzed patients only 4% of HOX11 positive patients (n=53) and 10% of HOX11 negative patients were methylated (M) in the analyzed promotor area. HOX11 negative patients were significantly more common associated with an unmethylated status (U) then HOX11 positive patients (57% vs 32%, p=0.003). The most prominent methylation phenotype in HOX11 positive patients compared to HOX11 negative samples was a mixed (MU) methylation status (55% vs. 27%, p=0.001). Interestingly, remission duration was significantly higher in pt. with the MU methylation status (M=49%, U=54% vs. MU=76%, p-logrank=0.0362). This translated also into a significant difference in the overall survival (M=55%, U=49%, MU=75%, p-logrank= 0.0202). However, in a multivariate analysis the methylation status could not be confirmed as an independent prognostic factor. The promotor-associated CpG methylation status was found to be remarkably heterogenous in the analyzed adult T-ALL patients with a predominantly unmethylated status in HOX11 negative samples and a mixed methylated/unmethylated picture in HOX11 positive samples. These findings contrast with our initial hypothesis that HOX11 expression is silenced in normal tissue by a promotor-associated CpG methylation and aberrantly reexpressed in leukemia cells by demethylation. However, various CpG residues associated with the HOX11 promotor might be of variable importance for the gene expression and intrinsic limitations of methylation-specific PCR might have influenced our analysis. Bisulphite sequencing techniques as well as the newly developed genome-wide cytosine methylation array could help overcome these issues. Understanding the role of promotor-associated methylation in HOX11 expression could clarify pathways in leukemogenesis and provide a valuable tool for better risk stratification in T-ALL.

An Immunoradiometric Assay for Factor VIII Related Antigen (VIIIRAg) Using Two Monoclonal Antibodies-Comparison with Polyclonal Rabbit Antibodies for Use in von Willebrand’s Disease Diagnosis

1984 ◽  
Vol 52 (03) ◽  
pp. 250-252 ◽  
Author(s):  
Y Sultan ◽  
Ph Avner ◽  
P Maisonneuve ◽  
D Arnaud ◽  
Ch Jeanneau
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Changes ◽  
Polyclonal Antibodies ◽  
Disease Diagnosis ◽  
Immunoradiometric Assay ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Structural Abnormalities ◽  
Antigenic Material ◽  
Von Willebrand

SummaryTwo monoclonal antibodies raised against FVIII/von Willebrand protein were used in an immunoradiometric assay (IRMA) to measure this antigen in normal plasma and plasma of patients with different forms of von Willebrand’s disease. The first antibody, an IgG1 was used to coat polystyrene tubes, the second one, an IgG2a, iodinated and used in the second step. Both antibodies inhibit ristocetin induced platelet agglutination and react strongly with platelets, megacaryocytes and endothelial cells. The IRMA test using these antibodies showed greater sensitivity than that using rabbit polyclonal anti VIIIRAg antibodies. A good correlation between the two tests was nevertheless found when VIIIRAg was measured in the majority of patient’s plasma. However 5 patients from 3 different families showed more antigenic material in the rabbit antibody IRMA than in the monoclonal antibody IRMA. It is suggested therefore that the monoclonal antibodies identify part of the VIIIR:Ag molecule showing structural abnormalities in these vWd patients, these structural changes remaining undetected by the polyclonal antibodies.

scholarly journals Genetic Deletion of Interleukin-15 Is Not Associated with Major Structural Changes Following Experimental Post-Traumatic Knee Osteoarthritis in Rats

2021 ◽  
Vol 11 (15) ◽  
pp. 7118
Author(s):  
Ermina Hadzic ◽  
Garth Blackler ◽  
Holly Dupuis ◽  
Stephen James Renaud ◽  
Christopher Thomas Appleton ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Subchondral Bone ◽  
Structural Changes ◽  
Cartilage Damage ◽  
Wild Type ◽  
Significant Difference ◽  
Post Traumatic ◽  
Interleukin 15 ◽  
Joint Trauma ◽  
Surgically Induced

Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease, leading to articular cartilage breakdown, osteophyte formation, and synovitis, caused by an initial joint trauma. Pro-inflammatory cytokines increase catabolic activity and may perpetuate inflammation following joint trauma. Interleukin-15 (IL-15), a pro-inflammatory cytokine, is increased in OA patients, although its roles in PTOA pathophysiology are not well characterized. Here, we utilized Il15 deficient rats to examine the role of IL-15 in PTOA pathogenesis in an injury-induced model. OA was surgically induced in Il15 deficient Holtzman Sprague-Dawley rats and control wild-type rats to compare PTOA progression. Semi-quantitative scoring of the articular cartilage, subchondral bone, osteophyte size, and synovium was performed by two blinded observers. There was no significant difference between Il15 deficient rats and wild-type rats following PTOA-induction across articular cartilage damage, subchondral bone damage, and osteophyte scoring. Similarly, synovitis scoring across six parameters found no significant difference between genetic variants. Overall, IL-15 does not appear to play a key role in the development of structural changes in this surgically-induced rat model of PTOA.

scholarly journals Cortical structural differences in major depressive disorder correlate with cell type-specific transcriptional signatures

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jiao Li ◽  
Jakob Seidlitz ◽  
John Suckling ◽  
Feiyang Fan ◽  
Gong-Jun Ji ◽  
...  
Keyword(s):  
Gene Expression ◽  
Major Depressive Disorder ◽  
Depressive Disorder ◽  
Structural Changes ◽  
Brain Regions ◽  
Cell Type ◽  
Major Depressive ◽  
Structural Differences ◽  
Neuroimaging Data ◽  
Cell Type Specific

AbstractMajor depressive disorder (MDD) has been shown to be associated with structural abnormalities in a variety of spatially diverse brain regions. However, the correlation between brain structural changes in MDD and gene expression is unclear. Here, we examine the link between brain-wide gene expression and morphometric changes in individuals with MDD, using neuroimaging data from two independent cohorts and a publicly available transcriptomic dataset. Morphometric similarity network (MSN) analysis shows replicable cortical structural differences in individuals with MDD compared to control subjects. Using human brain gene expression data, we observe that the expression of MDD-associated genes spatially correlates with MSN differences. Analysis of cell type-specific signature genes suggests that microglia and neuronal specific transcriptional changes account for most of the observed correlation with MDD-specific MSN differences. Collectively, our findings link molecular and structural changes relevant for MDD.

INSTANCE MIGRATION BETWEEN ONTOLOGIES HAVING STRUCTURAL DIFFERENCES

2011 ◽  
Vol 20 (06) ◽  
pp. 1127-1156 ◽  
Author(s):  
MAXIM DAVIDOVSKY ◽  
VADIM ERMOLAYEV ◽  
VYACHESLAV TOLOK
Keyword(s):  
Knowledge Management ◽  
Life Cycle ◽  
Structural Changes ◽  
Software Framework ◽  
Ontology Alignment ◽  
Transformation Rules ◽  
Structural Differences ◽  
Prototype Software ◽  
Descriptive Theories ◽  
Future Work

Ontology instance migration is one of the complex and not fully solved problems in knowledge management. A solution is required when the ontology schema evolves in the life cycle and the assertions have to be transferred to the newer version. The problem may become more complex in distributed settings when, for example, several autonomous software entities use and exchange partial assertional knowledge in a domain that is formalized by different though semantically overlapping descriptive theories. Such an exchange is essentially the migration of the assertional part of an ontology to other ontologies belonging to or used by different entities. The paper presents our method and tool for migrating instances between the ontologies that have structurally different but semantically overlapping schemas. The approach is based on the use of the manually coded transformation rules describing the changes between the input and the output ontologies. The tool is implemented as a plug-in for the ProjectNavigator prototype software framework. The article also reports the results of our three evaluation experiments. In these experiments we evaluated the degree of complexity in the structural changes to which our approach remains valid. We also chose the ontology sets in one of the experiments to make the results comparable with the ontology alignment software. Finally we checked how well our approach scales with the increase of the quantity of the migrated ontology instances to the numbers that are characteristic to industrial ontologies. In our opinion the evaluation results are satisfactory and suggest some directions for the future work.

scholarly journals Drug Targeting Mycobacterium tuberculosis Cell Wall Synthesis: Development of a Microtiter Plate-Based Screen for UDP-Galactopyranose Mutase and Identification of an Inhibitor from a Uridine-Based Library

2003 ◽  
Vol 47 (1) ◽  
pp. 378-382 ◽  
Author(s):  
Michael S. Scherman ◽  
Katharine A. Winans ◽  
Richard J. Stern ◽  
Victoria Jones ◽  
Carolyn R. Bertozzi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targeting ◽  
Enzyme Inhibitor ◽  
Microtiter Plate ◽  
Biosynthetic Enzyme ◽  
Cell Wall Synthesis ◽  
Chemical Library ◽  
Microtiter Plate Assay ◽  
Plate Assay

ABSTRACT A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.

scholarly journals Effect of S-PRG Eluate on Biofilm Formation and Enzyme Activity of Oral Bacteria

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Masahiro Yoneda ◽  
Nao Suzuki ◽  
Yosuke Masuo ◽  
Akie Fujimoto ◽  
Kosaku Iha ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Composite Resin ◽  
Fusobacterium Nucleatum ◽  
Microtiter Plate ◽  
Oral Bacteria ◽  
Gelatin Film ◽  
Glass Ionomer ◽  
Microtiter Plate Assay ◽  
Substrate Hydrolysis ◽  
Adherence Ability

Recently, the antibacterial activity of a composite resin containing prereacted glass ionomer (S-PRG) filler was revealed. We examined the effect of an S-PRG eluate on various biologic activities ofStreptococcus mutansandPorphyromonas gingivalis. Adherence ability ofS. mutanswas evaluated by microtiter plate assay; protease and gelatinase activities ofP. gingivaliswere examined by synthetic substrate hydrolysis and gelatin film spot assay, respectively. Coaggregation ofP. gingivaliswithFusobacterium nucleatumwas also examined. S-PRG eluate was found to suppress streptococcal adherence. S-PRG eluate inhibited the protease and gelatinase activities ofP. gingivalisand the coaggregation betweenP. gingivalisandF. nucleatum. These results indicate that S-PRG eluate suppresses streptococcal adherence and inhibits the protease and coaggregation activities ofP. gingivalis. These findings may prompt research into novel strategies for preventing caries and periodontitis.

Evaluation of the Role of Staphylococci in the Pathomechanism of Conjunctivitis

2021 ◽  
Author(s):  
Ewa Jasińska ◽  
Agnieszka Bogut ◽  
Agnieszka Magryś ◽  
Alina Olender
Keyword(s):  
Antibiotic Resistance ◽  
Epithelial Cells ◽  
Biofilm Formation ◽  
Methicillin Resistance ◽  
Microtiter Plate ◽  
Host Cells ◽  
Diffusion Method ◽  
Microtiter Plate Assay ◽  
Low Prevalence

Abstract Purpose: Determination of the association between ica genes and phenotypic biofilm formation in staphylococcal isolates involved in conjunctivitis, their antibiotic resistance as well as detection of selected virulence characteristics: adhesion to epithelial cells and in vitro cytotoxicity.Methods: The study included 26 Staphylococcus aureus (SA) and 26 Staphylococcus epidermidis (SE) isolates. The presence of icaAD genes and ica operon was determined by the PCR assay. Phenotypic biofilm formation was verified using the microtiter plate assay. Antibiotic resistance was performed using the disc diffusion method. Staphylococcal ability to attach to host cells was assessed by flow cytometry. Cytotoxicity on epithelial cells was evaluated by LDH assay.Results: The ica genes were detected in 26.9% of SE and in 42.3% of SA isolates. Only 15.3% of isolates (SE) were positive for both the icaAD and the ica operon. Phenotypically, 19.2% of SE isolates were strong biofilm producers, among which three were both icaAD- and ica operon-positive. 26.9% of SA isolates were strong biofilm producers. Methicillin resistance (MR) was detected in 34.6% of SE and 26.9% of SA isolates. 75% of MR isolates were multidrug resistant. SA isolates adhered to host cells more extensively than SE. SA isolates released higher level of LDH than SE.Conclusions: Adherence abilities were commonly observed in staphylococci associated with conjunctivitis. However, low prevalence of isolates positive for a complete and functional ica locus and low prevalence of strong biofilm producers was detected. SA adhered to a greater extent to eukaryotic cells than SE and were more cytotoxic.

A Cross-Sectional Study to Compare Differences on Clinical and Laboratory Findings in Children with Seropositive and Seronegative Lupus

2021 ◽  
Author(s):  
Shima Salehi ◽  
Rozita Hosseini Shamsabadi ◽  
Hassan Otukesh ◽  
Reza Shiari ◽  
Monir Sharafi
Keyword(s):  
Autoimmune Disease ◽  
Disease Diagnosis ◽  
Cross Sectional Study ◽  
Rapid Identification ◽  
The Body ◽  
Musculoskeletal Symptoms ◽  
Laboratory Findings ◽  
Cross Sectional ◽  
Significant Difference ◽  
History Of

Abstract Background: Lupus is an inflammatory and autoimmune disease that involves various tissues and organs of the body. Identification of diagnostic elements to rapid identification of seronegative lupus cases is very important in order to prevent morbidity and progression of disease. This study aimed to compare clinical and laboratory findings of seropositive cases with seronegative lupus patients. Methods: This cross-sectional analytic study was performed on 43 children (17 seronegative and 26 seropositive) with lupus who were admitted to Ali Asghar Hospital during 2007-2017. Seropositive patients had anti-nuclear antibody (ANA) titration >1/80, while seronegative patients had ANA titration <1/80 (at the time of disease diagnosis). Clinical and laboratory findings were compared between two groups.Results: Serositis in patients with ANA- was significantly higher than ANA+ (41.17% vs. 23.07%; p = 0.042). ANA- group had higher autoimmune disease history than ANA+ group (42.85% vs. 15.0%; p = 0.041). The family history of the disease in the ANA- group was greater than ANA+ group (50% vs. 23.52%). The percentage of hypertensive patients in ANA- group was higher than ANA+ group (52.94% vs. 26.92%; p = 0.037). Neurologic symptoms in ANA+ and ANA- groups were 38.46% and 17.64%, respectively (p = 0.043). The frequency of patients with thrombocytopenia in ANA+ group was significantly greater than ANA- group (32% vs. 12.5%; p=0.041). There was no significant difference in other clinical and laboratory findings between two groups. Conclusion: Seronegative lupus patients had higher percentage of musculoskeletal symptoms, autoimmune disease history, familial history of disease, and hypertension, while neurological and thrombocytopenia symptoms were higher in seropositive patients compared to seronegative cases. Therefore, evaluation of these factors can be helpful to diagnosis of seronegative patients.

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