Screening Of Plaques Research Articles

Deep learning-based fully automatic screening of carotid artery plaques in computed tomography angiography: a multicenter study

To develop and validate a deep learning (DL) algorithm for the automated detection and classification of carotid artery plaques (CAPs) on computed tomography angiography (CTA) images. This retrospective study enrolled 400 patients (300 in the Center Ⅰ and 100 in Ⅱ). Three radiologists co-labeled CAPs, and their revised calcification status (noncalcified, mixed, and calcified) was regarded as ground truth. Center Ⅰ patients were randomly divided into training and internal validation datasets, while Center Ⅱ patients served as the external validation dataset. Carotid artery regions were segmented using a modified 3D-UNet network, followed by CAPs detection and classification using a ResUNet-based architecture in a two-step DL system. The DL model's detection and classification performance were evaluated on the validation dataset using precision-recall curve, free-response receiver operating characteristic (fROC) curve, Cohen's kappa, and ROC curve analysis. The DL model had achieved 83.4% sensitivity at 3.0 false positives (FPs)/CTA scan in internal validation and 78.9% in external validation. F1-scores were 0.764 and 0.769 at the optimal threshold, and area under fROC curves were 0.756 and 0.738, respectively, indicating good overall accuracy for CAP detection. The DL model also showed good performance for the ternary classification of CAPs, with Cohen's kappa achieved 0.728 and 0.703 in both validation datasets. This study demonstrated the feasibility of using a fully automated DL-based algorithm for the detection and ternary classification of CAPs, which could be helpful for the workloads of radiologists.

Emerging nanomaterials to enhance electrochemical impedance spectroscopy for biomedical applications

Over the last few decades, electrical impedance-based sensors have been investigated for clinical translation to detect changes in tissue conductivities, including cardiac output and pulmonary function. Recently, electrochemical impedance spectroscopy (EIS) provides metabolic measurements that occur at the electrode-tissue interface, and the 3-D EIS can be reconstructed to generate electrical impedance tomography (EIT) for detecting the impedimetric properties of the vascular wall or fatty liver disease. In both EIS and EIT applications, the electrochemical properties of the interface electrodes are essential to address the signal-to-noise ratio or sensitivity of measurements in the biological environment. To enhance the conductive properties, we will survey a series of carbon-based nanomaterials as the emerging candidates for coating the electrodes of bioimpedance sensors. In this review, we will provide a theoretical background on impedance-based measurements and highlight the current state of EIS and EIT, including their applications for cancer screening and detection of vulnerable atherosclerotic plaques. Next, we will focus on the strengths of different nanomaterials when used as an electrode coating to optimize charge transfer across the electric double layers and to enhance measurement sensitivity. We will also identify some unmet clinical needs, such as the ability to adapt to different hemodynamic conditions and blood vessel geometries, that can be realized by the novel biomaterials for the future EIS-based sensors.

An Application of Mycobacteriophage Genome Engineering using Bacteriophage Recombineering with Electroporated DNA (BRED) and CRISPR Cas‐9 Systems

The increasing occurrence of antibiotic‐resistant pathogens remains a global threat to public health, spurring research to develop alternative treatments for infectious bacteria. Phage therapy, the targeted use of bacteria‐specific viral particles called bacteriophages, has emerged as a promising antibiotic alternative. Our lab studies mycobacteriophages – phages specific to bacteria of the genus Mycobacterium,which includes infamous, antibiotic‐resistant pathogens such as M. tuberculosisand M. abscessus.For the use of many mycobacteriophages in phage therapy treatments, gene deletions are often necessary to ensure host lethality. Previous methods for mutant generation used a technique called bacteriophage recombination with electroporated DNA (BRED), which used electrocompetent host cells equipped with recombination machinery, genomic phage DNA, and a synthetic DNA g‐block to generate mutants. This process proved inefficient with recalcitrant recovery of some deletions, requiring extensive screening of plaques. To improve the process of genetic modifications with mycobacteriophages, greater precision and improved screening has recently been achieved through the combined use of BRED with a mycobacteria‐efficient Streptococcus thermophilus CRISPR Cas‐9 gene knockdown system for selection against the parent phage (CRISPY‐BRED). Here we present an application of the CRISPY‐BRED technique for the deletion of the immunity repressor gene in the mycobacteriophage known as Mufasa. The Cas‐9 system involves a plasmid containing the modified, nuclease‐dead Cas9 gene, a sgRNA fused to an anhydrotetracycline inducible promoter, a kanamycin resistance gene, an E. colireplication origin, and an attPsite for chromosomal integration in mycobacteria. The identification of a Protospacer Adjacent Motif (PAM) within the mycobacteriophage immunity repressor gene allowed for the design of a 20 bp sgRNA targeting sequence from the phage DNA. With host cell transformation of this CRISPR Cas9 plasmid programmed to target the parent phage immunity repressor and inhibit overall parent phage gene expression, straightforward mutant recovery should be achievable.

Lipase C, Hepatic Type -250A/G (rs2070895) Variant Enhances Carotid Atherosclerosis in Normolipidemic and Asymptomatic Individuals from Brazil.

The common genetic variant in the promoter region of the hepatic lipase gene [LIPC -250G/A(rs2070895)] has an ambiguous association with cardiovascular disease. In this context, our study was performed to identify the relationships between the rs2070895 with carotid atherosclerosis, plasma lipids, and parameters of reverse cholesterol transport. A total of 285 normolipidemic and asymptomatic participants from an initial sample of 598,288 individuals (inclusion criteria: LDL-C ≤130 mg/dL and triglycerides ≤150 mg/dL; age: 20-75 years, both genders; confirmation of clinical, anthropometric and laboratory data; attended all visits; DNA was achieved to perform genetic analysis) were enrolled and the rs2070895 variant was genotyped by TaqMan® OpenArray® Plataform. Carotid intima-media thickness and the screening of atherosclerotic plaques were determined by B-mode ultrasonography. The rs2070895 genotype frequencies were 0.44, 0.41, and 0.15 (GG, GA, and AA, respectively). Logistic regression analysis showed that the risk of having plaques was increased in participants carrying the AA or AG genotypes (OR = 3.90; 95% CI = 1.54-10.33), despite an increase in high-density lipoprotein cholesterol levels, HDL diameter and apolipoprotein A-I, as compared to the GG genotype. Hepatic lipase and endogenous lecithin cholesterol acyl transferase activities were reduced (38% and 19%, respectively) and lipoprotein lipase was increased by 30% (AA vs GG). Our results provide evidence that the AA or AG genotypes of the rs2070895 were associated with carotid atherosclerosis in apparently healthy participants, probably as a consequence of reduced reverse cholesterol transport and accumulation of HDL subfraction 2 rich in triglycerides and depleted in cholesteryl esters that could become dysfunctional.

HEART's Original [臨床研究] 臨床研究当院における多列検出器CTを用いた冠動脈CTによる冠動脈スクリーニングの現状

HEART's Original [臨床研究] 臨床研究当院における多列検出器CTを用いた冠動脈CTによる冠動脈スクリーニングの現状

Intravascular photoacoustic imaging using an IVUS imaging catheter

Catheter-based imaging of atherosclerosis with high resolution, albeit invasive, is extremely important for screening and characterization of vulnerable plaques. Currently, there is a need for an imaging technique capable of providing comprehensive morphological and functional information of plaques. In this paper, we present an intravascular photoacoustic imaging technique to characterize vulnerable plaques by using optical absorption contrast between normal tissue and atherosclerotic lesions. Specifically, we investigate the feasibility of obtaining intravascular photoacoustic (IVPA) images using a high-frequency intravascular ultrasound (IVUS) imaging catheter. Indeed, the combination of IVPA imaging with clinically available IVUS imaging may provide desired functional and morphological assessment of the plaque. The imaging studies were performed with tissue-mimicking arterial vessel phantoms and excised samples of rabbit artery. The results of our study suggest that catheter-based intravascular photoacoustic imaging is possible, and the combination of IVPA with IVUS has the potential to detect and differentiate atherosclerosis based on both the structure and composition of the plaque.

Combined Measurement of Carotid Stiffness and Intima-Media Thickness Improves Prediction of Complex Aortic Plaques in Patients With Ischemic Stroke

We hypothesized that for the prediction or exclusion of aortic thrombi or plaques >or=4 mm, the combination of intima-media thickness (IMT) and distensibility (DC) of the common carotid arteries would be superior to the measurement of IMT alone. We prospectively included 208 stroke patients (mean age, 60 years) undergoing transesophageal echocardiography for screening of aortic plaques. IMT and DC were determined by ultrasound, and DC was quantified by measuring blood pressure and the common carotid arteries diameter change on M-mode ultrasound during the cardiac cycle. Negative predictive values of IMT <0.9 mm and DC >or=24x10(-3)/kPa for the exclusion of aortic atheroma >or=4 mm were similar (92.0% and 91.7%, respectively). However, negative predictive values increased to 98.2% and to 100.0% for the exclusion of aortic thrombi when both parameters were combined. Positive predictive values of IMT >or=0.9 mm and DC <24 were lower (46.3%, 41.1%; respectively), but they also increased in combination (54.3%). Our findings suggest that IMT and DC represent different vessel wall properties and that measuring both parameters provides optimized characterization of carotid atherosclerosis. Combining IMT and DC increases the predictive power of carotid ultrasound, making transesophageal echocardiography dispensable for assessment of the aorta for those with normal carotid arteries and indispensable for those patients with carotid atherosclerosis.

Tu-P7:116 Genomic screening of unstable plaques: Vitamin D receptor as a new target for atherosclerosis

Tu-P7:116 Genomic screening of unstable plaques: Vitamin D receptor as a new target for atherosclerosis

Enhanced isolation of low frequency herpes simplex virus recombinants using green-fluorescent protein and FACS

The generation of recombinant herpes simplex virus to study the effect of engineered mutations on viral biology relies on the isolation of recombinants from a mixed population of viruses following a marker transfer procedure. Currently, the E. coli lacZ or green-fluorescent protein ( GFP) genes are most frequently used as markers for isolation and isolation of recombinants relies on visual screening of plaques. Alternatively, novel restriction site changes can be inserted into a gene followed by screening of individual plaques for the novel change. These methods are inefficient when the frequency of recombinants in the pool of viruses is low. Using GFP as a selection marker, a FACS procedure that results in a substantial enrichment of the frequency of recombinants is described. Cells were infected at a multiplicity of infection (MOI) of 1.0 in the presence of acyclovir and at 10 h post-infection, either the GFP+ or GFP− cells were sorted by FACS, and the sorted cells were plated on fresh cells. After three rounds of selection, the frequency of GFP+ recombinants rose from 0.1 to 3–4%. A mutant virus with a GFP insertion in the US1 gene (α22 protein) was generated and then used to isolate a virus with a mutation, Y116C, in the α22 protein.

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease-specific epitopes from phage-displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease-specific phagotopes can be identified out of this pool through an 'antigen independent' procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease-related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

X-Neu5Ac: A novel substrate for chromogenic assay of neuraminidase activity in bacterial expression systems

A chromogenic substrate 1, 5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-α- d-glycero- d-galacto-2-nonulopyranosidonic acid (X-Neu5Ac), has been synthesized to facilitate the screening of bacterial colonies or plaques for the detection of either natural or mutant neuraminidase activity. Substrate 1 was hydrolyzed by neuraminidase isolated from Clostridium perfringens to release a halogenated indol-3-ol 2 that undergoes rapid aerobic oxidation to form the dark blue pigment, 5,5′-dibromo-4,-4′-dichloroindigo 3. Preliminary kinetic studies indicate that this compound is a good substrate ( K m 0.89 × 10 −3 M) for neuraminidase and is quite stable under identical conditions in the absence of enzyme. These results suggest that X-Neu5Ac 1 can be useful to screen for bacterially-encoded enzyme production directly on agar plates.

Methods for Cloning Key Primary Metabolic Enzymes and Ancillary Proteins Associated with the Acetone‐Butanol Fermentation of Clostridium acetobutylicuma

The unavailability of genetically defined mutants for complementation has intensified the problems inherent in cloning genes from C. acetobutylicum. The uniqueness of some of the pathways of this organism coupled with the relative inefficiency of transformation of clostridia and few characterized mutants in these pathways have made cloning these genes by traditional complementation methods impractical. Oligonucleotide hybridization techniques have been shown to circumvent many problems involved in detecting protein expression. The ease of hybridization screening of plaques allows phage libraries to be examined more readily than is generally the case with colony screening techniques. Recombinant lambda phages also contain more DNA per insert than most plasmid vectors can maintain, thus further decreasing the amount of screening necessary. Cosmid libraries, offering even greater length of individual inserts, can be screened in a similar manner, although such screening incorporates the limitations of colony screening techniques. It is true that the technique hinges on the ability to obtain an amino acid sequence from which an oligonucleotide can be designed. In the past, the ability to obtain sequences was limited because the quantity and number of purified proteins were limited or the proteins were amino-terminally blocked. However, recent technological advances in this area, such as high-resolution gel separation techniques coupled with microsequencing, have opened the door to proteins previously inaccessible. Deformylation methods have been developed to deblock amino-terminally formylated proteins, and successful internal amino acid sequence analysis by in situ protease digestion has also been reported using only picomolar quantities of proteins separated by one- or two-dimensional gel electrophoresis. Protein and DNA sequence data banks have been significantly upgraded in the past few years. A proposed oligonucleotide sequence can be evaluated to determine what other possible sequences have similar homology; moreover, protein similarity comparisons between related species might possibly supplant the need for protein isolation if regions of highly conserved amino acid sequences are found. To our knowledge, this represents the first reported use of oligonucleotide probe hybridization screening technology as a strategy for cloning solvent pathway genes of C. acetobutylicum. Despite the deleterious effects on hybridization inherent in the high A + T content of C. acetobutylicum gene specific-directed oligonucleotides, the technique has been shown to function with few modifications to previously recorded systems.

The major 45-kDa protein of the yeast mitochondrial outer membrane is not essential for cell growth or mitochondrial function

As part of an analysis of the function and assembly of the mitochondrial outer membrane, we have cloned and characterized the yeast gene encoding a 45-kDa polypeptide (OM45) which is a major constituent of this membrane. The nuclear gene was isolated by immunological screening of plaques of recombinant phage lambda gt11 containing fragments of yeast genomic DNA using an antibody against OM45. Determination of the nucleotide sequence of the DNA fragment isolated by this approach revealed a single open reading frame of 1179 base pairs which encodes a protein having a predicted molecular mass of 44.6-kDa. Disruption of the OM45 gene in haploid yeast cells eliminated the expression of OM45. The mutant strain showed no apparent defect in cell viability, growth, mitochondrial function, or mitochondrial protein import.

Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.

We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.

Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.

We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.

Screening of M13 mp bacteriophage clones for small inserts by primer extension and insert excision

The M13 bacteriophage was adapted by Messing for DNA cloning by insertion of sequences containing a β galactosidase gene with internal restriction sites. Cloning into these sites results in loss of β galactosidase activity, allowing for initial screening of plaques for insert DNA but sometimes producing false positives. In our hands, previously described methods for the further screening of phage for short inserts have limitations that are overcome by the method described here. In our method, a commercially available primer is annealed to single-stranded phage DNA at a sequence that lies immediately 5′ to the cloning site within the β galactosidase gene. Klenow fragment and 4 deoxynucleotide triphosphates are then employed to extend the primer, forming double-stranded DNA, which is cleaved at restriction sites flanking the cloning site. Agarose gel electrophoresis with ethidium bromide staining is used to separate phage from insert DNA, thus demonstrating the presence of cloned insert.

Cloned genomic DNA sequences from Mycoplasma hyorhinis encoding antigens expressed in Escherichia coli.

A library of cloned Mycoplasma hyorhinis genomic sequences was constructed by incorporation of EcoRI digestion fragments of mycoplasma DNA into the lambda Charon 4A bacteriophage vector. Immunological screening of recombinant phage plaques identified clones containing genes encoding mycoplasma antigenic structures expressed in an Escherichia coli host. Two such recombinant phage isolates, lambda Ch4A-MhrG1 and lambda Ch4A-MhrG28, were defined and found to contain distinct genomic sequences by analysis of restriction endonuclease fragments. Inoculation of mice with recombinant gene products from lambda Ch4A-MhrG1 yielded antiserum selectively recognizing a Mr 29,500 trypsin-sensitive mycoplasma constituent. This established a means for producing selected immunogenic mycoplasma component in a bacterial host. The cloned genomic sequences of M. hyorhinis encoding expressed mycoplasma antigens represent molecular probes that can be characterized both by specific DNA sequences and by the antigenic structure of corresponding gene products. These genomic fragments define initial physical markers of the M. hyorhinis genome and may be useful in assessing antigenic and molecular genetic relationships within the genus Mycoplasma and among other members of the class Mollicutes.

Structure of genes for human growth hormone and chorionic somatomammotropin.

A 2.6-kilobase (kb) EcoRI restriction endonuclease fragment containing human growth hormone (hGH; somatotropin) gene sequences and a 2.8-kb EcoRI fragment containing human chorionic somatomammotropin (hCS; choriomammotropin) gene sequences have been identified by hybridization to cloned cDNA. Human DNA was cleaved with EcoRI and fractionated by preparative agarose gel electrophoresis; DNA in the size range 2--3 kb was ligated to lambda gt WES.lambda B DNA and viable recombinant bacteriophage were recovered by in vitro packaging. After infection of Escherichia coli and screening of phage plaques, single isolates of hGH and hCS gene sequences were obtained. Restriction endonuclease mapping showed that the hGH gene contains three intervening sequences interrupting the coding sequence. Partial DNA sequence analysis of the hGH gene, obtained by the chain termination method, confirmed the location of the intervening sequences and the identity of the fragment.

25] Colony hybridization

Publisher Summary This chapter discusses colony hybridization, which allows bacterial clones to be rapidly screened by ribonucleic acid–deoxyribonucleic acid (RNA–DNA) or DNA–DNA hybridization for those containing specific DNA sequences. Bacterial colonies are grown on nitrocellulose filters lying on agar petri plates. Reference sets of colonies are made by replica plating and stored at 2°–4°. The cells on the filter are lysed, and their DNA is denatured in situ without dislodging the remnants of the cells from their initial site. Radiolabeled RNA or DNA probe is then hybridized with the filters. After washing away unhybridized probe, autoradiography reveals the DNA that prints on the filter and the colonies in the reference set that contain the DNA sequence in question. It is now also possible to screen phage plaques in a similar manner, with the added advantages of lambda phage cloning. It is possible to combine the advantages of lambda and plasmid cloning by the partial lysis of bacteria lysogenic for a thermoinducible phage. This allows a large number of colonies to be screened and obviates the need for replica plating a reference set. The colony hybridization procedure has also been adapted for the screening of yeast transformants and animal cell SV40-induced plaques.