Detection of Functional Significance of Coronary Stenoses Using Dynamic 13N-Ammonia Stress-PET/CT with Absolute Values of Myocardial Blood Flow and Coronary Flow Reserve

Objectives. The aim of the study was to compare the values of myocardial blood flow (MBF) at stress, MBF at rest and coronary flow reserve (CFR) obtained by 13Nammonia stress-PET/CT in patients with various degrees of coronary stenosis and in healthy patients. And thus to estimate the possible contribution of the stress-PET/CT quantitative data to the detection of functionally significant coronary stenoses in patients with coronary artery disease (CAD). Materials and methods. 63 patients (mean age 64±9 years) with known CAD underwent dynamic 13N-ammonia stress-PET/CT followed by calculation of MBF both at stress and at rest in absolute units and CFR. We compared quantitative values in two groups of patients with coronary artery stenosis: 1) ≥75% (n = 36) and 2) <75% (n = 27) confirmed by invasive coronary angiography and in group of healthy patients (n = 11). Results. MBF at stress was significantly lower in group with ≥75% diameter stenoses (median 1,44 [1,21; 1,85] mL/min per g) compared with group with <75% diameter stenoses (2,42 [1,75; 2,89] mL/min/g) and the normal group (2,54 [2,31; 2,86] mL/min/g), (p <0,001). There was no reliable difference in MBF at rest between the three groups (p = NS). CFR was significantly lower in the group of patients with severe ≥75% stenoses (1,85 [1,54; 2,31]) in comparison with patients group with stenoses of intermediate <75% severity (2,73 [2,19; 3,21]), and also in comparison with the normal group (3,12 [2,75; 3,23]), (p <0,001). Conclusion. The values of MBF at stress and CFR are significantly lower in patients with severe coronary arteries stenoses comparing with the group of patients with mild and moderate stenoses. The value of MBF at rest used independently has no diagnostic utility for detection of functional significance of coronary artery stenoses. Keywords: myocardial blood flow, coronary flow reserve, PET/CT, 13N-ammonia, coronary stenosis

11C-Choline Pet/Ct in the Detection of Prostate Cancer Relapse in Patients After Radical Treatment With Psa Level < 10 Ng/Ml

Purpose: To evaluate the usefulness of 11C-Choline PET/CT in the detection of recurrent prostate cancer (PCa) in patients with biochemical relapse after radical treatment. Materials and methods: This retrospective study included 217 PCa patients who underwent 11C-Choline PET/CT in the Department of Nuclear Medicine of Bakoulev Scientific Centre. All patients had biochemical relapse 3±2 years after radical treatment for locally advanced PCa (T1–3 N0–1 M0): radical prostatectomy (n = 159) and radiation therapy (n = 58). The mean PSA value in the group was 2.1±2.5 (0.2–9.7) ng/ml, median – 1.9 ng/ml. Imaging was performed on PET/CT scanner (Biograph-64, Siemens) 10 min after injection of 11C-Choline (400–550 Mbq). Results: Overall, according to 11C-Choline PET/CT results PCa relapse was detected in 56% (121/217) of cases: in 50% (80/159) after radical prostatectomy and in 71% (41/58) after radiation therapy. The mean PSA value in PET-positive cases was 3.1±2.2 (0.2–9.7) ng/ml, while in PETnegative cases – 1.8±1.7 (0.2–4.6) ng/ml. The majority – 68% (65/96) patients with PET-negative scan had low PSA levels (&lt; 2 ng/ml). PET/CT results were positive in 43% (50/115) patients with PSA of &lt; 2 ng/ml, in 63% (45/72) with PSA of 2 to 5 ng/ml, and in 87% (26/30) with PSA of &gt; 5 ng/ml. Local relapse was detected in 51% (62/121) patients, distant metastases – in 28% (34/121) of cases, both local and distant metastases – in 21% (25/121) of cases. Lymph node metastases were detected in 38% (86/217) of all patients included in the analysis, of which 28% (24/86) had lesions in lymph node of normal size (median 7 mm). Of all PET-positive patients bone metastases were detected in 33% (40/121), of which 60% (24/40) had isolated skeletal involvement. Importantly, that 27% (11/40) of PETpositive patients with bone metastases had no structural abnormalities on CT images (CT-negative cases), corresponding to isolated involvement of bone marrow. And half of these CT-negative patients (5/11) had single lesions. The mean PSA value in patients with bone metastases was 5.0±3.7 (0.4–9.1) ng/ml, median – 3.8 ng/ml. According to 11C-Choline PET/CT results oligometastatic PCa recurrence was revealed in 38% (82/217) of all patients, of which 62% (51/82) had local relapse only. Distant oligometastatic lesions were detected in 38% (31/82), of which 13% (4/31) were presented by normal-size lymph nodes and 19% (6/31) – by early bone marrow metastases. 48% (58/121) of PET-positive results were confirmed by data of repeated PET/CT examinations. Conclusion: 11C-Choline PET/CT has been shown to be a single noninvasive accurate technique for detection of recurrent PCa in patients with rising PSA after radical treatment, which allows to differentiate patients with local and distant metastases in one study, as well as identify oligometastatic process, and therefore was useful in determining the further personalized therapeutic approach. Keywords: prostate cancer, PET/CT, 11C-Choline, biochemical recurrence, PSA

In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology

Lung disease caused by ABCA3 mutations

Background Knowledge about the clinical spectrum of lung disease caused by variations in the ATP binding cassette subfamily A member 3 (ABCA3) gene is limited. Here we describe genotype-phenotype correlations in a European cohort. Methods We retrospectively analysed baseline and outcome characteristics of 40 patients with two disease-causing ABCA3 mutations collected between 2001 and 2015. Results Of 22 homozygous (15 male) and 18 compound heterozygous patients (3 male), 37 presented with neonatal respiratory distress syndrome as term babies. At follow-up, two major phenotypes are documented: patients with (1) early lethal mutations subdivided into (1a) dying within the first 6 months or (1b) before the age of 5 years, and (2) patients with prolonged survival into childhood, adolescence or adulthood. Patients with null/null mutations predicting complete ABCA3 deficiency died within the 1st weeks to months of life, while those with null/other or other/other mutations had a more variable presentation and outcome. Treatment with exogenous surfactant, systemic steroids, hydroxychloroquine and whole lung lavages had apparent but many times transient effects in individual subjects. Conclusions Overall long-term (>5 years) survival of subjects with two disease-causing ABCA3 mutations was <20%. Response to therapies needs to be ascertained in randomised controlled trials

Processing DNA molecules as text

Polymerase Chain Reaction (PCR) is the DNA-equivalent of Gutenberg’s movable type printing, both allowing large-scale replication of a piece of text. De novo DNA synthesis is the DNA-equivalent of mechanical typesetting, both ease the setting of text for replication. What is the DNA-equivalent of the word processor? Biology labs engage daily in DNA processing—the creation of variations and combinations of existing DNA—using a plethora of manual labor-intensive methods such as site-directed mutagenesis, error-prone PCR, assembly PCR, overlap extension PCR, cleavage and ligation, homologous recombination, and others. So far no universal method for DNA processing has been proposed and, consequently, no engineering discipline that could eliminate this manual labor has emerged. Here we present a novel operation on DNA molecules, called Y, which joins two DNA fragments into one, and show that it provides a foundation for DNA processing as it can implement all basic text processing operations on DNA molecules including insert, delete, replace, cut and paste and copy and paste. In addition, complicated DNA processing tasks such as the creation of libraries of DNA variants, chimeras and extensions can be accomplished with DNA processing plans consisting of multiple Y operations, which can be executed automatically under computer control. The resulting DNA processing system, which incorporates our earlier work on recursive DNA composition and error correction, is the first demonstration of a unified approach to DNA synthesis, editing, and library construction

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors

Exponential Megapriming PCR (EMP) Cloning-Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.National Institutes of Health (U.S.) (Grant GM077537

Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications