Small molecule-based detection of non-canonical RNA G-quadruplex structures that modulate protein translation.

Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.

Metal Ion-Directed Specific DNA Structures and Their Functions.

Various DNA structures, including specific metal ion complexes, have been designed based on the knowledge of canonical base pairing as well as general coordination chemistry. The role of metal ions in these studies is quite broad and diverse. Metal ions can be targets themselves in analytical applications, essential building blocks of certain DNA structures that one wishes to construct, or they can be responsible for signal generation, such as luminescence or redox. Using DNA conjugates with metal chelators, one can more freely design DNA complexes with diverse structures and functions by following the simple HSAB rule. In this short review, the authors summarize a part of their DNA chemistries involving specific metal ion coordination. It consists of three topics: (1) significant stabilization of DNA triple helix by silver ion; (2) metal ion-directed dynamic sequence edition through global conformational change by intramolecular complexation; and (3) reconstruction of luminescent lanthanide complexes on DNA and their analytical applications.

Cas9-guided haplotyping of three truncation variants in autosomal recessive disease.

An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5' and 3' variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5' and 3' variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.

RNA-based cooperative protein labeling that permits direct monitoring of the intracellular concentration change of an endogenous protein.

Imaging the dynamics of proteins in living cells is a powerful means for understanding cellular functions at a deeper level. Here, we report a versatile method for spatiotemporal imaging of specific endogenous proteins in living mammalian cells. The method employs a bifunctional aptamer capable of selective protein recognition and fluorescent probe-binding, which is induced only when the aptamer specifically binds to its target protein. An aptamer for ß-actin protein preferentially recognizes its monomer forms over filamentous forms, resulting in selective G-actin staining in both fixed and living cells. Through actin-drug treatment, the method permitted direct monitoring of the intracellular concentration change of endogenous G-actin. This protein-labeling method, which is highly selective and non-covalent, provides rich insights into the study of spatiotemporal protein dynamics in living cells.

Association between pulmonary oxygen uptake on-kinetics and acute cardiovascular responses to resistance exercise in patients with coronary artery disease: a preliminary study.

[Purpose] This study aimed to examine whether pulmonary oxygen uptake on-kinetics at the onset of moderate-intensity exercise can predict acute cardiovascular responses to resistance exercise. [Participants and Methods] The association between pulmonary oxygen uptake on-kinetics and acute cardiovascular responses to a single resistance exercise session was investigated in seven patients with low-risk coronary artery disease who underwent revascularization through percutaneous coronary intervention. The participants performed a cardiopulmonary exercise test on a cycle ergometer and a single resistance exercise session at 30% of maximum voluntary contraction on a bilateral leg-extension machine 1 week after surgery. We measured the ventilatory anaerobic threshold and pulmonary oxygen uptake on-kinetics during the cardiopulmonary exercise test; left ventricular ejection fraction at rest; and heart rate, systolic blood pressure, and rate pressure product during the single resistance exercise session. [Results] Pulmonary oxygen uptake on-kinetics showed a positive association with the amount of increase in systolic blood pressure and rate pressure product during the single resistance exercise session, but had no association with the amount of increase in heart rate. Ventilatory anaerobic threshold and left ventricular ejection fraction were not associated with these parameters. [Conclusion] These data suggested that pulmonary oxygen uptake on-kinetics can be a useful evaluation index for predicting acute systolic blood pressure and rate pressure product responses to low-intensity resistance exercise 1 week after percutaneous coronary intervention in patients with low-risk coronary artery disease.

Associations between High-Density Lipoprotein Functionality and Major Adverse Cardiovascular Events in Patients Who Have Undergone Coronary Computed Tomography Angiography.

The present study aimed to investigate the associations between high-density lipoprotein (HDL) functionality and major adverse cardiovascular events (MACE) in patients who have undergone coronary computed tomography angiography (CCTA). We performed a prospective cohort study and enrolled 151 patients who underwent CCTA and had a follow-up of up to 5 years. We measured cholesterol efflux capacity (CEC), caspase-3/7 activity and monocyte chemoattractant protein-1 (MCP-1) secretion as bioassays of HDL functionality. The patients were divided into MACE(-) (n = 138) and MACE(+) (n = 13) groups. While there was no significant difference in %CEC, caspase-3/7 activity or MCP-1 secretion between the MACE(-) and MACE(+) groups, total CEC and HDL cholesterol (HDL-C) in the MACE(+) group were significantly lower than those in the MACE(-) group. Total CEC was correlated with HDL-C. A receiver-operating characteristic curve analysis showed that there was no significant difference between the areas under the curves for total CEC and HDL-C. In conclusion, total CEC in addition to HDL-C, but not %CEC, was associated with the presence of MACE. On the other hand, HDL functionality with regard to anti-inflammatory and anti-apoptosis effects was not associated with MACE.

The Effects of Long-Term Nutrition Counseling According to the Behavioral Modification Stages in Patients with Cardiovascular Disease.

BACKGROUND: the behavioral modification stages (BMS) are widely used; however, there are no reports on long-term nutrition counseling for cardiovascular disease (CVD) according to BMS. AIM: to study the effects of long-term nutrition counseling based on the BMS in patients with CVD. METHODS: fifteen patients with CVD who participated in nutrition counseling were enrolled between June 2012 and December 2016. We provided BMS and dietary questionnaires to estimate the stage score (SS), salt intake, and drinking habits (non-drinking group (n = 7)/drinking group (n = 8)), and measured the blood pressure (BP), body mass index (BMI), and biochemical markers before and after hospitalization at 6 months, 1 year, and 1.5 years after leaving the outpatient department (OPD). RESULTS: a significant decreased salt intake and increase in SS were found at 1.5 years. It significantly decreased the BP and salt intake in the non-drinking group at 1.5 years. CONCLUSIONS: long-term nutrition counseling according to BMS improved salt intake and BP in the non-drinking group. However, in the drinking group, increased salt intake might weaken the BP improvement. Temperance and low-sodium intake are essential factors that control BP, especially in drinkers.

Association Between the Level of Low-Density Lipoprotein Cholesterol and Coronary Atherosclerosis in Patients Who Have Undergone Coronary Computed Tomography Angiography.

BACKGROUND: Although the Japan Atherosclerosis Society Guidelines 2017 recommend lower levels of low-density lipoprotein cholesterol (LDL-C, < 70 mg/dL or ≤ 100 mg/dL) to prevent secondary cardiovascular events, we cannot conclude that a low level of LDL-C prevents primary cardiovascular events in patients with suspected coronary artery disease (CAD). METHODS: We registered 1,016 patients who were clinically suspected to have CAD and who underwent coronary computed tomography angiography (CCTA) for screening of coronary atherosclerosis. We excluded 350 patients who were receiving anti-lipidemic therapies and finally analyzed 666 patients. The patients were divided into three groups according to the LDL-C level: < 70 mg/dL (n = 25, Low LDL-C), 70 - 99 mg/dL (n = 141, Middle LDL-C), and ≥ 100 mg/dL (n = 500, High LDL-C). A ≥ 50% coronary stenosis was initially diagnosed as CAD, and the number of significantly stenosed coronary vessels (VD), Gensini score and coronary artery calcification (CAC) score were quantified. RESULTS: There were no significant differences in age, high-density lipoprotein cholesterol, rates of hypertension, hemoglobin A1c, blood sugar or systolic blood pressure among the Low, Middle and High LDL-C groups. On the other hand, there were significant differences in rates of males, smoking, dyslipidemia and diabetes, diastolic blood pressure and triglyceride among the groups. The prevalence of CAD values in the Low, Middle and High LDL-C groups were similar, at 52%, 47%, and 46%, respectively. In addition, there were no significant differences in the number of VD, Gensini score or CAC score among the Low LDL-C, Middle LDL-C and High LDL-C groups. CONCLUSIONS: We showed that the level of LDL-C was not associated with the presence or severity of CAD, which indicates that we need to screen by CCTA to prevent primary coronary events even if patients without anti-lipidemic therapies show low levels of LDL-C.

Catalytic Amplification of Electrochemical Signal in Homogeneous Solution Using an Entropy-driven DNA Circuit.

The electrochemical signal from ferrocene on a DNA probe was successfully modulated in a homogeneous solution by the template-directed formation and dissociation of an inclusion complex with ß-cyclodextrin on another probe. The electrochemical response was amplified by combining with a DNA circuit, in which the target DNA served as a catalyst. This system did not require any modification of a complementary DNA with the ferrocene-modified probe on the electrode surface to separate the bound/free probe for the detection of 200 nM target DNA.

Associations Between High Levels of High-Density Lipoprotein Cholesterol and the Presence and Severity of Coronary Artery Disease in Patients Who Have Undergone Coronary Computed Tomography Angiography.

BACKGROUND: Although a recent study in a Japanese cohort indicated that extremely high-density lipoprotein cholesterol (HDL-C, ≥ 90 mg/dL) had an adverse effect on atherosclerotic cardiovascular disease mortality, we could not conclude that high levels of HDL-C were associated with the presence or severity of coronary artery disease (CAD). METHODS: We enrolled 1,016 patients who were clinically suspected to have CAD and who underwent coronary computed tomography angiography (CCTA). The number of significantly stenosed coronary vessels (vessel disease (VD), ≥ 50% coronary stenosis is diagnosed as CAD) and the Gensini score were quantified using CCTA, and the lipid profile was measured. The patients were divided into four groups according to the HDL-C level: < 40 mg/dL (n = 115, low), 40 - 59 mg/dL (n = 530, normal), 60 - 89 mg/dL (n = 335, high) and ≥ 90 mg/dL (n = 36, very-high). RESULTS: The percentage (%) of CAD in the low, normal, high and very-high groups was 69%, 55%, 42% and 25%, respectively (P for trend < 0.01). The Gensini score in the low, normal, high and very-high groups was 20 ± 25, 12 ± 16, 8 ± 12 and 4 ± 6, respectively (P for trend < 0.01). The very-high group showed the lowest triglyceride (TG) levels among the four groups. There were no significant differences in the level of low-density lipoprotein cholesterol or % use of statin among the four groups. Finally, the presence of CAD was independently associated with a low level of HDL-C, in addition to age, male, high systolic blood pressure and hemoglobin A1c, but not TG, by a multivariate logistic regression analysis. CONCLUSIONS: High levels of HDL-C at the time of CCTA for screening were associated with a reduced presence and severity of CAD.

A Strict Target for Low-Density Lipoprotein Cholesterol May not Be Necessary for Secondary Prevention of Cardiovascular Disease in All Elderly Patients With Dyslipidemia.

According to the Japan Atherosclerosis Society Guidelines for the Prevention of Atherosclerotic Cardiovascular Diseases 2017, standard statin therapy for hyper-low-density lipoprotein cholesterol cholesterolemia in elderly patients may be effective for the secondary prevention of coronary artery disease, as in non-elderly adults. On the other hand, high-intensity statin therapy may not be recommended in all elderly cardiovascular disease patients with dyslipidemia, and particularly in elderly patients aged ≥ 85 years. In any case, tailor-made medical care with use of statin is required that matches the background of each patient.

G-quadruplexes in mRNA: A key structure for biological function.

The last several years have seen exciting advances in the understanding of the structure and function of higher-order structures of RNA. Expression levels of some specific genes were shown to be directly regulated by environmentally-responsive formation of certain secondary structures such as stem-loops and pseudoknots. Even among these noncanonical structures, RNA G-quadruplexes, which form on the regions of guanine-rich sequences in mRNA, are highly stable structures that are involved in a variety of biological processes. However, many questions regarding the biological significance of RNA G-quadruplexes remain unsettled, mainly because it is difficult to locate the structures in mRNA. This review focuses on emerging methods that locate RNA G-quadruplexes in mRNA by computational and biochemical techniques. In addition, recent reports on the biological functions of RNA G-quadruplexes are also covered to highlight their various roles in cells, such as in regulating mRNA processing and translation.

A RuO2 Nanosheet as a Novel Quencher-free Platform for the Detection of Nucleic Acids in a Homogeneous Solution.

A fluorescent dye-labeled DNA probe was adsorbed and quenched on the monolayer of RuO2 nanosheets. Significant fluorescent recovery was observed upon the addition of complementary DNA due to desorption of the probe from the surface of the RuO2 nanosheet through duplex formation. The efficiency of fluorescence recovery was higher than that for graphene oxide, which was known as a quencher-free platform for the detection of nucleic acids in a homogeneous solution.

Catalytic formation of luminescent lanthanide complexes using an entropy-driven DNA circuit.

Two DNA conjugates modified with ethylenediaminetetraacetic acid and 1,10-phenanthroline were prepared as a pair of split probes. They were designed to form a duplex with their auxiliary groups facing each other, providing a microenvironment to accommodate lanthanide ions. The luminescent signal was amplified by catalytic duplex formation based on an entropy-driven DNA circuit.

Electrochemical Molecular Beacon for Nucleic Acid Sensing in a Homogeneous Solution.

Ferrocene (Fc) and ß-cyclodextrin (ßCyD) were modified at each end of stem-loop structured DNA as an electrochemical signal generator and its quencher, respectively, to give an electrochemical molecular beacon (eMB). A relatively high efficiency of signal quenching was achieved by an inclusion complex (ßCyD ⊃ Fc) formation that was induced on the stem structure of the closed form (= stem-loop structure) of eMB. With the addition of target DNA, the structure of eMB opened to form a linear duplex, where the Fc dissociated from the ßCyD to restore its intrinsic electrochemical signal. The signal contrast of the electric current for this off/on-type sensor was high, ca. 95. This technique did not require any modification of the electrode surface, and it realized the detection of the target nucleic acids in a homogeneous solution with a high sensitivity using high-performance liquid chromatography (HPLC) equipped with electrochemical detector.

Cooperative recognition of a repetitive sequence through consecutive formation of triplex and duplex structures.

Cooperative recognition of a repetitive sequence was performed with a short single DNA strand consisting of duplex- and triplex-forming regions modified with a ligand (benzoquinoquinoxaline) to stabilize a triplex structure. The former region was complementary with one unit of a repetitive sequence and the latter had a sequence that can bind with a cognate duplex formed by another DNA molecule bound on an adjacent site. The DNA binding to one unit of the repetitive sequence is expected to facilitate the second binding to an adjacent unit through cooperative triplex formation. The cooperativity was confirmed by evaluation of thermal stabilities of the complexes with a series of model repetitive sequences.

Multifunctionalization of Cells with a Self-Assembling Molecule to Enhance Cell Engraftment.

Cell-based therapy is a promising approach to restoring lost functions to compromised organs. However, the issue of inefficient cell engraftment remains to be resolved. Herein, we take a chemical approach to facilitate cell engraftment by using self-assembling molecules which modify two cellular traits: cell survival and invasiveness. In this system, the self-assembling molecule induces syndecan-4 clusters on the cellular surface, leading to enhanced cell viability. Further integration with Halo-tag technology provided this self-assembly structure with matrix metalloproteinase-2 to functionalize cells with cell-invasion activity. In vivo experiments showed that the pretreated cells were able to survive injection and then penetrate and engraft into the host tissue, demonstrating that the system enhances cell engraftment. Therefore, cell-surface modification via an alliance between self-assembling molecules and ligation technologies may prove to be a promising method for cell engraftment.

Catalytic Formation of Luminescent Complex Clusters Based on Autonomous Strand Exchange Reaction of DNA.

DNAs can act as flexible interfaces for arranging particular reactant partners such as biomolecules and other functional molecules modified on DNAs in close proximity to increase their effective concentrations. Here, we focused on dynamic programmability of the DNA structure based on sequence-specific autonomous strand exchange reactions triggered by an initiator DNA, i.e., DNA circuits, to achieve a catalytic reaction providing physical and chemical signals. For analytical applications, DNA-templated formation of luminescent lanthanide (Ln) complexes was combined with the described amplification system. An appropriate microenvironment for the accommodation of a lanthanide ion [Ln(III)] was constitutively generated by ethylenediaminetetraacetic acid (a chelator) and 1,10-phenanthroline (a sensitizer) tethered to the ends of assembled DNAs to form a luminescent complex. For DNA circuits, we used hybridization chain reaction and catalytic hairpin assembly to construct linear and cruciform DNA structures, respectively, as scaffolds of Ln cluster formation. Both systems were designed for complex formation at every site where the ends of constituent DNAs faced each other on the DNA scaffolds by addition of an initiator. After optimization of the reaction conditions, amplified luminescence of a Tb(III) complex was obtained, which implies formation of a large number of complexes after addition of the initiator DNA. The formation of lanthanide complex clusters can be simply governed by the thermodynamics of duplex hybridization, which can be rationally controlled by well-established parameters such as the DNA length and sequence, concentration, temperature, and ionic strength. The emission color of the Ln cluster can be easily changed by choosing Ln ions with the desired color. The principle behind this technique is simple; therefore, it can be applied to various catalytic DNA-templated reactions by replacing lanthanide complex ligands by other functional molecules and materials.

Risk Profile and 1-Year Outcome of Newly Diagnosed Atrial Fibrillation in Japan　- Insights From GARFIELD-AF.

BACKGROUND: Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective non-interventional study of stroke prevention in patients with newly diagnosed non-valvular AF (NAVF) that is being conducted in 35 countries. Methods and Results: A total of 52,081 patients with a new diagnosis of NVAF were enrolled prospectively in GARFIELD-AF. Of these, 4859 (9.3%) were recruited in Japan (2010-2016). In cohort 1 (2010-2011), few patients were on non-vitamin K antagonist oral anticoagulants (NOAC) globally. From cohort 2 onwards (2011-2016), however, there was a rapid increase in NOAC use around the globe, especially in Japan. By the last year of enrolment (2015-2016), 67.9% of patients in Japan and 43.1% of patients globally were on NOAC±antiplatelet therapy (AP). In Japan and globally, 17.0% and 12.2% of patients, respectively, did not receive stroke prevention treatment. Few patients in Japan (5.7%) received AP only. Compared with the other countries, the unadjusted rates of all-cause mortality and major bleeding were low, while rates of stroke/systemic embolism were similar after 1 year of follow-up. CONCLUSIONS: GARFIELD-AF continues to provide important information on the homogeneity and heterogeneity of baseline characteristics and treatment patterns in patients with newly diagnosed NVAF. This diversity reflects the differences in outcomes in Japan compared with the rest of the world.

Live-cell imaging of multiple endogenous mRNAs permits the direct observation of RNA granule dynamics.

Here, we developed two pairs of high-contrast chemical probes and their RNA aptamers with distinct readout channels that permitted simultaneous live-cell imaging of endogenous ß-actin and cortactin mRNAs. Application of this technology allowed the direct observation of the formation process of stress granules, protein-RNA assemblies essential for cellular response to the environment.