A New Method to Detect Variants of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Bioluminescent Assay in Real Time (RT-LAMP-BART).

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100-200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500-3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern.

Hidden chemical order in disordered Ba7Nb4MoO20 revealed by resonant X-ray diffraction and solid-state NMR.

The chemical order and disorder of solids have a decisive influence on the material properties. There are numerous materials exhibiting chemical order/disorder of atoms with similar X-ray atomic scattering factors and similar neutron scattering lengths. It is difficult to investigate such order/disorder hidden in the data obtained from conventional diffraction methods. Herein, we quantitatively determined the Mo/Nb order in the high ion conductor Ba7Nb4MoO20 by a technique combining resonant X-ray diffraction, solid-state nuclear magnetic resonance (NMR) and first-principle calculations. NMR provided direct evidence that Mo atoms occupy only the M2 site near the intrinsically oxygen-deficient ion-conducting layer. Resonant X-ray diffraction determined the occupancy factors of Mo atoms at the M2 and other sites to be 0.50 and 0.00, respectively. These findings provide a basis for the development of ion conductors. This combined technique would open a new avenue for in-depth investigation of the hidden chemical order/disorder in materials.

Effects of switching from liraglutide to semaglutide or dulaglutide in patients with type 2 diabetes: A randomized controlled trial.

INTRODUCTION: Few studies have examined the effects of glucagon-like peptide-1 receptor agonist switching, particularly in Japanese patients. Therefore, we aimed to investigate the effects of switching from liraglutide to semaglutide or dulaglutide on blood glucose, body weight, and the occurrence of adverse effects in clinical practice. MATERIALS AND METHODS: This was an open-label, prospective, randomized, parallel-group controlled trial. Patients with type 2 diabetes treated with liraglutide (0.6 or 0.9 mg) at Yokosuka Kyosai Hospital in Japan were recruited from September 2020 to March 2022 and, after obtaining informed consent, randomly assigned to the semaglutide or dulaglutide group (1:1). Changes in the glycated hemoglobin level from baseline to weeks 8, 16, and 26 were evaluated post-treatment. RESULTS: Initially, 32 participants were enrolled, of whom 30 completed the study. Glycemic control was significantly better in the semaglutide group than in the dulaglutide group (-0.42 ± 0.49% vs -0.00 ± 0.34%, P = 0.0120). Body weight significantly decreased in the semaglutide group (-2.6 ± 3.6 kg, P = 0.0153), whereas no change was observed in the dulaglutide group (-0.1 ± 2.7 kg, P = 0.8432). We found a significant difference in body weight between the groups (P = 0.0469). The proportion of participants who reported adverse events was 75.0% and 18.8% in the semaglutide and dulaglutide groups, respectively. One patient in the semaglutide group had difficulty continuing treatment due to severe vomiting and weight loss. CONCLUSIONS: Switching from once-daily liraglutide to once-weekly semaglutide 0.5 mg significantly improved glycemic control and body weight compared with switching to once-weekly dulaglutide 0.75 mg.

Detection of SARS-CoV-2 and the L452R spike mutation using reverse transcription loop-mediated isothermal amplification plus bioluminescent assay in real-time (RT-LAMP-BART).

The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-ß-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four ß-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six ß-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four ß-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four ß-lactamases.

23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method.

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

Separated quadrupole and shift interactions of 2H NMR spectra in paramagnetic solids by asymmetric pulse sequences.

Separated pure-quadrupole (PQ) and -shift (PS) spectra of 2H nuclear magnetic resonance (NMR) of paramagnetic solids are obtained and correlated by simple pulse sequences that can acquire the full magnetization under ideal conditions. Two-dimensional NMR signals obtained using an asymmetric π-pulse-inserted quadrupole-echo (APIQE) sequence yielded separated spectra through the skew operation of an affine transform (AT) before a Fourier transform. Modified APIQE sequences that acquire whole echo signals were fabricated, and separated PQ and PS spectra were obtained by applying a combination of AT, such as rotation and skew operations, to the signal data. These methods were demonstrated for diamagnetic Zn(CD3CO2)2⋅2H2O and paramagnetic Nd(CD3CO2)3⋅1.5H2O. Further, the dynamics of the D2O molecule and [Co(D2O)6]2+ ion in paramagnetic CoSiF6⋅6D2O was analyzed based on the temperature dependence of the separated spectra.

Comparison of Lipid-Lowering Effects of Anagliptin and Miglitol in Patients With Type 2 Diabetes: A Randomized Trial.

BACKGROUND: Recently, we reported that the level of lathosterol, a cholesterol synthesis marker, was suppressed after 1 month of treatment with anagliptin, a dipeptidyl peptidase-4 inhibitor. In this study, we administered either anagliptin or miglitol, an alpha-glucosidase inhibitor, for 3 months in patients with type 2 diabetes and compared the lipid-lowering effects of anagliptin with those of miglitol. METHODS: This study was a 12-week, open-label, prospective, randomized, parallel-group comparison trial. Fifty-two patients with type 2 diabetes who aged 20 - 70 years with a low-density lipoprotein cholesterol (LDL-C) level of over 120 mg/dL, and with no history of treatment with antihyperlipidemic drugs were enrolled. Patients were randomly assigned to either the anagliptin group or miglitol group. The 100 mg of anagliptin was administered twice a day for the anagliptin group and 50 mg of miglitol was administered thrice a day for miglitol group. The changes in lipids, cholesterol synthesis, and absorption markers were evaluated after 12 weeks. RESULTS: Fifty-two participants were initially enrolled in the trial, and 47 of them completed the protocol. There was no significant difference in LDL-C, cholesterol synthesis, and the absorption markers between anagliptin and miglitol groups. CONCLUSIONS: Anagliptin and miglitol are similarly effective on lipid and glycemic control.

Separation of 2H NMR spectra assisted by molecular dynamics in diamagnetic and paramagnetic solids.

The relaxation-assisted separation method was applied to overlapping 2H nuclear magnetic resonance (NMR) spectra of diamagnetic and paramagnetic solids to separate them based on the different relaxation behaviors caused by molecular motion. Carr-Purcell-Meiboom-Gill sequences for 2H NMR were adopted to build two-dimensional data sets from one-dimensional NMR experiments. For diamagnetic α-glycine, the 2H NMR spectrum collected at 198 K was separated into two components from static -CD2- and dynamic -ND3. In addition, for the paramagnetic Sm(NO3)3·6D2O, the asymmetric 2H NMR spectra obtained at 213 K due to the hyperfine coupling was also separated into two components, which corresponded to coordinated and crystal water molecules undergoing a 180° flip motion with different correlation times.

Deuterium off-magic-angle spinning NMR spectroscopy for pure-quadrupole spectra of paramagnetic solids.

We examined a simple two-dimensional 2H nuclear magnetic resonance spectroscopy for paramagnetic solids utilizing off-magic-angle spinning (OMAS). By adding a rotor-synchronized 180° pulse to rotational echo (RE) measurement, the effect of the shift interaction was removed from the indirect dimension. The obtained pure-quadrupole spectrum could be simulated by calculating a quasi-one-dimensional NMR signal without considering the shift interaction. The sensitivity of the proposed method was compared with that of previous static NMR methods.

Molecular and electron-spin structures of a ring-shaped mixed-valence polyoxovanadate (IV, V) studied by (11)B and (23)Na solid-state NMR spectroscopy and DFT calculations.

(11)B and (23)Na solid-state nuclear magnetic resonance (NMR) spectra of ring-shaped paramagnetic crystals of H15[V7(IV)V5(V)B32O84Na4]·13H2O containing seven d(1) electrons from V(IV) were studied. Magic-angle-spinning (MAS) and multiple-quantum MAS NMR experiments were performed at moderate (9.4T) and ultrahigh magnetic fields (21.6T). The NMR parameters for quadrupole and isotropic chemical shift interactions were estimated by simulation of the NMR spectra and from relativistic density functional theory (DFT) calculations. Four Na ions incorporated into the framework were found to occupy four distinct sites with different populations. The DFT calculation showed that d(1) electrons with effectively one up-spin caused by strong antiferromagnetic interactions were delocalized over the 12V ions.

2H NMR pure-quadrupole spectra for paramagnetic solids.

We report a simple two-dimensional NMR method for obtaining (2)H NMR pure-quadrupole spectra of paramagnetic solids. This method is based on a quadrupole-echo sequence inserted with 180° pulses, where the pulse spacings are incremented asymmetrically so that the (2)H magnetization evolves only by the quadrupole interaction in the indirect dimension. It is shown that when the sequence is carried out with strong radio-frequency pulses, the spectrum projected to the indirect dimension can be simulated using the quadrupole-echo sequence without considering the effect of the paramagnetic shift. The method was also used to investigate molecular dynamics by measurement and simulation of the temperature dependence of the (2)H NMR spectra.

Electron localization of polyoxomolybdates with ε-Keggin structure studied by solid-state 95Mo NMR and DFT calculation.

We report electron localization of polyoxomolybdates with ε-Keggin structure investigated by solid-state (95)Mo NMR and DFT calculation. The polyoxomolybdates studied are the basic ε-Keggin crystals of [Me3NH]6[H2Mo12O28(OH)12{MoO3}4] · 2H2O (1), the crystals suggested to have a disordered {ε-Mo12} core of [PMo12O36(OH)4{La(H2O)2.75Cl1.25}4]·27H2O (2), and the paramagnetic Keggin crystals of [H2Mo12O30(OH)10{Ni(H2O)3}4] · 14H2O (3). The spectra of (95)Mo static NMR of these samples were measured under moderate (9.4 and 11.7 T) and ultrahigh magnetic fields (21.8 T). From spectral simulation and quantum chemical calculation, the NMR parameters of the chemical shift and quadrupole interactions for (95)Mo were estimated. By the analysis based on the result for 1, it was found for 2 that although the {ε-Mo12} core is disordered, the eight d(1) electrons in it are not completely localized on four Mo-Mo bonds. Furthermore, it was shown for 3 that the d(1) electrons are localized to make the Mo-Mo bonds, while the unpaired electrons are also almost localized on the paramagnetic Ni(II) ions.

Riluzole does not affect hippocampal synaptic plasticity and spatial memory, which are impaired by diazepam in rats.

We have previously demonstrated that riluzole has anxiolytic-like effects in rats, without affecting spontaneous alternation performance in the Y-maze test. However, the effects of riluzole on hippocampal synaptic plasticity were still unclear. In this study, we showed that bath application of riluzole did not impair long-term potentiation and long-term depression, whereas a benzodiazepine anxiolytic, diazepam, significantly impaired them. Furthermore, the acquisition of spatial memory in the Morris water maze test was impaired in diazepam-treated but not riluzole-treated rats. We thus provide further evidence for the potential usefulness of riluzole as an anxiolytic that does not cause amnesia.

Compensation of effect of field instability by reference deconvolution with phase reconstruction.

A compensation method based on reference deconvolution is developed to obtain high-resolution NMR spectra under an unstable magnetic field. It is shown that the applicability of the original deconvolution method is limited for small fluctuation, and a process what may be called phase reconstruction is proposed to compensate large field fluctuation. We demonstrate the method using a probe with a coil that can generate a fluctuation field artificially. A high-resolution 1H NMR spectrum of ethylbenzene was obtained under the unstable field after compensation with this method.

High-resolution NMR with resistive and hybrid magnets: deconvolution using a field-fluctuation signal.

A method for compensating effect of field fluctuation is examined to attain high-resolution NMR spectra with resistive and hybrid magnets. In this method, time dependence of electromotive force induced for a pickup coil attached near a sample is measured synchronously with acquisition of NMR. Observed voltage across the pickup coil is converted to field fluctuation data, which is used to deconvolute NMR signals. The feasibility of the method is studied by (79)Br MAS NMR of KBr under a 30T magnetic field of a hybrid magnet. Twenty single-scan NMR signals were accumulated after the manipulation, resulting in a high-resolution NMR spectrum.

A soliton phenomenon in langmuir monolayers of amphiphilic bistable rotaxanes.

Surface pressure-area isotherms, light scattering microscopy, and atomic force microscopy have all been used to provide information about the stabilities and dynamics of Langmuir monolayers composed of amphiphilic bistable [2]rotaxane molecules. Superstructures that have the appearance of localized mobile solitons are formed during the compression of monolayers of the [2]rotaxanes below their collapse pressures. Solitons move solely in a linear trajectory in both directions across the film, perpendicular to the compression direction, without any apparent broadening or change in their shape.

Controllable donor-acceptor neutral [2]rotaxanes.

In pursuit of a neutral bistable [2]rotaxane made up of two tetraarylmethane stoppers--both carrying one isopropyl and two tert-butyl groups located at the para positions on each of three of the four aryl rings--known to permit the slippage of the pi-electron-donating 1,5-dinaphtho[38]crown-10 (1/5DNP38C10) at the thermodynamic instigation of pi-electron-accepting recognition sites, in this case, pyromellitic diimide (PmI) and 1,4,5,8-naphthalenetetracarboxylate diimide (NpI) units separated from each other along the rod section of the rotaxane's dumbbell component, and from the para positions of the fourth aryl group of the two stoppers by pentamethylene chains, a modular approach was employed in the synthesis of the dumbbell-shaped compound NpPmD, as well as of its two degenerate counterparts, one (PmPmD) which contains two PmI units and the other (NpNpD) which contains two NpI units. The bistable [2]rotaxane NpPmR, as well as its two degenerate analogues PmPmR and NpNpR, were obtained from the corresponding dumbbell-shaped compounds NpPmD, PmPmD, and NpNpD and 1/5DNP38C10 by slippage. Dynamic 1H NMR spectroscopy in CD2Cl2 revealed that shuttling of the 1/5DNP38C10 ring occurs in NpNpR and PmPmR, with activation barriers of 277 K of 14.0 and 10.9 kcal mol(-1), respectively, reflecting a much more pronounced donor-acceptor stabilizing interaction involving the NpI units over the PmI ones. The photophysical and electrochemical properties of the three neutral [2]rotaxanes and their dumbbell-shaped precursors have also been investigated in CH2Cl2. Interactions between 1/5DNP38C10 and PmI and NpI units located within the rod section of the dumbbell components of the [2]rotaxane give rise to the appearance of charge-transfer bands, the energies of which correlate with the electron-accepting properties of the two diimide moieties. Comparison between the positions of the visible absorption bands in the three [2]rotaxanes shows that, in NpPmR, the major translational isomer is the one in which 1/5DNP38C10 encircles the NpI unit. Correlations of the reduction potentials for all the compounds studied confirm that, in this non-degenerate [2]rotaxane, one of the translational isomers predominates. Furthermore, after deactivation of the NpI unit by one-electron reduction, the 1/5DNP38C10 macrocycle moves to the PmI unit. Li+ ions have been found to strengthen the interaction between the electron-donating crown ether and the electron-accepting diimide units, particularly the PmI one. Titration experiments show that two Li+ ions are involved in the strengthening of the donor-acceptor interaction. Addition of Li+ ions to NpPmR induces the 1/5DNP38C10 macrocycle to move from the NpI to the PmI unit. The Li+-ion-promoted switching of NpPmR in a 4:1 mixture of CD2Cl2 and CD3COCD3 has also been shown by 1H NMR spectroscopy to involve the mechanical movement of the 1/5DNP38C10 macrocycle from the NpI to the PmI unit, a process that can be reversed by adding an excess of [12]crown-4 to sequester the Li+ ions.

Motion of methanol adsorbed in porous coordination polymer with paramagnetic metal ions.

Molecular motions of methanol adsorbed in 1D nanochannels of pillared-layer coordination polymer with paramagnetic metal ions have been studied by (2)H NMR together with X-ray crystallography.