Real-time monitoring of enzyme-catalyzed phosphoribosylation of anti-influenza prodrug favipiravir by time-lapse nuclear magnetic resonance spectroscopy.

Favipiravir (brand name Avigan), a widely known anti-influenza prodrug, is metabolized by endogenous enzymes of host cells to generate the active form, which exerts inhibition of viral RNA-dependent RNA polymerase activity; first, favipiravir is converted to its phosphoribosylated form, favipiravir-ribofuranosyl-5'-monophosphate (favipiravir-RMP), by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Because this phosphoribosylation reaction is the rate-determining step in the generation of the active metabolite, quantitative and real-time monitoring of the HGPRT-catalyzed reaction is essential to understanding the pharmacokinetics of favipiravir. However, assay methods enabling such monitoring have not been established. 19 F- or 31 P-based nuclear magnetic resonance (NMR) are powerful techniques for observation of intermolecular interactions, chemical reactions, and metabolism of molecules of interest, given that NMR signals of the heteronuclei sensitively reflect changes in the chemical environment of these moieties. Here, we demonstrated direct, sensitive, target-selective, nondestructive, and real-time observation of HGPRT-catalyzed conversion of favipiravir to favipiravir-RMP by performing time-lapse 19 F-NMR monitoring of the fluorine atom of favipiravir. In addition, we showed that 31 P-NMR can be used for real-time observation of the identical reaction by monitoring phosphorus atoms of the phosphoribosyl group of favipiravir-RMP and of the pyrophosphate product of that reaction. Furthermore, we demonstrated that NMR approaches permit the determination of general parameters of enzymatic activity such as Vmax and Km . This method not only can be widely employed in enzyme assays, but also may be of use in the screening and development of new favipiravir-analog antiviral prodrugs that can be phosphoribosylated more efficiently by HGPRT, which would increase the intracellular concentration of the drug's active form. The techniques demonstrated in this study would allow more detailed investigation of the pharmacokinetics of fluorinated drugs, and might significantly contribute to opening new avenues for widespread pharmaceutical studies.

Dynamic nuclear polarization with photo-excited triplet electrons using 6,13-diphenylpentacene.

Dynamic nuclear polarization with photo-excited triplet electrons (Triplet-DNP) is demonstrated using 6,13-diphenylpentacene (DPPentacene). DPPentacene is soluble in various organic solvents, while pentacene, which is used in most of the triplet-DNP experiments, has limited solubility. An enhancement factor of 81 is obtained for 1H spins in the glass of ethanol-d6 : water = 80 : 20 (w/w) doped with 0.1 mM DPPentacene at 90 K in 0.67 T.

Dissolution Dynamic Nuclear Polarization at Room Temperature Using Photoexcited Triplet Electrons.

Dissolution dynamic nuclear polarization (DNP) has recently gained attention as a method to enhance the sensitivity of liquid NMR spectroscopy and MRI. We demonstrate dissolution of the sample hyperpolarized by DNP using photoexcited triplet electrons in 0.38 T at room temperature. The achieved polarization of 0.8% is 6100 times as high as that at thermal equilibrium under the condition. The result is an important step for DNP using photoexcited triplet electrons to become widely used in chemical and biomedical research.

Dynamic Nuclear Polarization using Photoexcited Triplet Electron Spins in Eutectic Mixtures.

Dynamic nuclear polarization using photoexcited triplet electrons (Triplet-DNP) is a method to significantly enhance nuclear spin polarization even in a low magnetic field and at room temperature. Pentacene has been practically used as an efficient polarizing agent for Triplet-DNP. In this study, we demonstrate room temperature 1H and 13C hyperpolarization of eutectic mixtures of deuterated benzoic acid doped with pentacene and a target molecule such as salicylic acid, nicotinic acid, or 2-naphthoic acid. These molecules are otherwise difficult to hyperpolarize by Triplet-DNP due to the low pentacene dopabilities of these molecules. The highest 1H polarization of 1.2% has been obtained for the eutectic mixture of salicylic acid in 0.39 T. The present sample preparation is a crucial method to widen the range of applications of Triplet-DNP to chemical and biomedical analyses.

Multiplanar evaluation of radiological findings associated with acetabular dysplasia and investigation of its prevalence in an Asian population: a CT-based study.

BACKGROUND: Acetabular dysplasia (AD) is a well-known cause of osteoarthritis (OA) of the hip, with its prevalence previously determined on plain radiography. The prevalence of preexisting AD was reported as 7.3% in a patient-based Asian population. Although computed tomography (CT) could evaluate AD in multiple planes, its prevalence using multiplanar CT images has not been reported. We investigated its prevalence with CT on coronal, axial, and sagittal planes and then determined if adding the axial and sagittal planes enhanced the investigation. METHODS: We retrospectively examined 52 consecutive Japanese individuals (mean age 59.4 years) who had undergone CT for conditions unrelated to hip disorders. The inclusion criteria of CT images were (1) reconstructed axial slice thickness of ≤1 mm and (2) normal pelvic rotations and tilt. Exclusion criteria were (1) age <20 years, (2) neither hip center could be clearly detected, (3) evidence of hip OA. The parameters used to define AD on the coronal plane were the center-edge angle, Sharp angle, acetabular index, acetabular depth ratio, and acetabulum head index. The anterior and posterior acetabular sector angles were used as axial parameters and the vertical-center-anterior margin angle as the sagittal parameter. AD prevalence was calculated using multiplanar images and then compared with the previously reported Asian prevalence using 95% confidence intervals (CI). In this study, we defined "prevalence" as the proportion of subjects who had AD in at least one hip. RESULTS: The mean prevalence of AD on coronal, axial, and sagittal planes was 16.9, 15.4, and 7.7%, respectively. The lowest prevalence found by combining the three planes was 25.0% (95% CI 15.2-38.2%). This prevalence was significantly higher than that in the previously reported Asian population (7.3%). CONCLUSIONS: At the lowest estimate, the prevalence of AD evaluated in three planes was more than twice as high as the previously reported prevalence in Asians when we investigated its prevalence using multiplanar images. The prevalence of AD in the axial and sagittal planes was not negligible. We therefore suggest that it is important to add axial and sagittal planes' data when investigating the prevalence of AD.

Room temperature hyperpolarization of nuclear spins in bulk.

Dynamic nuclear polarization (DNP), a means of transferring spin polarization from electrons to nuclei, can enhance the nuclear spin polarization (hence the NMR sensitivity) in bulk materials at most 660 times for (1)H spins, using electron spins in thermal equilibrium as polarizing agents. By using electron spins in photo-excited triplet states instead, DNP can overcome the above limit. We demonstrate a (1)H spin polarization of 34%, which gives an enhancement factor of 250,000 in 0.40 T, while maintaining a bulk sample (∼ 0.6 mg, ∼ 0.7 × 0.7 × 1 mm(3)) containing >10(19) (1)H spins at room temperature. Room temperature hyperpolarization achieved with DNP using photo-excited triplet electrons has potentials to be applied to a wide range of fields, including NMR spectroscopy and MRI as well as fundamental physics.

An ab initio MO study of heavy atom effects on the zero-field splitting tensors of high-spin nitrenes: how the spin-orbit contributions are affected.

The CASSCF and the hybrid CASSCF-MRMP2 methods are applied to the calculations of spin-spin and spin-orbit contributions to the zero-field splitting tensors (D tensors) of the halogen-substituted spin-septet 2,4,6-trinitrenopyridines, focusing on the heavy atom effects on the spin-orbit term of the D tensors (D(SO) tensors). The calculations reproduced experimentally determined |D| values within an error of 15%. Halogen substitutions at the 3,5-positions are less influential in the spin-spin dipolar (D(SS)) term of 2,4,6-trinitrenopyridines, although the D(SO) terms are strongly affected by the introduction of heavier halogens. The absolute sign of the D(SO) value (D = D(ZZ) - (D(XX) + D(YY))/2) of 3,5-dibromo derivative 3 is predicted to be negative, which contradicts the Pederson-Khanna (PK) DFT result previously reported. The large negative contributions to the D(SO) value of 3 arise from the excited spin-septet states ascribed mainly to the excitations of in-plane lone pair of bromine atoms → SOMO of π nature. The importance of the excited states involving electron transitions from the lone pair orbital of the halogen atom is also confirmed in the D(SO) tensors of halogen-substituted para-phenylnitrenes. A new scheme based on the orbital region partitioning is proposed for the analysis of the D(SO) tensors as calculated by means of the PK-DFT approach.

Triplet-DNP in magnetically oriented microcrystal arrays.

We explore dynamic nuclear polarization using electron spins in the photo-excited triplet state (Triplet-DNP) in magnetically oriented microcrystal arrays (MOMAs) of pentacene-doped p-terphenyl, in which the individual crystallites are magnetically aligned and UV-cured. In contrast to the conventional approach to Triplet-DNP in powder, which suffers from reduced nuclear polarization due to the averaged electron polarization and the broadening of electron-spin resonance, Triplet-DNP of the MOMAs offers as high dynamic polarization as that attainable in single-crystals. In the case of pentacene-doped p-terphenyl, the enhanced 1H polarization in the one-dimensional MOMA, prepared simply by leaving the suspension in a stationary magnetic field before UV curation, can be higher than that attainable in the powder sample by an order of magnitude and comparable to that in single crystals and in the three-dimensional MOMA made using a modulational rotating field. Triplet-DNP of the MOMAs may find potential applications, such as the polarization of the co-doped target molecules and dissolution experiments.

Tandem arrays of TEMPO and nitronyl nitroxide radicals with designed arrangements on DNA.

Herein we describe one-dimensional electron-spin arrays consisting of two different organic radicals with the designed arrangement based on the DNA sequence. Two mismatch-binding ligands carrying 2,2,6,6-tetramethylpiperidine N-oxide (TEMPO) and nitronyl nitroxide selectively bind to the predetermined sites on double stranded DNA. By using the two mismatch-binding ligands carrying the organic radicals as the glue for DNA, electron-spin assembly could be successfully synchronized with the hybridization. Periodically and tandemly arranged, two kinds of organic radical molecules at designed positions might be useful for an approach to build up scalable qubits of electron-spin-based quantum computing. The approach using DNA nanostructures as a scaffold to assembly functional small molecules can afford one of the promising ways for the future application of DNA nanostructures and nanotechnology.

Pulsed electron spin nutation spectroscopy of weakly exchange-coupled biradicals: a general theoretical approach and determination of the spin dipolar interaction.

Weakly exchange-coupled biradicals have attracted much attention in terms of their DNP application in NMR spectroscopy for biological systems or the use of synthetic electron-spin qubits. Pulse-ESR based electron spin nutation (ESN) spectroscopy applied to biradicals is generally treated as transition moment spectroscopy from the theoretical side, illustrating that it is a powerful and facile tool to determine relatively short distances between weakly exchange-coupled electron spins. The nutation frequency as a function of the microwave irradiation strength ω(1) (angular frequency) for any cases of weakly exchange-coupled systems can be classified into three categories; D(12) (spin dipolar interaction)-driven, Δg-driven and ω(1)-driven nutation behaviour with the increasing strength of ω(1). For hetero-spin biradicals, Δg effects can be a dominating characteristic in the biradical nutation spectroscopy. Two-dimensional pulse-based electron spin nutation (2D-ESN) spectroscopy operating at the X-band can afford to determine small values of D(12) in weakly exchange-coupled biradicals in rigid glasses. The analytical expressions derived here for ω(1)-dependent nutation frequencies are based on only four electronic spin states relevant to the biradicals, while real biradical systems often have sizable hyperfine interactions. Thus, we have evaluated nuclear hyperfine effects on the nutation frequencies to check the validity of the present theoretical treatment. The experimental spin dipolar coupling of a typical TEMPO-based biradical 1, (2,2,6,6-tetra[((2)H(3))methyl]-[3,3-(2)H(2),4-(2)H(1),5,5-(2)H(2)]piperidin-N-oxyl-4-yl)(2,2,6,6-tetra[((2)H(3))methyl]-[3,3-(2)H(2),4-(2)H(1),5,5-(2)H(2),(15)N]piperidin-(15)N-oxyl-4-yl) terephthalate in a toluene glass, with a distance of 1.69 nm between the two spin sites is D(12) = -32 MHz (the effect of the exchange coupling J(12) is vanishing due to the homo-spin sites of 1, i.e.Δg = 0), while 0 < |J(12)|≦ 1.0 MHz as determined by simulating the random-orientation CW ESR spectra of 1. In addition, we have carried out Q-band pulsed ELDOR (ELectron-electron DOuble Resonance) experiments to confirm whether the obtained values for D(12) and J(12) are accurate. The distance is in a fuzzy region for the distance-measurements capability of the conventional, powerful ELDOR spectroscopy. The strong and weak points of the ESN spectroscopy with a single microwave frequency applicable to weakly exchange-coupled multi-electron systems are discussed in comparison with conventional ELDOR spectroscopy. The theoretical spin dipolar tensor and exchange interaction of the TEMPO biradical, as obtained by sophisticated quantum chemical calculations, agree with the experimental ones.

A synthetic two-spin quantum bit: g-engineered exchange-coupled biradical designed for controlled-NOT gate operations.

A quantum gate: A system of two coupled electron spins that is useful for simple quantum computing operations has been prepared by synthesis of a biradical 1 and co-crystallization with an isomorphous host molecule. The two weakly exchange-coupled quantum bits (target qubit blue and control qubit red) span four electron spin states. The electron spin transition is denoted by two black arrows.

Development of an oncolytic mammalian orthoreovirus expressing the near-infrared fluorescent protein iRFP720.

Fluorescence-guided surgery (FGS) is a useful method for removing invasive tumor tissues. For this, near-infrared (NIR) fluorescence probes are suitable for visualizing cancer cells due to their low autofluorescence, and an oncolytic mammalian orthoreovirus (MRV) expressing an NIR fluorescent protein is expected to be a novel tool for FGS. In this study, we identified the optimal insertion site of the NIR fluorescent protein gene iRFP720 (915 nt) in the MRV genome. We constructed genome plasmids for the L1, M1, and S2 segments, where a gene cassette comprising iRFP720 and T2A self-cleaving peptide was inserted in the 5' or 3' region of each segment. Through virus recovery, the recombinant MRV with the gene cassette at the M1 segment's 3' end, T3D-L(M1/3'iRFP720), was capable of replication and passaging with bright NIR fluorescence. However, the replication of T3D-L(M1/3'iRFP720) was approximately 1,000-fold lower than that of the wild-type virus. T3D-L(M1/3'iRFP720) production improved due to the transfection of a fusion-associated small transmembrane protein gene of fusogenic reovirus. Further, fluorescence signals were detected in T3D-L(M1/3'iRFP720)-infected human gastric and pancreatic cancer cells. Thus, the M1 segment's 3' end tolerates the expression of the long iRFP720 gene, which may propel the development of recombinant MRV vectors for FGS.

Cytokine Profile From the Ligamentum Flavum in Patients with Ossification of the Posterior Longitudinal Ligament in the Cervical Spine.

STUDY DESIGN: Histological, immunohistochemical, and suspension array analyses of cytokine expression in human cervical ossification of the posterior longitudinal ligament (OPLL). OBJECTIVES: The aim of this study was to determine whether changes in the cytokine profile reflect the maturation of chondrocytes and osteoblasts are associated with OPLL development. SUMMARY OF BACKGROUND DATA: OPLL progresses gradually over a prolonged period and may lead to serious spinal cord complications. However, treatment methods only include conservative therapy for neurological symptoms or surgical decompression, whereas preventive therapy for OPLL remains nonexistent. METHODS: Ligamentous samples were harvested from 24 patients with OPLL who underwent spinal surgery, and five control samples from cervical spondylotic myelo/radiculopathy patients without OPLL. Tissue sections were used for immunohistochemical studies and primary cells were cultured from the ligamentous samples for cytokine profiling. Using a suspension array system, concentrations of 27 inflammatory cytokines or growth factors were measured to generate the cytokine profiles. RESULTS: Suspension array and immunoblot analysis revealed significant increments in the levels of interleukin (IL)-6, IL-1α, basic fibroblast growth factor, and RANTES in patients with OPLL. Immunohistochemical analysis further revealed that these factors were present in mesenchymal cells within the degenerative portion of the ligamentous matrix. CONCLUSION: Our findings suggest that specific changes in the cytokine profile during ossification promote osteoblast differentiation, thereby providing new insights into OPLL pathogenesis. Moreover, this work supports the development of a new therapeutic method for preventing OPLL progression by regulating the cytokine profiles.Level of Evidence: 3.

Scalable spin amplification with a gain over a hundred.

We propose a scalable and practical implementation of spin amplification which does not require individual addressing nor a specially tailored spin network. We have demonstrated a gain of 140 in a solid-state nuclear spin system of which the spin polarization has been increased to 0.12 using dynamic nuclear polarization with photoexcited triplet electron spins. Spin amplification scalable to a higher gain opens the door to the single spin measurement for a readout of quantum computers as well as practical applications of nuclear magnetic resonance spectroscopy to infinitesimal samples which have been concealed by thermal noise.

Ab initio and DFT studies of the spin-orbit and spin-spin contributions to the zero-field splitting tensors of triplet nitrenes with aryl scaffolds.

Spin-orbit and spin-spin contributions to the zero-field splitting (ZFS) tensors (D tensors) of spin-triplet phenyl-, naphthyl-, and anthryl-nitrenes in their ground state are investigated by quantum chemical calculations, focusing on the effects of the ring size and substituted position of nitrene on the D tensor. A hybrid CASSCF/MRMP2 approach to the spin-orbit term of the D tensor (D(SO) tensor), which was recently proposed by us, has shown that the spin-orbit contribution to the entire D value, termed the ZFS parameter or fine-structure constant, is about 10% in all the arylnitrenes under study and less depends on the size and connectivity of the aryl groups. Order of the absolute values for D(SO) can be explained by the perturbation on the energy level and spatial distributions of π-SOMO through the orbital interaction between SOMO of the nitrene moiety and frontier orbitals of the aryl scaffolds. Spin-spin contribution to the D tensor (D(SS) tensor) has been calculated in terms of the McWeeny-Mizuno equation with the DFT/EPR-II spin densities. The D(SS) value calculated with the RO-B3LYP spin density agrees well with the D(Exptl) -D(SO) reference value in phenylnitrene, but agreement with the reference value gradually becomes worse as the D value decreases. Exchange-correlation functional dependence on the D(SS) tensor has been explored with standard 23 exchange-correlation functionals in both RO- and U-DFT methodologies, and the RO-HCTH/407 method gives the best agreement with the D(Exptl) -D(SO) reference value. Significant exchange-correlation functional dependence is observed in spin-delocalized systems such as 9-anthrylnitrene (6). By employing the hybrid CASSCF/MRMP2 approach and the McWeeny-Mizuno equation combined with the RO-HCTH/407/EPR-II//U-HCTH/407/6-31G* spin densities for D(SO) and D(SS), respectively, a quantitative agreement with the experiment is achieved with errors less than 10% in all the arylnitrenes under study. Guidelines to the putative approaches to D(SS) tensor calculations are given.

Incidence of Cranial Adjacent Segment Disease after Posterior Lumbar Interbody Fusion Using the Cortical Bone Trajectory Technique for the Treatment of Single-Level Degenerative Lumbar Spondylolisthesis; More than a 2-Year Follow-Up.

INTRODUCTION: Posterior lumbar interbody fusion (PLIF) is a widely used effective, safe, and established treatment for degenerative spinal disorders. Adjacent segment disease (ASD) is one of the serious concerns governing the clinical results following spinal fusion surgery. Cortical bone trajectory (CBT) is an alternative and less-invasive technique for lumbar pedicle screw placement. Its unique medial and caudal entry point has the potential to prevent an iatrogenic facet joint violence leading to the ASD; however, the incidence of ASD following PLIF using the CBT technique (CBT-PLIF) remains unknown. METHODS: Among patients surgically treated with CBT-PLIF in our institute, 52 consecutive patients (13 males, 39 females) with single-level degenerative lumbar spondylolisthesis (DLS) who were followed up for at least 24 months were exclusively enrolled. Their clinical and radiological features, including the incidence of radiographical and symptomatic ASD and significantly associated factor for the developing radiographical ASD, were retrospectively measured. RESULTS: In the present study, we could confirm significant neurological improvement and reduction of the spondylolisthesis with mean follow-up period of 43 months. Radiographical and symptomatic ASD was observed in 14 (27%) and 2 (3.8%) cases, respectively. We compared these two groups and found that the latest lumbar lordosis was significantly different between the two groups, but not in age, body mass index, and Japan Orthopaedic Association score. Two patients with symptomatic ASD required additional surgical treatment around 1 year following the initial surgery. CONCLUSIONS: The present study, even though it is preliminary, revealed that CBT-PLIF can achieve a neurological improvement and an effective reduction of spondylolisthesis for the treatment of single-level DLS. The CBT technique is capable of reducing the incidence of ASD compared with the traditional technique; however, we must keep in mind that appropriate postoperative lumbar lordosis should be achieved. Larger, longer-term follow-up studies are required to elucidate the clinical output of CBT-PLIF.

Room-temperature hyperpolarization of polycrystalline samples with optically polarized triplet electrons: pentacene or nitrogen-vacancy center in diamond?

We demonstrate room-temperature 13C hyperpolarization by dynamic nuclear polarization (DNP) using optically polarized triplet electron spins in two polycrystalline systems: pentacene-doped [carboxyl-13C] benzoic acid and microdiamonds containing nitrogen-vacancy (NV-) centers. For both samples, the integrated solid effect (ISE) is used to polarize the 13C spin system in magnetic fields of 350-400 mT. In the benzoic acid sample, the 13C spin polarization is enhanced by up to 0.12 % through direct electron-to-13C polarization transfer without performing dynamic 1H polarization followed by 1H-13C cross-polarization. In addition, the ISE has been successfully applied to polarize naturally abundant 13C spins in a microdiamond sample to 0.01 %. To characterize the buildup of the 13C polarization, we discuss the efficiencies of direct polarization transfer between the electron and 13C spins as well as that of 13C-13C spin diffusion, examining various parameters which are beneficial or detrimental for successful bulk dynamic 13C polarization.

Triple-stranded metallo-helicates addressable as Lloyd's electron spin qubits.

We have first achieved the synthesis of triple-stranded metallo-helicates composed of 4,4':2',2'':4'',4'''-quaterimidazole (Qim) and Mn(II) or Zn(II) ions, which serve as synthetic electron spin qubits (quantum bits). In the crystal structure, a hydrogen-bonding network through counteranions and/or crystal solvents was constructed by the outward N-H hydrogen-bonding functional groups intrinsic to the imidazole skeleton. Importantly, these helicates showed high stability even in a solution state at room temperature. These salient features of triple helicates of Qim are different from those of reported metallo-helicates. These chemical properties of the Qim-based triple helicates allow us to synthesize magnetically diluted single crystals composed of Mn(II) (S = 5/2) and diamagnetic Zn(II) complexes of Qim in an appropriate Mn(II)/Zn(II) ratio. The magnetically diluted crystals can afford to build up the prototype of electron-spin qubits of Lloyd's one-dimensional periodic system, which gives a practical approach to scalable quantum computers/quantum information processing systems (QCs/QIPSs). The experiments have proven the practical capability of oligo(imidazole)s as a component of Lloyd's system which has nonequivalent g-tensors within the helicate (g-engineering). The helical symmetry plays an important role in giving a prototype of the synthetic spin qubits of the formidable Lloyd model. This result links supramolecular chemistry to the field of QCs/QIPSs.

Spin-orbit contributions in high-spin nitrenes/carbenes: a hybrid CASSCF/MRMP2 study of zero-field splitting tensors.

Zero-field splitting (ZFS) tensors (D tensors) of organic high-spin oligonitrenes/oligocarbenes up to spin-septet are quantitatively determined on the basis of quantum chemical calculations. The spin-orbit contributions, D(SO) tensors are calculated in terms of a hybrid CASSCF/MRMP2 approach, which was recently proposed by us. The spin-spin counterparts, D(SS) tensors are computed based on McWeeny-Mizuno's equation in conjunction with the RODFT spin densities. The present calculations show that more than 10% of ZFS arises from spin-orbit interactions in the high-spin nitrenes under study. Contributions of spin-bearing site-site interactions are estimated with the aid of a semi-empirical model for the D tensors and found to be ca. 5% of the D(SO) tensor. The analysis of intermediate states reveal that the largest contributions to the calculated D(SO) tensors are attributed to intra-site spin flip excitations and delocalized π and π* orbitals play an important role in the inter-site spin-orbit interactions.